key: cord-0010268-5skyttto
authors: nan
title: Oral abstracts
date: 2010-06-25
journal: Vox Sang
DOI: 10.1111/j.1423-0410.2010.01343_1.x
sha: a0eb0b01b6f523b0492ef3c66889d743461d9db2
doc_id: 10268
cord_uid: 5skyttto

nan

It has become well accepted, that the immune system plays an important role in the success and/or failure of allogeneic stem cell transplantation. Modern transplantation approaches therefore try to harness the graftversus-leukemia effects to eradicate tumor cells, while at the same time trying to reduce the risk for graft-versus-host diseases and viral infections. In children with high risk leukaemia, who are devoid of any HLA-identical stem cell donor, the transplantation of megadosis (>10 · 10E6/kgBW) and fully T-cell depleted stem cell grafts has allowed to provide haploidentical stem cell transplantations from either parent. However, the incidence of graft rejection and delayed immune reconstitution has led to the further development of stem cell grafts that are depleted of CD3-T-lymphocytes and B-lymphocytes, while at the same time retaining other immune effector cells, such as monocytes and NK cells. These cells are expected to facilitate engraftment, speed up immune reconstitution and provide also anti tumour activity in vivo. Here we present the preliminary results of our pilot study in children and adolescents who were grafted from their haploidentical parents or from unrelated donors using CD3/CD19-depleted peripheral stem cell grafts. Since the recurrence of viral infections (such as e.g. CMV and EBV), as well as aspergillosis, are a major cause of post transplant mortality, the management of such complications is crucial for the success of haploidentical transplantations. Here, new technologies allow the adoptive transfer of highly purified, antigen specific donor lymphocytes. Hence, stem cell transplantation has developed to a sophisticated immune therapeutic regime that takes advantage of the different immune effectors cells provided with the graft to eradicate leukaemia and fight viral infections. The use of mesenchymal stroma/stem cells (MSC¢s) will further allow reducing the intensity of the conditioning regimen.

2C-S01-02

Oepkes D Leiden University Medical Centre, Leiden, Netherlands Fifty years ago, hemolytic disease of the newborn was one of the most common causes of perinatal mortality. Early delivery and neonatal exchange transfusions were the only treatment options, until in the 1960s, intrauterine transfusion became possible. Freda and colleagues performed open fetal surgery, transfusing the fetus using a vein in the exteriorized leg. A major breakthrough was the development of percutaneous intraperitoneal transfusion under X-ray guidance by William Liley. However, hydropic fetusus did not take up the transfused blood from the peritoneal cavity very well. In addition, the technique was not feasible before 27 weeks'gestation. In the 1980s, Charles Rodeck first described the technique of intravascular transfusion by needling the umbilical artery under direct fetoscopic guidance. Shortly afterwards, Jens Bang in Denmark and Ferdnand Daffos in France introduced fetal umbilical venous blood sampling under ultrasound guidance. In the last two decades, their approach is still used worldwide as the standard technique for intrauterine transfusions. The main indication for intrauterine blood transfusion still is fetal anemia due to red cell alloimmunization. However, in any fetal disease with severe anemia, intrauterine blood transfusion can be considered. Successful fetal treatment has been reported in human parvovirus B19 infection, massive fetomaternal hemorrhage, placental chorioangiomas, twin-to-twin transfusion syndrome, homozygous alpha-thalassemia and others. Intravenous access to the fetus also allows treatment of other conditions, including alloimmune thrombocytopenia by platelet transfusions, and cardiac arrhythmias by injecting antiarrhythmic agents. The procedure of intrauterine transfusion is identical in all these diseases, independent of the indication. In this lecture the focus will be on intrauterine transfusion for red cell alloimmunization, current technique, success rates and complications, with a brief discussion on a few particular aspects of transfusion for other reasons. Nowadays monitoring of pregnancies complicated by anti-Rh immunization is mainly performed by non-invasive techniques. Thus the high risk of boosting antibodies by invasive procedures which have been previously applied for prenatal Rh testing could be reduced significantly. However, the foetal Rh type cannot be tested with serological methods from maternal blood in early pregnancy. Instead the analysis of cell free foetal (cff) DNA sequences in maternal plasma is applied to predict the foetal Rh type according to the presence or absence of characteristic blood group polymorphisms. Most frequently a service for the diagnosis of the foetal RhD type is offered but some institutions also validated assays for the prediction of the foetal C, c and E status. The non-invasive prenatal diagnosis of the foetal Rh-type can be performed early in pregnancy. However, if the first test before week 12 of gestation reveals a Rh negative result, the test should be repeated on a second sample because during the first trimester in some individuals the concentration of cff DNA might be below the detection limit. Based on our data I suggest separating plasma from cells within 2 days after phlebotomy as a reasonable compromise between laboratory and logistic requirements. If the separation is delayed, the amount of maternal DNA is increasing dramatically due to the release of DNA from maternal leucocytes. In this lecture different DNA extraction will be discussed which have already been validated for the isolation of cff DNA. Target sequences have to be amplified and amplification products can either be analyzed with real time PCR or Matrix Assisted Laser Desorption Ionization -Time Of Flight Mass Spectrometry (MALDI-TOF). Both methods are patented. At least two exons of the RHD gene should be included in the amplification/detection method in order to detect D-variants. An assay which reacts negative with the RHD pseudogene (RHDw) should be included if the patient population does not consist predominantly of Whites or Asians. Primers specific for RHD exon 10 may lead to many false positive results in Asians and Blacks because of the relatively high frequency of RHD-CE-D hybrid alleles in these ethnic groups. Recently, the technology was improved by the introduction of methylation sensitive restriction enzymes which allow confirming the presence of fetal DNA in the absence of target sequences. As an alternative multiple polymorphisms can be investigated and the results compared with the maternal polymorphisms. The main task in the field today and in the near future is the introduction of prenatal RhD testing in all D-negative women to target anti-D immunoglobulin for the prevention of haemolytic disease of the newborn to those, who carry a D-positive foetus. Targeted Rh prophylaxis is offered as an option to pregnant women in some countries, whereas it is becoming mandatory in others. Since a RHD-specific international reference material has been established and regular workshops have been implemented by ISBT members appropriate external quality control is already available for such a new screening on high-throughput. The fetal and neonatal alloimmune thrombocytopenia results from maternal immunization against a specific platelet alloantigen paternally inherited by the foetus. This syndrome is the platelet counterpart of haemolytic disease of the neonate, although it frequently affects the first infant. The incidence of NAIT has been estimated in unselected Caucasian population to be 1/800-1/1000 live births by prospective studies. The most feared complication is intracranial hemorrhage (ICH) in the setting of severe thrombocytopenia. The morbidity has been estimated to be 20% of the reported cases and mortality up to 15%. Since the first description of these conditions in the 1950's by Harrington, significant progress has been made in the laboratory diagnosis and management of this condition. During pregnancy, fetal thrombocytopenia should be suspected in a variety of circumstances: recurrent miscarriages, or ICH which may be diagnosed by sonography. In the neonate, alloimmune thrombocytopenia (NAIT) is the commonest cause of early onset isolated thrombocytopenia in an otherwise healthy newborn. NAIT is usually discovered incidentally when a full-term neonate born to a first time pregnant healthy mother has petechiae, purpura or, less frequently, overt visceral bleeding at birth or a few hours afterwards. ICH may be present at birth or can occur as long as the newborn is severely thrombocytopenic. The diagnosis of NAIT is suspected when other causes of thrombocytopenia are excluded. However, the infant may be asymptomatic, with thrombocytopenia discovered incidentally. Therefore, unexpected or unexplained neonatal thrombocytopenia or early onset of severe thrombocytopenia in both pre-term and term babies should raise the possibility of NAIT and guide investigations accordingly. The risk of lifethreatening hemorrhage necessitates prompt diagnosis and effective therapy. The laboratory diagnosis is of utmost importance for the management of the affected infant and the subsequent pregnancies. The diagnosis is straightforward when a maternal alloantibody is detected directed against the offending antigen present in the infant. The molecular basis of the platelet alloantigens has been elucidated and a number of genotyping methods have been developed: PCR-RFLP, PCR-SSP, real time PCR and more recently microarrays. Although genotyping is widely used, unknown genetic variants may alter the results, and ethnic diversity is of importance. The detection of the alloantibodies could be challenging. Currently the most widely techniques used are the antigencapture assays with mouse monoclonal antibodies (MAIPA technique). Alloantibody heterogeneity should be taken into consideration. Detection of alloantibodies directed against low frequency antigens is somehow more complicated. New techniques are developed to overcome these problems. When the diagnosis is equivocal, retesting is recommended with new samples. Difficulties in laboratory diagnosis should not delay therapy when there is bleeding tendency or severe thrombocytopenia. Severe neonatal thrombocytopenia necessitates platelet transfusions. Antenatal management has been developed due to the high rate of recurrence for subsequent incompatible fetuses, with usually a more severe condition. Maternal therapy with weekly IVIG with or without corticosteroids and reduction of invasive procedures are considered as first line approach. A patient who has the onset of fever, rash, diarrhea, and pancytopenia within days or weeks of a transfusion should be considered to have Transfusion-associated Graft-versus Host Disease (T-A G-v-H D) until proven otherwise. The diagnosis may be assisted by skin biopsy of the rash but finding different HLA antigens in the blood from the HLA antigens of other tissues, e.g. bucal smear, makes the case for T-A G-v-H D. As T-A Gv-H D is usually lethal, prevention is the key. Patients at risk of T-A G-v-H D may be immunodeficient or immunocompetent. The immunodeficiency is usually cellular, or combined, and may be congenital or acquired. However, HIV infection, even with full blown AIDS, does not place a patient at risk for T-A G-v-H D. Groups of patients at risk of T-A G-v-H D include neonates, especially those who are premature and have had intrauterine and/or exchange transfusions at birth. Congenital immunodeficiency states like Severe Combined Immunodeficiency Disease (SCID), Wiskott-Aldrich Syndrome, and Nezeloff's Syndrome place patients at risk. Hematologic malignancies like acute leukemia and lymphomas place patients at risk due to their disease as well as the therapy they receive. After any type of stem cell transplant, patients are at risk due to their ongoing immunosuppression to keep the new marrow from being rejected. When patients receive HLA-matched components, this places them at increased risk, but especially if the HLA-matched components are from blood relatives or populations with limited HLA diversity, e.g. Japanese. Donors with homozygous HLA alleles in common with a patient, present the highest risk of engraftment of stem cells in the transfusion.. Patients at very minimal or only theoretical risk include those with aplastic anemia, unless immunosuppressed, and those post organ transplants like kidneys and livers. There are four approaches to prevention of T-A G-v-H D: leukoreduction, pathogen inactivation, irradiation, and the use of only autologous units. Leukoreduction reduces but does not eliminate the risk. Pathogen inactivation, e.g. using a chemical plus ultraviolet light, where approved, obviates the need for irradiation or leukoreduction. Irradiation is the standard way to reduce effectively the risk of T-A G-v-H D. Cobalt 60 and Cesium 137 sources are the basis for gamma irradiation but now X-ray irradiators are becoming increasingly used. A conversion from a Cesium gamma irradiator to an X-ray one is expensive, plus X-ray irradiators require more maintainance, but not a radioactive materials license (at least in the USA). One must ensure that irradiation is carried out to prevent T-A G-v-H D. Simple labels are available for both gamma and X-ray irradiators and for ultraviolet lights used in pathogen inactivation. In essence, T-A G-v-H D must be prevented and irradiation of cellular components for at risk patients should be performed, unless a pathogen inactivation process is in place. Appropriate treatment of bleeding patients with complex hemostatic defects requires both, adequate transfusion of hemotherapeutics and close monitoring of their effects. While improvement or restoration of oxygen transport upon replacement therapy with packed red blood cells (RBC) is easy to examine in anemic patients, administration of fresh frozen plasma (FFP), prothrombin complex and/or coagulation factor concentrates, and platelet units requires close control. This is best achieved by longitudinal laboratory assessment within short-term intervals. Screening tests of coagulation, i.e. prothrombin time (PT), activated partial thromboplastine time (APTT), fibrinogen, and as measure of fibrin cross-linking such as D-dimer/fibrin degradation product should be performed together with platelet counting. In addition, evaluation of whole blood platelet responses using point-of-care (POC) methods such as platelet function analysis (PFA-100) as a measure of bleeding time in vitro, impedance platelet aggregometry (Multiplate), or performing global hemostasis testing by rotational thrombelastography (ROTEM) will provide useful information to guide hemotherapy and document its effects. Thus, instead of ''empirical'' substitution, simply controlled by clinical observation, such as cessation of bleeding, a rational, efficient, and also economic hemotherapy can be achieved, replacing specifically those components that are depleted in the individual patient. This procedural approach has several advantages: (i) It strictly follows the guidelines of modern hemotherapy by administrating cellular or plasma products targeted and adjusted to the individual needs of a given patient (''as much as required, as less as needed''), (ii) prevents from overtreatment, (iii) indicates therapeutic failure, and (iv) allows early detection of imminent side-effects or complications, e.g. disseminated intravascular coagulation (DIC) in due time. Hemostasis-driven hemotherapy also requires an ongoing synopsis of clinical and laboratory findings. Thus, a comprehensive clinical follow-up is mandatory. The best way to achieve this requirement is that a clinically experienced hemostasis consultant examines the patient at the bedside and discusses clinical and laboratory results along with the therapeutic consequences directly with the physicians primarily responsible. To illustrate how these principles of hemostasis-guided hemotherapy are realized in daily clinical practice, typical cases will be presented with special emphasis on the hemotherapeutic management and laboratory monitoring. rscharf@uni-duesseldorf.de World Health Assembly ''urged all Member States to promote the development of national blood transfusion services.''''It is the responsibility of governments to ensure a safe and adequate supply of blood!''So donor association should be funded by government Replications of an efficient donor organization 1. The patients will benefit from safe treatment 2. Donors and volunteers get higher self-esteem and respect, 3 . Young people get a possibility to contribute to their society and train participation in society 4. You develop a culture of cooperation and solidarity 5. Volunteers bring resources from the outside to the blood service Sustainability Well functioning donor associations with strong networks make it possible to sustain a constant inflow of donors. New donors are found by direct personal contact. Present donors are the best to recruit new donors. Voluntary organizations are low cost = economical sustainability. Essential to have full support of your government All health ministers have subscribed to the Resolutions of the World Health Assembly. and have direct contact to national blood supplies Blood banks must be up-to-date to attract voluntary donors (necessary funding for the blood service). Legislation outlawing paid blood is also necessary Good quality and efficiency in blood services is a must Efficient service in the blood bank (elements to be given) with NO WAITING !!!! There must be no waste and minimal outdating The safety of the donor is essential with proper medical help with accidents -and insurance Donor recruitment costs money, but only direct costs need to be covered Each blood center should have a local donor association, run by volunteers, and all donors should belong to the local donor association Seek help with recruitment from patient's organizations Donors should be well informed 1. 100% correct 2. Use internet for quick information up-date 3. Have a comprehensive media approach (blood service and donor association) Be visible (examples to be given) Donors should be recognized continuously A donor organization should be modern and well run! It will also need professional staff Campaign towards young people: (examples to be given) Use local and national networks ! Take contact with other voluntary organizations as blood donation is a good side-activity. Two out of three donors are recruited by direct personal contact Why use volunteers in blood donor recruitment and retention?

1. They have networks to scout-groups, sports-organizations, tradeunions, Rotary, staff of large companies etc. 2. Bring in fellow volunteers with different prospect of society 3. Paid recruiters tend not to remain and you can not recruit by telephone! You need direct personal contact = need many people who can contact potential donors directly 4. (Paid donation gives the act of blood donation LOW status, and paid blood is unsafe) The act of blood donation should be respected, and praised by role-models Efficient work and close cooperation of government, blood bank staff and volunteer associations is the key to success in blood donor recruitment and retention! 3B-S02-03 ESTABLISHED WAYS TO KEEP DONOR'S INTEREST ALIVE a relatively small increase of BD return. It is the task of Blood donation services (BDSs) to elaborate specific and adequate measures to increase the BD¢s likelihood to return. Successful BD retention programmes are viable to ensure a sufficient supply with blood and blood components at present and the upcoming years. Aim: To give recommendations for BD retention strategies based on a survey of potential and established measures how BD¢s interest could be kept alive. Methods: With focus on the last decade, literature about internal and external influences on BD¢s intention to regular blood donation and their actual return behavior was reviewed. Furthermore, a special aspect was drawn on published articles about established or potential measures to increase BD¢s return-rate. Based on this information, different ways how BD¢s interest could be kept alive were suggested. Results: Overall, individuals of younger age (<30-40 years), females, those with a lower education level are less likely to return to blood donation. External influences of friends, family or co-workers are import for starting a BD career. To become a committed BD, however, a high level of intrinsic motivation is needed. To keep BD¢s interest alive for a long time, BDSs should focus on the following to increase the satisfaction of the BD: Make blood donation a good experience and as convenient as possible, reduce adverse events and anxiety, and train and motivate your staff. This could be further supported by an intensive and active communication with the BDs right from the start, the application of loyalty builders to establish BD identity, and the appropriate use of incentives. Finally, temporarily deferred BDs should ask to return personally and advertisement programmes for repeat BDs should appeal on personal motivation and moral norms. However, BDS should always try to adapt their measures on their target population considering that people are different all around the world. Moreover, some promotion programmes should be even tailored for distinct subgroups of BDs to have a successful outcome. Summary/conclusions: There is quite a number of ways to keep BDs interest alive and to start a career as a regular and committed BD. In this context, the self-identification as a BD is definitely of major importance. BDSs are challenged to support this developmental process. They have to make sure that blood donation is associated with a good experience for the BD making him or her feeling good and happy. Background: The immune system is educated to detect and react with foreign antigens and to tolerate self-antigen. Transfusion of blood cells and plasma and pregnancies challenge the immune system by the introduction of foreign antigens. The antigens may cause an immune response, but in many instances this is not the case and the individual is not immunised after exposure to blood group antigens. Aim: The aim of the presentation is to dissect some immune responses to blood group antigens in order to understand the mechanism of immunisation. Methods: The results of immune responses to blood group antigens can be detected by the presence of antibodies to the antigens. If the antibodies are of IgG class, the activated B cells have received help from antigen specific T cells. Both antibodies, B cells and T cells can be isolated from immunised individuals and studied in the laboratory. Also B-cell receptors and T-cell receptors as well as MHC molecules on antigen presenting cells can be studied and models of the immune synapses can be created in vitro.

The most classic immune responses in transfusion medicine and in incompatible pregnancies are immune responses to the RhD antigen on red cells, HLA class I molecules on white cells and platelets and human platelet antigens. The nature of these antigens are different; RhD antigens are part of a large complex, present on red cells from RhD positive individuals and completely lacking on red cells from RhD negative individuals. It is likely that many peptides derived from this antigen complex may stimulate T cells and B cells. HLA antigens are highly polymorphic and the antigens are known to induce strong alloimmune responses. The HPA antigens are created by one amino acid difference in allotypes based on a single nucleotide polymorphism at the genetic level. HPA 1a induce immune responses in 10% of HPA 1b homozygote pregnant women. The result of these immune responses is destruction of blood cells with clinical consequences connected to the effect of transfusions or the outcome of pregnancies. Summary/conclusions: Even though there is emerging knowledge about the immune responses to some of the blood group antigens, more information must be gained in order to understand the complete picture. The action of the innate immune response initiating the adaptive immune response to blood group antigens is not well understood. A detailed understanding of both the innate ad the adaptive part of the immune response is necessary to identify individuals at risk for immunisation and to prevent immunisation to blood group antigens.

Cartron JP Institut National de la Transfusion Sanguine, Paris cedex 15, France

In the past years, the discovery of blood groups has significantly contributed to our knowledge of human genetics. Currently, numerous studies in biochemistry, genetics and molecular biology conducted over two decades have shown that blood group gene products exhibit a variety of potential functions which can be schematically classified into functional groups such as transporters and channels, receptors, adhesion molecules, enzymes or structural components, with a further level of complexity as a same molecule may exhibit mutiple functions. This is not surprising since although many blood group molecules were ''fished out'' by their antigenic properties on the red cell surface, most are also expressed in non-erythroid tissues. As structures present on red cells, blood group and blood grouprelated molecules may contribute to the structure and function of the red cell membrane, and may help to clarify unresolved biological processes (for instance water and gas transport through biological membranes). Alternatively, these molecules may simply be the witnesses of a residual persistence of membrane components during erythroid cell differentiation with no significant function. Moreover, several are passively acquired from plasma. Investigation of blood group and blood group-related molecules as structures present on epithelial or endothelial cells of tissues and organs has revealed other features of their potential physiological function and led to the discovery of some unexpected functions (for instance Fy-mediated transcytose). Increasing our knowledge on both aspects is of interest not only for transfusion medicine, but also for transplantation and for understanding the normal physiology and physiopathology of several diseases. The biological function of blood group molecules, first based mostly on structural criteria, can now be grasped in a variety of ways, including experimental methods designed with native cells, purified components or recombinant molecules in expression systems coupled to site-directed mutagenesis. Physiological studies in humans are difficult or not feasible, but studies of genetic variants in humans and in animal models, or homologues in lower organisms, provide another way to approach blood group function in vivo. All these studies have illustrated the large diversity of functional activities reported, and the potential physiological roles of blood group and blood group-related molecules cover large areas of human physiology from cerebral to renal and reproduction biology. As specific deficiencies of these molecules have only a minor or no detrimental effect in most instances, they are either dispensable for cell function or redundant. In some instances, however, a function becomes apparent under stress or in pathological conditions. Alternatively, such molecules may represent evolutionary vestiges devoid of a significant function. How common and rare blood group polymorphisms are maintained and may impact function is largely unknown, although a few examples clearly point to selective pressure exerted by pathogenic micro-organisms. Despite significant progress, much remains to be discovered to clearly delineate how blood group molecules, alone or as molecular complexes in erythroid and non-erythroid cells, may act in health and disease, to understand the underlying mechanisms, and, ultimately, how these findings might eventually be translated into clinical applications. Background: Most blood group phenotypes are caused by polymorphisms in genes that directly encode protein antigens expressed in the red cell membrane (e.g. Rh, Kell, Duffy and Kidd systems) or in genes encoding glycosyltransferases giving rise to carbohydrate antigens (such as those in the ABO, P, Lewis and H systems). However, there are examples of individuals with unusual blood group phenotypes which cannot be explained by either of these mechanisms. These include individuals with abnormal expression of several antigens from genetically distinct blood group systems like the InLu type of Lu(a-b-) which also has weakened In b and P1 antigens and a patient with an unusual congenital dyserythropoietic anemia whose red cells are In(a-b-), Co(a-b-). In addition, rare families have been described in which a gene on the X-chromosome controls the expression of Lutheran or Kidd antigens, yet these blood group proteins are known to be encoded by genes on autosomes. Aim: To determine the molecular basis of the InLu type of Lu(a-b-) and subsequently the molecular basis of two rare phenotypes with particular features in common with InLu; the X-linked form of Lu(a-b-) and a unique form of CDA which has normal Lutheran expression but is Co(a-b-) and In(a-b-). Methods: Genomic DNA was extracted from either archived blood samples, cultured erythroblasts or EBV-transformed lymphoblastoid cell lines derived from the blood of individuals with the phenotype of interest, and from unaffected family members where possible. The proximal promoter and coding exons of the EKLF and GATA1 genes were amplified by PCR and sequenced using standard methods. Results: We find that individuals with the InLu form of Lu(a-b-) have inactivating mutations on one allele of the gene encoding the erythroid transcription factor EKLF (Singleton et al. Blood 2008 Blood ,112:2081 whilst the individual with an unusual CDA also has a mutation in EKLF but in this case the mutation is not inactivating. The CDA patient has a heterozygous G973A mutation resulting in a Glu325Lys change in a conserved DNAbinding residue (Singleton et al. Blood 2009,114:72 (abstract) ). An individual with the X-linked form of Lu(a-b-) has a mutation in the gene for the erythroid transcription factor GATA-1. This mutation occurs in the termination codon (Stop414Arg) and is predicted to result in a protein with an extra 41 amino acids at the C-terminus. (Singleton et al. Blood 2009,114 :783 (abstract)). Conclusions: These are, to our knowledge, the first descriptions of mutations in erythroid transcription factors associated with unusual blood group phenotype expression and consequently identify a new area for blood group research. Extensive examination of blood group phenotype in cases of unusual anemia may reveal subtle changes in surface antigen expression indicative of mutations in molecules involved in regulatory pathways of erythropoiesis. Our observation that different mutations in EKLF can give rise to selective loss of different blood group proteins (Lutheran glycoprotein or AQP1) suggests that mutations in regulatory molecules may provide an explanation for the many reports of transient data on the function of this molecule. While structurally homologous proteins like those of the butyrophilin family are located in the major histocompatibility complex (MHC) locus on chromosome 6, ERMAP resides on chromosome 1. Aim: To investigate whether ERMAP can modulate cytotoxic T cell activity. Methods: Recombinant soluble ERMAP was expressed in HEK cells and used to stimulate primary CD8+ T cells alone or in combination with IL-2. The cytokine secretion profile of CD8+ T cells was characterized in presence and absence of ERMAP. T cell proliferation assays were performed in the presence or absence of ERMAP. Cytotoxic assays were performed using allogeneic B-lymphoblastoid cell line (B-LCL) cells as target cells and nonstimulated CD8+ T cells or CD8+ T cells pre-stimulated with ERMAP as effector cells.

Results: Stimulation of CD8+ T cells with ERMAP plus IL-2 caused a significant increase of IFN-gamma secretion (1098.4 pg/ml) in comparison with IL-2 stimulation alone (456.4 pg/ml). Stimulation with ERMAP alone or in conjugation with IL2 induced an increase of IL-1b secretion to 38.7 pg/ml and 37.19 pg/ml respectively, in comparison to IL-2 alone (13.2 pg/ml). IL-5 secretion strongly increased to 107.4 pg/ml upon IL-2 plus ERMAP stimulation in comparison to 7.5 pg/ml produced by CD8+ T cells stimulated with IL-2 only. ERMAP stimulation also led to five-fold higher secretion of IL-6 (13.9 pg/ml) in CD8+ T cells than stimulation with IL-2 (2.4 pg/ml). The proliferation rate of CD8+ T cells was increased by up to 30% upon ERMAP stimulation compared to non-stimulated CD8+ T cells. Moreover, in the presence of ERMAP the cytotoxic capacity of CD8+ T cells against allogeneic B-LCL cells increased by up to 50% compared to non-stimulated CD8+ T cells. Accordingly, a five-fold upregulation of Granzyme B mRNA levels was observed in the effector cells after ERMAP stimulation.

Conclusions: This study demonstrates for the first time that ERMAP induces a potent cytotoxic T cell response, identifying ERMAP as an important effector molecule in cell-based immunity.

by PNGaseF treatment) were vizualized by immunoblotting. Immunoprecipates of non-biotinylated cell surface proteins where tryptically digested. Peptides where purified by C18 pipette tips and then separated by a reverse phase C18 column and analyzed by linear ion trap fourier transform ion cyclotron resonance mass spectrometry (LTQ-FTICR-MS). Proteins were identified by searching MS/MS spectra against filtered human entries in the SwissProt database. Subsequently, nonsynonymous mutations in the gene SLC44A2 encoding choline transporter-like protein 2 (CTL2) were examined by sequencespecific PCR with DNA of 54 individuals serologically typed for HNA-3a. A population study of 3700 individuals was performed by DNA micoarray analyses. Parts of the first extracellular domain of CTL2 were heterologous expressed as GST-fusion-proteins in E. coli BL21 and purified with glutathion sepharose. Fusion-proteins were used for affinity chromatography of HNA-3a antibodies from human plasma. Results: HNA-3a was found to be expressed on CTL2. Ten CTL2-peptides were identified and were only present in the HNA-3a precipitates but not in the control precipitates. CTL2 is a 80-97 kDa glycoprotein predicted to have ten transmembrane domains and three N-linked glycosylation sites. The protein is reduced to 64 kDa after deglycosylation.The single nucleotide polymorphism (SNP) 461 G>A resulting in an amino acid substitution from arginine to glutamine at position 154 was fully concordant with the HNA-3a/b phenotypes of 54 individuals. The SNP is located on the first extracellular domain of CTL2. Of 3200 tested individuals, 176 (4.8%) were homozygous for A461 (HNA-3b), 1188 (32.1%) were heterozygous (AG461), and 2336 (63.1%) were homozygous for G461 (HNA-3a). Heterologously expressed CTL2-fragments bind HNA-3a alloantibodies in immunoblot-and enzyme-linked immunosorbent assays. By fluorescence activated cell sorting (FACS) analyses it could also be shown, that by these fragments affinity purified HNA-3a antibodies bind to granulocytes, monocytes and lymphocytes only of HNA-3a positive individuals. Conclusions: The molecular characterization and recombinant expression of the HNA-3a antigen provides the basis for large scale screening of blood donors for HNA-3a antibodies. This will lead to an improvement in transfusion safety. Background: TACO, characterized by new respiratory distress and hydrostatic pulmonary edema within 6 h after transfusion, is a serious, potentially fatal condition. Its incidence has been reported as high as 8 per 100 transfused patients (Bierbaum et al. J Bone Joint Surg Am 1999) and it has been linked with very young and old age as well as preexisting cardiac conditions. Despite substantial overall morbidity attributable to its frequency, TACO has been the subject of relatively little research. Aim: This study aims to augment the current body of knowledge about TACO. We sought to identify clinically relevant risk factors and determine the mortality attributable to TACO. Methods: As part of a case-control study of TRALI conducted at University of California San Francisco (UCSF) and Mayo Clinic, Rochester (MN) hospitals, 82 patients with severe TACO were identified during a two-step, active surveillance process that linked transfusion status with arterial blood gas data (Finlay et al. Am J Clin Pathol 2005) . From among all transfused patients without hypoxemia, we enrolled 164 controls into number of blood units transfused strata defined by TRALI cases. Relevant data on subjects and clinical care were collected via chart review; data on infusion rates were unavailable. We calculated odds ratios (OR) and 95% confidence intervals adjusting for site and number of blood units transfused using logistic regression. Results: Both cases and controls had similar distributions of age, prior hospital length of stay and number of units transfused, but TACO cases were more likely to be female and on surgical services. Female gender (OR = 2.1), past history of CHF (OR = 5.6), history of hemodialysis (OR = 3.5), recent surgery (OR = 2.3), mechanical ventilation before transfusion (OR = 2.7), recent administration of vasopressors (OR = 9.7) were all significantly associated with TACO (all 95% CI excluded unity). Age and body mass index appeared to have little association with TACO. After adjustment for age, sex, hospital and transfusion intensity, TACO was associated with more than three-fold increased hospital mortality compared to controls (OR = 3.80, 95% CI 1.39-10.20). Conclusions: Risk factors for TACO include female sex, history of cardiac disease, chronic renal failure, recent surgery, mechanical ventilation and vasopressor use. These data, if confirmed by other studies, could be used to construct predictive algorithms for TACO. Blood product dose or infusion rate could then be reduced in high risk patients. Supported by NHLBI grant P50-HL-81027. The practice of transfusion medicine has become complex in recent decades and despite the technical progress, transfusions are not without risk and may trigger acute reactions. Haemovigilance consists of voluntary reporting transfusion reactions in order to prevent their recurrence and to lower the risk associated with transfusions. The University Hospital Complex of UNICAMP is formed from three units with high complexity of medical care, totaling about 550 beds. The Blood Center of Campinas (BCC) ensures the blood supply, monitors the transfusions and coordinates the local Haemovigilance System. This study aims to evaluate the prevalence and characteristics of acute transfusion reactions (ATR) that was reported and unreported. The communications of ATR is spontaneous and made by the nurse and/or physician attendees. All ATR reported are evaluated immediately by a hematologist and if necessary laboratory investigation is carried out according to established protocol. A preliminary classification according to the established conventions of the AABB was made and after the second and final classification was repeated by only one of authors (MAC) based on registered records. We evaluated the transfusion of blood products in patients admitted in hospital complex during 2007 and 2008 (24 months) using a visit by authors (TG, RADN, KAP) in the after day of transfusion, with evaluation of medical and nursing records and, if possible, direct questioning the patient about the occurrence of signs or symptoms during or up to 4 h after transfusion which could be characterized as transfusion reaction. A total of 52,135 blood components were transfused during the study period, in which 34,815 (66.8%) were considered evaluable (excluding transfusions during surgical procedures). Of these, 16,945 (48.7%) events were assessed and information about presence or absence of signs or symptoms related to transfusion has been identified. During the study period, 208 events were reported spontaneously (0.40% of transfusions) with: 144 febrile non-hemolytic transfusion reactions (FNHTR) (0.28%), 48 urticarial reactions (UR) (0.09%), seven anaphylactic reactions (AR) (0.01%) and eight other types (0.02%). No transfusion associated circulatory overload (TACO) was reported spontaneously in this period. A total of 146 ATR (0.28% of transfusions) were identified that had not been reported spontaneously with: 84 FNHTR (0.16%), 26 UR (0.05%), seven AR (0.01%), 28 TACO (0.05%) and one other. Therefore, the prevalence of ATR identified with the deployment of active haemovig-ilance is 0.68% of transfusions. Noting, TACO was not reported spontaneously. In conclusion, underreporting of ATR is a reality in our service, with the majority of cases of TACO. The active search for ATR is feasible and has the potential to develop actions aimed at improving transfusion safety by allowing the dissemination of the concepts of vigilance in health and encouraging the communication of ATR improving the management of risks associated with transfusions. Background: Granulocyte associated antibodies in blood components can be a potential cause for severe pulmonary transfusion reactions (TRALI). The main problem in the investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence (GIFT) and granulocyte agglutination test (GAT) is time consuming. Especially the large number of test cells required for an investigation asked for a different approach. The novel Flow-GIFT method, (flow cytometric granulocyte immunofluorescence) allows a rapid detection of granulocyte antibodies, marked by an automation in pipetting of samples and flow cytometry. The aim of the study was to show the distribution of granulocyte antibodies in a general blood donor population using the automated Flow-GIFT method. Material and methods: We investigated 4349 sera from female (n = 2488, 57%) and male (n = 1861, 43%) whole blood donors. 35% of the female blood donors had a history of pregnancy. Leucocytes from two HNA-and HLA-typed donors were isolated using cell sedimentation in a ficoll density gradient. For testing sera were incubated with isolated test cells in 96-deep well plates. Antibody binding to test cells was detected using FITC-conjugated antibodies and analysed on the flow cytometer FC 500 MPL. 7-AAD was used to exclude dead cells. All pipetting steps were automated using the Biomek NXp Workstation. Positive sera were retested by standard GIFT and GAT as reference methods. For the detection of HLA class I and II IgG antibodies, AB screen ELISA assay was used. Results: In 432 (28.3%) of 1528 females with a history of pregnancy, specific antibodies against granulocyte-antigens (n = 20; 1.31%), HLA class I (n = 156; 10.2%), HLA class II (n = 107; 7%) and both HLA class I as well as class II (n = 149; 9.8%) could be detected. The granulocyte-antibodies in females with history of pregnancy were determined as anti-HNA-1b (n = 3), anti-HNA-2a (n = 5), anti-HNA-3a (n = 3); anti-CD16 (n = 3), anti-CD11a (n = 2), four antibodies had unclear specificity. In 4.1% (n = 39) of 960 females without history of pregnancy antibodies against granulocyte-antigens (n = 7; 0.73%) with anti-HNA-1b (n = 1), anti-CD16 (n = 2), anti-CD177 (n = 2), anti-CD11a (n = 2), HLA class I (n = 24; 2.5%) and HLA class II (n = 8; 0.83%) could be found. Notably, in 2.2% (n = 41) of 1861 males granulocyte antibodies (n = 11; 0.6%) with anti-CD177 (n = 7), anti-CD11a (n = 3), CD16 (n = 1) and HLA class I (n = 30; 1.61%) could be also detected. Conclusions: The automated Flow-GIFT method enables a fast and feasible detection of granulocyte antibodies with the advantage of using less donor test cells in comparison to GAT and GIFT. Therefore, this high throughput method enables the screening of granulocyte antibodies in large donor populations. This will reduce or avoid immune TRALI due to transfusion of blood components. Aim: Since TRALI is a frequent cause of transfusion-associated major morbidity and mortality in the Western world, we established and compared methods for geno-and phenotyping of HNA-3a and -3b. Methods: A polymerase chain reaction using sequence-specific primers (PCR-SSP) for genotyping of HNA-3a and -3b alleles was designed. Genotyping results were compared with phenotypings in 40 individuals as well with findings in six TRALI cases associated with HNA-3a antibodies. Phenotyping was performed by granulocyte and lymphocyte immunofluorescence and granulocyte agglutination using established typing sera for HNA-3a and two recently found sera with alloantibodies directed against HNA-3b. Gene frequencies of HNA-3a and -3b in 398 randomly selected Germans were compared with gene frequencies reported by van Leeuwen et al. in 1964. Results: Genotyping and phenotyping results correlated perfectly and were in accordance with alloantibody formation and binding in TRALI cases associated with HNA-3a alloantibodies. The gene frequencies of HNA-3a and -3b were found to be 0,79 and 0,21 in the German population. Genotyping revealed 64.1% homozygous individuals for the HNA-3a allele, 5.5% for the HNA-3b allele and 30.4% heterozygous individuals. These findings are in accordance with the Hardy-Weinberg equilibrium and with the gene frequencies of 0,819 and 0,181 reported in 1964, confirming that HNA-3 is a biallelic system. Calculations suggested immunization rates of about 7% for HNA-3a and 0,5% for HNA-3b. Conclusions: The PCR-SSP method allows reliable determination of the HNA-3a and -3b genotypes. HNA-3 is a biallelic system. HNA-3a is a high frequency antigen in the European population and is more immunogenic than HNA-3b. Results: At no time point, significant differences in overall DNMT activity and DNA methylation could be detected between the groups. The proportion of methylated lymphocytes varied between 95% and 99% in the active group and between 93% and 98% in the control group. Moreover, flow cytometric analysis of lymphocyte subpopulations revealed no differences in the extent of DNA-methylation between CD3+ T-cells and CD19+ B-cells.

Conclusions: In our prospective study no evidence was found for the induction of DNMT activity or enhanced DNA-methylation after rhG-CSF administration in healthy stem cell donors. Background: Transplantation of bone marrow (BM) is an effective treatment for some non-malignant disorders like acquired or congenital bone marrow failure syndromes (BMF), hemoglobinopathies or immunodeficiencies. In the recent years there was a clear trend towards increasing use of peripheral blood progenitor cells (PBSC) instead of BM in many indications. PBSC grafts are associated with higher rates of chronic graft-versus-host disease (cGVHD). This adverse effect may be offset by lower rates of leukemia relapse in some settings. In contrast, there is no perceived benefit of cGVHD in non-malignant disorders. We performed a retrospective analysis of the DRST dataset to (i) study impact of stem cell source on both HLA-identical-sibling and matched unrelated donor (MUD) SCT and (ii) to analyze outcome after MUD compared to sibling-SCT in non-malignant indications. Results: DRST registered transplants in Germany since 1998. As of December 2009, 455 allogeneic transplants for acquired or congenital BMF, 94 transplants for hemoglobinopathies and 261 transplants for immunodeficiencies or other inborn disorders are registered in the DRST. Use of PBSC was much lower in SCT for BMF (53%), hemoglobinopathies (33%) and for immunodeficiencies and other inherited disorders (46%) as compared to AML/ALL (87% /74%). The donor type differed substantially among the various non-malignant disorders. HLA-id siblings were the donors in 46% of SCT for BMF, 52% for hemoglobinopathies and only 20% for immunodeficiencies (for comparison: AML/ ALL: 36% /29%). The influence of stem cell source and donor type is demonstrated by comparing 182 sibling-SCT and 114 MUD SCT for acquired aplastic anemia (AA). Median time between diagnosis and transplant was 98.5 days vs 511.5 days; P < 0.001; median age 28.5 years vs 30 years (P = 0.41). 5year probability of survival was 84.6% (95%-CI:79.3-90.3%) after sibling SCT and 70.1% (95%-CI:61.8-79.6%) after MUD (P < 0.003). In the period 2003-2008 2-year probability of survival was 81.6% (95%-CI:72.9-91.3%) after sibling SCT (n = 80) and 75.6 (66.1-86.5%) after MUD SCT (n = 73) (P = 0.34). After sibling SCT survival was significantly better with BM as compared to PBSCT (5-year. prob. 95.3%; 95%-CI:90.9-99.9%; n = 89 vs 74.1%, 95%-CI:65.1-84.2%; n = 92; P < 0.001) and cumulative incidence of cGvHD was significantly higher with PBSCT as compared to BM (47.0% vs 18.4%; P < 0.01). Cumulative incidence of acute GvHDII-IV did not differ significantly between BM and PBSC. In contrast, stem cells source did neither significantly affect overall survival nor cumulative incidence of acute or chronic GvHD after MUD. In multivariate analysis of sibling SCT older age (>30 years) and use of PBSC were significant risk factors for mortality (Hazard Ratio (HR) 3.4 (1. [2] [3] [4] [5] [6] [7] [8] [9] . 3 ) and HR 4.0 (1. 3-12.2) . For MUD SCT none of these variables were significant in a multivariate model. The failure to harvest a CD34+ cell target dose of 2 · 10 6 /kg for an autologous transplantation remains a significant problem for many transplant centres. Recently, Plerixafor, a chemokine receptor antagonist in combination with G-CSF has shown to be safe and more effective than placebo + G-CSF in mobilizing hematopoietic stem cells. On a namedpatient basis, Plerixafor has been used for stem cell mobilization in patients failing to mobilize by conventional means. Aim: We present our initial experience with Plerixafor in 33 mobilisation attempts in 30 patients with myeloma (n = 8), lymphoma (n = 19) or germ cell cancer (n = 3), who had previously failed to mobilize a transplantable stem cell dose. Methods: Patients had a median number of 8 (range, 3-27) chemotherapy cycles and at least one previous mobilization attempt which failed or resulted in an insufficient CD34+ cell dose for transplant. Four patients had a pre-existing CD34+ dose collected from bone marrow (range 0.16-0.89 · 10 6 /kg). As recommended, patients received four days of G-CSF at a dose of 10 lg/kg followed by Plerixafor 240 lg/kg subcutaneously, 10-11 h prior to first apheresis. G-CSF and Plerixafor were repeated daily until a defined cell dose (at least >2 · 10 6 /kg CD34+ cells) was obtained if possible. Results: The CD34 blood levels from day +4 (4th day of G-CSF, before 1st Plerixafor administration) to day +5 (after 1st Plerixafor) showed a median increase of fivefold (range, 1-12), achieving 24 (range, 5-61) CD34+ cells/ ll ( Figure 1 ). Twenty-one of 30 patients achieved a transplantable stem cell dose (median 4.15, range 2.03-8.07 · 10 6 /kg CD34+ cells) after one (n = 20) or two (n = 1) courses of G-CSF + Plerixafor mobilization cycles, resulting in a total success rate of 70%. Common side effects included abdominal discomfort (3), diarrhoea (5) , and paresthesia and (1). Thirteen patients have progressed to transplant. The median transplanted cell dose was 3.19 · 10 6 /kg CD34+ cells. All patients have achieved sustained neutrophil and platelet engraftment (median 12 days to neutrophils >0.5 G/l; median 15 days to platelets >20 G/l).

Conclusions: In our experience, Plerixafor appears highly effective in the mobilisation of PBSC for transplant from patients failing to mobilize by conventional means, with generally acceptable toxicity and a high rate of success. Background: Peripheral blood stem cell (PBSC) transplantation is performed in an increasingly older population. In this situation, related donors, if available, are preferred due to a presumed favorable risk profile compared to unrelated donors. We evaluated related PBSC donations performed between 1/1997 and 9/2009 in our clinic with regard to mobilization efficacy and side effects. Design and methods: PBSC collections from 465 healthy related (264 male, 201 female) donors were prospectively documented in a database. For PBSC mobilization, 7.5 lg/kg rhG-CSF (lenograstim) were administered for 5-6 consecutive days. Leukaphereses were performed on day 5 and 6, if necessary. 4-5 times the donor's total blood volume was processed by a continuous-flow blood cell separator (Cobe Spectra, Caridian BCT, Lakewood, Co.). Peripheral blood CD 34 counts were measured before each leukapheresis. CD 34 yield of every PBPC product was calculated in absolute numbers and per kg body weight of the recipient. Questionnaires for side effects were completed by all the donors immediately after the last apheresis. The results were compared with the data from 2775 unrelated donors who donated at the same time in our institution. Statistics: All parameters are presented as median values and range. Wilcoxon-Mann-Whithney test was performed. Results: The median peripheral CD 34 count at day 5 was 51/ll in related and 62/ll in unrelated donors (P = 0.000). CD 34 yield was 6.1 (0.7-55) · 106/kg recipient weight in the related donor group and 8.2 (0.22-142.5) in the unrelated donors (P = 0.000). A second apheresis was necessary in 30.9% of the related, but only in 21.6% of the unrelated donors. Low CD34-yields (<4 · 106/kg) were obtained in 6.6% of the unrelated, but in 16.4% of the related donors. Inadequate CD34-doses (<2 · 106/kg) were collected only in 0.6% of the unrelated but in 2.7% (n = 12) of the related donors. Bone pain due to the treatment with G-CSF was reported by 90.5% of the unrelated, but only by 75.1% of the related donors. Headache occurred in 44.5% of the unrelated and 36.5% of the related donors. During leukapheresis paresthesia were observed in 78.5% of the unrelated and 62.3% of the related donor group. Conclusions: PBPC mobilization with a dose of 7.5 lg/kg/day lenograstim proved to be effective to collect sufficient cell doses for allogeneic transplantation in most of the related donors. The peripheral CD34 counts and apheresis yields were significantly lower in related than in unrelated donors. This difference still persists if the higher proportion of women in the related donor group (43.2% vs 30% in the unrelated donors) is considered. The higher age (median 48 vs 33 years) of the related donors is the main reason for the difference in mobilisation efficacy. Side effects of G-GCF administration and leukapheresis were less pronounced in related than in unrelated donors. The risk of poor and very poor PBSC mobilization is higher in related than in unrelated donors. These data should be borne in mind during donor selection for older patients. Haemovigilance systems continue to report bacterial contamination as an important residual risk of transfusion. The spectrum of bacterial contamination ranges from fatal sepsis to detection of bacteria on routine component surveillance testing without apparent consequence. Measures introduced in recent years by blood services and hospitals have targeted multiple stages of the collection, manufacturing and transfusion processes in order to reduce the likelihood of serious clinical consequences. Interventions at the blood collection stage have included careful donor selection and health screening, improvements in skin cleansing procedures and use of diversion pouches (to avoid the initial collection volume, which may contain residual skin flora after phlebotomy site preparation, reaching the collection bag). Improvements in component manufacturing in a good manufacturing environment incorporate use of closed system kits, environmental monitoring, proper storage and handling procedures (including attention to time and temperature restrictions at all stages of the process) and product quality control testing. Some countries have adopted routine pre-release screening of platelets, promoted the use of apheresis platelets to minimise exposure to donor skin flora, and/or introduced pathogen reduction technologies designed to eliminate bacteria from the final product. Most platelet screening programs use a culturebased method, typically with very high sensitivity, designed to detect small numbers of bacteria present in the initial inoculum. However, these systems may have substantial rates of initial reactive results. It can be difficult to compare data due to differences in method and timing of sampling, volume inoculated, use of aerobic with or without anerobic culture, different hold or quarantine periods of components after testing, and definitions of confirmed positive results. Bacterial screening of platelets does not eliminate contamination risk, and false negatives do occur, sometimes with serious clinical consequences. New methods for routine screening or for use immediately prior to transfusion are in development or already available, including using flow cytometry and molecular techniques. Some examples of new testing strategies will be addressed during this presentation. Availability of international standards suitable for use in the transfusion setting will assist comparisons between laboratories and in the evaluation of new technologies. The bacterial subgroup of the ISBT working party on transfusion-transmissible infections has coordinated an international validation study on candidate blood bacteria standards, which will be presented separately during this meeting. The combination of measures to reduce bacterial contamination of blood components must also be matched by efforts to promote more appropriate and evidence-based clinical practice, and to improve awareness and management of transfusion-related sepsis. Aim: We initiated a look-back for blood donors who at post-donation information notified the blood bank of a proven C. burnetti infection after unknowingly donating whole blood within the incubation period, in order to assess the risk of blood transfusion transmission of C. burnetti. Methods: All components from whole blood donors who notified a proven C. burnetti infection (confirmed elsewhere by serology or PCR) within 3 weeks after their last blood donation were recalled. If these blood components (RBC or pooled platelet concentrates [PPC]) had been transfused to a recipient, a look-back was performed. Donors with proven C. burnetti infection were deferred from further blood donation. Results: A total of eight blood donors notified the blood bank of a recent Q-fever infection, which was confirmed elsewhere by serology only (IgM/ IgG) in six, by PCR in two and by both serology and PCR in two donors. Mean interval between last donation and first day of clinical Q-fever was 13 days (range 5-22 days). From all donors a C. burnetti PCR was performed on repository samples of their last donation: all PCRs were negative except for 1 donor. Outcome of the look-back in donors and recipients is shown in Table 1 . In six recipients C. burnetti serology and/or PCR tests were performed after transfusion; 2 of 6 (30%) had positive serology (IgG) for C. burnetti, but negative PCR. One of these recipients had recurrent positive C. burnetti serology (positive IgG, negative IgM), but no clinical signs of Q-fever infection. As this recipient lived in a region with endemic Q-fever, exposure from animal sources provides an alternative source for the positive serology. No C. burnetti serology or PCR tests were performed in this recipient before transfusion. In this case transmission of C. burnetti by transfusion therefore remained undetermined. The other recipient with positive serology for C. burnetti, was already known with a Q-fever pneumonia in the medical history before transfusion. The recipient who received blood from the donor with a positive PCR for C. burnetti in the repository sample was not tested (being a patient with terminal cancer). Background: Transfusion-transmitted bacterial infections and transfusion reactions due to bacterial contamination are still a problem in transfusion medicine, especially with platelet concentrates (PCs). The FDA approved culture methods are sensitive and reliable, however, many blood products are already transfused before the required incubation time is reached and furthermore false-negative results have been reported. In order to overcome the shortcomings of blood culture, rapid approaches are needed to identify bacterial contamination. Aim: We developed and extensively validated a highly sensitive Light-Cycler real-time assay based on generic amplification of bacteria using hybridization probes. Methods: The validation study of bacterial identification with real-time PCR has been performed based on ICH (International Conference of Harmonisation)-guidelines for analytical procedures. Half-logarithmic diluted reference material of P. aeruginosa and S. aureus was used to determine the lower limit of detection (LOD) by probit analysis. Specificity was tested with an extended panel of selected bacteria. In addition, a prospective study (n = 450) on single donor PCs screening was performed to evaluate the routine use of the real-time assay against the BacT/ALERT. Results: The 95% LOD was 5.2 (CI 10.2-3.9) cfu/ml for P. aeruginosa and 16.8 (CI 44.1-11.4) cfu/ml for S. aureus. Based on the bacterial validation panel, no significant differences in sensitivity for gram-positive and gram-negative bacteria were found. Three of 450 (0.66%) samples in the prospective study were initially culture positive and two of these samples confirmed as Propionibacterium acnes. The units indicated a BacT/ALERT positive result after 5 respectively 7 days and gave a PCR negative result. Conclusions: We conclude that the developed real-time assay is appropriate for the screening of bacterial contamination in platelet concentrates. The high sensitivity of the BacT/ALERT system could not be attained due to lower sample input. However, broad range 16 sec PCR is able to generate a result within a short time and is therefore superior to culturing methods because prevention of septic reaction is only available if the screening result is generated before product transfusion. Background: Hemovigilance to prevent transfusion of bacteria contaminated platelet concentrates is of continuing importance. The use of culture methods, as a predictive measure of bacterial contamination at the point of platelet transfusion, has not eliminated transfusion transmitted bacterial sepsis. In a recent study, a significant proportion of contaminated platelets were transfused before the first indication of a positive signal from automated culture systems. In addition to failing to provide timely results for existing day 1 testing of platelet concentrates, culture systems, with their reported variable time lag to result, cannot be applied to pre-transfusion testing or other proposed screening strategies involving late testing at day 5 of storage and beyond. Rapid detection has been identified as the solution for pre-transfusion and late testing of platelet concentrates. Aim: We have developed laser scanning technology (Bac-Detect) as a rapid detection solution for bacteria contamination which meets the requirements of speed and simplicity for platelet screening. Methods: Apheresis or pooled platelet concentrates were spiked with bacterial species, Escherichia coli, Staphylococcus epidermidis or Bacillus cereus at three concentrations (10 -2 cfu/ml, 10 -3 cfu/ml and 10 -4 cfu/ml) determined from McFarland standards. Samples (1 ml) were prepared for scanning by addition of a solution to lyse and denature platelets and cell debris. Contaminating bacteria were collected by centrifugation and labelled with a DNA intercalating fluorescent stain (Sybr green). Scanning is carried out in a proprietary micro-slide with six wells. The laser scanning cytometer uses a 488 nm laser and fluorescence filters to detect 500-530 nm wavelengths. Discrimination and enumeration of bacteria in the sample is carried out automatically by dedicated software based on discrimination of shape, size, colour, and fluorescence intensity. Samples of platelets not spiked with bacteria were used as controls. Bacterial counts in spiked and non-spiked platelets were confirmed by agar spread plate culture assays (cfu). Results: We have produced a protocol based on chemical lysis of platelets and fluorescent labelling of bacteria with a time to analysis of <30 min and scanning time of 20 sec per sample. Software discrimination of the results effectively eliminated user input in the analysis of the scanning data. A detection threshold of 100 cfu/ml resulted in no false positive results from the control platelet samples. Results of spiked platelets with Bac-Detect showed good agreement with the colony forming assay. Conclusions: Bac-Detect using laser scanning technology, provides a sensitive rapid screening method for bacterial contamination of platelets. As such, it is suitable for application in pre-transfusion and late testing protocols.

3B-S07 -Evidence based medicine 3B-S07-01

Flanagan P New Zealand Blood Service, Auckland, New Zealand Current practice in Transfusion Medicine has evolved over many years and has played an important role in supporting the evolution of modern medicine, particularly in the fields of surgery and haemato-oncology. This view is not disputed. Nonetheless close scrutiny of the evidence base suggests that there are many unanswered questions and that the evidence base for current practice in transfusion medicine is not strong. The introduction of evidence based medicine provides a real opportunity to reconsider many of our current beliefs and in doing so progress towards a better understanding of the science underpinning our everyday activities. In recent years significant progress has been made in understanding the evidence base relating to the clinical use of blood components. Systematic reviews have clarified what we know and most importantly identified a number of important questions for study. This has lead to the development of 'evidence based clinical guidelines'. Increasingly a more participative approach, involving input from clinical users of the components is being adopted. This is a positive development and will potentially lead to more informed and appropriate use of blood components. Benefit will also arise from the application of the principles of evidence based medicine in the collection and manufacturing areas of transfusion medicine. Changes in population demographics present significant challenges to blood services. Many of our current donor selection criteria have been in place for many years. Close scrutiny often reveals, at best, only limited evidence to support their continuation. Donor age, recording of blood pressure are two good examples where a more informed and evidence based approach will potentially improve donor numbers and reduce stress on shrinking donor bases. The current debate relating to blood donation by men who have had sex with other men is another example where a variation in national standards, at least in part, reflects a weak evidence base for current restrictions. Similar issues can be identified in relation to the specification for blood components. Platelet components are a good example. How do we define an acceptable platelet component? What constitutes an adult therapeutic dose of platelets? What are the most appropriate in vitro and in vivo measurements to determine clinical effectiveness? These questions become evermore important as we assess feasibility of extending platelet shelf life and the true benefits of pathogen reduction systems. Blood components currently available for clinical use undeniably play an important role in modern medical practice. Real opportunities to improve outcomes for patients however continue to exist and the application of an evidence based approach will assist in achieving this. Background: Reduction of anti-A and/or anti-B levels by plasmapheresis or immunoadsorption, combined with drug therapy, can prevent hyperacute rejection in ABO incompatible (ABOi) renal transplantation (RTx). This practice is now commonly used in many transplant centres, with good reported clinical outcomes. Clinical protocols depend on results of anti-A/B titrations, which are monitored until at a pre-defined safe level for transplantation. Current literature suggests lack of standardisation in laboratory techniques, making it difficult to transfer clinical protocols between transplant centres.

Aim: An exploratory pilot exercise was distributed by UK NEQAS with three Aims: (i) to assess the variability in titration values and methodology; (ii) to determine routine policy relating to testing and reporting antibody levels for ABOi RTx; (iii) to demonstrate the requirement for EQA in this area. Methods: Fifty-two participants were recruited into the Pilot from 15 countries. Four group O plasma samples from standard donations were distributed for titration of anti-A and anti-B against A1 and B red cells provided. Titration results and data relating to procedures and policy were collected using an on-line survey tool. Results: Forty-nine sets of results were analysed from 46 centres, 48 using titration and one flow cytometry. 41% undertake ABO titration for the purposes of ABOi RTx. In the RTx group, 80% used an indirect antiglobulin test (IAT) (25% using DTT treatment), 80% used direct agglutination at room temperature (DART), and 65% used both IAT and DART. 37% used a tube technique and 63% column agglutination technology (CAT) for DART, whilst 19% used tube and 81% CAT (20 DiaMed and 1 BioVue) for IAT. There was no standardisation within or between these techniques relating to diluents, incubation time, end-point, plasma:red cell ratio or red cell concentration. One of the titrations showed a reduction in median IAT titre with DTT treated cells. There was a wide range of reported titration values, particularly by tube technique, e.g. results for sample 1 anti-A ranged from 8 to 512 by tube DART and 16-64 by CAT DART. Numbers were generally too small to establish an association between any variable and titration value. Of 11 institutions in the RTx group which provide advice on a safe titration level for transplantation, 6 cite an IgG titre only, 3 IgG plus IgM, and 2 do not distinguish antibody class. Safe levels cited for levels of IgG, range from <4 to 16. Conclusions: Both tube and CAT techniques (mainly DiaMed) were employed for titration of anti-A and anti-B and there was no standardisation within either technique. There was a wide range of results, particularly within the tube group. There appears to be no standard clinical policy for ABOi RTx with respect to the class of antibody or the safe level reported. An EQA scheme is required to provide ongoing assessment of proficiency for this test. It may be necessary to develop some standardisation in methodology and clinical policy, in order to fully realise the benefit of EQA and to maximise efficacy and safety for patients undergoing ABOi RTx. btlp@ukneqas.org.uk Introduction: Traumatic hip fracture is a common problem in the elderly population and a frequent indication for blood transfusion. Patients are often elderly and frail. Recent reductions in the threshold for transfusion have largely been based on evidence from patients in ITU. It has been suggested that patients with hip fracture might benefit from higher Haemoglobin (Hb) through faster rehabilitation, reduced readmission rates after initial discharge, and through reduced mortality. Our policy is to transfuse if the Hb falls to <8 g/dl or if the patient is symptomatic. We reasoned that if discharge with a lower Hb led to more deaths and readmissions then we would see a lower mean discharge Hb in those patients. We therefore carried out a retrospective study of discharge Hb and the combined outcome of readmission or death. Patients and methods: All patients admitted with traumatic hip fracture during 2007 were identified from the hospital administration system (n = 316). Demographic details, dates of admission, discharge, and readmission and or death within 60 days of initial discharge were captured from the same database. We excluded patients of <60 years age, those admitted for <2 days, those who died during the first admission and those transferred to a ward other than for rehabilitation. The pathology database was used to extract all Hb levels and details of any blood transfused. Sufficient information was available on 307 patients. The NHS tracing service was used to check on deaths in the community within 60 days of discharge. Results: After exclusions 216 patients were eligible. Results are shown in the table. There was no difference (P = 0.51, 2 tailed student's t-test) in discharge Hb between patients who were discharged without readmission or death (group A; discharge Hb 11.1 g/dl) and those discharged but who died or who were readmitted (group B: 11.3 g/dl) . Nor were there any differences in admission Hb, % patients transfused or pre-transfusion Hb between groups A and B. In addition we found no differences in admission Hb and pretransfusion Hb for groups A and B combined and those excluded because of death or transfer during first admission.

We found no evidence to suggest that transfusing patients to a higher Haemoglobin in hospital after surgery for traumatic hip fracture would affect rates of death or readmission. Background: The usage of fresh frozen plasma (FFP) is unacceptably high in many european countries, this being in contrast with the endeavour of founding its use only on strict pathophysiological reasons. A clinical setting in which the use of blood products needs a more rational approach is the Obstetric and Gynaecologic area. Aim: HELLP syndrome is a severe form of multi-organ disease occurring in 4-10% of pregnant women with pre-eclampsia/eclampsia characterised by thrombocytopenia haemolysis and elevation of liver's enzymes (LDH, AST, ALT). FFP was commonly used in this clinical setting, even in the form of Plasma-Exchange (Martin,1994) . Martin (1997) showed that dexamethasone (Dex) therapy results in better maternal outcome. The benefit is more clear with i.v. high dose of corticosteroid (Martin,2003) . We evaluated the pattern of FFP usage in pregnant women having HELLP according to the treatment with and/or without dexamethasone. Methods: A retrospective study was conducted on women consecutively diagnosed as having HELLP in the period 1995-2009. FFP usage and patients' outcome were compared according to the treatment they had and the HELLP class. Since 1998 in our Institution dexamethasone has been associated to FFP infusion and since 2000 dexamethasone is considered the first line treatment, using FFP only on pathophysiological bases (e.g. occurrence of DIC A disproportionate number of laboratory errors reported to SHOT occur ''out of hours'', and in manual testing systems. However, UK NEQAS 2009 questionnaire data shows that of the 218 laboratories with access to full automation and undertaking testing outside core hours, 12 (6%) always, and 33 (15%) sometimes revert to manual testing out of hours. Of those using EI, 6% are not using full automation. 36% using EI (cf. 26% overall and 31% using full automation) omit the reverse group on patients with a previous group, with one also omitting the reverse group on new patients. SHOT data shows several incidences of inappropriate use of EI, including a case of an ABO incompatible transfusion, where EI was based on an incorrect ABO result entered manually whilst the automation / LIMS interface was down. An ideal protocol would comprise EI for all suitable patients (identified by reliable computer algorithms), with PTT meeting national guidelines, and performed on securely interfaced fully automated systems in use 24/7. Reverting to manual testing and/or manual data transfer out of hours is a significant risk, as is not performing a reverse group on a new patient. 99% of laboratories use grouping and screening techniques suitable for automation, with increasing use of SPMP antibody screening following introduction of full automation using this technology. Not all laboratories are currently in a position to implement EI, mostly due to lack of automation and/or suitable IT, or in a few cases an unsuitable case mix. It is important that any laboratory using (or considering using) EI is aware of the potential risks involved, and the serological / IT requirements to ensure safe provision of blood. The trend for increased use of EI mirrors the increased use of automation, and has so far been driven by time and staffing pressures. With laboratories working towards the UK Transfusion Collaborative recommendations, this increase is likely to continue. UK NEQAS need to continue to monitor trends and to reinforce guidance for best practice in PTT. At Blood Systems, the most serious injuries related to blood donation occur to donors who are young, who give whole blood instead of via apheresis and who have reactions after they leave the donation site. An upright donor who faints has an increased risk of injury. Designing interventions to reduce injury requires knowing when the important reactions and the injuries occur. Therefore the whole blood donation process has been studied from the time the donor enters the site to the time of venipuncture (period 1), through the donation (period 2), the time in the refreshment area (15 min post needle removal) (period 3) and until the donors have reported all reactions experienced after donation (period 4) to the blood center. The adverse event we measured in this study is loss-of-consciousness (vasovagal syncope) because this is an objective measure. It should provide a basis for comparison and this reaction may be associated with a higher likelihood of injury. The Blood Systems 2007 data base, containing 977,591 donor registrations, 536,907 complete whole blood donations and 17,606 incomplete whole blood donations was used for this study. During this time period there were 1520 episodes of VVS. The time course of vasovagal syncope events is described. Between arriving at the donation site and the venipuncture, there are very few fainting reactions. The VVS rate is 1/20,000 during period 1. During period 2 (phlebotomy-complete 540 ml whole blood donation), the VVS rate is 0.002/1000 venipunctures at the beginning of phlebotomy but increases to 0.03/1000 in the period between 1 and 2 min before needle removal. Then at the time the needle is removed the staff record a VVS rate of 0.28/1000 (approximately 11% of the total number of VVS events occur during this 1 min time period.). Forty-eight percent of reactions occurred between needle removal and 5 min post. During the 10 min after needle withdrawal, the VVS rate varies from 0.1/1000 to 0.17/1000 for each 1 min period. This rate falls over the next 5 min period to a level of 0.04/1000. The VVS rate continues to fall until the last reaction is reported at 265 min post needle withdrawal. Many donors leave the donation site by 15 min after the phlebotomy was terminated. After the donor leaves the donation site, there is no active donor surveillance; thus the prevalence of off-site syncope is probably underestimated. Seventy-eight percent of reactions occurred within 15 min, 96% within 60 min and 99% within 120 min of needle withdrawal. Among reacting donors, a higher percentage of female reactions occur late than male reactions. Ninety-five % of male reactions began within 19 min of needle withdrawal, but 50 min was required before 95% of the female reactions had begun. Eleven percent of vasovagal syncope reactions occur away from the donation site. Off-site and late VVS reactions are more common in females. Having a baseline data base allows analysis of the mechanisms of reaction and creates the possibility of assessing the impact of interventions. Tremendous progress has been made in the past 45 years with the treatment of hemophilia: from an almost untreatable disease with a short life-expectancy to a disease with an almost normal life-expectancy. Progress can also be seen in the treatment of the complications, like the development of inhibitors. Viral infections (HCV, HIV) have put their shadow over this success, but also in the treatment of these viral complications (esp. HIV) progress can be reported. Hemophilia products (plasma-derived as well as recombinant) have now a good safety profile. In some hemophilia treatment centers in the Western world, there is now a considerable number of patients above the age of 60. So, in contrast with the general picture, hemophilia is not a disease of young people anymore. However, these older patients have to deal with serious orthopedic problems due to the lack of treatment in their youth and co-morbidity due to their high age. Despite this track record, there are still a number of triggering issues to resolve. First of all, treatment of hemophilia is only an option for some 25-30 percent of the total world population and this percentage has not changed that much in the past decades. So here lies a big challenge for the respective national blood transfusion services and the pharmaceutical industry to come up with new technologies or new proposals for cooperation. Especially, the utilization of factor VIII and IX from plasma sources that are hardly used anymore in countries who made a switch from plasma to recombinant factor VIII and IX products, is a serious option. Another challenge is the need for cheaper products, especially in a period of less economic growth. Hemophilia patient groups should be the ''natural'' drivers for seeking answers for these challenges, for instance through gene therapy or transgenic animals. Therefore, they maybe should look much more to new models of product development that take place in the area of rare diseases and orphan drugs. All of the above mentioned issued will be explored in more depth in this presentation *Dr. Cees Smit has severe hemophilia A and has been the co-ordinator of the Netherlands Hemophilia Society from 1987-1998. Background: Patients with inhibitory antibodies against coagulation factor VIII (FVIII) pose a major problem in hemophilia therapy. Several strategies to bypass FVIII are in clinical use to treat acute bleedings. However, substances available so far, such as FVIIa, only have short halflives and raise safety concerns especially in a prophylactic therapy setting. Aim: Our aim is to develop FIX variants which do not require FVIII and can therefore be used as zymogene treatment alternatives. We further want to demonstrate that FIX mutein therapy is effective, safe and that FIX mutations do not pose an immunogenic risk which could prevent their applicability. Methods: In an initial in vitro screening of recombinant FIX variants, three possible muteins were identified as possible therapeutics: One single mutant protein (K265T) with 6.6% clotting activity at physiological levels (FIX antigen 100%) in FVIII deficient plasma, a triple variant (V181I/ K265T/I383V) with 17% activity in absence of FVIII, and a quintuple variant (V181I/K265T/R338A/S377W/I383V) with 22% clotting activity in absence and 1600% activity in presence of FVIII. Bypassing activity was confirmed in plasma of patients with high titers of inhibitory antibodies. The three different muteins were introduced into a non-viral vector system and stably expressed in different mouse models. Results: At FIX levels ranging from 7500 to 19000 ng/ml partial normalization of the aPTT and of blood loss following tail clip assay (1.5 and 3 mm) were observed in all three variant groups (n = 5-9 mice/group, P < 0.05-0.005), while wild-type FIX expressing mice did not differ from untreated animals. Similar results were obtained in mice with high titers of anti-FVIII antibodies. Further, the efficacy of the FIX variants was confirmed following laser induced injury of a cremaster arteriole by in vivo imaging technology. To address the thrombogenic risk, FIX was expressed in hemostatically normal mice. Below 5 lg/ml (normal FIX plasma level) no change in TAT levels was observed for the wild-type, the single, and the triple variant groups (n = 6 mice/group). Only at highest plasma levels (5 to >50 lg), elevated TAT levels were measured. In contrast, the quintuple variant with additional 16-fold higher FIX specific activity increased TAT levels already at expression levels below 5 lg/ml. None of the mice showed signs of DIC or thrombosis (drop in platelet counts, high D-dimers). Further, untreated mice and mice tolerized to wild-type FIX were challenged with wild-type and mutant FIXs. While all untreated mice exhibited a strong immune response against all forms of FIX, no immune response was observed in mice tolerant to the wild type protein. Introduction: Di-ethyl-hexyl-phthalate (DEHP) is a frequently-used plasticizer in PVC blood bag systems. An advantage is that DEHP protects the red cells from hemolysis, so they can be stored better. A side effect is that DEHP could leak into the blood product and can be converted into toxic mono-ethyl-hexyl-phthalate (MEHP). Aim: To measure the amount of DEHP in blood products, particularly those intended for pediatric use, such as pedipacks (aliquots of regular red cell concentrates: RCC) and reconstituted whole blood (WB) for exchange transfusions (RCC + plasma). Methods: The amount of DEHP was measured in pedipacks and exchange transfusions, intended for newborns and children up to 4 years old. Routinely produced pedipacks were stored in regular bags (manufacturer A and B; because Sanquin blood banks are using two different brands) and sampled directly after preparation and at day 28, 35 and 42 after donation. Exchange transfusions, prepared in routine, were sampled directly and 24 h after preparation. DEHP extraction was performed in all samples. Analysis of DEHP was performed by HPLC. These results were compared with historical DEHP data of routinely produced RCC. DEHP was also measured in FFP, immediately after thawing and during storage at 4°C. Results: In total, 12 pedipacks and four reconstitued WB for exchange transfusions were investigated. The amount of DEHP in pedipacks increased during cold storage, in bag A to significantly higher levels 24.9 ± 5.5 ppm) than in bag B (20.8 ± 2.6 ppm). These levels were comparable with adult-dose RCC from a former study, which contained 19.4 ± 2.3 ppm on Day 35 of storage. The amount of DEHP in exchange transfusions increased with 50% during 24 h of storage at 4°C from 8.3 ± 1.2 to 13.3 ± 0.5 ppm. This is faster than in pedipacks, due to better solubility of DEHP in plasma as compared to SAGM. DEHP levels in FFP were low (about 16 ppm) immediately after thawing, but showed a rapid increase during storage at 4°C, with at least 50% increase per 24 h. Conclusions: With a transfusion of 20 ml to a baby of 4 kg, the amount of DEHP transfused is 40-125 lg/kg/day (depending on the storage time). For an exchange transfusion with 300 ml reconstituted whole blood this amounts to 600 lg/kg/day. These amounts are similar (pedipacks) or lower (exchange transfusion) as reported in literature. The recently determined Tolerable Daily Intake (TDI) for DEHP is 48 lg/kg/day, but this is based on feeding rats during three generations with DEHP. The daily exposure (mainly from food) for DEHP is 2-8 lg/kg/day, thus blood transfusion is causing peak values in the exposure of DEHP. For adults in case of massive plasma transfusion (for example exchange transfusion for TTP) the amount of DEHP transfused can easily come in the mg/kg range. Background: FFP is frequently prescribed for non-bleeding patients with a minimally elevated INR (1.2-2.0) prior to an invasive diagnostic procedure or for warfarin reversal in bleeding patients, many of who have higher INR's (>3.0). Evidence based medicine does not support the use of FFP in the former situation and use of intravenous vitamin K may be more suitable in many of the latter cases. Aim: To reduce inappropriate FFP use after an education program emphasizing evidence based use of FFP followed by the enforcement of practice guidelines. Methods: An educational program consisting of lectures and printed material was initiated over a 3-year period (2000) (2001) (2002) . During this time, physicians were given information regarding appropriate use of FFP but no orders were interdicted. This was followed by a 7 year intervention and enforcement period (2003) (2004) (2005) (2006) (2007) (2008) (2009) ) in which all FFP orders were screened by the blood bank technologists for pretransfusion INR's. Physicians were advised not to give FFP if the INR was <2.0 in non-bleeding, non-warfarinized patients, and to use vitamin K either p.o. or IV if the patient was on warfarin, depending on the degree of urgency and the extent of clinical bleeding, if any. Physicians who expressed concern were referred to the Medical Director. Data were tabulated for each year for the total non-apheresis FFP units transfused, total allogeneic red cells transfused, calculation of the RBC:FFP ratio (R:P Ratio) and the number of hospital discharges linked to the mean acuity index (severity of sickness) of the discharged patients. (2000) (2001) (2002) , was not associated with any observable reduction in FFP use but after the implementation of guidelines and enforcement (2003-2009), a significant decline in FFP use is observed in the order of approximately 85%. The number of discharges showed minimal change (10% or less) from year to year and disease acuity showed a trend for sicker patients particularly in the period 2006-2009. Hence this reduction in FFP is not attributable to a change in patient volume nor patient severity of illness. No clinical complications were reported as a result of this practice change. Conclusions: Education programs, while important, appear to be of limited effectiveness in changing FFP prescribing patterns indicating that these prescribing practices are ingrained. However, a program of active prospective intervention can be dramatically effective in reducing inappropriate FFP use. The data add support to existing data that a large fraction of FFP is unnecessarily transfused.

3D-S10 -Quality control of blood components/cells 3D-S10-01 THE RED BLOOD CELL RBC PROTEOME A most memorable scene in ''Bram Stoker's Dracula''of Francis Ford Coppola sees Count Vlad II of Wallachia procl Aim: ''The blood is the life!''It is impossible to evaluate how much the Irish novelist, writer of the 1897 book the film was based upon, was influenced by the developments in blood transfusion that characterized the end of the 19th and the beginning of the 20th century, but the word ''transfusion''figures prominently in his manuscript. Past centuries had been characterized by successful blood transfusions between animals, but success in human transfusions had been limited prior to Landsteiner's discovery of the red blood cell (RBC) membrane ABO antigens (1901) that permitted the establishment of safe blood transfusions. The application of refrigeration and anticoagulants to blood storage in the 1910s then paved the way to blood banking. Since those early days, developments in the RBC field have been tightly associated with technologies that changed science, allowing researchers to home into the detail, and had a huge impact on the quality of life. Today, proteomics technologies can be used to tackle important but neglected aspects of transfusion medicine, such as determining changes at the peptide and protein level during storage of blood products. Studies have demonstrated that stored leucocyte-undepleted whole RBC populations release over hundred proteins compared to the few released by leucocyte-depleted blood units. The leucocyte-poor RBC predominantly released carbonic anhydrase and thioredoxin peroxidase B. The presence of oxygen, in this context, gives rise to extensive RBC surface modifications, which can be prevented by adding protease inhibitors. Proteins released during storage of leucocyte-depleted RBCs were found in form of microvesicles and nanovesicles enriched not only in integral membrane proteins, but specifically in oligomerized band 3, suggesting a preferential release of damaged cell components from otherwise functional RBCs. Flow cytometry was used in combination with proteomics to study storage of younger versus older RBCs. In leucocyte-depleted RBC stored for 42 days, phosphatidylserine exposure at the RBC membrane external surface did not increase and none of the surface markers studied decreased significantly, while the copy numbers of CD44, CD58, CD147 and glycophorin decreased and annexin release increased in RBC stored with leucocytes. A recent review summarizes the contribution of proteomics to transfusion medicine and puts forward a possible approach in form of a workflow for monitoring the quality of blood-based therapeutics by pro-teomics. This monitoring may be essential as more detailed analyses using state of the art mass spectrometry tools are likely to provide an even better insight into storage and environment-induced changes in the RBC that may be critical to optimize the quality of transfused RBC. Increasing our knowledge of the RBC protein make-up (low abundant proteins in particular); of their changes in health and disease and in the interplay with other blood cells/endothelial cells will also be beneficial in assessing changes in their quality, while the study of intra-species differences will be instrumental in the light of the passage from animal experimentation to clinical trials. White blood cells, particularly lymphocytes in blood components have been shown to contribute to variety of adverse reactions in recipients of blood components one of which is Transfusion Associated Graft versus Host Disease (TA-GvHD). It is especially severe for immunocompromised recipients and is caused by donor T-lymphocytes. Leukoreduction of blood components or inactivation of leukocytes is therefore necessary, depending on the type of post-transfusion reaction. Gamma-irradiation is currently used to inactivate leukocytes in blood components to prevent TA-GvHD. This study compares the effects of irradiation and PRT treatment (Mirasol* Ò ) on lymphocyte survival and inactivation in non-leukoreduced platelet concentrates (PCs). PRT treatment is a pathogen reduction technology that targets nucleic acids after exposure to riboflavin and UV-light. We analyzed 7 untreated (C), 7 PRT treated (M) and 7 irradiated (RD) platelet concentrates. Non-leukoreduced buffy coats (mean volume 65 ml) were obtained from non-remunerated donors. The PCs were prepared by pooling 15 buffy coats (ABO identical) suspended in three plasma units; the pool was then divided into three equal-weight units into comparable bags. M units were illuminated after adding 35 ml riboflavin solution. Saline solution (35 ml) was added to C and RD units. All PCs were then stored for 5 days at 22°C with agitation. Samples were analyzed on days 1, 3 and 6. The lymphocyte survival rate was determined by 7AAD (7-amino-actinomycin D staining of dead cells, Becton Dickinson) and their activation by anti-CD69-APC staining (Becton Dickinson). Samples were also stained with anti-CD45-PE antibodies to identify and gate on lymphocytes. Samples were analyzed on the Becton Dickinson Cytometer FACSCanto I. The number of dead cells did not increase during 6 days of storage in C. However, after 3 days of storage, the percentage of dead cells in M was significantly higher than in C and RD (Student t-test, P = 0.004 and P = 0.03, respectively). After 6 days, the percentage of dead cells in M was 72% vs 30% following irradiation. The percentage of 7AAD-positive cells was significantly higher compared to C samples, both in M (P = 0,001) and in RD (P = 0,004). The percentage of dead lymphocytes was also observed to be statistically higher in M than in RD samples (P = 0,001). Lymphocyte activation analysis was performed on live (7AAD-negative, CD45-positive) cells only. In all tested samples (C, M, RD), the CD69 expression was 20-40%) during 6 days of storage. On days 1 and 3, Mirasol treatment significantly reduced lymphocyte activation as shown by the ratio (test/control) of %CD69-positive cells (P = 0,004 and P = 0,001, respectively) and mean fluorescence expression intensity of CD69. Worth noting is that after 6 days of storage, lymphocyte activation was significantly higher in RD samples than in C and M (P = 0,03 and P = 0,02, respectively). After 6 days of storage, the number of dead lymphocytes significantly increased in PRT-treated PCs; two-fold compared to gamma-irradiated PCs. During 6 days of storage, lymphocyte activation decreased in PRT-treated PCs. PRT is therefore more effective for leukocyte inactivation than gamma-irradiation. Background: The hollow-fibre blood separation can be done with a disposable device for whole blood collection and following separation into plasma and red cells, using gravity force. In this work, the use of the close system ErySep Ò is presented. This device is capable of whole blood collection, whole blood leukofiltration by in-line leukofilter, and separation of blood components by hollow-fiber filter. Aim: Manufactured leukodepleted RBCs and plasma were tested for various hematological and biochemical parameters and compared with components obtained by standard centrifugation technique. Material and methods: Twenty-eight whole blood units were collected and turned into leukodepleted RBCs and plasma. Forteen units were collected, leukodepleted and separated by disposable device ErySep Ò LMB with the hollow fibre technology, 14 units were collected into blood collection set Leucoflex Macopharma with in-line leukofilter and separated by centrifugation. Both groups of RBCs were tested for hemolysis, osmolality, pH, K, P, NH3, LDH and 2,3-DPG in day 0 and day 42 after storage in additive solution. Moreover, number of leukocyte and the loss of hemoglobin during separation, were measured and calculated in day 0. In both groups of plasma, activity of F VIII was tested before and after freezing, and total protein, leucocytes, erythrocytes and platelets were measured. Results: Measured values are displayed in Table 1 and Table 2 . Background: Red Blood Cells (RBC) have special flow-affecting properties (FP) that are major determinants of blood flow: deformability, self aggregability, and adherence to vascular endothelial cells (hereafter ''adherence''). Blood-banking procedures, particularly cold-storage and cirradiation (GIr) are associated with progressive impairment of RBC FP, implying that transfusion of such blood-banked RBC (bbRBC) can introduce a circulatory risk to recipients. Therefore, procedures for reduction or reversal of blood banking-induced damage to RBC FP are very desirable. Aim: To comprehensively explore the effects of routine cold storage and c-irradiation on RBC deformability, aggregability and adherence, as well as their attenuation and/or reversal by pre-storage leuko-reduction (LR) and/ or post-storage ''rejuvenation''treatment. Methods: Human blood units were subjected to pre-storage LR, c-irradiation, or post-storage ''rejuvenation''. RBC FP were monitored before and after the above procedures as function of cold-storage duration. RBC deformability, aggregability and adherence were determined as a function of shear stress ('), using a computerized cell-flow-properties analyzer (CFA) developed in our lab, enabling the direct visual monitoring of RBC number, cell and aggregate shape, and spatial organization. The CFA image analysis provides integrative parameters of the RBC FP, and their distribution in large RBC population. RBC aggregability is determined by aggregate size distribution and its dependence on shear stress, expressing the aggregate resistance to flow-induced disaggregation. RBC deformability is determined by the elongation ratio (ER), expressing the change in cell axial ratio under shear stress, where ER = 1 (circle) for rigid cells that are not stretched under flow. RBC adherence is determined under increasing shear stress by the number of RBC that remain adherent to cultured endothelial cells at each shear stress. Results: All RBC FP were progressively altered during cold-storage, showing significant impairment as early as the second storage week. Twenty-eight days of cold-storage induced an 8-10-fold increase in aggregability, eight-fold increase in adherence, and ten-fold increase in the percent of undeformable RBC (having ER £ 1.1). GIr did not affect RBC aggregability or adherence. However, at any time point during cold-storage, GIr resulted in an immediate drastic increase in RBC rigidity, enhancing the portion of undeformable cells from 2% to 42%. Blood banking-induced impairment of RBC FP were attenuated by pre-storage LR and reversed by post-storage ''rejuvenation''. Conclusions: RBC FP appear to be especially sensitive to cold-storage, as they are impaired long before expiration date, as well as to GIr inducing a marked reduction in deformability, and these are effectively attenuated and reversed by LR and rejuvenation, respectively. The current criteria for quality control of bbRBC are their survival in recipients, while their hemodynamic behavior is ignored. Since transfusion of RBC with impaired hemodynamics facilitates circulatory disorders, this study, together with others', strongly demonstrate the need for monitoring blood-banked RBC FP and their improvement for transfusion therapy.

3D-S10-05

Shabayek S, Moftah F, Ahmed A Egyptian National Blood Transfusion Center, Cairo, Egypt Background: The Egyptian NBTS is spreading the error management culture among its staff. This is accomplished by successive training workshops on main quality concepts & error management system. taken the responsibility to report errors as some of NBTS technicians starts to report errors they detect by themselves. Conclusions: Awareness to the importance of error reporting as a tool of quality improvement is increasing among the NBTS staff. In-depth analysis of important incidents will bring greater benefits in improvement of quality system in NB TS Development of error management culture is the resultant of a variety of organizational efforts. Norms of compliance with SOPs, resources available for training and maintenance, and relations between management and NBTS Staff. Clinical trials for therapeutic angiogenesis use blood-or marrow-derived transplants containing endothelial progenitor cells (EPCs), myelomonocytic cells and mesenchymal stromal cells (MSCs) to support vascular regeneration. Safety concerns have emerged since all three cell types can also contribute to atherosclerosis. We therefore asked whether monocytes, EPCs or MSCs do in fact accumulate lipid droplets (LDs) and form foam cells in vitro as a surrogate marker for potential pro-atherogenic side effects of therapeutic angiogenesis. LD accumulation was quantified by flow cytometry, confocal laser scanning microscopy and cholesterol measurement in each of the cell types. The impact of an initial three-day pro-angiogenic culture on subsequent foam cell formation was studied to mimic a relevant setting already used in clinical trials. The phosphorylation state of intracellular signaling molecules in response to pro-angiogenic stimulation was determined to delineate the operative mechanisms and to establish a basis for interventional strategies. Foam cells developed from monocytes but not from EPCs or MSCs after pro-angiogenic induction. The mitogen-activated protein kinase (MAPK) p38 phosphorylation related to foam cell development and stress-induced stimuli was enhanced in monocytes after pro-angiogenic stimulation. MAPK p38 inhibition almost abrogated intracellular LD accumulation in vitro. These data raise serious concerns that cellular therapy with hematopoietic cell preparations containing monocytes may be counterproductive or even aggravate atherogenesis in patients with cardiovascular diseases. We therefore support the argumentation that the role of transplanted cells in the various aspects of vascular homeostasis, regeneration and therapeutic angiogenesis must be re-examined prior to further clinical trials. Background: Cell therapy with MSC is in the focus of intense clinical research. Knowledge on trafficking and homing of MSCs after ex-vivo expansion and transplantation is limited. The optimal application mode (i.v. local injection, local on scaffold) is unknown. Thus a method enabling in vivo observation of the MSCs is desirable. MRI is a method suitable for this (no ionizing radiation; high spatial resolution at near cellular level). A prerequisite is labeling of the MSCs with a MR contrast agent not influencing the phenotype, proliferation, clonogenicity and adhesion of MSCs. Methods: Ex-vivo expanded MSCs were labeled with the iron-PLLA-particles. Using transmission electron microscopy, flowcytometry, confocal laser scanning microscopy, Prussian blue staining, CYQUANT Ò prolifera-tion assay, CFU-F assays, cell adhesion under shear stress and long term cell observer studies we studied intracellular uptake, persistence, viability, proliferation, surface marker expression, trifunctional differentiation, clonogenicity, cell adhesion, and particle distribution during cell division. MR phantom studies with particle labeled MSCs were performed in a 3 Tesla MR scanner. Labeled and unlabeled (negative control) MSCs were surgically implanted subcutaneously in a male Whistar rat mixed in a collagen scaffold and also injected in PBS intramuscularly and followed up via 3 Tesla MRI up to 25 days. Results: Particles were taken up rapidly by MSCs and persisted at least up to 14 days after particle removal (mainly in endosomal / lysosomal compartment) in vitro. In MRI 96 h after particle removal 2 · 10 5 labeled MSCs could be clearly discriminated from 5 · 10 4 MSC and from unlabeled MSC in an agarose phantom. Particle-labeled MSCs maintained their differentiation potential and showed excellent viability. Standard surface markers, proliferation capacity and clonogenicity were not influenced by the labeling. No difference between labeled and unlabeled MSC was observed regarding adhesion to HUVEC under low and high shear stress in a parallel flow chamber system. In cell observer studies it could be shown that in the majority of cells particles are distributed almost equally among daughter cells. 1 · 10 6 labeled cells/ml subcutaneously implanted in rat in a collagen scaffold can be imaged with high signal intensity up to 25 days after implantation. Intramuscular injected labeled MSCs can be followed up at the injected site up to 4 days. Conclusions: Iron-PLLA particles are suitable for MSC labeling for MR imaging in vivo, as they do not change MSC behavior observed with different methods in comparison to unlabeled cells. MSCs implanted in a Whistar rat in a collagen scaffold remain stable at the implantation site whereas intramuscular injected labeled MSC in PBS leave the injection site within 4 days after injection. Further animal studies testing different MSCs, different application modes and scaffolds are currently ongoing (Supported by EU 7th FP CASCADE; N°223236 and REBORNE, No241879.) 3D-S11-04 THE ROLE OF CXCR1 AND CXCR2 RECEPTORS ON EXPANSION AND DIFFERENTIATION OF UMBILICAL CORD BLOOD CD133+ CELLS Background: Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for transplantation. However, low number of cells and delayed hematopoietic cells engraftment is a problem that limits wide use of UCB transplantation. Some studies have been shown that chemokines regulate hematopoiesis. The aim of this study was to survey of role of two chemokine receptors, CXCR1 and CXCR2 on expansion and differentiation of UCB CD133+ cells. Methods: Umbilical cord blood CD133+ cells were separated by magnetic cell sorting method. Purified CD133+ cells were cultured in a serum free medium supplemented with IL3, IL6, TPO and stem cell factor (SCF) for 7 and 12 days as control cells. To investigate the role of CXCR1 and CXCR2 inhibition on expansion and differentiation, CD133+ cells were cultured in the present of anti-CXCR1 and/or anti-CXCR2 neutralizing antibodies. Total Cell number was counted after 7 and 12 days and cell viability evaluated using dye exclusion assay and 7-AAD. Flowcytometric analysis was performed for CD41 and CD61 as megakaryocytic markers. Results: The results showed that inhibition of CXCR1 or CXCR2 receptor alone didn't have any significant effect on expansion of UCB CD133+ cells on day 7 and 12. Interestingly, total count of grown cells on day 7 and 12, significantly decreased after inhibition of CXCR1 and CXCR2 together compared with control group (P < 0.05). The mean percentage of CD41+ and CD61+ cells after treatment with anti-CXCR1 and anti-CXCR2 significantly increased (P < 0.05). Conclusions: Inhibition of CXCR1 and CXCR2 receptors simultaneously, inhibit the stimulatory effect of cytokines on ex vivo expansion of CD133+ cells but promote differentiation to megakaryocytic progenitors. Attention to the regulatory effect of CXCR1 and CXCR2 receptors on megakarypoiesis could be useful for solving complication of UCB transplantation. Background: The growth factors are biological natural mediators which present specific and powerful actions on the regulations of proliferation, death, motility and the differentiation of component cells that lead to the repair and regeneration tissues. These molecules are universal initiators of any healing process in the body. Actually, in regenerative medicine, there are some autologous therapies with platelets growth factors (PLT-AGF) in sores, this PLT-AGF can be obtained from peripheral blood by venous puncture recollected in citrate tubes, centrifuging to obtain the plasma and treated with calcium for the formation of a coat that contains it. Aim: To analyze the rate of regeneration, the efficiency and the improvement of the patient¢s sores treated in situ with activated PTL-AGF. Methods: Five male and two female consent-informed patients were included. The patients were between 55 to 91 years old (media 71.3). In this study, the process to obtain PLT-AGF was using tubes without additives and without calcium avoiding contact whit another substances, only promoting the platelet activation with heat for release of PLT-AGF. All sores was treated with 300 ll of activated PLT-AGF once a day every day direct to the sores up to the complete cicatrization. Results: The complete cicatrization of the sores measure by Visitrak system Ò was observed in a maximum of 11 weeks. The rate of regeneration account from 0.06 to 1.5 cm 2 /week and efficiency completes of 31.3% for the week 4, 25% in the week 7, 12.5% in the week 6, 12.5% in the week 9 and 12.5% in the week 11. (Image 1). Table 1 :

Conclusions: The use of growth factors obtained from heat-activated platelets, is as useful as those obtained with methods using calcium in order to form a clot of fibrin. Our technique represents by itself a cheaper, safer, faster and easier method for treatment of patients with sores. Background: Mesenchymal stem/stromal cells (MSC) are adult-derived cells that have multipotent lineage potential, an immunoprivileged status and therapeutic potential for repair of tissue injury. We have previously shown that in both healthy (unperturbed) and inflammatory (myocardial infarct model) settings only small numbers of murine MSC show specific migration and engraftment after intravenous injection. Aim: To show that enhanced chemokine receptor expression on mMSC is correlated with enhanced in vitro migration with preincubation with proinflammatory cytokines in response to CXCL12 and CCL7. Furthermore, to demonstrate that mMSC show increased specific in vivo migration after pre-incubation with pro-inflammatory cytokines in a skin biopsy/stitch injury and in a model of AMI. Methods and results: To determine if this was due to an inability to respond to chemotactic stimuli, chemokine expression was analysed at various time points by qRT-PCR in mice after acute myocardial infarction (AMI). Many chemokines were up-regulated after AMI, with the highest at 24 h being CXCL12 and CCL7. We next analysed mRNA and protein expression of MSC chemokine receptors (CRs). As we have previously found with human MSC, murine MSC had low levels of CR expression, both at the mRNA level and surface protein level. However, it was noted that the majority of chemokine receptors studied could be detected as proteins at an intracellular level. Therefore, although, MSC express specific chemokine receptors for chemokines released after AMI, they appear to be tightly regulated at both the mRNA and at the cell surface level. This seems to have functional consequences as MSC migration towards CXCL12 and CCL7 in vitro showed low migration efficiency. Using UpCellTM tissue culture plastic for gentle dissociation of MSC and by pre-treating MSC with pro-inflammatory cytokines, we found enhanced surface expression and up-regulation of mRNA for chemokine receptors CXCR4, CCR1, CCR2, CCR3 and CCR5. The chemotactic response of MSC to CXCL12 and CCL7 was significantly higher in cells pre-treated with TNF-a, IFN-c, IL-1a and IL-6, and to CCL7 was significantly higher in cells pre-treated with IFN-c, IL-1a and IL-6. In vivo, MSC pre-treated with TNF-a and IFN-c showed significantly increased migration to site of skin wound injury at 24 h (P = 0.0006) compared to untreated MSC. In the AMI model, MSC pre-treated with TNF-a showed significantly increased migration to the infarcted myocardium at 3 h (P = 0.05) compared to untreated MSC, suggesting pro-inflammatory treatment strategy for enhanced migratory efficiency to AMI. Conclusions: We have shown that MSC preferentially migrate to inflamed tissues in vivo after pre-treatment with the pro-inflammatory cytokines TNF-a and IFN-c. This may provide a means to increase the therapeutic potential of IV delivered MSC. Further experiments will be required to show if pre-treated MSC can provide therapeutic benefit. However, the relatively small numbers of engrafted MSC found so far indicate that further manipulation of MSC may still be required for functional efficacy.

3D-S12 -Hepatitis associated to blood transfusion 3D-S12-01

OCCULT HEPATITIS -STILL A RISK FOR TRANSFUSIONS?

Velati C Sondrio Hospital, Sondrio, Italy NAT testing has been firstly introduced in blood screening to identify HCV infections due to the wide capacity to reduce the window period in comparison with EIA tests. Public concern about safety in blood transfusion, particularly for the risk of infectious diseases transmission, has strongly influenced the decision to implement NAT blood screening for HCV and HIV and, in fact, the residual risk has been dramatically decreased. NAT screening for HBV has been introduced only when multiplex technology was available, only in Countries where the endemic of HBV was on medium/high level and where the screening with other serological tests, like anti-core, was impossible without impair selfsufficiency in blood procurement. The first goal of NAT testing introduction in blood screening was to increase the sensibility of screening tests in order to impair transfusion of blood units from subjects in initial acute phase of infection with no other serological markers (window period shortage). But after HBV NAT implementation the great majority of NAT HBV positive/HBsAg negative subjects are represented by repeat donors, 50-55 years old, with other serological markers of previous contact with HBV (anti-core and anti-HBs), with low viral load and totally silent from the clinical point of view. This picture in blood donors is defined as Occult hepatitis B virus Infection (OBI). In the 2001-2008 period 7,436,996 blood units were tested, in Italy, for HBV DNA and 383 resulted positive: 20 of them (1:372.000) were subjects in acute phase of the infection and seroconverted, 363 (1:20.000) were OBI. Similar pictures are observed in other Mediterranean and East Europe Countries. These data could induce all mathematical models, finalized to calculate the residual risk, to consider HBV as the main infectious risk in transfusion field. But we can observe, where HBV DNA blood screening is in progress since some years, a downward trend of OBI blood donors: in Italy 62 · 106 subjects have been observed in 2005 and 42 · 106 in 2008 (P < 0.05). On the contrary, acute cases are stable. In addition, there are some doubts about the actual capacity of OBI subjects to infect the recipient: some experiences showed that HBV DNA of OBI subjects is less efficient in transmitting the infection than DNA of acute phase subjects. Nevertheless, HBV transfusion transmitted infections from blood donors with low viral load are still reported. In conclusion blood screening with both HBsAg and HBV DNA test is, at the moment, the best way for HBV safe transfusion; in the meantime it limits donors deferral only to those who are really at risk of transmitting a HBV infection. In the future the occurrence of blood donors with OBI will decrease due to progressive deferral of these subjects and to the vaccination campaigns against HBV in progress in Countries at medium/high level of HBV endemic. Background: In spite of anti-HBc and voluntary HBV NAT screening in Germany, the risk to transmit HBV by blood products is circa 10 times higher than for HCV or HIV. NAT screening for HBV DNA is not obligatory in Germany and many other countries. How often donations from donors with persistent occult HBV infection transmit the virus and how sensitive NAT screening for HBV should be is unclear. Aim: We wanted to find out how infectious anti-HBc only positive donations were and which kinds of HBV variants were transmitted. Methods: Anti-HBc positive donors with detectable viremia were identified by look back in donors of infected recipients. Recipients of donations from a donor who was later found to be HBV DNA and anti-HBc positive were tested for anti-HBc and asked for symptoms of acute hepatitis. HBV DNAs from the viremic donors and infected recipients were amplified, the PCR products cloned and 10-20 clones sequenced. Results: We identified persistently infected five donors with negative HBsAg screening tests, positive anti-HBc, and low but detectable levels of HBV DNA (9-240 genomes/ml). Four donors were anti-HBs negative, one had mutated HBsAg and anti-HBs. Among 55 recipients who could be followed, three were infected and died of fulminant hepatitis B. Two of these were immunosuppressed at the time of transfusion, one suffered from sepsis after the operation. Transmission was proven by sequence identity and the time course of the infection. Twenty-five recipients were anti-HBc positive, but were HBsAg and HBV DNA negative and did not report to have had hepatitis. No pre-transfusion serology was available in these cases. Since the prevalence of anti-HBc is 7% in Germany, most of the positive recipients were probably infected by the donations. Recipients of plasma units were more often anti-HBc positive (11/11) than those who received the red cell units from the same donor (5/13). This suggests that >1/100 ml but <1/ml infectious doses were present in these plasmas and that only a minor part of the HBV DNA containing particles were infectious. Surprisingly no case of acute hepatitis B was observed in these recipients. The S gene sequences of the occult viruses were sequenced. In four of the five donors, they were very heterogeneous, changed over time and showed many known and novel escape mutations. Variability was highest in the HBsAg loop (4.4-14.4%) and lower in the preS region (0.8-2.9%) or in the overlapping reverse transcriptase sequence (2.5-4.8%). Conclusions: The data suggest that the HBV mutants were continuously selected to escape neutralisation by anti-HBs although at the time of analysis anti-HBs was detectable in one donor only. Normal hepatitis B vaccines may possibly not protect against these heavily mutated variants. The high transmission rate and the fatal courses suggest that the mutants are viable in immunodeficient patients, but impaired in inducing full blown infection in immunocompetent recipients. HBV NAT in individual donations is probably able to detect most infectious donations from persistent occult HBV infections but further studies are necessary to prove this point. Wolfram.h.gerlich@viro.med.uni-giessen.de Background: Occult hepatitis B virus infection (OBI) is defined as the persistence of HBV genome without detectable HBV surface antigen (HBsAg) excluding the window period and is generally accompanied by anti-HBc and/or anti-HBs. Donor blood with OBI carries a risk of HBV transmission to recipients. The main mechanism of genotype D OBI is a faulty immune control leading to high frequency of multiple amino acid substitutions in the major hydrophilic region (MHR) of the surface protein (HBs). Such previously unreported mutants were hypothesized to escape OBI donors' anti-HBs and detection by commercial HBsAg assays. The immune reactivity of recombinant mutant HBs from OBIs was studied. Methods: Recombinant HBs of 20 genotype D strains (two wild type controls and 18 OBI mutants) were expressed in yeast and purified as spherical microparticles. Recombinant HBs proteins included a Flag epitope used for purification and detection by ELISAs standardized at 1 lg/ml. Critical mutations were corrected back to wild type by site-directed mutagenesis. Results: Only 1/14 autologous OBI plasmas reacted with its own mutated HBs, strongly supporting the escape mechanism for the other 13 mutants. Most mutants were recognized by polyclonal heterologous antibodies from vaccinated volunteers, except HBs with mutated C124 and/or C137. Mutant proteins were tested with 10 commercial HBsAg assays (five microtiter EIAs, three rapid tests and two quantitative assays). The percentage of detected mutant HBs ranged between 33 (6/18) and 83 (15/18).

No assays detected mutant BR432 with C124Y and C137Y. Mutants including a single mutated cysteine (124, 137 or 147) were poorly or not recognized by most assays. Assay reactivity was recovered by re-establishment of these cysteines by site-directed mutagenesis. Other mutant HBs including the associations M133L/Y134H or T127M/T131K or P120R/ T125M/T127P/T131A reacted with 1, 2 and 2/10 assays, respectively. Probing identified linear and conformational epitopes with six monoclonal and one polyclonal antibodies confirmed that disruption of HBs epitope presentation by mutated cysteines was the most effective strategy of immune escape. Conclusions: HBs mutants observed in virtually every genotype D OBI appear generated by escape from immune control. Some mutants, particularly those involving MHR cysteines, escape self anti-HBs, vaccine-induced anti-HBs and capture/detection antibodies utilized in qualitative and quantitative commercial HBsAg assays. Undetectability of HBsAg defining OBI may in some cases originate from qualitative defects of screening assays unable to detect a range of new mutant HBs. Screening HBsAg assays can be assessed with panels of recombinant mutant HBs proteins. OBI mutants should be added to previously described mutants generated by vaccination, passive immunotherapy and anti-viral therapy. New monoclonal or broadly reactive polyclonal antibodies may be needed to identify this new class of HBsAg mutants. Background: Hepatitis C Virus (HCV) is estimated to affect 130-180 million people worldwide. Previous attempts to estimate spatiotemporal parameters of the global and regional spread of the virus from sequence data suggested that the HCV epidemic started in 1900 and expanded steadily until the late 1980s although historical data suggests a more recent expansion. Aim: To re-evaluate the time and route of the global spread of HCV genotype 1a and 1b. Methods: All 1a and 1b HCV nucleotide sequences publicly available at the beginning of the study (September 2007) collected in 21 and 29 countries were assembled and analyzed. The E2-P7-NS2 genomic region of HCV was used for phylodynamic and phylogeographic analyses to estimate the spatiotemporal parameters of the global expansion of the most prevalent HCV subtypes 1a and 1b. Results: We empirically showed that E2-P7-N52 is phylogenetically more informative than other HCV genomic regions including NS5b. The virus is evolving 2-4 times faster than previously thought explaining a more recent expansion of the pandemic. The nucleotide sequences were proven representative of the 1a and 1b global epidemics. Significant improvements of the underlying experimental and theoretical phylodynamic methods helped determining that the current global epidemic of subtypes 1a and 1b ''exploded''between 1940 and 1980; HCV 1b spread preceding 1a by 16 years. More specifically, the transmission of HCV subtype 1a occurred at a low rate between 1906 and the 1960s, at which time there was an explosive increase in its transmission rate. Similarly, subtype 1b transmission occurred at a low rate between 1922 and the late 1940s then increased exponentially. From 1980 onwards, the transmission rate of both subtypes stabilized at a high level. Phylogeographic analysis of all available NS5B sequences suggested that the United States and other developed countries were the epicentres of the HCV 1a and 1b epidemic and that the route of dissemination was through injections of blood or plasma derivatives in the developed world. Conclusions: The HCV global spread coincided with the widespread use of transfused blood products and the expansion of intravenous drug use, but slowed prior to the wide implementation of anti-HCV screening. Primary prevention measures such as blood donor selection, viral inactivation of clotting factor concentrates and prohibition of plasma pooling, led to a deceleration in the expansion of a poorly infectious blood borne pathogen through human activity. Background: HBV surface antigen (HBsAg) is the established serological marker routinely used for the diagnosis of acute or chronic hepatitis B virus (HBV) infections and the screening of blood or organ donors. Natural variation and mutations in HBV S gene can induce HBsAg conformational modifications that may affect the performance of HBsAg assays and thus the blood safety. Aim: To assess the performance of HBsAg assays currently used for the blood screening for the analytical sensitivity and the clinical sensitivity on HBsAg mutants. 1. A high analytical sensitivity level compared to european guidelines requiring a detection threshold below 0.13 IU/ml for the CE marking. 2. Despite this high analytical sensitivity level, disparities on HBsAg mutants detection were observed. This allows to classify assays in three categories: (i) assays in which mutations have no impact on HBsAg detection, (ii) assays provided false negative results on HBsAg mutants, (iii) assays able to detect mutations but at a lower level than wild-type strains. The emergence of HBsAg mutants could have an impact on blood safety if the blood donations screening is done only on HBsAg detection. Despite an unknown frequency of this phenomenon, the vigilance is required. Background: Red cell alloantibodies can cause haemolytic disease of the fetus and newborn (HDFN). Especially anti-D and -K, but also anti-c, -E and -C, can induce severe HDFN. In the last years, non-invasive fetal typing using cell-free fetal DNA present in maternal plasma was introduced. As a national reference laboratory, we have been offering noninvasive fetal blood group typing for clinical purposes since the beginning of 2003 using a stringent diagnostic algorithm with the inclusion of fetal DNA identifiers. The objective of our study was to review the diagnostic usage of non-invasive fetal blood group antigen typing.

Methods: All consecutive patients tested from 2003 up to 2010 were included. Fetal K typing was started in 2006 and fetal RHc and RHE typing was introduced in 2007. Blood samples were received preferentially after week 9 of pregnancy. In case of a negative fetal blood group typing PCR result, the presence of fetal DNA was ascertained through the use of the Ychromosome-specific SRY PCR (only positive in case of a male fetus) or 24 biallelic insertion/deletion polymorphisms or any other paternally inherited blood group antigen. Only if the presence of fetal DNA was confirmed, a fetal typing result was reported. Because the fetal K typing assay had a lower sensitivity compared to the control PCR assays, a negative fetal K typing was advised to be repeated later in pregnancy; preferentially >week 18. In all cases, a cord blood sample or typing result was asked as test performance quality control. Results: In total, blood samples from 365 pregnant women were tested for the following fetal blood group antigens: RHD: n = 175; K: n = 48; RHc: n = 53; RHE: n = 78; RHC: n = 9). In 355 women a conclusive result was obtained and reported within 1 week upon receipt of the sample. In six cases (four fetal RHD and two fetal K typing assays), no test result was issued because the presence of fetal DNA could not be confirmed. In one fetal K typing case, tested as one of the first clinical samples, K typing results were considered as inconclusive because of aspecific amplification from maternal DNA, whereas the child was found after birth to be K negative. Furthermore, fetal typing was not possible in one alloimmunised RhD-negative woman carrying the DOL variant and in two others who were found positive for an RHD-null allele. Another six alloimmunised RhDnegative women carried an RHD/-gene; in all a test result was issued, only one of these women was carrying an RHD-negative child. In two RhDnegative women, an unexpected low level of RHD-exon -5 or exon-7 amplification was observed and paternal typing results pointed to the presence of an RHD variant in the fetus (DIVa and DAU-5, respectively). Conclusions: Non-invasive fetal blood group typing can routinely be performed for RhD, c, C, E and K. There is no longer a need for invasive procedures to determine these fetal blood groups. Molecular genetic test results are usually obtained after PCR amplification of certain gene sequences and single nucleotide polymorphisms (SNPs). However, test interpretation can fail, if insufficient amplification of fetal DNA occurs (a negative test result is expected, if the fetus is RHD negative but may also occur if the amplification of the fetal DNA fails). To discriminate fetal and maternal DNA and to obtain a positive internal control for fetal DNA in the test sample we evaluated the use of 26 SNPs as internal positive controls (IPC) in our PCR settings. Methods: DNA from 128 plasma samples (fetal & maternal DNA) was screened for RHD exons 3,4,5,7 in a multiplex PCR setting including 26 SNPs divided into two primer pools. Additional PCR pools were tested with sequence specific primers for KEL, RHE and RHc instead of RHD. Detection of amplicons were done using SBE (single base extension) and the Gene-Scan Method in an ABI310. In addition, 26 paternal samples were examined. Results of D-screening were compared to standard RHD genotyping of amniocentic fluid (n = 51) or real-time PCR of fetal DNA from maternal plasma (n = 77). SNP frequencies were calculated and compared to ALFRED (allele frequency database). Results: With the exception of two SNPs (rs914165, rs2056277) the estimated frequencies were in concordance with the German population data given in ALFRED. 86% of all samples showed differences in maternal and fetal SNP patterns, 14% lacked these differences because of identical genotype or due to failure of fetal DNA extraction. In 5/40 samples previously typed as RHD negative and 13/88 samples previously typed as RHD positve an internal positive control was not detectable. Another six samples gave false positive results for D-typing. Comparison with the independent results did not reveal a single false-negative case among samples with positive internal control and negative fetal RHD typing. Conclusions: Co-amplification of SNPs and RHD-specific sequences allows fetal blood group determination with a positive control for the presence of fetal DNA in more than 85% of cases. The method may even be used if a paternal blood sample is not available. Detection of amplicons with single base extension gives also the opportunity to replace RHDspecific sequences with other blood group polymorphisms, eg. KEL, RHE, RHc. Background: RhD, Rhc or RhE and Kell antigens can cause maternal alloimmunization that occasionally leads to severe haemolytic disease of the fetus and newborn (HDFN). Noninvasive blood group genotyping of the fetus from maternal blood is the latest possibility for pregnant women with antibodies to determine whether the fetus is compatible with the mother. Such patients do not need invasive diagnostics and treatment associated with the risk of fetal loss or additional alloimmunisation. Since 2000, cell free fetal DNA (cff-DNA) analysis has been developed at the Institute of Haematology and Transfusion Medicine (IHTM) in Warsaw, firstly for RHD, next for RHCE*c and RHCE*E detection in the plasma of pregnant women, and then introduced into the clinical practice. Currently we are developing the noninvasive determination of KEL*1 allele. Aim: To sum up the 10-year diagnostic experience with non-invasive fetal blood group genotyping. Methods DNA was isolated automatically (easy-Mag) from plasma of 480 pregnant women (432 RhD negative, 22 Rhc negative, 21 RhE negative, 5 K negative) and examined by RQ-PCR method (ABI Prism 7700) using TaqMan probes for three fragments of RHD (intron 4, exon 7 and exon 10), RHCE exon 2 or exon 5 and KEL*1(with LNAmodified primers) respectively. CCR5 was determined as an isolation control. To confirm the fetal negative result, the presence of fetal DNA in plasma was tested by RQ-PCR with a panel of biallelic polymorphisms. Results: In 353 cases the RhD status of the newborn was checked and the results were confronted with noninvasive fetal RHD determination. In five cases the RHD gene variant was present in maternal genome (RHD(IVS3+1G>A) or D weak) and the noninvasive determination of RHD of the fetus was impossible. In five cases the RQ-PCR signal was very weak in the first trimester of pregnancy so the investigation was repeated in the second trimester. In all 348 cases the results of noninvasive RHD determination were correct: in 260 cases RHD positive, and in 88 cases RHD negative. The presence of fetal DNA in the sample was confirmed in 87/88 cases: in 51 by SRY detection (the newborns were boys) and in 36 by detection of polymorphism inherited from the father and absent in the mother. In one case such polymorphism was not found and we were not able to confirm the presence of fetal DNA. In 22 Rhc negative and 21 RhE negative pregnant women the results of noninvasive diagnostics of the fetus showed full concordance to the phenotypes of the newborn. Preliminary data of fetal KEL*1 allele detection in 5 K negative women were also correct. Summary/conclusions: Our experience with noninvasive determination of fetal Rh status indicates that RQ-PCR based method is currently highly reliable since the second trimester of pregnancy. However, for diagnostics of SNP-based antigens or in the earlier pregnancy our protocols need to be further improved by more efficient fetal-DNA separation from maternal plasma. Fetal genotype results were compared with the phenotype of the red blood cells of the babies at birth. Results: 1. For genetic counseling for future pregnancies of allo-immunized woman with anti-D, 18 determination of the paternal zygosity at the RHD locus were done. Forteen fathers were found homozygous RHD/RHD and four fathers were found heterozygous rhd/RHD. 2. To determine fetal RHD status, 1378 non invasive fetal RHD genotype from maternal blood were done: 192 from allo-immunized anti-D woman (142 positive fetuses, 48 negative and two undetermined) and 1378 from non allo-immunized woman (847 positive fetuses, 323 negative and 16 undetermined). Two hundred and eight six invasive fetal RHD genotype from chorionic villus or amniotic cells were done: nine from alloimmunized anti-D women (seven positive fetuses and two negative) and 277 from non allo-immunized women (182 positive fetuses, 94 negative and one undetermined). 3. To determine fetal RHE status, two invasive fetal RHE genotype from amniotic cells of allo-immunized anti-E woman were done (one positive and one negative fetuses). 4. To determine fetal RHC status, one invasive fetal RHC genotype from amniotic cells of allo-immunized anti-C woman was done (positive fetus). 5. To determine fetal RHc status, two invasive fetal RHc genotype from amniotic cells of allo-immunized anti-c woman were done (one positive and one negative fetuses). 6. To determine fetal Kell status, 16 invasive fetal Kell genotype from amniotic cells were done (seven positive fetuses and nine negative). Conclusions: Molecular biology is a powerful tool to diagnose a fetomaternel red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring. In non immunized RHD-negative pregnant woman, it allows to rationalize prophylaxis indicated only for women expecting a RHD-positive baby.

3D-S13-05

Background: Maternal plasma analysis for determination of the fetal RHD status is an exciting new tool for the management of RhD-negative pregnant women, specially sensitized women. We assessed the accuracy of fetal RHD genotyping by analysis of maternal plasma in a multi-ethnic population. Methods: We analyzed plasma samples from 64 RhD-negative pregnant women between 11 and 39 weeks of gestation, median age of 28 years old to determine the fetal RHD genotype. This population was from Southeastern Brazil with high mixed ethnic Background. Eighteen patients (18%) had anti-D alloantibody. We used Taqman primers and probes to detect by real-time PCR, exons 4, 5 and 10 of RHD as previously reported (Finning et al, Methods Mol Biol, 2009). As internal controls we used primers/probes sets to SRY and CCR5. Peripheral or umbilical cord bloods from respective nenonates were collected during delivery and hemagglutination was performed. Results: Forty-two samples (65.6%) were genotyped as RHD+, nineteen samples (29.7%) showed completely absence of RHD and three samples (4.7%) presented the RHDw. All the results agreed with the neonatal typing including the three fetuses with the RHDw, phenotyped as RhD-negative. Thus, the accuracy of the fetal RHD genotyping in this mixed population was 100%. The earliest pregnancy in which fetal RHD was detected was 11 weeks. Conclusions: Our findings indicate that the accuracy of RHD gene using three regions (exons 4, 5 and 10) can be sufficient for clinical application in a multi-ethnic population. This knowledge helped us on the development of a feasible protocol for fetal RHD genotyping on DNA from maternal plasma and should become practice in the near future.

3D-S13-06

Methods: In order to select the optimal DNA extraction method an automated procedure was compared to a manual procedure. The automated procedure utilized the MagNA Pure LC instrument (Roche) in combination with the MagNA Pure LC Total Nucleic Acid Isolation kit -Large Volume and the manual procedure utilized the QIAamp DSP Virus kit according to the manufacturer's instruction. The PCR amplification was done using the ABI 7500 Real-Time PCR System. The PCR assay was designed for the multiplex detection of RHD exon 4 and GAPDH DNA. Total sample DNA content in each sample was estimated by comparing the obtained GAPDH Ct value in the sample to the GAPDH Ct value obtained in the positive control with known DNA concentration. In the primary evaluation 170 samples from 121 pregnant women, in gestation week 11 (median, range 6-38) were included. From additional 41 women blood samples were obtained in the second and third trimester. From 25 women three EDTA-samples were requested in early pregnancy, the first separated and freezed within 24 h, the second after 36-48 and the third after 60-72 h. Results: In 25 samples drawn before gestation week 15, extraction was done with both the manual (QIAamp) and automated (MagNA Pure) methods before PCR analysis. The manual method gave three false positive and the automated one false negative result, which turned positive when re-analysed. Based on these results the automated method was chosen for the study and each sample was analysed in triplicate. Thirty-one samples were excluded in the correlation calculation due to extraction failure (n = 5), too low or too high DNA concentration (n = 6), blood group serology data not available (n = 16) or RhD positive mothers (n = 4). In 138/139 samples the results corresponded with blood group serology in the newborns. In one sample the RHD gene was detected but the phenotype determined serologically was RhD negative. In early pregnancy the Ct value for RHD was 39,2 + 3,1 (GAPDH 32,5 + 2,4) compared to 33,9 + 2,5 (GAPDH 33,9 + 2,4) in the second or third trimester. The EDTA samples could be stored for 72 h before centrifugation and freezing of plasma, without influence on the results. Summary: We have developed an assay with high sensitivity and specificity for the detection of fetal RHD DNA in maternal plasma in early pregnancy with >99% concordance with the blood group serology results. The analysis was added as a routine laboratory test for all RhD negative women from September 2009. Until now we have analyzed 1386 samples in combination with administration of antenatal anti-D prophylaxis to those carrying an RHD positive fetus. 60.2% have been reported RHD positive. Twenty-six newborns have been born and followed-up, with 100% corresponding results. An up-dated result will be presented at the conference.

3E-S14 -Blood component preparation 3E-S14-01

During whole blood donation a number of essential parameters must be determined as Blood establishments are ultimately responsible for the quality and safety of blood and blood components collected but also for the safety and health of the blood donor. First of all, all relevant information must be provided to prospective donors such as: accurate educational materials that can be easily understood. Furthermore, the reasons for the requiring of a medical assessment and the testing of all donations must be explained to the donor adequately. All blood donors must undergo a screening process to assess their suitability, since only healthy people with a good medical history can be accepted. The age limits for blood donors are a minimum age of 18 years and a maximum age of 65 years. However in the recent past the upper age limit has been extended to 68 or even to 71 years at some Blood establishments. Besides the exclusion because of obvious signs of infection, i.e. temperature above 38°C, it is essential to determine infectious disease parameters for the following diseases: HIV/AIDS, Hepatitis B, Hepatitis C and Syphilis. Whole blood donations must not be collected from persons weighing <50 kg. A maximum of six standard donations per year can be collected from male and up to four donations from female individuals. A minimum interval of 2 months between standard collections not exceeding 500 ml has to be observed. The hemoglobin concentration or hematocrit must be measured every time a donor attempts to donate. The minimum values to allow a donation are 125 g/l hemoglobin (0.38 hematocrit) for female and 135 g/l haemoglobin (0.4 hematocrit) for male donors. Individual donations below these values will only be permitted after consulting the responsible physicians. For special blood donations there are additional parameters that have to be taken into account. For plasmapheresis donations, the volume of plasma collected at each donation must not exceed 750 ml and a minimum of 33 plasmapheresis procedures may be performed per donor per year and not more than 1.5 l of plasma is supposed be collected per week. Furthermore, protein analyses, especially albumin must be performed at least annually and the total protein count must not be below 60 g/l. The following requirements have to be taken into consideration for donors undergoing platelet-apheresis. It must not be performed on individuals with a platelet count below 150 · 109/l. Also, donors must not be exposed to plateletapheresis procedures more often than once every 2 weeks. Exceptions can only be considered if they are HPA or HLA matched donations. In summary the requirements for the determination of essential parameters during blood donations reflect not only the necessity to protect the donor's health but also to guarantee high quality standards (specifications) for modern up-to-date blood products.

3E-S14-02

This presentation is part of a Satellite Symposium on Blood Component Preparation. The basic elements of blood component production from either whole blood donations or apheresis including the general characteristics of the products will be reviewed. Continuous improvement in the area of blood component production has brought an increased emphasis on process control hand in hand with the creation of processes that provide a greater degree of standardization of the products. This work has included the systematic characterization of products produced using routine high throughput systems in order to understand which elements of the production process give rise either to non-conforming products or to high levels of variation in products. This presentation will review the recent progress in blood component production, including new developments in automation. The areas where we still lack a good understanding of the factors contributing to overall component quality will also be identified as future targets for research effort.

3E-S14-03

Introduction: An increasing demand for blood components is opposed by a decreasing donor availability for the collection of the required blood components. Furthermore, current stem cell transplantation regiments require the collection of more than one similar or different component from one donor or patient. One strategy for maintaining the patients' supply with the required blood components can be the concurrent collection of more than one component from one donor by apheresis, thus multicomponent apheresis. Scope of multicomponent combinations: Combinations are possible for nearly every kind of blood components. In one session it is possibledepending on the apheresis device -to collect 1. up to four plasma units alone, 2. one or more plasma units and one or two RBC units, 3. one or more plasma units and one or more platelet units, 4. one or more plasma units and one or two RBC units and one or more platelet units, 5. one or two RBC units and one or more platelet units, 6. two RBC units alone, 7. one or more platelet units. Also in leucocytapheresis the collection of more than one blood component has become a routine procedure. Performing allogeneic stem cell apheresis can lead to a cell dose for two transplantations or to one unit of PBSCs and concurrently collected and frozen mononuclear cells used for DLI therapy. Autologous mononuclear cell products (e.g. PBSCs or monocytes for dendritic cell generation) usually are cryoconserved before use and require additional plasmaproteins for cryoconservation. The latter can be obtained by the concurrent collection of plasma during leucocytapheresis. Thus, the combination of cell and plasma units or of cell units of different dose or for different purpose are further examples for the implementation of multicomponent apheresis in tumor therapy. Conclusions: The understanding of apheresis technologies facilitates the use of multicomponent apheresis in the vast application field for tailoring the kind, quantity, and quality of blood components for patient care.

3E-S15 -Platelet transfusion 3E-S15-01

Over the past 20 years there have been more than 20 randomized controlled trials (RCTs) that have investigated various aspects of platelet transfusion therapy in Hematology/Oncology patients. These studies have focused on the best platelet product, the importance of ABO compatibility, pathogen inactivation of platelets, platelet triggers and the optimal platelet dose. Three RCTs have informed practices around platelet transfusion trigger with the largest study by Rebulla et al., being the primary study that has changed practices worldwide, with a move towards a lower prophylactic platelet transfusion trigger of 10 · 10 9 /l. Two groups (Germany and Oxford UK) are currently investigating whether we can push the boundaries of prophylactic platelet transfusions even further by eliminating this form of therapy. Preliminary results from these studies have been published but we will await the final results to determine whether this research will indeed change practice. Over the past year there has also been two major studies (one by the BEST Collaborative, and the second by the US Transfusion Medicine /Hemostasis Network), that provide new information to guide platelet dosing. The Study by the BEST Collaborative (SToP) compared low dose platelets to standard dose platelets with WHO bleeding greater than or equal to grade 2 as the primary outcome. The US study (PLADO) compared three doses (low, medium and high) and measured the same outcome (WHO bleeding ‡Grade 2). Although all of these studies further our knowledge to prescribe platelet transfusions, they also raise some interesting questions about the clinical relevance of the outcomes that we are currently using for these studies. The trend over the past decade has been to use bleeding as the primary outcome; however, bleeding is a complex composite outcome (Grades 2, 3 & 4) comprised of some surrogate components (Grade 2 and 3). It is also an outcome that may be difficult to measure and grade in a consistent and reliable manner. The clinical relevance of this outcome is also complex and may vary depending on the perspective from which it is viewed. In this presentation our current knowledge related to prescribing platelet transfusions will be revisited along with some ideas of where clinical research in this area may be heading. Background: Neonatal alloimmune thrombocytopenia (NAIT) is most commonly caused by transplacental passage of maternal HPA-1a antibodies leading to fetal platelet destruction. Recently, we could demonstrate that the F(ab')2 fragment of monoclonal antibody (mab) SZ21 specific for HPA-1a epitope is capable to prevent human platelet clearance induced by anti-HPA-1a in an in vivo NOD/SCID mouse model. It is known, however, that F(ab')2 fragment can not pass across the placenta which limits the therapeutic use of this agent to postnatal treatment only. There is growing evidence that cleavage of a N-linked sugar moiety bound to the Fc region via Asn297 residue plays a crucial role in FcRg mediated phagocytosis. Interestingly, such modified IgG is still capable to bind FcR neonate (FcRn) and might be transported through the placenta. Aim: In this study, we investigated the inhibitory capacity of N-glycan modified (NGM) SZ21 on HPA-1a-mediated platelet clearance. Methods: Purified mAb SZ21 was treated with recombinant Endo F enzyme. The removal of N-Glycan from NGM-SZ21 was confirmed by lectin bloting using lens culinaris agglutinin (LCA) and by MALDI-TOF mass spectrometric analysis. An in vitro phagocytosis assay was employed to test the capability of NGM-SZ21 to inhibit PLT-phagocytosis induced by HPA-1a antibodies. The competition between NGM-SZ21 and HPA-1a antibodies was analyzed using Surface Plasmon Resonance (SPR) technology. The ability of NGM-SZ21 to protect human platelets from anti-HPA-1a mediated clearance was analyzed by injection of resting human PLTs from healthy blood donors in NOD/SCID mice with or without NGM-SZ21 prior to the injection of purified HPA-1a antibodies. Mouse blood samples were taken periodically and the survival of human platelets in the mouse circulation was analyzed by flow cytometry. Results: LCA-lectin blotting revealed a complete absence of N-glycan in our NGM-SZ21. MALDI-TOF analysis documented the specific cleavage of N-glycan moiety attached to Asn297. In SPR, both NGM-and untreated-SZ21 presented similar Kd values (344 nM and 350 nM, respectively), indicating that the N-glycan removal did not impair the binding properties of mab SZ21. In contrast, the phagocytic activity of platelets treated with NGM-SZ21 was declined compared to unmodified mab SZ21 (7% vs 89%, respectively). Furthermore, PLT-phagocytosis induced by anti-HPA-1a antibodies (n = 4) was efficiently inhibited by NGM-SZ21 (mean: 62 ± 5% vs 12 ± 1%, P < 0.0001). The inhibitory capability of NGM-SZ21 could also be confirmed in vivo. While a significant inhibition of human platelet clearance induced by HPA-1a antibodies was achieved by pre-injection of NGM-SZ21, no prevention was observed when NGM-isotype control antibody was injected (PLT-survival after 120 min: 62% ± 3% vs 33 ± 1%).

Conclusions: This study demonstrates that the removal of N-glycan attached to Asn297 of IgG antibodies inhibits the Fc-dependent effector function without altering its paratope(s). Moreover, NGM-SZ21 represents an effective inhibitor of anti-HPA-1a-mediated PLT-destruction both in vitro and in vivo, indicating its potential use as a therapeutic agent in preventing NAIT. Further study is ongoing to prove the ability of NGM-SZ21 to passage across the placenta. Aim: To make the critical points for effective supply of HLA-PCs, we reviewed the cases of HLA-PC transfusion. Material and methods: Patients who are refractory to transfusion of random platelet concentrates are screened for antibodies to HLA class I antigens with a flow-cytometry method using antigen-coated microbeads (FlowPRA, One Lambda), followed by Lymphocyte Immunofluorescence Test-Flow Cytometry (LIFT-FCM) for determination of HLA specificity. HLA genotyping is carried out for the seropositive patients. The donors of HLA-PCs are selected from the list of the HLA-typed donor pool. The HLA-PCs are subjected to the crossmatch test (LIFT-FCM) before transfusion. Results: Among the PTR patients, about 70% of females and 40% of males were positive for HLA antibodies. FlowPRA was used for screening, because the method is sensitive enough to yield very few false negative. Antibody specificity in the patients' sera is an important factor on donor selection in addition to HLA compatibility between donor and patient. We were able to evaluate the transfusion effects of HLA-PCs (n = 3,542) for 163 patients during the period of Apr 2007 and Sep 2009. HLA-PCs were effective (1 h CCI ‡ 0.75 · 10 4 /ll) for 151 patients while ineffective for 12 patients including seven terminal and five reason unknown patients. No PTR case due to existent HLA antibodies was recognized during the period, while there had been several cases of PTR due to such HLA antibodies in the previous years when the less sensitive lymphocyte cytotoxicity test was used as screening. However, in a few cases, newly formed HLA antibodies were detected by the crossmatch test in the patients' sera that had been obtained for monitoring of HLA antibodies. In such cases, newly prepared HLA-PCs were supplied with effective results. In one case, 20 h CCI dropped down from 2.39 (·10 4 /ll) to 1.00 (·10 4 /ll) after the HLA-PC transfusion. The crossmatch test became positive with the patient's serum soon after the HLA-PC transfusion in question. Most of HLA-PCs were supplied to the patients in 2~4 days after the order reception. Summary: Following three points are important for effective transfusion of HLA-PCs; (i) quick, sensitive and specific HLA antibody screening in the patients' sera, (ii) occasional monitoring of patients' sera during the supply period and (iii) information about outcome of HLA-PC transfusion in the patients.

3E-S15-04

Schubert P, Chow V, Culibrk B, Devine DV Canadian Blood Services, Vancouver, Canada

Background: Our studies of platelet storage revealed translation of glycoprotein (GP) IIIa and evidence of synthesis of several other proteins. This investigation found a remarkable stability of the GPIIIa mRNA of 2.9 days in stored platelet concentrates. In the absence of a nucleus, protein translation in platelets must be primarily determined by post-transcriptional regulation. In mamalian nucleated cells, a known control mechanism involves the formation of RNA granules defined as aggregates comprising mRNA and mRNA-binding proteins whose formation is induced by various forms of stress. Aim: We investigated whether blood platelets express components of stress granules and, if so, whether they assemble during platelet storage to protect mRNA from degradation. Methods: Arsenite was used to stress platelets by incubation either with citrate-glucose-saline (CGS) buffer alone or with 1 mM arsenite in CGS for 1 h. For stored platelets, samples were collected on day 1 and day 7. All platelets were washed with CGS and lysed in a Triton-containing buffer spiked with protease inhibitors. Lysate was further subjected to crude fractionation by differential centrifugation resulting in a low-spin (LSP) and a high-spin pellet (HSP) and a high-spin supernatant (HSN), which are enriched for the actin cytoskeleton, the membrane skeleton, and the cytoplasm, respectively. Immunoprecipitations (IP) were carried out by incubation of an aliqout of the lysate with a specific antibody, then IP samples and unprecipitated lysates were separated on SDS PAGE for immunoblot analyses. The signal strength of the protein bands was quantified by densitometry using the LICOR software Odyssey. Results: Immunoblot analyses of whole platelet lysates revealed that blood platelets express common components of stress granules including Ras GTPase-activating protein-binding protein 1 (G3BP-1), Poly(A)-binding protein 1 (PABP-1) and T-cell intracellular antigen 1 (TIA-1). Surprisingly, crude fractionation analyses revealed that G3BP-1 and TIA-1 are localized in the LSP and not in the cytoplasm as is characteristic for nucleated mammalian cells. G3BP-1 showed no change in localization during storage or after treatment of platelets with arsenite, a common initiator for stress granule formation. Another common stress granule component, eIF2a, relocated from the HSN to LSP during storage as well as during the arsenite treatment. Assembly of stress granules during storage is supported by the formation of a complex between either G3BP-1 or TIA-1 with eIF2a as demonstrated by co-immunoprecipitation. Lastly, time-dependent studies of arsenite treatment showed no effect on platelet activation; however, a 67% reduction of responsiveness to ADP was seen in an extent of shape change assay. Similar findings were seen if platelets were treated with the translational inhibitor puromycin indicating that stress granule formation in platelets might be involved in regulating protein translation. Summary/conclusions: We report for the first time evidence for the formation of stress granules in blood platelets during storage. This finding might explain the stability of a subset of platelet mRNAs and further supports the hypothesis of post-transcriptional control of protein expression in anucleated blood platelets. The role of this process in the maintenance of platelet integrity during storage remains to be determined. Background: Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT) are rare inherited abnormalities of the megakaryocyteplatelet system characterized by quantitative or qualitative defects of specific plasma membrane receptors. BSS is caused by abnormalities of the glycoprotein (GP) Ib-IX-V complex, while GT results from deficient or defective expression of GPIIb-IIIa (also known as integrin aIIbb3). Both congenital disorders are associated with hemorrhagic, sometimes lifethreatening complications. Standard hemotherapy of bleeding episodes or prevention of hemorrhages in patients with BSS or GT comprises platelet transfusions. However, this treatment can be complicated by antibody formation to GPIb-IX-V or GPIIb-IIIa, and/or alloimmunization by HLA, thus limiting the responsiveness to future platelet transfusions. Aim: We have evaluated the efficacy and safety of recombinant activated factor VII (rFVIIa) as first-line therapy in patients with congenital platelet receptor defects. Patients: Four males and six females (age ranging from 14 to 34 years) with prediagnosed BSS (n = 4) or GT (n = 6) were included in this study. The patients were referred for hemotherapy prior to elective surgery. All of them presented without manifest mucocutaneous bleedings but had abnormal closure times (>300 sec), as determined by platelet function analyzer (PFA-100) in response to collagen/epinephrine and collagen/ADP due to their congenital platelet defect.

Therapy Protocol: Treatment included a bolus of rFVIIa (110 lg/kg) administered 15 min prior to surgery followed by two doses of 90 lg/kg at 2 h intervals postoperatively. During the first 3 days after surgery, rFVIIa 90 (lg/kg) was given every 8 or 12 h. Tranexamic acid (3 · 1 g/day) was started postoperatively and continued for 10 days in all 10 patients. Results: None of the patients experienced any intra-or postoperative bleeding episodes. By contrast, hemostasis and wound healing were nearnormal. No platelet transfusions were required in any of the 10 patients. Conclusions: Perioperative administration of high-dose rFVIIa in combination with antifibrinolytics such as tranexamic acid can be indeed an effective and safe therapeutic alternative for prophylaxis of bleeding episodes in patients with BSS or GT undergoing elective surgery. Although this is an off-label use at present, administration of rFVIIa should be considered instead of platelet transfusions, specifically in young patients with BSS or GT in order to prevent alloimmunization and the risk of refractoriness to future platelet transfusions whenever required.

3E-S16 -Regulation of plasma derivatives 3E-S16-02

Human blood is the source of a range of therapeutic products, either obtained from the processing of single donations of blood or plasma, generally known as blood compo-nents, or obtained by an industrial fractionation process of large numbers of plasma units that combines protein purification and viral inactivation and removal steps. The strategy to ensure safety of blood products includes a combination of measures to minimise infectious agent content in the starting material, together with validated manufacturing processes, including steps to inactivate or remove potential contaminating agents. With the implementation of Good Practices (GP) during plasma collection procedures, a systematic approach should ensure e.g. the application of donor selection criteria for each donation, prevents the occurrence of errors during testing, or supports traceability by adequate documentation of all important steps, and that the whole chain of processes in the production of blood products, i.e. correct processing, labelling, storage and transportation are covered by relevant, reliable quality assurance systems. During the (industrial) manufacturing process itself the Good Manufacturing Practice (GMP) is already a well accepted basic element involved in ensuring technical quality and safety of medicinal products; it is also applicable to its starting materials. A specific importance of GMP in relation to blood products needs to be mentioned with regard to the virus inactivation steps: As powerful as the contribution of properly validated and im-plemented steps for virus inactivation and removal has been shown to be, it remains es-sential to limit the virus load at the stage of the donation by avoiding, through donor se-lection and donation screenings, the inclusion of a high-titre infectious donation. Only with strict adherence to GMP the efficiency of a validated virus inactivation step is ensured. Furthermore, GMP helps to establish a well defined contract while regulating duties and responsibilities of the contract giver (plasma supplier) and the contract acceptor (fractionator). Using a GMP approach provides a manufacturing model that allows for a documented system of incorporating quality throughout the entire manufacturing process and describes the activities and controls needed to consistently produce products that comply with specifications and are safe for use. There are no doubts that the aim of providing safe and high quality product to the patients should be the same for all products independent of its use as blood component for direct transfusion, or as industrially manufactured product. In conclusion, it would be difficult to justify if the GP for collection in the blood establishment and GMP for manufacturing would not ensure equivalent levels of quality and safety. Global initiatives to further promote the implementation of harmonised Good Manufacturing Practices for the collection in blood establishments and a stringent regulatory control are ongoing. This would further contribute to the global availability of plasma-derived medicinal. which is a documentation related to the starting material human plasma for fractionation. The PMF is a stand alone documentation which is separate from the medicinal product dossier for a Marketing Authorisation. It is a compilation of all the required scientific data (Commission directives, scientific guidelines, European Pharmacopoeia monographs, WHO and CPMP/CHMP statements) on the quality and safety of human plasma which is used for the manufacture of plasma-derived medicinal products, medical devices incorporating plasmaderived medicinal products and investigational products that use human plasma in their manufacture. These data cover all aspects from collection of plasma to the manufacture and testing of the (manufacturing) plasma pool. The PMF procedure is a two step system: In the first step the documentation is evaluated resulting in a PMF certificate and in the second step the PMF is linked to respective marketing authorisation(s). The idea behind the introduction of the PMF was to avoid multiplication of documentation, to avoid multiple assessments of this documentation, and to improve and harmonise within the EU/EEA the assessment of the documentation related to the starting material ''plasma for fractionation''. For medicinal products derived from human blood or plasma the dossier requirements mentioned in ''Information related to the starting and raw materials'', for starting materials made of human blood/plasma may be replaced by a Plasma Master File certified in accordance with Directive 2003/63/EC. This procedure is similar to the evaluation system of an application for a Marketing Authorisation under the ''centralised procedure''at The Agency (EMA). Following the satisfactory outcome of the evaluation, The Agency issues a PMF certificate of compliance with Community legislation, which is valid throughout the European Community. This certificate is accompanied by the evaluation report. This PMF certification procedure is the 1st step. The PMF is re-certified on an annual basis. If a PMF corresponds only to blood/plasma-derived medicinal products of which the marketing authorisation is restricted to a single Member State, then the scientific and technical evaluation of the respective PMF shall be carried out by the national competent authority of that Member State. In the so called 2nd step it is the responsibility of the Marketing Authorisation Holder to update its medicinal product licence(s) and to incorporate the certified PMF in its Marketing Authorisation(s). The Competent Authority that will grant or has granted the marketing authorisation will take into account the effect of the certification or recertification of the PMF on the concerned medicinal product(s).

3E-S16-04

therapy is needed to avoid improper usage. Therefore a second symposium was organized to re-evaluate existing guidelines, establish a network of experts to optimize hemotherapy from the clinical and economic perspective, and to initiate further studies needed for this process. Methods: A questionnaire concerning guidelines, quality management in clinical use of blood products, provision of products in the individual countries and revision of the Wildbad Kreuth recommendations from 1999 was completed in advance to the April 2009 meeting by European and international experts in transfusion medicine, regulators and regulatory authorities, representatives of the European Committee on Blood Transfusion, and the WHO. After key lectures and specific presentations describing national experiences, the symposium was structured in four workshops: (i) blood products: red cells, platelets, fresh frozen plasma, albumin, (ii) clotting factor concentrates and haemophilia treatment, (iii) quality management in clinical use, (iv) efficacy in terms of outcome including Health Technology Assessment and cost-effectiveness. Discussions were based on a series of reseraches which had been performed prior to the symposium. In the respective workshops previous recommendations as well as their state of implementation were revisited and revised where necessary. Results: One hundred and ten participants attended the meeting. Seventysix experts completed the questionnaire. Approximately 63% of responders were from transfusion services, 30% clinicians participating in daily routine care, 16% from governmental/regulatory authorities, 5% from other organizations (e.g. WHO). International guidelines are applied by 74% of experts; in addition 68% have national guidelines for clinical use of blood products. 79.7% collect quantitative information on blood use, 83.3% demonstrate clearly allocated responsibilities for blood transfusions and 88.5% execute standard operating procedures. With regard to provision of blood products, 77% of the responders have already encountered shortages and 41% expect price increases. 29% stated that they do not have a structured education system for professionals involved in transfusion. For the following years responders indicated that supply is the major priority, followed by education, technical improvement, and research. Experts showed a clear commitment to measure clinical outcomes in transfusion medicine. However, the few available publications on outcomes show that appropriate study designs, study endpoints and statistical methods necessitate further elaboration. Conclusions: Results of this survey carried out prior to the 2nd Wildbad Kreuth meeting 2009 show that recommendations from the first Wildbad Kreuth-1999 initiative meeting were still valid. However, often they were still not transposed into daily clinical routine. Critical discussions aimed at identifying reasons for insufficient implementation. Furthermore, the interviewed experts showed a clear commitment to measure clinical outcomes in transfusion medicine. A clear need for education in transfusion medicine was identified. Though based on European data, the results of the Wildbad Kreuth initiative could be a valuable source supporting the goal of optimal use of blood and blood products globally.

3E-S17 -Cellular immunotherapy 3E-S17-02

Yang J Shanghai Blood Centre, Shanghai, China Background: As the important regulator of immunity, tolerogenic dendritic cells (tDC) were potent to induce and maintain tolerance based on their distinct characters from conventional DCs, such as low expression of MHC and co-stimulatory molecules, high IL-10 and TGF-b production.

Specifically, tDCs had the ability to generate and expand regulatory T cells (Tregs) and promote apoptosis of effector T cells. Recent reports showed that donor or host tDCs could promote allograft survival in mice. In this study, the efficacy of third-party tDCs to prevent aGVHD was determined. Methods: In vitro, tDCs derived from bone marrow (BM) of DBA/1(H-2Kq) mice were induced by GM-CSF, IL-4 with IL-10 and TGF-b1. At day 9, CD11c+ cells are harvested, termed ''tDC''. The phenotypes of these tDCs were analyzed by flow cytometry and the expressions of cytokines and function associated molecules were assessed by RT-PCR. In the suppression assay, CD4+T cells from B6 (H-2Kb) as responder cells were activated by mature DC from DBA/2 (H-2Kd) alone or in the presence of tDCs at different ratio. CFSE progressive dilution was used as readout of responder cells proliferation. Meanwhile, the aGVHD mouse models were established. DBA/2 mice as hosts were given total-body lethal irradiation and injected with 3-107 BM and 107 splenocytes from B6 donors after 24 h. In addition, various doses of tDCs were transferred, along with the same counts of BM and SC as the aGVHD model. Then the survival, body weight, GVHD scoring, histopathological specimens and serum cytokine analysis in the aGVHD group were compared with those in the tDC-treated groups. Results: Compared with mature or immature DCs, tDCs had lower expression of MHC ? molecules (IA-IE) and co-stimulatory molecules (CD86/80). They also secreted low IL-12p70 production and express high levers of ''immunosuppressive'' molecules, such as IL-10, TGF-b, FasL, indoleamine 2,3-dioxygenase (IDO) and arginase. Functionally, tDCs suppressed proliferation of allo-CD4+T cells in a dose-dependent manner. In the B6'DBA/2 mouse model, all aGVHD mice died within 14 days. Remarkably, if 104 third-party tDCs from DBA/1 were transferred, 60% mice survived at least 60 days without clinical signs of GVHD except fur turned white gradually. Surprisingly, when the dose of tDCs was reduced to 103 cells, only 20% of mice survived past day 60 and when increased to 105, 100% died within day 47 after BMT. It was also found that the levers of IL-10 in serum were significantly higher in the mice treated 104 tDCs than in mice transferred other doses of tDCs. Furthermore, the percentage of Foxp3+ cells in the 104tDCs-treated mice increased to 18.46% at day 60. Conclusions: These results indicated that the third-party tDCs played a crucial role in reducing the severity of aGVHD in the dose-dependent manner, and this reduction was associated with high IL-10 secretion and Foxp3+ regulatory T cells expansion. And special attention should be paid to the optimal range of tDCs for preventing allograft rejection. Our findings represented a significant advance in the development of DC-based immunotherapy and raised the possibility of using third-party tDCs for therapeutic applications. Heme oxygenase (HO)-1 is the inducible isoform of the first and ratelimiting enzyme of heme degradation. It has been shown in HO-1 knockout mice and in human genetic HO-1 deficiency that this enzyme has potent anti-inflammatory and immunomodulatory functions. Specifically, HO-1 knockout mice are highly sensitive to a lipopolysaccharide (LPS)-mediated experimental sepsis. Although HO-1 is known to be highly inducible by LPS via a toll-like receptor (TLR)-4 dependent signaling pathway, details of this regulatory mechanism are largely unknown. In the present study we demonstrate that Bruton's tyrosine kinase (Btk) is involved in LPS-induced activation of the HO-1 gene in macrophages. Btk is a central cytoplasmic tyrosine kinase of B cell signaling and mutations in the Btk gene lead to Xlinked agammaglobulinemia (XLA). A specific pharmacological inhibitor of Btk alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), blocked LPS-induced HO-1 induction at the mRNA and protein level. Furthermore, LFM-A13 and a dominant negative mutant of Btk blocked the LPS-induced activation of a reporter gene construct with the HO-1 gene promoter in transfected RAW264.7 macrophages. Luciferase reporter gene studies also revealed that the redoxregulated transcription factor Nrf2 was involved in Btk-specific upregulation of HO-1 gene expression and LFM-A13 blocked the LPSinduced nuclear translocation of Nrf2. Finally, because specific blockage of Btk was also involved in HO-1 induction by other TLR ligands such as TLR7 and TLR9, we suggest that this tyrosine kinase may be a general regulator for TLR-mediated HO-1 activation in macrophages. In summary, the data indicates that Btk-dependent HO-1 induction in macrophages may play a crucial role for the innate immune response. Moreover, this pathway may be involved in the increased incidence of bacterial infections in XLA patients. Background: Stress-inducible HSP70 has gained plenty of attention as a potent adjuvant capable to induce antigen-specific CD8+ and CD4+ T-cell response. The capability of HSP70/peptide complexes to elicit CTL responses by cross-presentation of exogenous antigen via HLA class I is of great interest in immunotherapy. By connecting the sequence of the HLA class I signal peptide to the N-terminal sequence of HSP70 a soluble from of the chaperone secreted into the supernatant of HEK293 cells was generated and used for the induction of antigen-specific CD8+ CTLs. Aim: We examined the role of HSP70/CMVpp65495-503 peptide-complexes (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for the ex vivo expansion of CMV-specific CTLs from unfractionated PBMCs. Methods: Therefore, PBMCs of HLA-A*0201 CMV-seropositive donors were stimulated with HSP70/CMV-PCs. Increase of CMV-specific CTLs was visualised by pentameric HLA-A*0201/CMVpp65495-503 complexes. For control, PBMCs were cultured in the presence of CMVpp65495-503 peptide or HSP70. Results: Antiviral T-cells expanded for 21 days using the HSP70/CMV-PCs were more than 93% CMV-specific and showed a high IFN-c expression and cytotoxic activity. In contrast only 85% CMV-specific T-cells were found after stimulation with the CMV peptide alone. We were able to induce antiviral CTLs appeared to be sufficient for adoptive immunotherapy and the clinical use. Conclusions: We show that chaperoned viral peptides elicit a very strong specific T-cell response. Thus, we propose that HSP70 acts as an excellent guide for cross-presentation of chaperoned peptides. The fast, cost effective and efficient protocol can easily be adapted to GMP conditions for translational purposes. Background: We have recently started cytotoxic T lymphocyte (CTL) therapy as ''an advanced therapy''; one considered to have more specificity for tumours than adoptive immunotherapy using lymphokine-activated killer cells. This, however, needs approval from the Japanese government and a special hospital facility known as a cell processing room. We obtained approval and started CTL therapy as a new treatment in August 2007. Here we report on our analysis of the relationship between treatment outcomes, and CTL markers and functions. Methods: Over two years, from August 2007 to July 2009, we carried out CTL therapy in 22 cases. From these, seventeen received one complete course of CTL therapy comprising four sets of CTL administration and were eligible for clinical outcome evaluation. For these cases, CTL surface markers, including CD2, CD4, CD8, and CD56, were examined using flow cytometry. To determine reactivity of CTLs against tumour cells, IFN-c production by CTLs was measured using the ELISA method under the coexistence of autologous tumour cells for five cases. Correlation between these data and clinical outcome was analyzed. Most cases received CTL therapy as outpatients at Aichi Medical University Hospital. Results: Clinical outcomes were evaluated as complete remission (CR), partial remission (PR), no change (NC), or progression of disease (PD) in 3, 0, 4, and 10 cases respectively. Three CR cases (17.6%) were found to have head and neck cancer, including floor of the mouth and tongue cancers. Surface marker examination showed that 99% were CD2+ in all cases, and, of these, about 60% (15-87%) were CD8+. Actual CD8+ cells administered ranged from 0.5 to 20-10 8 cells, averaging 9-10 8 cells. When we compared the number of CD8+ cells and clinical outcome, the number of CD8+ cells decreased in the order of CR>NC>PD, indicating that a greater number of CD8+ cells administered resulted in a better clinical outcome. A further examination of IFN-c production of CTLs at the first and fourth administrations and clinical consequence in five cases showed no apparent correlation. Conclusions: Among seventeen cases treated with CTL therapy, three (17.6%) with head and neck cancer resulted in complete remission. When we analyzed correlation between clinical outcome and in vitro data of CTLs administered, we found some evidence of a tendency for larger numbers of CD8+ cells to result in better clinical outcome. However, no apparent correlation between IFN-c production and clinical consequence was found.

To determine the most appropriate indicator of clinical outcome for CTL therapy, not only in vitro data but also in vivo data including immunosuppressive mechanism by regulatory T cells and further collection of data may be needed. Most if not all arboviruses have a relatively short viremic phase during the course of infection, but perhaps more importantly asymptomatic viremia may precede the onset of clinical disease by 1-2 days and therefore deferral of donors with potential unrecognized infection is undoable. Further to that arboviral infection is often asymptomatic and in the absence of suitable donor screening assays transfusion risk of infection may be considerable. Arboviruses like other RNA viruses have remarkable genomic plasticity and propensity to adapt when infecting different mosquito vectors; a single mutation alanine substituted by valine (A226V) in the E1 envelope glycoprotein of Chikungunya virus (CHIKV) significantly increased the viral replication and dissemination in Ae. Albopictus thus enhancing the transmission efficiency of infection with that vector. Similar process of adaptation, although not of the same importance has been observed once WNV became established in US; a new dominant genotype of the virus named WN02 became increasingly prevalent since 2002 displacing the original NY99 strain possibly due to increased transmission efficiency of the new genotype with domestic North American mosquitos. There are also observations that some current Asian strains of Dengue virus (DENV) may be more virulent for humans compared to strains circulating in South America. The above mentioned evidence of the adaptive capacity of different arboviruses obviously increases the risk of their global incursion. Arbovirus transmission through transfusion of blood products from asymptomatic donors has been documented for West Nile Virus (WNV), Dengue Virus, Tick-borne encephalitis (TBEV) virus, Colorado Tick Fever (CTFV) virus, and recently Yellow Fever virus (vaccine strain). Arbovirus transfusion transmitted infections pose a credible risk to the safety of the blood supply and although it is impossible to predict which pathogen may be on the top of the priority list currently of greatest concern are Dengue virus, CHIKV, O'nyong nyong virus, Rift Valley Virus and Japanese encephalitis virus which are considered exotic arboviruses for the temperate climatic zones. A worrisome trend is that arboviral infections are on the rise both in terms of magnitude of affected populations as well as in variety of incursions of individual viruses rarely heard before; a good example is Usutu virus emergence in Europe or Zika virus in Micronesia. Public health will play a major role in preventing and/or sustaining arboviral incursions when they occur. Key to development of real-time warning system for vector borne diseases is adequate surveillance. Examples of excellent networks specifically targeting arbovirus ecology are ArboNet in US, the European Mosquito Control Association (EMCA), the European Network for Diagnostics of Imported Viral Diseases (ENIVD), EpiSouth, a network for the Mediterranean region and the Balkans, the National Arbovirus and Malaria Advisory Committee (NAMAC) in Australia. Background: Dengue (DEN) virus (DENV) is the most important arbovirus in the world; its range is expanding. DENV is transmitted naturally by the bite of an infected mosquito but also is transfusion transmitted. One in 1376 blood donors were viremic using transcription-mediated amplification (TMA) to detect RNA during the latter half of the 2005 DEN outbreak in Puerto Rico (PR), a DEN hyper-endemic region. Aim: In 2007, a larger DEN outbreak occurred in PR from which donation samples were retained for RNA and antigen (Ag) testing, and from which recipients of positive (P) donations were traced and tested. Methods: Samples in a 2007 repository were tested individually (ID) by TMA (Gen-Probe) or NS1 Ag (Bio-Rad). For TMA, initially reactive (IR) samples were retested by the original TMA or an alternate TMA by ID and at a 1:16 dilution to model pooling. TMA-repeat reactive (RR) samples were considered P. The NS1 Ag algorithm involved repeat testing ·2 of all IRs and any sample in a 50% grey zone; all RRs were further tested. Virologic, infectivity and serologic testing performed at the CDC Dengue Branch in PR included serotype-specific qualitative and quantitative PCR, mosquito cell culture and IgM testing. Hospitals receiving components from RR donations were contacted for recipient follow up including a risk factor and symptom questionnaire. The study was IRB approved. Results: A total of 15,350 samples were tested for RNA by TMA with 35 IR and 29 RR (P) samples for a prevalence of 1:529. A 1:16 dilution detected 14/29 (42%). Of 4401 samples tested for NS1 Ag, 5 were IR and 3 were RR (1:1467); all 3 RRs confirmed as P. Supplemental testing demonstrated DENV 1, 2 and 3 (corresponding to those circulating in PR); 12/29 (33%) TMA-P samples were PCR P with 11 having viral titers of 10^5-10^9 copies/ml. All 11 high-titer PCR-P samples infected C636 mosquito cell cultures and 9/11 (82%) were detected at a 1:16 dilution. Six of 22 (27%) samples tested for IgM were P, only two of which had quantifiable virus (10^6 and 10^8). Of the 3 Ag RR samples, all three were DENV 3 P with viral loads of 10^6-10^7 and IgM negative. Specificity for TMA was 99.95% and for Ag was 100%. Information on all 32 (29 + 3) recipients receiving P donations was obtained but only 3 were tested; 1 recipient in PR transfused with RBCs containing 10^8 DENV 2 developed dengue hemorrhagic fever (DHF) 4 days post transfusion. DENV 2 of the identical sequence (envelope region) was found in the donor and recipient by the CDC. Conclusions: High rates of donor viremia in PR were detected by TMA and Ag (1:529-1:1467) of which more than half of the viremic samples lacked IgM, with significant numbers (30-100%) having high viral loads and capable of infecting mosquito cells in culture. This study is the first to prospectively document DENV transmission resulting in DHF in a recipient. Thus, screening of donors should be considered in DEN-endemic areas. Background: Since its first identification in the US in 1999, WNV has established an endemic pattern, reoccurring for the past 11 years. WNV is estimated to have caused about 4 million human infections, with close to 1,200 human reported deaths. Viremia in healthy people is frequently asymptomatic, facilitating transmission of WNV by organ and tissue transplantation and by blood transfusion. Screening of donated blood and tissue for WNV RNA in the US since 2003 has prevented thousands of potential transmissions. Reoccurring outbreaks of WNV suggest viral adaptation though genetic mutations that have the potential to alter viral phenotype and virulence, degrade the performance of diagnostic and screening assays, and interfere with the effects of vaccines and therapeutic agents. We performed molecular epidemiological studies of the degree of variability of the full genome of WNV in the US from 2002-2009 using human specimens collected during the epidemic seasons. Methods: Viral RNA extracts from 32 isolates from infected blood donor specimens were subjected to complete genome amplification using a set of 30 specific primers. The amplified products were subjected to sequencing. Analyses of phylogenetic relationships were based on distance, parsimony, and likelihood algorithms using MEGA software. Secondary structures of the 3'NCR region of some isolates with mutations were predicted by the mfold software. In order to determine whether the observed genetic variations had biological relevance, selected isolates were cultured in susceptible cell lines and phenotypical characteristics and viral replication were studied. Case report: A 66-years-old patient, a kidney transplant recipient with a history of colon carcinoma, was admitted in our hospital with a respiratory infection. H1N1 infection was ruled out on admission by Polymerase Chain Reaction of material obtained from oropharyngeal swab. During the hospitalization period, the patient was transfused with red blood cell (RBC) concentrate. Four days after the transfusion our Department of Transfusion Medicine and Blood Bank was informed by the collecting facility that the RBC concentrate administered was from a donor who reported that he had been diagnosed with H1N1 flu after the donation. The assistant doctor was informed and a new screening for H1N1 was then performed. The PCR test was positive though the patient had no flu clinical criteria and never developed symptoms. These findings raise the possibility that this infection was transfusion transmitted. However the possibility of other sources of infection cannot ruled out. Conclusions: The risk for transfusion transmissible infections still remains a constant threat for a transfusion department, especially for blood components which cannot be inactivated. Clinicians should be alert to the possibility of infection with emerging infectious agents, because preventive measures may not be available in all cases.This risk can be drastically reduced due to a proper selection of the donor with a good interview, more sensitive methods for testing and use of procedures for a more restrictive transfusion policy. Background: In Italy, the first cases of equine West Nile virus (WNV) infections were detected in 1998, but no human cases were detected until the 2008, when a total of eight human cases of WNV neuroinvasive disease and one case of West Nile Fever were reported by two regions: Emilia-Romagna and Veneto. In 2009, 16 human cases of WNV disease were reported in three regions: Emilia-Romagna, Veneto and Lombardia. The Venezia Trasfusional Department (VTD) was involved in WNV NAT detection on donor tissue and blood 15th September 2009, after the alert by Regional Centre for Transfusional Activity due to a human case of WNV neuroinvasive disease identified in a subject living in the district of Venezia. The screening ended on November 2nd. Herein we report the results obtained on blood and tissue/organ donors from the above period. Methods: WNV NAT screening on blood and tissues/organs donors was performed employing Procleix Ò WNV Assay (Novartis Diagnostics) on full authomated Tigris Ò System following the manufacturer instructions. Results: WNV NAT was promptly introduced to test all blood and tissue/ organ donors living in the area of Venezia. Test validation and LIS communications for WNV NAT results with each blood bank afferent to VTD were performed in <48 h from the alert, without any impact with blood inventory for Venezia district. Table shows the data obtained in WNV NAT screening. We obtained only one positive case, confirmed by other PCR methods and for the presence of IgM, few invalid tests were due to fibrin contamination; re-tested they were negative. Conclusions: The reported experiences show that new emerging pathogens may be a threat to public health not only because of their impact on the population but also because they have a relevant potential to threaten blood safety and could be a challenge to the maintenance of blood inventory. We need to collect more data in order to define the possibility of introduce this test as a routin tool for donors screening. Helpful could be a sieroprevalence study in the district involved in WNV diffusion outside WNV season. Respective approaches avoid needless anti-D immunizations and have taught a lot about RHD genetics in the past years. Aim: A review of respective publications in the field of molecular testing of D-blood samples was compiled. This with special attention to molecular techniques used (e.g. pooled versus single donor DNA testing, and which genetic sites to be tested on RHD), and to ethnic evidence provided in numerous studies (e.g. African, Asian and Caucasian descent). Methods: D-RHD alleles reported in the different studies were counted, and their expected frequencies in the different ethnic groups were estimated. Genetic sites to be tested on RHD were deduced primarily considering their ability to deliver all relevant RHD positive samples among Dindividuals screened. Results/conclusions: Observations on the geographical distribution of various RHD alleles favours a general versus an allele-excluding prescreening. Unsurprisingly, analysis of data revealed, that a minimal and exemplary RHD exon 1, 5 and 10 pre-screening of DNA samples is best followed by a complete molecular resolution of emerging D-RHD alleles. The suggested 2-3-step resolution ideally uses a selected set of SNP-specific analytical methods (''tools''), determines RHD zygosity, and resolves remaining questionable cases by full RHD gene sequencing approaches. SNP-specific tools should be capable of telling the kind of RHD-CE-D hybrid genes and additionally recognize RHDpsi and Cdes, both commonly found among individuals of African origin. RHD DEL samples with minute expression of D to be identified by the SNP-specific tool are RHD(K409K), (IVS3+1G>A) and (M295I). Depending on the costs for full RHD gene sequencing, additional Caucasian alleles RHD(X418L), (147delA), (W16X), (IVS3+2T>A) and the Asian (710delC) may be considered for inclusion in the SNP-specific tool. Decisions of wheter to chose a pooled-, or single-DNA approach are mainly driven by costs. However, there is a pronounced technological advantage for reliable D chimera detection, and ''DNA recycling-prospects''when opting for single-DNA approaches. There is an ongoing debate whether platelet concentrates (PCs) prepared from either whole blood donations (by the buffy-coat method or the platelet-rich plasma method (PRP)) or from plateletpheresis are superior. In summary, there is some advantage of pooled platelets prepared by the buffy coat method over the PRP PCs. However, in comparison between apheresis PCs and pooled buffy coat-derived PCs the data suggest equivalence of the products in non-allosensitized recipients. A clear advantage of apheresis PCs can only be demonstrated in allosensitized patients with HLA-and or HPA-antibodies who receive antigen-compatible apheresis PCs. On this basis it was recommended to base the product choice mainly on availability and medical indication. Usage of these two product types varies greatly between countries and individual institutions. Recent data on therapeutic instead of prophylactic transfusions require a new position-fixing on when platelet transfusions should be given. We will discuss benefits and risks of a merely therapeutic transfusions strategy. Given the attempt to optimize patient safety and to avoid life-threatening bleeding complications the available data still suggest using a prophylactic approach in routine settings outside of clinical trials for treatment of thrombocytopenia in the context of chemotherapy and impaired platelet production. Various studies addressed the effect of platelet dose in prophylactic transfusions in patients with hypoproliferative thrombocytopenia on number of platetelets transfusion, total number of transfusions, transfusion interval and bleeding. We will review these studies and discuss implications for clinical practice. Some patients fail to achieve the appropriate platelet count increment after transfusion. This is still a challenging situation for clinicians and tranfusions services. Approaches to identify the cause of platelet refractoriness as well as therapeutic algorithms will be proposed. All these topics mentioned above are controversial. The standard approach with regard to these aspects can differ substantially between various countries or different institutions. We will present how ''we'' (i.e. the DRK Blood Donor Service Baden-Wrttemberg -Hessia). The ISBT meeting is a good opportunity to discuss the pros and cons for our view with the international audience.

Pathogen reduction is a promising technology to reduce the residual risk of transmission of infectious agents. Reduction of various contaminating bacteria, viruses and parasites by few to several log steps, efficiency to prevent GVHD has been shown. Presently two companies offer technology for the treatment of platelets: Amotosalen (InterØcept, Cerus, Concord, USA), Vitamin B2 (Mirasol, Caridian, Denver, USA). For both technologies, different groups of investigators have shown acceptable in-vitro results with respect to functional and storage data for platelets stored for up to 5 days after production and before transfusion. Initial clinical studies showed no inferiority of the treated platelets in comparison to untreated controls in thrombocytopenic patients. However for both techniques a tendency towards lower CCI has been reported, which may be more pronounced in the platelets treated with the Intercept process. For introduction of pathogen reduction many countries require not only CE mark but licensing with the respective authorities (in Germany: Paul-Ehrlich-Institute (PEI) and local authorities: Bezirksregierung). The local authorities give permission to the manufacturer only if functional and storage parameters have been demonstrated at site to be within the required range (German, European Guidelines). Treatment for pathogen reduction is regarded as creating a ''new''blood product; PEI requires in vitro and additional clinical studies before granting the license. Regarding early clinical studies, it seems recommendable to increase the lower limit of platelet content of the product to 2.5 · 10 11 . Particularly for the Intercept system, where a considerable amount of platelets is lost in the purification of the product from Amotosalen a change in the production process of platelets to increase the platelet yield may be necessary. Compared to conventional manufacturing of platelets by apheresis or pooling of buffy coats pathogen reduction requires additional labor, space, and quality control. Both companies provide refined and highly standardized systems for the respective pathogen reduction process: The addition of the pathogen reducing compound is standardized. The irradiation process is automated, controlled. All steps of the processes are well documented in both systems. Quality control should not only comprise the standard parameters for platelets: efficiency of the process should be assesed, for the Intercept process the residual amount of Amotosalen should be measured (at least in the number required for residual leukocytes with respect to leukocyte depletion). In most countries the shelf life of platelets is limited because of the risk of bacterial contamination (in Germany presently to 4 days). Whether a prolongation to five or more days will be possible remains topic of future studies; our data show a tendency to better functional and storage parameters for the Mirasol process. First results of the clinical application in a small study group (Miracle trial) detected fewer alloimmunisation for Mirasol treated platelets. Experiences in lager clinical trials it will be necessary to evaluate whether pathogen reduction keeps its clinical promises (with respect to reduction of transmission of infections, less alloimmunisation) and to calculate the actual costs (which may be compensated for by longer shelf life). Chronically transfused patients usually suffer from a disease characterized by ineffective erythropoiesis. As the latter causes increased intestinal iron uptake, these disorders are called ''iron loading anemias''. However, the most important cause of iron overload in such patients is chronic transfusion therapy. Most of our knowledge regarding the detrimental effects of transfusional iron overload stems from experience in bthalassaemia major. However, as the thalassemias and other hereditary iron loading anemias are rare in central Europe, we see secondary hemochromatosis mainly in patients with myelodysplastic syndromes (MDS). For diagnosing iron overload, the test with the most favorable cost-benefit ratio is measurement of the serum ferritin level; this can, however, be elevated also in the setting of inflammation. A suspected diagnosis of secondary hemochromatosis should, therefore, be confirmed with imaging studies. This is particularly true because even a liver biopsy is not reliable as a gold standard, as hepatic iron accumulation is markedly inhomogeneous. Furthermore, the biopsy procedure can be complicated by hemorrhage. After extensive development, magnetic resonance imaging (MRI) with so-called T2* weighting can now reliably and non-invasively quantify iron in the liver and heart. There is no linear correlation between hepatic and cardiac iron overload. In general, severe iron overload of the myocardium arises only after the iron storage capacity of the liver has been exceeded. The diagnosis of iron overload with MRI requires special expertise and is not yet reimbursable in Germany. Several guidelines recommend iron chelation for transfusion-dependent MDS patients with a reasonable life expectancy who have a serum ferritin exceeding 1000 or 1500 ng/ml. In fact, there is no universally applicable ferritin threshold for triggering iron chelation because the intensity of the individual patient's transfusion regimen must be taken into account. For decades, deferoxamine (DFO) has been the first-line therapy. As the drug has a short plasma half-life, slow subcutaneous infusions 3-7 times weekly, or twice daily subcutaneous bolus injections are required. Since the administration of DFO is inconvenient and causes injection site reactions and pain, this treatment is fraught with poor compliance. For many years, the only alternative was the oral iron chelator deferiprone (DFP), which is effective in removing iron from the heart but carries the risk of agranulocytosis. DFP has been successfully combined with DFO to treat patients with severe cardiac iron overload. In September 2006, the oral iron chelator deferasirox (DFX) was approved for use in Germany. Deferasirox has undergone extensive clinical trials, mainly in patients with thalassemia major, but also in other transfusion dependent anemias including MDS. A dosage of at least 20 mg/kg/day is needed to achieve a negative iron balance. In order to prove that deferasirox results in clinical benefit for patients with MDS, a prospective, randomized, double-blind, placebo-controlled trial has just been started. The goal is to demonstrate in low/Int-1 risk MDS patients treated as per standard practice the clinical superiority of deferasirox to placebo in terms of overall survival, cardiac function and liver function, which potentially can be affected by iron overload complications. Background: Sickle cell disease (SCD) is a monogenic red blood cell disorder resulting from a single amino acid substitution in the hemoglobin (Hb) b-chain. This abnormal Hb, named HbS, polymerizes under deoxygenated conditions leading to less deformable sickle red blood cell (SS RBC) formation. Vaso-occlusion crises (VOC) are the main acute complication of SCD and are the consequence of blood microvessels obstruction by SS RBCs. In addition to SS RBCs, clinical observations suggested a role for leukocytes in the pathophysiological scheme of SCD. Indeed, leukocytes could adhere to the vessel wall through interactions with endothelial proteins and could interact with circulating SS RBCs leading to aggregates formation, potentially enhancing the incidence of VOC. Aim: In this study we noticed that enrichment of Peripheral Blood Mononuclear Cells (PBMCs) by density gradient separation (ficoll-histopaque) of SS whole blood was accompanied by an abundant and abnormal presence of RBCs in the cell layer containing the PBMCs fraction. Following this observation, we postulated that this abnormal co-selection of both cell types could result from SS RBC aggregation with PBMCs. Methods: To test this hypothesis, blood samples were collected from SS patients (n = 17) and healthy subjects (n = 5) and analyzed by classical and imaging flow cytometry after density gradient separation. The identity of the cells in the PBMC layer was determined using antibodies directed specifically against white (anti-CD45) or red (anti-Glycophorin A) blood cells. Results: Imaging flow cytometry analysis gave a strong evidence of the abnormal RBC-PBMC interactions that could occur in SCD patients' whole blood as they visualized for the first time circulating PBMC-RBC aggregates. The aggregation rate was ten-fold higher in SS patients than in controls. The RBC population involved in the aggregates was a mixture of mature RBCs and reticulocytes, whereas monocytes seemed to be the main PBMCs aggregating with RBCs. Inhibition assays using specific antibodies and soluble proteins were performed to identify the proteins involved in aggregates formation. This indicated that erythroid Lu/BCAM was implicated in these aggregates through its interaction with a4b1 integrin on PBMCs. Analyzing patients treated with hydroxyurea (HU), lower aggregation rates were observed as compared to untreated patients. Summary/conclusions: In conclusion, our study gives visual evidence for the existence of RBC-PBMC aggregates in SCD patients and shows that the aggregation rate is decreased during HU treatment. Considering the potential of such aggregates in promoting vaso-occlusion in SCD patients our data could explain, at least in part, the clinical benefit of HU in reducing the frequency of VOC. Our results strongly suggest that erythroid Lu/BCAM is implicated in these aggregates through its interaction with a4b1 integrin on PBMCs. Future investigation should help characterize the mechanisms leading to this interaction and evaluate the impact of these aggregates in VOC occurrence, which could generate new therapeutic perspectives. Background: Alloimmunization to red cell antigens is an immune response often stimulated by ''transfusion of blood products. It is one of the complications of blood transfusion that ''limit the availability of further safe transfusion and can be associated with hemolytic ''transfusion reaction. Patient's age at the start of transfusion is one among several ''factors that affects the development of red cell alloimmunization as the immune ''response may be affected by the patient's age at the start of transfusion.'' Aim: To study the relationship between the incidence of erythrocyte alloimmunization and the patient's age at the start of transfusion among multi-transfused patients who received ''regular transfusions.'' Methods: A total of 139 Chronically transfused patients (128 thalassemia patients, 11 Sickle cell ''Diseased patients''); ranging from 1 month to 40 years; who received regular ''transfusions'' in the NBTC clinic were included in this study. Clinical and laboratory data were collected. The patient's age of first blood transfusion and the occurrence of red cell ''alloimmunization of those patients had been recorded.'' Patients were divided into three groups according to age of start of transfusion: '' -Group 1 includes patients aging <1 year'' '' -Group 2 includes patients aging from 1-5 years.'' '' -Group 3 includes patients more than 5 years.'' The percentage of positive antibody screening was recorded in each group.'' Results: 1. Patients who had an older age had a higher alloimmunization rate, a finding ''approaching statistical significance (P = 0.042)'' Group 1 patients: Out of 65 patients, 13 (20%) had developed alloantibodies.'' Group 2 patients: Out of 48 patients, 14 (29.17%) had developed alloantibodies.'' Group 3 patients: Out of 26 patients, 12 (46.15) had developed alloantibodies.'' 2. Alloimmunization is a less significant problem in patient whose transfusion is initiated ''before the age of 3.5 years (The cutoff between positive and negative screening was ''''3.5)'' Conclusions: Alloimmunization is affected by the patient's age at the start of transfusion; the older ''the patient receives the blood transfusion the more the probability of forming ''alloantibodies.'' Transfusion at an early age (<3.5 years old) may offer some immune ''tolerance and protection against alloimmunization in chronically transfused patients.'' Background: In thalassaemia major, long-term transfusion from early childhood remains the cornerstone of treatment. For maximal effectiveness of transfusion therapy, an adequate supply of safe blood is required. Blood requirements depend on the molecular basis of thalassaemia, the patient's age and the clinical condition. Usually, concentrated red cells are administered every 2-4 weeks, aiming to maintain the pre-transfusion haemoglobin above 90-105 g/l. This moderate transfusion regimen allows normal physical activity, improves growth and prevents bone marrow malformation and cardiomegaly. It also allows effective prevention of iron loading and permits spontaneous pubertal development, in contrast to the hypertransfusion regimens once favoured. Chronic transfusion exposes the patient to various risks, notably alloimmunisation and disease transmission. Haemosiderosis is a serious and irreversible complication. All patients need iron chelation therapy to prevent progressive and ultimately fatal organ damage. The Greek experience: Effective treatment has greatly improved the survival of thalassaemic patients. In Greece their median age has increased from 10.5 years in 1983 to 34.5 in 2008. Patients receive RCC's, as fresh as possible, matched for ABO, Rh (CcDEe) and Kell systems, leucoreduced to <1 · 10 6 leucocytes per unit (35% with pre-storage and 65% with bedside filtration). Screening for irregular antibodies is performed before each transfusion. A better match policy is applied for 28% of the patients. Currently, haemovigilance data demonstrate that in 1,321 patients receiving 523,116 blood units, the total transfusion reaction rate was 0.3% (0.2% with pre-storage leucodepletion, 0.8% with bedside filtration). The commonest reactions were NHFTR and allergic (both 41%). Alloimmunization was detected in 3.1% of patients. Antibodies of the Rh system, JK a , Kp a , Le a+b , Fy a+b and S were commonest. Autoimmunization of IgG type was diagnosed in 1.6% of the patients. One sickle thalassaemia patient died of hyperhaemolysis syndrome. HIV was transmitted to one patient during the window period. Anti-malarial antibodies were detected in three patients and possible transfusion-transmitted Yersinia enterocolitica 13. HBsAg prevalence is 1.8%, anti-HIV 0.3%, anti-HCV 54%, anti-HTLV 0.8% and ParvoB 19 IgG antibodies 52%. In a ten-year follow up of 1,600 patients for HIV infection it was shown that infections occurred in 1983-1984. 63% of seropositive patients progressed to AIDS, after about 4 years, and 50% died about 5 years after diagnosis on average, mostly in CDC stage C3. The longest survival after infection was 12 years. Low serum ferritin was associated with survival. Fourteen years later, seven patients are still alive; one without antiretroviral treatment has not proceeded to AIDS and in another patient anti-HIV has become negative. Occult hepatitis B was detected in 2/154 patients (1.3%). Both suffer from chronic HCV and receive anti-viral treatment without satisfactory response. Immunologic response to vaccination may vary because of escaping mutants of anti-HBs and HBsAg complexes suppressing HBV-DNA below the detectable level. Antiviral therapy may also account for suppression of HBV viraemia. Chronic HCV is developed in 60% of seropositive patients. 22% progressed to cirrhosis and three died of hepatocellular carcinoma. Conclusions: It is vital to continue to improve laboratory and clinical practice towards greater safety of blood and to find ways of reducing blood requirements and the number of donor exposures. patients and related perioperative therapy have been associated with increased morbidity (including increased rates of perioperative infection) and mortality. Clinical care pathways for patients in these settings have been developed by the Society for Blood Management (SABM) and the Network for the Advancement of Transfusion Alternatives (NATA). These consensus recommendations emphasize: (i) preadmission testing, including complete blood counts (CBC) that should occur as close as possible to 30 days before the scheduled surgery date; (ii) any anemia identified should be evaluated and managed before surgery; (iii) evaluations and laboratory testing should be performed to rule out nutritional causes (particularly iron deficiency), chronic kidney disease (CKD), and/or anemia of inflammation; and (iv) management of anemia should include consideration of IV iron therapy and/or therapy with erythropoiesis stimulating agents (ESA). Interventions to decrease the need for allogeneic transfusion can be categorised under four main headings 1. adequate preoperative preparation 2. surgical techniques to reduce blood loss 3. anaesthetic techniques to reduce bleeding 4. pharmacological agents to enhance clotting and minimise bleeding Surgical intervention will always cause a degree of blood loss. Even the most minimally invasive surgery will cause bleeding but rarely of any significance unless there are technically difficult aspects. The most important factor is a surgeon who has the necessary dissecting skills and knowledge about various blood conservation techniques. Successful dissection will depend upon good visibility within the operative field and relies upon suction and swabbing of blood . Equally meticulous ligature, clip and diathermy use will minimise bleeding and so make visibility better. There are many different ligatures and a variety of clipping devices available. The diathermy devices available have diversified and the use of harmonic scalpels and laser dissecting devices all decrease blood loss and aid surgical dissection. The use of intra-operative red cell salvage allows recycling of spilt blood and can be used in many surgical procedures to minimise anaemia perioperatively. Post operative blood salvage can also be used in specific circumstances. Anaesthetic techniques themselves can also play a part as reducing mean arterial pressure which will lead to decreased blood loss when vessel integrity is breached. Regional techniques are reported to have similar effects of blood loss by reducing vessel tone. The unconscious patient has to be positioned to minimise pressure effects on skin and nerves during prolonged surgery, but also to decrease venous pressure in the operative field. When the operative field is higher than the heart it allows adequate venous drainage. Care needs to be exercised with this positioning so that dangerous air embolism does not occur if large veins remain open and able to entrain air. Normothermia is important particularly as ambient temperature will affect heat loss in the anaesthetised patient. However if humidity and temperature of the theatre are too high it becomes uncomfortable for the surgical team to operate with their additional sterile clothing, therefore patient centred warming becomes necessary. If the patient becomes hypothermic then normal coagulation is affected. The use of heating devices to minimise heat loss are now widespread and the use of a heat and moisture exchange filter in the anaesthetic breathing circuit is routine. Few pharmacological agents are available in 2010 to control and/or to decrease blood loss and transfusion in the perioperative setting. Desmo-pressin (DDAVP) is not effective in the common bleeding patient and should only be used in mild Willebrand (vWF) type I patients or minor haemophiliac in close collaboration with the haemostasis lab. It carries a real prothrombotic risk related to its remaining vasopressive effect and to the induced-increase in factor VIII and vWF factor. Aprotinin has been withdrawn in 2008 after the warning issued from the BART study in high risk cardiac surgery patients. The compound is no more available on the world market. Recombinant activated factor VII is a fascinating and very potent agent. However, most of the prospective studies using this molecule are negative, and an increase in the induced-thrombotic risk cannot be ruled out. Of note, compassionnal case reports have been published showing a positive response to this agent in about 70% of the patients. Tranexamic acid is a weak agent which has only been developped in a limited number of clinical settings, mainly major hip and knee surgery. Furthermore, as it has replaced aprotinin in many cardiac surgery centers, an increased number of seizures has been reported since this molecule is used more extensively. New antiplasmin agents are under development, mainly in phase 2 trials, as the new small protease inhibitor CU-20101. Confirmatory studies are awaited. Some re-enginering of the rVIIa molecule are also on the way with either more potent activities or longer half lives. Even if intra operative bleeding is less frequent nowadays, pending the huge progress of surgical techniques and anaesthesiology, we still need a safe and effective compound to control bleeding and limit transfusion, without any significant increase in the thrombotic risk. Although improved donor selection criteria and tests of increasing sensitivity have dramatically reduced the risks of transfusion-transmitted infectious disease, multiple potential means exist for a pathogen to escape detection and injure a recipient: a window period remains in which an infectious donor cannot be detected; some transmissions (e.g. bacterial contamination of platelets) have detection methods that are far from the desired capabilities; and ''emerging''pathogens continue to represent a risk to recipients until effective testing strategies are developed and implemented. Pathogen inactivation of labile blood components represents a means of addressing all three of these shortcomings simultaneously as they have for plasma derivatives. Multiple effective means of pathogen inactivation have been validated and put into use for plasma and for platelet components. While not yet universally implemented, these appear to have an acceptable toxicity profile and retain a clinically usefulalthough slightly diminished -degree of efficacy that is not associated with increased usage. Some of these techniques, such as solvent-detergent treatment of plasma, appear to offer benefits beyond avoidance of infectious disease; in this case, the pooling involved in the process, while creating a risk for dissemination of non-enveloped viruses, appears to reduce the risk of TRALI considerably. Promising systems for pathogen inactivation of red blood cells and whole blood are under development and would complete the spectrum of treatment across all labile components. While some jurisdictions, most notably the U.S., interpret the completed clinical trials as not yet presenting adequate benefit to balance perceived risks, the primary impediments to implementation would appear to fall more into the logistic than scientific category. Is there sufficient benefit to warrant implementation of pathogen inactivation for plasma and platelets before red cell treatment is feasible? How can the processes be accomplished while minimizing cost to the system? How will each country's healthcare system accommodate the inevitably increased expense for this additional production step? What impact will a fully pathogeninactivated blood supply have on clinicians' conception of transfusion risk and thus blood utilization? This review provides an overview of data available from clinical trials and attempts to identify a path toward improving transfusion recipient safety. Background: Pathogen reduction technologies (PRT) present an excellent opportunity to reduce the risk of pathogen transmission in stored platelets products, especially the not insignificant risk of bacterial contamination beyond 5 days of storage, as well as to extend the storage limits in this context. Three platelet PRTs are currently marketed or in trials; however, clinical studies suggest that increased safety may be paired with a partial loss of efficacy even without extended storage. In vitro studies have demonstrated that increases in CD62P and loss of responsiveness to ADP occur more rapidly in stored PRT treated platelets. Since all platelet PRT methods use UV irradiation, it is possible that platelet protein synthetic repair mechanisms are damaged by PRT with a concomitant acceleration of platelet storage lesion development. Aim: Using proteomic and biochemical tools, we sought to determine whether treatment of buffy-coat platelets with the Mirasol system, a UV-A/ riboflavin method, caused alterations in the platelet proteome after treatment as well as during subsequent storage. Methods: In a two-arm pool-and-split study, buffy coat platelet concentrates were stored either untreated or after treatment with UV-A/riboflavin. Samples were collected before and after initial irradiation and on day 2, 5 and 7 of storage. Platelets washed in a citrate-dextrose-saline buffer were lysed in a triton-containing lysis buffer supplemented with protease inhibitors. Protein separation was carried out by SDS PAGE and proteins were either visualized by silver-staining or subjected to immunoblot analyses. Quantification of the signal strength of the protein bands was determined using the LICOR software Odyssey. Results: Differences in the platelet proteome comparing samples with and without UV-A/riboflavin treatment were assessed initially by gel electrophoresis. Irradiated samples showed an overall~10% decrease in protein expression as determined by densitometry scanning immediately after preparation. This observation was reversed after storage with irradiated units stored at day 2 and 5 showing a total protein increase of 8% and~6%, respectively, compared to untreated controls and samples on day 7 exhibiting only minor differences of 2-3%. For more detailed protein analyses, we assessed proteins previously identified to change concentration during storage. A~2-fold increase in protein expression for the GTPases Rap1 and RhoA as well as the GTPase-regulating protein RhoGDI was triggered by the initial treatment process. Subsequently, a decrease compared to untreated units in the concentration of these proteins of 1.5-2.5-fold by day 2 and an increase of 2-3-fold by day 5, was seen, resulting in a protein expression level at the end of storage similar to that at the beginning. The initial change for RhoGDI mediated by irradiation is in good agreement with a recent proteomic study analyzing differences in the platelet proteome resulting from UV-C, UV-B and gamma irradiation. Summary/conclusions: Pathogen reduction technologies are a trade off between increased pathogen safety and decreased platelet function. Our preliminary data reveal alterations in the platelet proteome triggered by UV-A/riboflavin treatment. Analysis of these changes is important in order to evaluate the changes to platelet function caused by irradiation and any impact on viability for transfusion. Background: A novel system using UV light and riboflavin (Mirasol System) to inactivate nucleic acids has been developed to treat whole blood (WB) instead of individual components. This treatment may be used for pathogen reduction and wherever leukoreduction, pathogen testing, or gamma-irradiation are unavailable. Aim: The IMPROVE Study evaluated the Mirasol System for Whole Blood by measuring in vitro parameters and in vivo 24-h recovery and survival of autologous, 42-day stored, treated RBCs in healthy subjects. Methods: Consented, healthy volunteers were assigned to one of three test groups. The subjects in each group donated a unit of non-leukoreduced WB, which was treated with the Mirasol System for Whole Blood, at one of the three assigned test energies (22, 33 or 44 J/ml RBC). The units were separated into components and the resulting platelet concentrate (PC), fresh frozen plasma (FFP), and red blood cell (RBC) units stored for 5, 28, and 42 days, respectively. In vitro evaluations for the assessment of blood quality included CBC (complete blood count), blood gases, clinical chemistry, ATP (adenosine 5'-triphosphate), mean osmotic fragility, activity of clotting factors, and extent of shape change (ESC). White blood cell (WBC) inactivation was assessed by proliferation and functionality assays before and after treatment. On day 42, treated, autologous RBCs were radiolabelled with chromium-51 and infused into the healthy volunteers. Samples were removed from the volunteers at 24 h post-infusion to determine 24-h recovery, and at 2, 3, 7, 14, 21, and 28 days to determine RBC T 50 . Results: Eleven subjects completed the in vivo re-infusion and evaluation of recovery and survival of RBCs. No SAEs were observed. Differences between groups were observed at 42 days of storage for potassium and sodium levels, but not for the majority of CBC parameters, RBC phenotyping, anti-IgG/anti-C3, plasma crossmatch, mean osmotic fragility or other RBC, platelet or plasma quality variables. WBC functionality as measured by proliferation, activation and cytokine production was decreased at all energies with the most complete inactivation at the highest energy. No significant differences were identified between the three energy groups, with regard to the primary endpoint of 24-h recovery, after 42 days of storage. Spearman Correlations of >0.7 were identified between 24-h recovery values, 42-day hemolysis and ATP. The highest correlations (Rs > 0.7) with T 50 survival results were found with ATP and pCO 2 (Day 42 values). Conclusions: The IMPROVE study showed that the key RBC quality parameters, hemolysis and ATP concentration may be predictive of their 24-h recovery and T 50 survival. These variables can now be used to assess modifications to treatment with the Mirasol System in the pre-clinical laboratory, including storage duration, storage temperature, and appropriate energy dose for treatment. concentrates and plasma. However, PI of all three blood components remains desirable to reach the full possible benefit for patients and PI of red blood cell RBC concentrates will lead to new standards of infectious safety in blood transfusion. The S-303 pathogen inactivation (PI) process in developed by Cerus uses S 303 to form crosslinks with nucleic acids and prevent replication of contaminating pathogens and leukocytes in RBC concentrates prepared for transfusion. In conjunction, the system uses the natural antioxidant, glutathione (GSH) to potentiate quenching of unreacted S-303. Aim: The purpose of the study was to assess the quality of pathogen inactivated buffy coat-depleted RBC concentrates, or conventional RBC concentrates stored in SAGM storage solution. Methods: Twelve whole blood donations of 500 ml (range: 450-515 ml), held overnight at room temperature, were centrifuged at 4000g for 15 min and separated into a buffy coat (60 ml), a red cell concentrate which was subsequently leukodepleted and to which SAGM storage solution was added, and plasma. Pairs of ABO, Rh(D) identical red cell concentrates were pooled and split in two halves with mean volumes of 280 ml. One product was processed with the pathogen inactivation process (Exchange process) according to standard protocols using S-303 and glutathione and one was kept unprocessed. Quality attributes were compared after the PI process. RBC concentrates were stored at 4 ± 20°C and samples for quality control were removed weekly for analysis of standard parameters reported below. Results: The PI process did not result in detectable loss of volume (mean: 280 ml), red cell content (mean: 1.85 · 1012) or total hemoglobin/unit (53 g) compared to the unprocessed RBC concentrates. The red cell quality parameters that were not statistically different between PI treated and control groups during storage included MCV, haemoglobin concentration, hemolysis and extracellular potassium concentration. MCHC, hematocrit, ATP concentration and pH were statistically different in PI treated RBC concentrates compared to control. Statistically significantly improved values were observed for free potassium, residual glucose and lactate production in PI compared to control products. The results obtained from this study conducted at a large transfusion service using the overnight hold of whole blood and buffy-coat depletion are similar to results previously presented for the S-303 pathogen inactivation system (A. Erickson, et al. Vox Sang. 96(S1):222). 

The in vitro quality of pathogen inactivated RBC using the S-303 treatment process was analyzed in RBC concentrates prepared by buffy coat depletion and storage in SAGM. Results obtained from this study showed similar trends as observed in prior studies without overnight hold of whole blood and buffy coat depletion. The results from this study demonstrate that PI using the S-303 system can be implemented in a transfusion service and is suitable for generating a red cell concentrate fulfilling European guidelines for the quality of red cell products.

Thiele T 1 , Steil L 2 , Mohr H 3 , Mü ller T 3 , Vö lker U 2 , Greinacher A 1 1 Institut fü r Immunologie und Transfusionsmedizin, Abteilung Transfusionsmedizin, Greifswald, Germany 2 Interfaculty Institue for Genetics and Functional Genomics, Greifswald, Germany 3 German Red Cross NSTOB, Springe, Germany

Purpose: Platelet concentrates (PC) bear an enlarged risk of bacterial growth due to storage at 22°C. Pathogen inactivation procedures are increasingly applied to reduce this risk. We monitored changes of the platelet proteome induced by a new pathogen reduction technology based on UVC-irradiation and compared protein alterations provoked by UVCtreatment with standard gamma-irradiation. Methods: Four identical buffy coat PCs were treated either with UVC-light (0.4 J/cm 2 ), gamma-irradiated (25Gray), or by combined UVC-and gamma-irradiation. The control PC was not irradiated. PCs were stored under blood bank conditions and aliquots were taken 2, 5 and 9 days after production. Samples were analyzed by Differential In-Gel-Electrophoresis (2D-DIGE) and mass spectrometry (LC-ESI-MS/MS). Results: One thousand and two hundred and sixty-four protein spots were quantitatively monitored in all four groups during 9 days of storage, revealing that UVC induced less changes (14 protein spots) on the platelet proteome compared to gamma irradiation alone (40 spots), or combined UVC and gamma irradiation (26 spots) at day 1. After nine days of storage, only nine spots differed between the control PC and the gamma-irradiated PC; while 110 spots of the UVC-treated PC and 196 of the UVC+gamma treated PC differed from the control. Conclusions: Changes caused by 25 Gy gamma-irrdiation seem to be reversible and differences to the control decrease during storage, whereas in UVC-irradiated platelets differences to the control proteome increase. Clinical studies are needed to evaluate the biological relevance of these findings. BTS have witnessed tremendous developments. Modern mile stone has happened in early 1980s following AIDS panic. Developments included technical, social, economical and financial aspects. All these lead to growth of safety and efficacy of blood components offered to many patients and casualties.This progress needed a huge financial support and strong infrastructure to meet the expenses. Introduction: A modern, efficient and robust BTS have placed a lot of financial burden on budgets of many countries. Developed countries with strong health insurance system and well established infrastructure, have managed to accommodate these budgets. Countries with limited resources faced a huge challenge to meet this pressure of providing same safety and efficacy. These challenges included provision of funds, transfer of know how, insuring relevant HR, and procurement logistics. Subject: Egypt as has gone through a very tough experience during the last decade to establish a well organized blood transfusion services. Ministry of Health in Egypt seeked help of Swiss government to conduct a project between the two countries titled restructuring of blood services in Egyptian MoH. The project aims at; 1. Creating national blood policy, plan, standards and guidelines. 2. Phasing out the family donors to be replaced by VNRBD. 3. Establishing a network of regional blood centers to be sole provider of blood components. 4. Converting all existing hospital blood banks into storage facilities. 5. Implementing a national quality system and heovigilance. 6. Implementing ACUB. 7. Implementing a blood management system using robust software, hardware, and networking. 8. Establishing a national blood regulator. The project was designed to be executed in two phases during the period from 1997 to 2011. Many challenges have faced the project implementation unit [PIU] as follows;

9. Reasonable sustainable funds. 10. HR development. 11. Acceptance by health authorities, personnel and society. 12. Enforcing the policy and standards. 13. Mobilizing the community to become VNRBD. 14. Educating clinical and medical staff to follow the ACUB guidelines. 15. Resistance by other health sectors inside MoH. 16. Changing the concepts and cultures of the society. The managing team handled these challenges in a professional way which needed a lot of patience, advocacy, evidence based results, and consistency. Results and conclusions: In 12 years the NBTS of MoH in Egypt has become a model in the Middle East for a strong organization in spite of the previously mentioned challenges and the other underdeveloped health aspects. The NBTS has successfully implemented a plan that lead to one organization for blood provision in MoH institutions. The main challenge that is facing this organization at the moment is the IT project and provision of enough blood supply. Recommendations: Limited resources countries in need of a well organized blood transfusion services are advised to follow following recommendations; Background: Blood transfusion services in Egypt that are part of the Ministry of Health (MoH) which used to be comprised of fragmented blood banks acting independently with almost no standardization. At that point, restructuring of blood transfusion services became mandatory in light of epidemiological and demographical changes. A program for restructuring the national blood transfusion services was initiated through an agreement between the Egyptian and Swiss Governments to address these challenges. The Swiss Red Cross was mandated to support the program implementation. Aim: The overall aim of this program is to upgrade, enhance and extend the National Blood Transfusion Services (NBTS) of the Ministry of Health in Egypt. The objectives of the program includes the development of a national blood policy; implementation of national quality management standards, construction and rehabilitation of buildings for blood transfusion centers; provision of laboratory and IT equipment; strengthening of the blood donor program to improve public education for the motivation, recruitment and retention of voluntary non-remunerated blood donors from low risk populations; ensuring the appropriate clinical use of blood; capability building of the NBTS staff; and implementation of Blood Management System to ensure timely and efficient blood supply to hospitals. Methods: In order to pursue the overall aim and objectives of the program, a Technical Assistance Program (TAP) was created to compliment the Organizational Capabilities Restructuring covering the following:

1. General Management Activities (incl. leadership & management development) 2. Quality Management (QM) 3. Appropriate Clinical Use of Blood 4. Laboratory Services 5. Blood Donor Recruitment (incl. marketing/ communication) 6. Information Technology (IT) 7. Procurement and Logistics A Project Implementation Unit (PIU) chaired by the director general of the NBTS and a group of technical advisors assigned to each capability of the TAP was established. Furthermore, the Human Resources (HR) of the NBTS is instrumental in recruiting the right people for the program as well as for NBTS. Results: The achieved results are multifold: development of a National Blood Policy, centralization of testing, education on appropriate clinical use of blood, implementation of standard procedures and forms across all centers. Furthermore, one key accomplishment helping to achieve the overall objective is that the NBTS through the PIU and technical advisors has developed a professional culture (e.g. education, quality management, HR). The work of this team has also provided a solid foundation (e.g. defined business process, established collaboration between physicians, IT and QM professionals) for the implementation of the blood management system. Conclusions: The establishment of the PIU and its team supported by HR has proven to be the right organizational measure to develop the required capabilities to reach the objectives of the program up till now. Furthermore, it is an important means for reaching the remaining objectives (e.g. implementation blood management system) and the overall aim of the program in a sustainable way. Background: Estimates revealed that average growth rate of blood consumptions in China reached 18-20% for recent years, which had been taken seriously by the Health Ministry. To be the first city in China to have realized all blood collected from voluntary non-remunerated blood donors, Shenzhen was able to put more time and efforts to blood safety and effectiveness of clinical use. Target With a suggestion of safe blood given by Shenzhen Blood Center, Shenzhen Health Bureau had integrated blood managements into medical quality administration since 2006. Methods First, to set up Expert Committee of Clinical Blood Transfusion and worked out practical guidelines for clinical use of blood, and all hospitals established Hospital Committee of Blood Transfusion (HCBT). Second, blood management was taken into consideration of comprehensive evaluation on hospital quality administration by inspections and retrospective investigations on medical records of clinical transfusion. Thirdly, the results would be openly reported, and the problems should be reformed and adjusted within a given time and also re-estimated. Fourthly, blood booklets compiled by the expert committee were distributed to physicians in regulating performance of clinical transfusion. And finally, continuing medical educations were held at all levels ranked from high trainings for all key directors to low one for nurses. Statistics showed that there were over 4000 personals receive the education in nine trainings in the latest 3 years. Aim: Objective of the study was to design, implement and evaluate a series of problem based learning sessions for the junior medical officers directly involved in patient care and clinical transfusions to understand the pathophysiology, diagnosis and management of transfusion related adverse reactions. Methodology: Five real life case scenarios including an acute haemolytic transfusion reaction, anaphylactic reaction, transfusion related acute lung injury (TRALI), febrile non haemolytic transfusion reaction and transfusion associated circulatory overload (TACO) which took place in Base hospital, Wathupitiwala, Sri Lanka and National Hospital of Sri Lanka were used to develop PBL case scenarios. 3C3R PBL problem design model was used in developing the case scenarios with the assistance of an expert in medical education. PBL sessions were carried out by a trained facilitator with two groups comprising of 23 junior medical officers/ intern house officers starting clinical training. A pre and post test with the use of 15 best of 5 response MCQs assessing pathophysiology, diagnosis and management and a questionnaire on participant perception was used to assess the effectiveness of the PBL sessions. Results: After the PBL sessions improvement from pre to post tests was shown in all three areas on adverse reaction pathophysiology (35.4%), diagnosis (23.2%) and management (18.1%). Nineteen (82.6%) of the medical officers felt that the PBL sessions have met the learning criteria and 18 (78.2%) agreed that cases helped to identify some insufficient areas of core knowledge in immunology and medicine. Seventeen (73.9%) medical officers agreed that the PBL case scenarios encouraged them to engage in further self directed learning. 100% agreed that the sessions have contributed positively in improving their confidence in managing patients with transfusion related adverse reactions. Conclusions: A problem based approach using actual case scenarios designed and implemented based on sound educational principles can be used successfully to educate junior doctors on managing transfusion related adverse reactions. Background: In Pakistan, About 50% of transfusions are being given in the private sector. Inappropriate use of blood is estimated up to 25-45% without separation into its components, with 80-85% of blood being used as whole blood. The large and medium size hospitals have their own blood banks which cater to the hospital needs. In addition, private blood banks also operate with varying standards of service. Transfusion Medicine, as a field of study, does not exist in Pakistan and is considered synonymous with Hematology at best and sometimes even with Pathology. As a result, the physician who prescribes Blood is not educated in Component therapy and Rationale Clinical Use Of Blood. Aim and objectives: The aim of this study was to find out the orientation of Hospital Blood Prescribing physicians of Different specialties in Blood Components and to analyze there prescribing practices and the impact of teaching and training. Study was done at Husaini Haematology and Oncology Trust between September to December 2009 on the major prescribers (first ten hospitals only). The training to Hospital Physicians was done on site at the major prescribing both Public Private and Federal Govt hospitals (total 29). The specialties covered were gynaecology, oncology, pediatrics, hematology, casualty, cardiology, surgery, and medicine and burn units. Methods and data information: 1. Data was studied by Blood bank management software (oracle based), each customer/ hospital prescriptions were analyzed/ monthly. (no of each product prescribed). 2. Analysis was initially done on first ten major prescribers of blood and blood products. 3. Similar hospital dataset were analyzed after three months of extensive training programs in blood component therapy, Rationale Clinical use on site in the hospitals with all the prescribers extensive participation before that all prescribers were asked to fill a questionnaire regarding basic knowledge of components therapy. 4 Background: Kenya's ground coverage is 582,650 sq. km, Population 36,913,721 million, annual growth rate 3.09%, 56% of population living below poverty line, 53% aged 16-65 years, 80% live in rural areas and Life expectancy at birth is 47 years. With massive constantly growing population, the country has adopted marketing as a strategy to donor recruitment Aim: To recruit, educate, retain voluntary non remunerated blood donors thus reducing high Sero -prevalence of TTI's. Methods: To achieve the above, NBTS used a marketing approach in segmenting the donors by demographics by Age -Youth groups, societies and clubs. Gender -women groups and men groups. Occupation -formal and informal sectors and Religions further narrowed down to Faith and community based, Work places and High Schools. In targeting, a scientific marketing approach with reference to the Ansoft matrix (Growth Strategies) was used as follows. (i) Penetration -Segments identified were already active with donors but needed to be maintained. However, to further tap in to these segments, we chose not to change any of our offerings but sought to further penetrate by regularising donations to at least thrice annually as opposed to once a year as was previously the case. (ii) Development and Diversification -These was necessitated by the need to develop un tapped individuals with in the population by generating new segments that would ensure an adequate supply of blood and thus facilitate the shift from over reliance on high school children to the adult population thus a diverse blood donor base. (iii) Positioning-Blood donor recruiters positioned in the mindset of the above segments that blood donation is a safe voluntary humanitarian act that aims to save life. This has served to instill pride and satisfaction to the donors that their benevolent act saves lives. With this in place, it was important to further strengthen and reinforce this approach by placing great emphasis on private public Sector partnerships with the aim of getting full or partial Sponsorship of items such as Media, Transport Somatic cells which have been reprogrammed back to an induced pluripotent state (iPS) will likely become a key source for the in vitro generation of patient-tailored hematopoietic stem cells (HSCs) in future gene therapy. This opens avenues for efficient selection of molecularly characterized, ''safe'', gene-corrected clones at the pluripotent, undifferentiated level. Improvements in directing differentiation of pluripotent stem cells to HSCs, in vitro, are pivotal to advance the use of iPS cells in patient-specific cell replacement therapies. We and others have previously shown that ectopic expression of a member of the human homeodomain containing family of transcription factors, HOXB4, mediates HSC expansion both of mice and humans, in vitro and in vivo, and also enhances the in vivo long-term multilineage repopulation ability of in vitro differentiated mouse ES-cells in a dosage dependent manner. Despite this significant progress towards the in vitro development of HSCs, full maturation to an adult-type of HSC which completely corresponds to bone marrow-resident HSCs has not yet been achieved. Moreover, it is not yet known how HOXB4 promotes the conversion of pluripotent cell derivatives towards transplantable HSCs. Thus, determining the identity of maturing ESCs during pluripotent stem cell differentiation in culture is crucial for the rational design of new strategies of their directed maturation towards a fully functional adult HSC. The CD41 hi ckit + population marks the earliest definitive hematopoietic progenitors in the mouse embryo. The expansion of such cells which emerge during mouse ES-cell differentiation, in vitro, is enhanced by ectopic expression of HOXB4. In our approach, we analysed the expression of surface markers known to be associated with fetal and adult hematopoiesis, in vivo (Tie2, CD41, CD45, ckit, CD34, CD150, CD48 and CD201) on ES-cell derived hematopoietic cells (ES-HCs) that were generated in bulk cultures of dissociated, day 6 EB cells ectopically expressing HOXB4, after cultivation in the presence of cytokines for 10 days, with and without stromal cell support. Our findings allowed us to further improve HSC generation and expansion. Thus, we expect to significantly increase the amount of mature, multilineage long-term repopulating HSCs/HPCs, in vitro. Thus, in combination with these improved differentiation conditions, the use of HOXB4 protein or of ''HOXB4-mimetic''drugs may also support the de novo generation and expansion of safe (gene-corrected) hematopoietic stem and differentiated effector cells derived from patient-specific induced pluripotent stem cells, entirely in vitro. Background/Aim: Mesenchymal stromal cells (MSC) are discussed to be a potential tool in the therapy of various chronic and acute diseases like acute and chronic GvHD, chronic wounds, and colitis. We established a standardized protocol for ex-vivo expansion of MSC isolated from BM in a system compliant to GMP conditions. As manipulation of MSC should be minimized, we use a single step protocol starting with unmanipulated BM to produce clinical doses of MSC within 12-17 days. Methods: Heparin anti-coagulated BM (n = 6) was obtained from healthy volunteers by iliac crest puncture. Unprocessed BM was seeded on 5chamber CellSTACK (Corning) at a density of 1.2 · 10 4 MNC/cm 2 in MEMalpha (LONZA), supplemented with 10% platelet lysate (PLP; IKT Ulm) and 2 I.U. heparin per ml and incubated under standard cell culture conditions. After 2-4 days, non-adherent cells were washed with PBS, fresh medium was added and cells were incubated under standard conditions. Twice a week 45% of medium were replaced. After 12-17 days cells were harvested using TRYPZEAN (LONZA). CFU-F were set up in T25 flasks (NUNC) from the starting material. Standardization and implementation of a GMP-grade protocol was also applied to the production of platelet lysate from human pooled platelet concentrates in additive solution and characterization of this supplement as alternative for fetal calf serum. Results: The period between iliac crest puncture and seeding ranged from 7-41 h. The average aspiration volume ranged from 27-54 ml with an average MNC content of 1.0 · 10 7 (range: 4.9 · 10 6 -1.9 · 10 7 ) MNC/ml BM, which corresponds to 0.53 ± 0,32 CFU-F/cm 2 (range: 0.18-0.86). The cell density at harvest was between 1.2 · 10 3 /cm 2 and 4.4 · 10 · 4 /cm 2 (mean: 1.3 · 10 4 /cm 2 ± 1.5 · 10 4 /cm 2 ). Estimating a maximum of eight CellSTACK (equaling a full load of a 210 liter incubator) for ex-vivo expansion, our protocol can result in a total harvest between 1.6 · 10 7 and 1.1 · 10 9 MSC/BM-aspirate (mean: 3.0 · 10 8 ± 3.8 · 10 8 ). The average number of CFU-F/10 6 MNC was 53 (range: 15-72). Notwithstanding of the different aspiration volumes, MNC/ml BM counts and variations in the number of CFU-F/10 6 MNC, the number of population doublings (mean: 13.3 ± 1,6; range: 11,1-15,8) and the doubling time (mean: 24 ± 3 h; range: 20-29 h) were similar. Expanded MSC preparations passed the following controls: osteogenic, chondrogenic, adipogenic differentiation, BACTAlert, test for antibodies against HBsAg, HBc, HCV, HIV, Treponema pallidum, PCR for HBV, HCV, HIV, endotoxin testing (Ph.Eur.2.6.14), PCR for mycoplasma (Ph.Eur.2.6.7). Summary/conclusions: We established a standardized GMP compliant system for ex-vivo expansion of MSC from BM. Our system does not contain any reagent of animal origin, except heparin, a licensed drug for use in humans. The variations in overall cell harvest are due to a combination of different volumes of starting material (27-54 ml) and to variations in BM quality (MNC/ml BM). Our data demonstrate that heterogeneity of harvest reflect heterogeneity in quality of starting material. Further studies should aim to improve standardization of quality of starting material in order to assure generation of a clinical dose for an average weight adult in a one-step procedure without about 2 weeks. (Supported by EU 7thFP CASCADE;N°223236). platelets are introduced as alternatives to replace fetal bovine serum (FBS). We recently demonstrated that pooled blood group AB human serum (HS) supports the expansion of adipose tissue-derived MSCs (ASCs). Differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence of ASCs cultivated in both supplements. ASCs were isolated and expanded with either FBS or HS. A whole genome microarray was performed on six ASCs from passage two and data verified by RT-qPCR and protein analyses. Subsequently, ASC adhesion to plastic and different extracellular matrix molecules under static conditions was measured. Under flow conditions adhesion to a histamin-stimulated HUVEC-monolayer was monitored. We further analysed the migration and transmigration towards tumor-conditioned medium as chemoattractant. For in vivo homing assays, ASCs cultivated in either FBS or HS were co-injected to NOD/SCID mice. After 2 h mice were sacrificed and organs analysed for the ratio of ASC-FBS and ASC-HS. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASC-FBS compared to HS. 90 of these genes were higher expressed in FBS cultures (fold change ‡2). Confirmative assays analyzing transcript or protein expression with selected genes supported these results. A cluster of adhesion and extracellular matrix associated molecules appeared to be differentially expressed. To check potential effects on homing abilities, we first analyzed the adhesion. In static conditions, ASCs cultivated in HS demonstrated higher adhesion to plastic and different extracellular matrix molecules. The adhesion to endothelial cells however was reduced. Under flow conditions (1 dyne/cm 2 ) no significant differences were observed: Compared to a melanoma cell line, ASCs showed low tethering and rolling with few cells strongly adhering to an endothelial monolayer. Migration and transmigration assays towards tumor-conditioned medium revealed strong donor-specific differences, but no significant effect of the supplement. Co-injecting differentially labeled ASCs of both conditions and analyzing the proportion of cells in different organs after 2 h to the current date suggested no differential allocation in the organs. Major changes in the manufacturing process by introducing alternate supplements may impact the final product. Accordingly comparability studies are demanded. Our comparative data indicate that FBS modifies gene and protein expression in ASCs enhancing gene expression of homing factors. However, comparing the migratory capacities revealed no significant differences in ASCs cultivated in either FBS or HS, assaying in vitro adhesion, migration and transmigration in vitro and homing in vivo. Accordingly, we suggest the use of HS as safe alternative supplement for clinical-grade ASC expansion. Aim: Many studies have demonstrated that resquimod represents a powerful immune response modifier but little is known regarding the influence on DC differentiation and maturation. Methods: Human monocyte-derived DC were generated in the presence of GM-CSF and IL-4 for 5 days. DC differentiation and maturation were analysed by flow cytometry. Results: Resiquimod efficiently inhibited downregulation of CD14 and upregulation of CD1a expression, indicating suppression of conventional DC differentiation. In order to dissect the contribution of TLR7 and TLR8 signalling on DC differentiation we performed additional experiments with synthetic TLR7 ligands loxoribine and gardiquimod showing no inhibition of CD14 downregulation and CD1a upregulation. To directly prove that resiquimod exerts its effects via TLR8 we performed coculture experiments with resiquimod and inhibitory oligonucelotides suppressing either TLR7 (iODN 20958), TLR7+8 (iODN 20959) or TLR7+8+9 (iODN 2088). These experiments revealed that resiquimod's inhibitory effects were antagonized only in the presence of TLR8 inhibiting ODNs (20959, 2088) but not iODN20958 inhibiting only TLR7. Interestingly, resiquimodderived DC exhibited significantly higher T-cell costimulatory molecules CD40, CD80, CD86, MHC class II expression, de novo expression of CD25 (IL-2 receptor) and CD123 (IL-3a receptor) and higher T cell proliferation suggesting potent immunomodulatory capacity being different from conventional DC. Conclusions: TLR8-dependent modulation of DC differentiation and maturation through resiquimod represents a novel pathway to manipulate the T cell activatory activity of DC. The T-cell content of products did not differ. CD14+ monocytes, however, were higher in group 2 (CD14+. 19.7% vs 24.3%, P = 0.047). Leukapheresis products were analysed due to buffy coat (BC) volumes of the COM.TEC leukocyte harvests. The donors¢ pre-donation WBC subset counts did not differ significantly. Reduction of BC volume, BC: 6.3 ± 0.8 ml (group 1) vs 8.4 ml ± 0.79 ml (group 2) reduced product volume significantly (107 ml vs 134 ml, P = 0.006). Product data: WBCs: 55157/ll vs 43614/ll (P = 0.165); CD3+ T cells: 65.3% vs 60.7% (P = 0.064); CD3+4+ Thelper (Th) cells: 30.9% vs 30.4%; Tregs: 6.36% vs 7.05%. Only the platelet concentration differed significantly (6.98 · 10e6/ ll vs 5.90 · 10e6/ll, P = 0.023). Conclusions: In healthy non-cytokine stimulated blood donors the percentage of Tregs of lymphocytes was found to be around 8% and no difference was found due to the age of donors. The leukapheresis products contained on average 748/ll CD3+CD4+CD25+ Tregs. Reduction of the BC volume reduced the product volume and increased the platelet concentration significantly but did not influence the T-cell content. Introduction: Platelets are the major source of soluble CD40 ligand (sCD40L) (1) in the blood. It has been demonstrated that CD40L is cleaved from the surface of activated platelets to release sCD40L. sCD40L is well known to show immun-modulatory functions and high concentrations in blood products so that it is supposed to trigger adverse transfusion reactions like TRALI (2). Therefore we examined sCD40L concentrations in stem cell apheresis. Material and methods: In four patients suffering from multiple myeloma and undergoing autologous stem cell apheresis, sCD40L concentrations were measured in peripheral blood samples before, during and after apheresis procedure and in the respective stem cell product. sCD40L concentrations were determined by a commercially available ELISA kit (R&D Systems). In an additional approach, platelet-rich plasma (PRP) from healthy volunteers (n = 5) was incubated with different pharmacological inhibitors (MMP-2/MMP-9 Inhibitor I, MMP-9 Inhibitor I, MMP-2 Inhibitor I, recombinant ADAM 10, and recombinant ADAM-17) during platelet activation, and sCD40L was measured as described before.

Results: During stem cell apheresis, a decrease in platelet count could be observed from 93,052/ll ± 59,123/ll at the beginning to 55,342/ll ± 28435/ll at the end of the procedure. The thrombocyte loss was accompanied by a significant lowering of sCD40L concentrations in peripheral blood samples from 235 pg/ml ± 145 pg/ml to 129 pg/ml ± 73 pg/ml (dependent on platelet count, linearly correlated, r = 0.95). In stem cell products, sCD40L concentrations were manifold elevated (range from 2239 to 3641 pg/ml) in comparison to concentrations of peripheral blood samples.Using the MMP-9 inhibitor (100 nM) and the MMP-2/9 inhibitor (3 lM) sCD40L release by platelets could be inhibited by >60%. Interestingly, the MMP-2 inhibitor (17 lM) completely prevented the shedding of sCD40L from activated platelets. Conclusions: During stem cell apheresis, sCD40L concentrations in peripheral blood were mainly influenced by alterations of platelet count. As known from platelet concentrates, sCD40L accumulates also in stem cell products reaching clearly elevated levels underlying again the importance of sCD40L release by platelets. Additionally, these data support the hypothesis that MMP-2 might be the protease, primarily responsible for sCD40L cleavage from platelet surface. Hematopoietic stem cell transplantation (HSCT) is used worldwide to mainly cure patients with hematological malignancies or inherited diseases. In Europe, more than 25,000 HSCT were reported to the EBMT registry from a total of 613 centers in 42 countries in 2007. Of these, approximately two thirds were autologous and one third allogeneic transplantations using peripheral blood (98% autologous versus 71% allogeneic) instead of bone marrow as main source. HSCT has become a professional medical activity, which takes place in highly specialized transplantation units that collaborate closely with clinical departments (internal medicine, pediatrics) and most frequently a donation center experienced in the GMP-compliant pharmaceutical manufacture of stem cells and other cellular therapeutics. The European Union Directive 2004/ 23/EC which came into force in April 2006 defined standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. These standards must be implemented through national legislation by all EU member states. In addition, since January 2004 the Joint Accreditation Committee of the ISCT and EBMT (JACIE) offers an inspection program to improve the quality of HSCT, to maintain the high-quality practice achieved and to ensure further EU harmonization. Besides hematopoietic stem cells, donor lymphocytes infusions and the co-transplantation of mesenchymal stem cells are also covered by the JACIE standards. In real life, HSCT is used to reconstitute the patient's hematopoietic and immune system after high-dose chemotherapy or myeloablation. Therefore, quality and safety of stem cell products are of utmost importance. In addition, individual and practicable issues have to be addressed and carefully balanced with the patient's own risk-benefit assessment and personal intention. Thus, representative surrogate markers for the in vivo stem cell homing should be established as in-process controls in order to characterize and quantify the pharmaceutically active cellular components, e.g. the amounts of CD34-positive cells and of ''contaminants'' like Tlymphocytes which might attribute to both transplant-related adverse events such as graft-versus-host disease (GvHD) and additional therapeutic effects such as graft versus leukemia (GvL). There is still an urgent need to correlate the results obtained by in vitro assays with the hematopoietic short-term and long-term engraftment in vivo and the frequency of transplant-related adverse events. Nowadays, manufacturers have to employ an GMP-based quality management system that includes standard operating procedures (SOPs) describing very precisely the collection and processing steps and also minor or major cell manipulations (a differentiation made by FDA) with possible impact on hematopoietic stem cell quality. Furthermore, cleanroom microenvironment for sterile production, qualified medicinal equipment and well-trained personnel are essentials which have to be in agreement with the mandatory requirements of the international guidelines and official authorities. The purpose of this presentation is not to provide a historical overview of donor pre-selection, stem cell collection and graft processing, but to summarize recent trends and new challenges in standardizing stem cell preparations, for example ex vivo centrifugation, immunomagnetic selection of cells by antibodies, cryopreservation and long-term storage. It thus aims to identify potential risk factors with major impact on product quality.

The harvest, production, storage and release of cellular products are regulated by international guidelines like the EU guidelines 2004/23/EC or 2006/17/EC. In addition world-wide effective guidelines are existent with the FACT-JACIE standards. As cellular products such as hematopoietic progenitor cells (HPC) are usually applied to critically ill patients requirements for release must be well defined and high. The determination of criteria for release requires a prior definition of specifications of parameters for the product. Such specifications and the question of release of products with parameters out of specification will be addressed exemplarily for autologous and allogeneic HPC grafts. The harvest of cellular products must be preceded by an examination of the healthy donor or the autologous patient. Apart from the health history and the clinical condition of the donor or patient results of the testing of infectious disease markers ((IDM) HCV, HBV, HIV1,2 and Syphilis at the minimum) are decisive for the judgment on the donor's or the patient's eligibility for donation. In case of an autologous donor, even positive test results for the IDM do not necessarily exclude a donation. After harvest and processing of the cellular product the processing records must be complete and parameters like the content of the medically relevant component, i.e. CD34+ cells for HPC grafts, the content of possible cellular contaminants such as CD3+ cells in the allogeneic setting or tumor cells in the autologous setting are relevant and need to be specified. For CD34+ cells a minimum amount of 2 · 10E + 06 or 4 · 10E + 06 cells is requested for autologous or allogeneic grafts, respectively. This might sometimes only be achieved by more than one harvest and product. Regarding CD34+ cells the quality of the cells defined by viability and optional by analysis of clonogenic growth has to be evaluated. Such parameters need to be measured especially after manipulations such as cryopreservation or selection or depletion procedures. In addition, microbial contaminations, i.e. potential contaminations by aerobe or anaerobe bacteria or fungi for HPC products have to be examined. Cellular grafts undergoing incubation steps need to be additionally examined for contaminations by mycoplasma or viruses. Furthermore, incubation or activation steps of cellular products require examinations regarding functionality, genomic stability and potential malignant transformation of the cultivated cells. The final decision to release or not to release a cellular product despite parameters out of specifications must be guided by the question whether benefits outweigh potential risks for the recipient when administering the product. Furthermore the kind of the product, HPC or other product, and its importance for the patient's medical situation, i.e. the documented urgent medical need might have an influence on the decision of release.

The transplantation of human tissues and cells (musculoskeletal tissues, corneal tissue, cardiovascular tissue, skin and skin substitutes) is a strongly expanding field of medicine which offers great opportunities for the treatment of tissue defects. The quality and safety of these substances should be guaranteed, particularly in order to prevent the transmission of diseases. As tissue and cell therapy is a field in which an intensive worldwide exchange is taking place, it is desirable to achieve worldwide standards. The European Community should therefore endeavor to promote the highest possible level of protection to safeguard public health regarding quality and safety of tissues and cells. The Directive 2004/23/EC of the European Parliament and of The Council on Setting Standards of ''Quality and Safety for the Donation, Procurement, Testing, Processing, Preservation, Storage and Distribution of Human Tissues and Cells''and whose complementary Directives 2006/17/EC and 2006/86/EC are now implemented into national law in the countries of the European Union. Mainly, the regulations in the recent Tissue Acts have significantly changed the working situation for tissue establishments. The transmission of viral and non-viral infectious pathogens continues to be the most serious of the potential adverse effects of allogenic tissue transplants. Therefore, tissue establishments must focus on comprehensive and robust quality assurance in the areas of donation, testing, processing and preservation. While serologic testing for anti-HIV-1 and -2, anti-HCV, HBsAg, anti-HBc, and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic amplification testing (NAT) to detect human immunodeficiency virus, hepatitis B and C virus (HIV, HBV, HCV) infection. Reports on viral transmission events, tissue-specific issues, manufacturing and inactivation procedures should be evaluated regarding the significance of HIV, HCV and HBV detection by NAT in donors of various types of tissues. In view of numerous synergy effects with transfusion medicine it would be advantageous for tissue establishments to cooperate with blood bank laboratories in performing virological tests. Whenever possible, a validated inactivation procedure should be included in the manufacturing process of tissue transplants. Tissue establishments should follow a documented quality management system on the basis of good technical practice procedures which is maintained to the current standards. Deviations from the stipulated quality and safety standards must result in documented investigations which include decisions on options for correctional and preventive measures. Donor tissue can only be released if defined criteria are fulfilled. Any suspicion of severe undesired reactions and events for the recipient of a tissue transplant must be registered with the authorities. In order to properly identify donors, secure traceability of all donated material and provide information about characteristic features and properties of tissues and cells, the European Union demands a single European coding system for cells and tissues.

4D-S27-01

Several hemostyptic agents as well as cellular and plasmatic blood products are available for treatment of bleeding complications. In addition to fresh frozen plasma coagulation factor concentrates can be applied to bleeding patients. Selective use of these compounds according to the patients hemostatic disturbances is superior to standard application of cellular concentrates and fresh frozen plasma. Near patient testing of whole blood coagulation and fibrinolysis has been suggested for goal directed therapy. Recent data show reduction of consumption of blood products using algorithms and tailored therapy. To apply the right compounds to the right patients comprehensive knowledge of efficiency and safety is of utmost importance.

Pape A J.W. Goethe University Hospital, Frankurt, Germany Point-of-care (POC) monitoring of coagulation can be helpful to early recognise non-surgical reasons of bleeding and to initiate a targeted treatment in a variety of coagulation disorders (e.g. dilutional coagulopathy, isolated factor deficits, hyperfibrinolysis). In contrast to conventional coagulation tests (PT, aPTT, platelet counts), POC-tests are performed in whole blood allowing the assessment of interactions between cellular components (platelets, red blood cells) and plasmatic coagulation factors. POC-systems like rotation thrombelastography (ROTEM Ò ) and multiple electrode aggregometry (MEA, Multiplate Ò ) have been designed for the analysis of coagulation directly at the bedside, which may help to gain time in situations of critical bleeding. Moreover, these analyses provide data about the functional state of the coagulation system including platelet function and the dynamic of clot formation and lysis and the mechanic stability (i.e. clot firmness). The analysis of the viscoelastic properties of the clot with ROTEM Ò is based on the principle that a mobile pin suspended into the blood sample rotates through an angle of 4.75°. Due to beginning coagulation and the formation of fibrin strands these movements are impeded while the rotational amplitude is simultaneously translated into a trace. ROTEM Ò provides screening tests for the intrinsic and extrinsic pathway of the coagulation cascade (InTEM Ò , ExTEM Ò ), as well as specific tests for heparin effects, fibrin-polymerisation and (hyper)fibrinolysis (HepTEM Ò , FibTEM Ò and ApTEM Ò ). ROTEM Ò is increasingly used to in clinical situations with inherent risk of coagulation disorders: dilutional coagulopathy in massive hemorrhage, assessment of hypo-or hypercoagulable states in patients taking anticoagulant drugs (monitoring), hyperfibrinolysis in cardiac surgery, orthotopic liver transplant, sepsis, trauma, urology and gynecology/obstetrics. Indeed, the institution of POC-based transfusion algorithms seems to reduce perioperative blood transfusions. The Multiplate Ò device exerts impedance aggregometry for assessment of platelet function. Basically, two pairs of silver-coated conductive copper wires are suspended into the blood sample, which is mixed with the testspecific agent. Following activation, platelets expose receptors on their surface and attach to the surface of the test-wires. The resulting increase of impedance is translated into arbitrary ''aggregation units''(AU) which are plotted versus time. The slope and the area under the curve represent the velocity and overall potency of platelet function. Depending on the assay, platelets are activated by standardised concentrations of collagen (COLtest), arachidonic acid (ASPI-test), adenosine diphosphate (ADP-test), ristocetin (Risto-test) or thrombin-receptor activating peptide (TRAP-test). Inasmuch, Multiplate Ò can provide information regarding the extent of platelet inhibition (e.g. with acetylsalicylic acid or clopidogrel) as well as information regarding the functional state of native thrombocytes. Despite their predictive value regarding transfusion requirements a major limitation is that ROTEM Ò and Multiplate Ò hardly assess primary hemostasis are not apt to detect von Willebrand's syndrome. Further critiques address the need for additional personnel and resources for maintenance and performance as well as for supervision and quality control at POC. In summary, ROTEM Ò and Multiplate Ò are suitable tools for POC to identify hemostatic disorders and to guide pro-and anticoagulant therapies. Their appropriate use, however, requires strict quality controls and trained personnel to ensure optimal accuracy and performance. Background: It has been well recognized that in many patients who undergo cardiovascular surgery, blood transfusion is required so as to avoid tissue hypoxia due to anemia and massive bleeding due to low platelet counts and/or platelet dysfunction. However, the recent studies have suggested that transfusion of red blood cell concentrate (RCC) or platelet concentrate (PC) can be associated with postoperative ischemic events and mortality in patients undergoing cardiac surgery. (Circulation 2007; 116: 2544-52, Transfusion 2004; 44: 1143-1148) Aim and methods: To evaluate risk factors for thromboembolic events in patients undergoing cardiovascular surgery, we analyzed a database, which we obtained as part of a multicenter prospective cohort study to investigate the incidence of immune heparin-induced thrombocytopenia. A total of 1,444 adult patients scheduled for cardiovascular surgery were enrolled at 11 institutions during a one-year period and were followed for 30 days after surgery. Of these patients, 1,429 were eligible for analysis. We examined the patients' demographic data, type of operation, changes in platelet count, timing and period of heparin administration, episodes of transfusion, complications, thromboembolism, and death. Thromboembolic events were evaluated objectively only when clinically suspected. We took blood samples before surgery and on postoperative days 7 and 14 to test for anti-platelet factor 4 (PF4)/heparin antibodies. On the day of surgery, 372 cases received transfusions of ‡10 units of RCC (in our country, one unit is equivalent to the amount of red cells derived from 200 ml of whole blood) while 289 cases received units of PC (any number).

Results: Fifty-seven patients suffered from thromboembolic events, including both arterial and venous thromboses, during surgery and/or the postoperative period. The incidence of thromboembolic events was higher in patients undergoing aortic (7.0%) or combined surgery (7.9%) as compared to those undergoing coronary artery bypass graft (2.5%) or valve surgery (0.9%). Among patient characteristics, hypertension, renal dysfunction, and a history of stroke/transient ischemic attack or arteriosclerosis obliterans were identified as risk factors for thromboembolisms by univariate logistic regression analysis. However, the presence of anti-PF4/ heparin antibodies detected by an enzyme-linked immunosorbent assay was not associated with thromboembolic events. Multiple logistic regression analysis revealed that the incidence of thromboembolism was higher in patients who received transfusion of Background: Recombinant activated factor VII (rFVIIa, Novoseven) is approved for the treatment of spontaneous and surgical bleeding in patients with haemophilia A or B and with antibodies to either factor VIII or factor IX. However rFVIIa has increasingly been used for indications outside the approved areas as a last resort treatment in trauma, cardiac surgery and other critical bleeding episodes despite the absence of strong randomized clinical trial (RCT) data. Aim: The aim of the Haemostasis Registry was to collect data (including dose, adverse events and outcome) on the off-license use of rFVIIa in Australia and New Zealand. Methods: Monash University established the Haemostasis registry in 2005 with an educational grant from NovoNordisk Pharmaceuticals. More than 100 hospitals (including all major users of rFVIIa in Australia and New Zealand) contributed retrospective data to the Registry over a ten year period (2000-2010). Results: More than 3400 off-license rFVIIa cases have been reported to the Registry. The number of cases peaked in 2006 and remained constant thereafter. There were 65% males and the median age was 56 years. Major areas of use were cardiac surgery (~43%), other surgery (~18%) and trauma (~13%). The majority (~77%) of patients received a single dose of rFVIIa with a median (IQR) dose of 91 (73-103) mcg/kg. Sixty-eight percent of cases documented a response (decrease or cessation) to bleeding following a single dose of rFVIIa. Response to bleeding increased after two or more doses (~74%). Overall, the median number of red blood cell units transfused was reduced from 6 to 2 following administration of rFVIIa (P < 0.001) and an 81% reduction in total blood products was observed (P < 0.001). Patient mortality at 28 days was less if bleeding was reportedly stopped or decreased (88% or 82% vs 39% no change, respectively) (v 2 2 = 533, P < 0.001). Stepwise logistic regression analysis identified the number of units of RBC's, pH and context of bleeding as significant independent predictors of patient response to rFVIIa administration and age, pH, context of bleeding, units of FFP, use of protamine and APTT level were the best predictive model of patient outcome at 28 days. No changes in the size of dose, number of doses, blood components transfused, response to rFVIIa administration or mortality were observed over the data collection period. However the type of cases treated with rFVIIa broadened over time. Thromboembolic adverse events were reported in 7.5% of patients yet risk-adjusted adverse event rates were not significantly different from those seen in non-rFVIIa treated cardiac patients recorded in the ASCTS registry (www.ascts.org). Conclusions: The Haemostasis Registry is the largest dataset of its kind and provides critical observational data on the importance of pH, temperature and coagulopathic state in determining the response to rFVIIa. Although the role of rFVIIa in critical bleeding is still not clear, the Haemostasis Registry has been an invaluable resource for rigorously tracking adverse events and helping to guide clinical practice in an arena unlikely to be supported by new RCT data. Background: The worldwide shortage of immunoglobulin G (IgG) is affecting, among others, patients in developing countries where such preparations are not available in sufficient amounts or not affordable. In addition, it is recognized that substitutive therapy in immunodeficient patients is best achieved using IgG preparations made from local plasma collected from donors exposed to the same environment. Therefore, the development of a robust technology to prepare IgG from a small volume of plasma -without the need to build, qualify, validate and operate a sophisticated manufacturing pharmaceutical facility -should help to increase the access to these essential medicinal products . Aim: Recently, we have developed a procedure for the preparation of virally-inactivated plasma, cryoprecipitate, and cryo-poor plasma using an integrated disposable bag and filtration system. We have now evaluated a similar approach for the fractionation of a plasma-derived IgG-enriched preparation using caprylic acid and fully disposable equipment. Methods: Batches of 20 plasma donations obtained from whole blood from volunteer donors were thawed at 1-3°C to isolate the cryoprecipitate. The supernatant was pooled aseptically (about 4 liters) and subjected, or not, to an adsorption step on DEAE-Sephadex A-50 in a closed sterile bag to capture the prothrombin complex The unabsorbed fraction containing IgG was recovered, transferred into plastic bag and treated for at least 1 h with 5% caprylic acid at pH 5.5 to precipitate non-Ig proteins and to inactivate viruses. The precipitate was removed by centrifugation. The IgG supernatant was then concentrated by ultrafiltration and subjected to five times wash by pharmacological preparation of normal saline to remove any residual caprylic acid in the concentrate. The washed IVIg was sterile filtered by a set of clarifying and 0.2 micron filters and dispensed aseptically into final bags for transfusion. The final preparation was subjected to a set of quality controls to assess protein composition (total proteins, IgG, IgM, IgA, and albumin), turbidity, purity (zone electrophoresis and SDS-PAGE), molecular size distribution (HPLC), proteolytic activity (chromogenic substrate S-2288) and residual caprylic acid (GC). Results: IgG of good purity was obtained with a mean recovery of 50-65% from cryo-poor plasma, corresponding to about 4.5-6 g/l. IgG was at a mean concentration close to 30 g/l and albumin was <3 g/l. Mean IgA and IgM were 2.7 and 0.78 g/l, respectively. High-molecular-weight proteins/ aggregates were <3%. There was no detectable proteolytic activity. Residual caprylic acid in the final preparation <30 ppm. Conclusions: The concept of fractionating IgG from small plasma pools using disposable equipment and under conditions providing high yield and satisfactory purity is technically achievable. This opens new perspectives for countries (i) to prepare IgG from domestic plasma at affordable costs, and (ii) to virally-inactivate pooled convalescent plasma to treat emerging infections. Further developments are in progress (i) to validate the extent of viral inactivation during the caprylic acid treatment and (ii) to obtain a preparation with low IgA content intended for patients with IgA deficiency. The reported higher prevalence of HIV infection in FRD is largely reflecting age difference from VNRBD. In Sub-Saharan Africa, VNRBD median age ranges between 18-20 years while FRD median age is 25-35 year, the age of maximum HIV risk but, when adjusted for age, prevalence of HIV infection is similar in both donor groups. (iii) Despite considerable efforts to induce repeat donation in VNRBD, 70-80% of VNRBD in sub-Saharan Africa are first-time donors and only this group is comparable to FRD. Recent data collected in three Sub-Saharan African countries (Cameroon, Ghana and Guinea) evidenced an absence of significant difference in prevalence of HIV, HBV or HCV between FRD and first-time VNRBD and VNRBD carrying significantly more HIV and HBV infections than FRD. A unanimous conclusion was that first-time VNRBD and FRD were epidemiologically undistinguishable and that only repeat donation significantly increased blood safety. Importantly, there is clear evidence that a unit of sub-Saharan African FRD blood costs $12-18/unit while a VNRBD blood unit costs $25-$100. Additional costs stem from recruiters salary, mobile collection equipment and running costs, advertisement, compensations and rewards given to donors. External funding has recently been widely dispensed to many resource poor countries with the proviso of developing only VNRBD in centralized systems largely at odd with the structure and means of poor countries without consideration of the negative consequences of establishing a long-term unaffordable system when funding dries out. Sub-Saharan and other resource poor areas are largely structured around extended family and communities addressing individual issues and making family blood donation a culturally natural act to be taken advantage of rather than eliminated. Most resource-poor countries collect 2-10 units of blood/1000 inhabitants for an estimated need of 10-20 U/1000 to cover emergency transfusion. Unless all blood sources are utilised, restricting blood donation to VNRBD may significantly contribute to blood shortages detrimental to saving patient's lives.

In conclusion, based on safety, cost and availability, resource-poor countries can legitimately take a pragmatic rather than dogmatic view on strategies to insure adequate blood supply at an affordable cost relying on both VNRBD and FRD as a single donor pool and on repeat donation to improve blood safety. Background: The safety and availability of blood largely depends on committed, repeat donors who donate regularly over years and have a low risk of infectious diseases. Altruism is often reported by those repeat donors to be their major motivation. It is, however, unclear whether altruism is also relevant at the beginning of a donor career and whether altruistically motivated first-time donors are more likely to return than donors who were initially activated by health check-ups or by other nonmonetary incentives. Aim: The aim of the study was to investigate how the initial factors involved in motivating people to donate blood affected the probability of donor return and the number of donations within the following 24 months. Methods: A self-administered questionnaire was completed in 2009 by 3,077 non-remunerated whole blood donors, who first donated in the year 2005 through the German Red Cross Blood Bank in southwest Germany. Retrospective questions about motivation for the initial donation were used to calculate a score to assess the relevance of altruism, health check-ups and nonmonetary incentives. Firstly, a logistic regression model was run to check whether motivation scores were associated with the probability of return for at least one further donation within a 24-month period. Secondly, an overdispersed Poisson regression model was used to determine the contribution of the scores to the number of donations in this time period of those donors who returned at least a second time. Results: In the 24 months following initial donation, 74.1% of the participating first-time donors returned at least a second time. On average, each donor made 2.0 additional donations (standard deviation: 1.9). Altruistic motives were most often mentioned as important for the decision to donate the first time, followed by health check-ups and other nonmonetary incentives. The results of a logistic regression model showed that the odds of donor return increased significantly when altruistic reasons were given as the motivating factor for the initial donation (OR: 1.06 for the altruism score) and decreased if health check-ups were reported (OR: 0,96 for the health check-up score). Activation by nonmonetary incentives had no significant effect on donor return. The number of donations within the 24-month period of those donors who returned at least a second time was positively associated with initial activation by nonmonetary incentives whereas activation by health check-ups and altruistic motives were not. Conclusions: Altruistic motivation at the time of the initial donation is an important determinant for donor return. Therefore, first-time donor campaigns should continue to appeal to altruism. Health check-ups may be an additional incentive for committed donors, but are not adequate for recruiting first-time donors who are likely to become repeat donors. Activation by nonmonetary incentives is not associated with donor return. But interestingly, donors who were initially activated by nonmonetary incentives are more likely to become regular donors when returning a second time. Background: In today's globalized world, blood collection agencies are faced with new challenges and opportunities. As immigrant populations grow, the recruitment of new blood donors and the need to involve minority groups in the blood donation cause are increasingly pressing.

Agencies must ensure that blood banks' diversity reflects the medical needs of an increasingly pluralistic society. Furthermore, minority groups represent a potential donor base that has not traditionally been targeted. According to the 2006 Canadian census, 16.5% of Montreal's population belongs to a minority group. Héma-Québec encourages partnerships with minority communities and their blood donors. Nonetheless, only 1% of volunteer mobile drives held annually are conducted with such groups. Aim: This study seeks to examine the socio-cultural aspects of blood donation for minority groups in Montreal, in order to better recruit minority donors and ensure diversity within the blood bank. Methods: Seventy-five semi-structured qualitative interviews were carried out in the Greater Montreal Area with six members of Héma-Québec personnel, nine minority blood drive organizers, 37 minority leaders not formally involved in organizing blood drives, and 23 minority blood donors. Most informants were originally from Asia, the Middle East, Latin America, Africa and the Caribbean, and from an array of religious Backgrounds. Results: Donor motivations include religious beliefs, perceived health benefits of giving blood, previous exposure to blood donation, and pride in contributing to saving a life. Though most agree that giving blood is a generous act, many also describe it as a moral duty or a civic obligation. The workplace and school play a significant role in triggering blood donation for minority donors, in part because of the accessibility of drives but also because of peer pressure. For non-donors and potential partners, obstacles to blood donation include: little or no exposure to the cause, no knowledge of practices in Quebec, the perception that there is no imminent need for blood, a feeling of exclusion from society, believing that their blood does not qualify and therefore might be discarded, and socio-cultural beliefs pertaining to each minority. Existing blood drive collaborators are generally associated with religious centers. The most successful drives are by well-organized communities with a strong sense of identity and a broad mobilization capacity, who hold blood drives for commemorative purposes. Conclusions: Interviews with potential blood drive organizers show that cultural intermediaries are essential in building trust. Because of the lack of information concerning blood donation practices in Quebec, outreach programs need adjustment to address the concerns of minority groups. Blood collection agencies need to be operationally ready to establish longterm partnerships with minority groups and foster the integration of minority donors. Awareness campaigns should use ethnic media rather than be aimed at the general population. By targeting minority donors through their religious activities or cultural centers, agencies actively involve communities, benefit from religious leaders' clout, and expose minority communities to the continuous need for blood. Schools and workplaces offer convenience and visibility in social environments that encourage blood donation; they must therefore remain important places to recruit minority donors. Methods: A survey was carry out in three study groups: 100 persons up 18 years of age from the general population, 100 students and 100 health care professionals. Through the clustering technique, a representative sample of 50 blood banks was taken to assess sociodemographic variables of replacement donors and voluntary altruistic donors. Results: Of the total respondents, 34.3% had donated to a family member or a friend and 19.2% had donated altruistically, of this, 40% were health care professionals, 38% students and 22% general population. The reasons for donating were: request of a relative or friend 29%, solidarity 26%, to save lives 22%, generosity 12% and social duty 11%. The reasons for not donating blood were: not complying the requirements 29%, fear of infection 28%, lack of information 11%, lack of time 11%, fear of fainting 8.5%, indifference 5%, laziness 3.5%, health affection 2%, to prevent blood trade 2%. Nationwide the replacement donors in the 75% of cases were male, between 24 and 34 years old, 63.8% were married and basic school level, 80% were masons, or merchants. Voluntary altruistic donors were young people aged 19 to 28 years old, university students, academic and single. Conclusions: Investment in education and awareness about the culture of altruistic voluntary donation of blood is necessary; the subject should be inserted into the public agenda through mass communication media. The development of appropriate promotional campaigns for healthcare care professionals and for the general population will help to eliminate the myths related to it.

Introduction: Although the idea of connecting personality and influence of communication messages has been appealing for a long time, it hasn't received the adequate research attention in the field of voluntary blood donation. Bjorn Z. Ekelund's tricolor typology of personality communication styles has a successful application in the field of social marketing. It describes three types: ''blue people''that are concrete and practical, driven by structured, logical, precise and factual data; ''red people''that are characterized by social orientation, caring for others, building cooperation and harmony, and ''green people''that are engaged in ideas, alternative solutions, future, vision, and global values. Aim: The aim of the paper was to explore the relations between Ekelund's tricolor personality communication styles and stimulating power of arguments for voluntary blood donation, i.e. whether ''blue'', ''red'' and ''green'' people are more prone to be affected by certain arguments for voluntary blood donation. Methods: A sample of 215 students (age range 18-29 years, 31% donated blood) answered DI instrument for assessment of tricolor personality communication styles. Stimulating power of arguments was assessed based on a scale that consisted of arguments taken from leaflets and other communications material for voluntary blood donation. Respondents rated each argument based on the probability that it would stimulate them to donate blood on a five-point Likert-type scale, from 1 -highly improbable, to 5 -highly probable. Statistics: correlation analyses between red, blue and green communication styles and stimulating power of arguments. Results: The respondents with people oriented, ''red'' communication style would be affected by the largest number of arguments (significant positive correlations with probability of being stimulated to donate blood by 10 arguments). The largest observed correlations were between ''red'' types and arguments ''With a single blood donation you can save up to four lives'' (r = 0.241, sig = 0.000), ''Donating blood voluntarily one saves lives'' (r = 0.224, sig = 0.001). Argument ''In some serious accidents, blood transfusion can help a critically injured patient to stay alive long enough for their loved ones to reach the hospital to see them for the last time'' had positive power for the ''red'' type (r = 0.203, sig = 0.003) and negative power for the ''blue'', facts oriented type (r = -0.144, sig = 0.038). The stimulating power of the argument ''With a single blood donation you can save up to four lives'' was negatively correlated with the ''blue'' type (r = -0.232, sig = 0.001). Stimulating power of an argument ''When you give blood you will do one of the most amazing things anybody could dream of -saving a life'' was negatively correlated with ''green'', vision oriented type (r = -0.238, sig = 0.001). Conclusions: Some tested arguments had no stimulating power for explored personality types, and some had different effect on different types of people. Messages and slogans for voluntary blood donation should be pre-tested for their stimulating power for different personality communication styles. In order to influence all the potential voluntary blood donors segmented approach would secure better results. Financial support: Ministry of Science and Technology, Projects 149018D and 149047D. Background: In China, all the blood and blood components supplied for clinical use are donated by volunteers since 1998 Blood Donation Law of the People's Republic of China was put into force. In Beijing, the clinical consumption of blood has been increasing progressively at the annual speed of 10-20% since 2000. However, shortage of donor blood happens every winter and summer, and during emergencies. The age of blood donors must be within 18-55 years old according to the law, and most of the blood donors in Beijing are mainly university students. Aim: The aim of this study is to provide a better understanding of the psychological and environmental factors which are associated with blood donor recruitment and retention. Methods: Subjects are 1953 Beijing citizens, including 922 donors and 1031 non-donors. Survey questionnaire was used in the study. First, we interviewed randomly 100 citizens with open-ended papers about blood donation to identify the factors which may affect blood donation. In this research, 146 items were analyzed. These factors reflect motivation, beliefs, knowledge, and service in regards to blood donation. These items were compiled into a questionnaire and administered to the subjects. Results: (i) The results of independent sample T tests show that, compared with non-donors, donors reported higher level of extraversion, agreeableness and conscientiousness, but a lower level of neuroticism; (ii) The results showed that benevolence (not altruism, kinship and hedonism) is the greatest motivator for blood donors; (iii) Path model predicting donor intentions from the beliefs showed that kinship support, self-efficacy, selfcontrol and identity predict intention of blood-donation positively; (iv) The results of independent sample T tests show that blood-donors score higher in knowledge about blood-donation than non-donors; (v) The zero-order correlations showed that the service characteristics of blood organizations (such as kindness, considerateness and respect) predict positively the intention of blood-donation. Conclusions: Based on the findings mentioned above, we conclude that (i) Blood-donation is associated with some specific personality characteristics such as extraversion, agreeableness and conscientiousness and neuroticism. Outgoing, agreeable and conscientious individuals are more likely to be blood donors, whereas nervous individuals are more unlikely to donate blood; (ii) If blood-donors are supported by their close friends or relatives, or feel helpful by donating blood, they will become life-long donors; (iii) Blood donation motivation is better described as ''benevolence''rather than ''altruism''. Thus, we suggest that the messages in blood campaigns should focus on benevolence rather than altruism; (iv) donors retention is greater when the donors feel they are being treated with kindness, consideration, and with respect during blood donation. Keywords: Blood-donation; intention of blood-donation; altruism; extraversion; service quality. Background: AVIS is the most important Italian Association of blood donors, with more than 1 million members. Our regional organization in Campania counts over 55,000 members, who regularly donate at our collection centers unselfishly and free. Aim: With this survey we want to evaluate the degree of satisfaction, related to the donation at our centers, of our associates. We have used questions to analize the reasons that drive our members to give away blood. This survey aims to improve the perceived quality of the services we offer to our members and to prepare plans for recruitment of new donors wich take account of the results of this study. Material and methods: We prepared a phone service, whe have trained five of our volunteers for this porpouse. In collaboration with external specialised consultants it has been developed a special questionnaire, wich consist of five simple questions to allow a rapid understanding of respondents. To contact donors we have used the phone numbers that them provided at the time of the last donation. They were carried out approximately 10,000 telephone requests for investigation and 5,670 donors agreed to participate to the survey. Results: Fifty-seven percent of the sample judged the service recived excellent, referring both to mobile collection centres and fixed centres. 33% gave a good judgement, 8% sufficient and only 2% gave insufficient respond. 98% of the interviewees have received at home, by postal service, the entire health and associative documentation regarding donation and they have expressed positive opinions about this organizational aspect. 99% of interviewees gave their agreement to be contacted for our future promotional and associative programmes. More than 90% of donors interviewed by telephone expressed their satisfaction about how AVIS came into contact with them. It emerged clearly that, for almost all of the interviews, the main reason that drives volounteers to donate is not for a personal need, but because of their awareness of the social importance of the associated and periodic donations. Conclusions: Telephone has been appropriated for collect satisfaction of our users. The research has achieved results regard the motivational aspects exceeding our initial expectations. Interviews showed the desire of our associated to show their wishes of social participation, altruism and solidarity. In addition to what we requested by converses. It has emerged the need to involve young people, also because of the increasingly raising of the age of our population. It might be successful the involvement of those who, because of their age, are no longer active donors, but show desire to continue to make part of the life of the Association and of the Society.

4D-S29-01

The HLA complex is located on a chromosomal segment on the short arm of chromosome 6 (6p21.31). It is also referred to as the human major histocompatibility complex -MHC, a cluster of genes encoding molecules that have immunological functions and govern histocompatibility. Such a MHC has been found in all vertebrates studied so far. In humans, it is arranged in two chromosomal regions named HLA class I and HLA class II; the chromosomal region in between those two has been designated ''HLA class III''. A complete gene map for a single, artificial HLA haplotype has been assembled and its contiguous nucleotide sequence has been determined. In this refined analysis the HLA complex has turned out to be one of the most gene-dense regions of the human genome, as it contains at least 300 expressed loci. In transfusion medicine, the HLA system is relevant for the following problems:

1. Platelet refractoriness mainly induced by antibodies against HLA class I gene products, 2. TRALI due to antibodies against HLA and HNA gene products, 3. Febrile non-hemolytic transfusion reactions (FNHTR) often caused by antibodies against HLA class I gene products, 4. Transfusion-associated graft-versus-host disease (TA-GvHD) induced by the transfusion of HLA compatible T lymphocytes. In transplantation and cell therapy, the importance of the HLA system can be seen in the following topics: 5. Solid organ transplantation, especially renal transplantation, 6. Stem cell transplantation, 7. Cancer research and immunotherapy : dissemination of metastases, therapies by dendritic cells, NK cells or cytotoxic T lymphocytes, 8. Vaccination: presentation of antigens by HLA gene products. The various problems connected with these topics will be discussed. In future, the significance of HLA for transfusion medicine will not increase as the connected problems can be handled easily: avoidance of the induction of HLA antibodies and of FNHTR by leukodepletion, avoidance of TRALI by HLA and HNA antibody negative donors and of TA-GvHD by irradiation of the products. The importance of HLA in transplantation and cell therapy, however, will increase due to a better understanding of permissible incompatibilities and of the influence of other genetic systems, e.g. KIR; furthermore, a better knowledge of the function of the HLA gene products will be helpful for the selection of cells for therapeutic purposes in malignant diseases. Background: On behalf of the Working Party for Platelet Immunology of the ISBT, a platelet immunology workshop is held every second year as a regular training and quality assurance programme on an international base. It is the special focus of this workshop to include new developments in the field and to allow for setting standards in platelet immunology. Aim: Major aims of the 15th Workshop were platelet auto-and alloantibody testing and quality assurance in human platelet alloantigens (HPA)genotyping. This is the first ISBT Workshop to include platelet autoantibody testing with a standard protocol. Methods: After two announcements to previous participants and via the ISBT home page, blinded samples were distributed to all enrolled laboratories. This workshop included autoantibody testing in two serum samples with a standard protocol and standardized reagents (monoclonal antibodies, buffers, and substrate), detection and titration of HPA-1a alloantibodies in two samples, and detection of HPA-3a alloantibodies, some of which had been characterized as ''labile''(difficult to detect in some assays). Finally, DNA samples were distributed for standard genotyping (HPA 1-5 and 15) and, in addition, quality assessment of HPA-6 and HPA-9 genotyping. Samples with unusual genotypes were also included. Results: In total, 35 laboratories from 24 countries in America, Asia, Australia, and Europe were enrolled. Results are to be reported until March 31, 2010, and will be analyzed by the local organizing committee. Overall outcome, major advances and the most frequent problems will be summarized. Summary/conclusions: Results of the 15th Workshop will hopefully allow evaluating the chance of standardizing platelet autoantibody detection in order to support the clinical diagnosis of immune thrombocytopenia (ITP). Comparison of HPA-1a alloantibody titration with standardized protocols will contribute to the ongoing discussion if and how mothers at risk for giving birth to a baby with fetal-neonatal alloimmune thrombocytopenia should be monitored. HPA-3a antibody detection will alert participants to the risk of overlooking ''labile''anti-platelet alloantibodies with certain assays and may lead to a recommendation how such antibodies should be assessed. In the genotying exercise, samples with unusual genotypes will remind participants about limitations of certain typing approaches. Overall conclusion with interest to a broader public will be presented. Background: Candida albicans is the most common cause of nosocomial bloodstream fungal infections in neutropenic patients. Since innate immune cells including neutrophils, monocytes, macrophages can recognize and eliminate invasive C. albicans, leukocyte transfusion has been shown to be a promising treatment for these patients. However, the mechanisms of host neutrophils recognize C. albicans and activate anti-Candida responses are not well understood. b-glucan is the major structure component of C. albicans cell wall. It has been demonstrated that Dectin-1 as the principal C-type lectin pattern-recognition receptor (PRR) can recognize fungal b-glucan and induce immune responses. Aim: In this study, we sought to clarify whether human neutrophils could recognize insoluble b-glucan from the cell wall of C. albicans (CaIG) through Dectin-1 and induce anti-Candida immune responses and to determine the underlying mechanisms. Methods: Human neutrophils were challenged with CaIG in vitro. The mRNA expression of Dectin-1, Toll-like receptors (TLR2) and b-defensin-2 was assayed by real time reverse transcription polymerase chain reaction (RT-PCR). Dectin-1 expression was measured via flow cytometry. The amount of b-defensin-2 was measured by ELISA. H 2 O 2 release was determined by microplate fluorescent assay. Neutrophils fungicidal activity was analyzed. Western blotting was used to analyze IjB-a phosphorylation and degradation. Results: Exposure of neutrophils to CaIG led to increased gene and protein expression of Dectin-1, whereas the mRNA level of TLR2 was not altered. CaIG induced the secretion of b-defensin-2 and H 2 O 2 in a dose-dependent manner. Human neutrophils challenged with CaIG resulted in the activation of NF-jB in a dose-dependent manner. Dectin-1 inhibitor laminarin blocked the CaIG-induced production of b-defensin-2 and H 2 O 2 and the ability of killing C. albicans in human neutrophils, but no such effect was observed in pretreatment with anti-TLR2 neutralizing antibody and the LPS inhibitor (polymyxin B). Conclusions: These data suggest the interaction of Dectin-1 and insoluble b-glucan from the cell wall of C. albicans may contribute to the recognition and killing of C. albicans by human neutrophils. These findings may have important implications for the transfusion of leukocyte to cytopenic patients. (1-89) , including 12 children (<16 years old). Underlying diseases were: acute leukemia n = 41, lymphoma n = 9, MDS/MPS n = 8, aplastic anaemia n = 7, solid tumors n = 5 and others n = 13. In all cases platelet genotype (PCR-SSP) and the presence of anti-HLA and anti-HPA abs (ELISA & MAIPA) were investigated; if the HLA antibody screening was positive, platelet cross-matching was performed (Capture-P; Immucor), and platelet aphaeresis from a compatible donor was transfused. In selected cases, requiring prolonged support, the patient was HLA typed (PCR-SBT) and the anti-HLA abs specificity investigated (Luminex Single Antigen), to expand the availability of donors. Results: Anti-HLA abs were found in 49 cases (59%), with a median positivity of 85% (4-100%). Noteworthy, 75% of the women had anti-HLA abs versus 35% of the male patients (P = 0.001). Antibodies reactive with several platelet glycoproteins were identified in eight patients (9%) whereas specific anti-HPA abs were found in nine cases (11%) (HPA-1b n = 6, HPA-5b n = 1, HPA-15a n = 1 and HPA-15b n = 1). In 15 of these later cases, anti-HLA abs were also present. Anti-HPA abs were more frequently found in women as well (16% vs 3%; P = 0.001). The study was entirely negative in 32 patients (39%). Several non-immune factors could justified the platelet refractoriness observed in all of these patients; at least two factors were present in >80%, and three or more non-immune factors were present in >40%. Platelet crossmatching was carried out in 45 cases; no compatible donors could be identified in seven cases; <10% of the donors were compatible in eight cases; between 10% and 50% in 20 cases, and >50% in 10 cases. The degree of anti-HLA reactivity inversely correlated with the probability of finding matched donors (P = 0.007). In 10 patients, HLAbased selection from a panel of HLA and HPA typed donors was undertaken. An improved response to transfusions was observed by selecting crossmatch-compatible or HLA-matched platelets in more than 60% of the allo-immunized patients with available donors. Summary: Antibodies against HLA were identified in 59% of the total patients studied. Considering only the female patients, this incidence reached 75%. Anti-HPA specific antibodies were found in 11% of the patients and a further 9% showed pan-reactive antibodies, which could also justify the refractoriness observed. Background: Foetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against foetal platelet antigens, where anti-HPA-1a antibodies are the most frequent cause. Recent prospective screening studies reveal that maternal HPA-1a alloimmunization most often occurs in association with delivery. This finding suggests that alloimmunization may be prevented by administration of anti-HPA-1a antibodies to HPA-1a-negative women following delivery. A similar prophylactic strategy is currently used to prevent haemolytic disease of the newborn by administration of anti-RhD antibodies to RhD-negative women in connection with pregnancy. To generate a limitless source of antibodies for prophylactic treatment of FNAIT, we aimed to generate high-affinity human monoclonal antibodies (mAbs) specific for HPA-1a by immortalizing memory B cells from an alloimmunized woman. We rea-soned that naturally selected antibodies derived from immunized women may be more suitable for preventing immunization compared to human anti-HPA-1a mAbs made by phage display. Aim: The aim of the study was to generate human monoclonal anti-HPA-1a antibodies by immortalization of memory B cells from a naturally HPA-1a alloimmunized woman. Methods: Memory B cells were isolated by a combination of magneticand fluorescence-activated cell sorting (MACS, FACS), and immortalized by Epstein-Barr virus (EBV) transformation. Anti-HPA-1a antibodies were detected in cell culture supernatants by the monoclonal antibody immobilisation of platelets assay (MAIPA). Immunoglobulin variable region genes were amplified by reverse transcription PCR and sequenced using standard sequencing technique. Polyethylene glycol-mediated fusion was used to generate hybridomas. Background: Patients receiving multiple transfusion of non leuko-depleted platelet components, have a high risk for developing antibodies against platelets. In Jakarta, patients mostly maintained by transfusion with platelets derived from buffy coat (~90%), and rarely by platelet apheresis component (0.4%). Most of our patients come from internal medicine department with hemato-oncology cases which need frequent platelet transfusions. Therefore, we assume that some of these multiple transfused patients would have developed platelet alloantibodies. Aim: The goal of this study is to identify the specificity of these platelet alloantibodies and to estimate their frequency in patients receiving multiple non leuko-depleted platelet transfusions. Methods: We analyzed sera or plasma from 53 multi-transfused patients (27 males and 26 females) for platelet antibodies by whole-platelet ELISA using a panel of platelets (n = 40) to detect platelet reactive antibodies. Antigen capture assay, MAIPA, was performed to determine HLA or/and HPA antibodies. The specificity of HLA antibodies was then identified by the use of LCT against a lymphocyte panel (n = 60), and confirmed by cross-match analysis. Results: Among total samples, 12 sera (23%) reacted in whole-platelet ELISA. Two of them were derived from male patients, and the rest were from female patients who have history of pregnancy. All 12 patients had hemato-oncology problems such as acute myeloid leukemia, aplastic anemia, immune thrombocytopenia, thalasemia, myelodysplastic syndrome, and disseminated intravascular coagulation. Furthermore, we found by MAIPA, eight samples with HLA class I antibodies alone (21%) and one sample (2%) with HPA antibody alone, most probably against HPA-3a. In three samples, antibodies against HLA class I as well as antibodies against platelet GPIIb/IIIa and GPIb/IX (6%) were detected. In seven samples containing HLA class I antibodies, four patients developed multi-specific HLA antibodies, all of which developed alloantibody against HLA-A2, which was confirmed by positive cross-match with HLA-A2 homozygous cells. Other three samples had non-specific antibody, one sample suspected for IgM-antibody against HLA. Conclusions: Alloantibodies against HLA were the most frequent antibody developed in our multi-transfused patients especially with non-leukodepleted platelet transfusion history. However, alloimmunization against HPA may also occur. ELISA with platelets is effective for platelet antibody screening, followed by MAIPA and LCT for identification of HPA and HLA antibodies, respectively. Thus, the problem of refractory states in multitransfused patient must be concerned in the current platelet transfusion practice in Jakarta. Background: Genotyping techniques are routinely used for platelet antigen typing. Among these, PCR amplification using allele-specific primers, either in multiplex or uniplex, is a widely used approach. Here we report a case with a discrepancy in the results obtained by two different genotyping techniques, due to a point mutation that might had led to a false HPA typing result in a neonatal alloimmune thrombocytopenia case. Aim: To identify the cause of this discrepancy and characterize the GPIBA allelic variant in this carrier. Methods: Platelet genotyping for the HPA systems 1-3, 5 and 15 was initially performed by PCR-SSP, in a multiplex reaction in the case of HPA-1,-2 and -3. Extended HPA genotyping with the DNA-Chip platform BLOODchip Ò v.2.0, was carried out to discard a potential incompatibility between the mother and the newborn for a low-frequency or rare HPA antigen. A 370 bp fragment of the GPIBA gene, spanning the HPA-2 polymorphism and adjacent sequences, was amplified and sequenced using the Big Dye Terminator v.1.1 kit. Results: A single incompatibility between the mother and the newborn was found in the original HPA genotyping. The mother was typed HPA-2b2b, the father HPA-2a2a and the newborn HPA-2a2b, although platelet alloantibodies were not detected in the mother's serum. Later studies of extended HPA genotyping, investigating possible additional incompatibilities for previously untested low-incidence HPA antigens, revealed a discrepant HPA-2 typing result in the mother's sample. The HPA-2 genotype assigned by the BLOODchip Ò v. 2.0 was HPA-2a2b. We then suspected a mutation in the GPIBA gene, that might have impaired the specific amplification of the mother's HPA-2a allele by PCR-SSP. To identify this hypothetical mutation, a maternal genomic fragment corresponding to GPIBA exon 2 was amplified and sequenced. The sequence detected at the polymorphic HPA-2 position (nt 482) confirmed the heterozygous HPA-2a2b genotype, in concordance with the BLOODchip Ò results. A new mutation which does not induce an amino acid change was also found at nucleotide position 468 (C>G), in heterozygous state. This mutation is located within the sequence recognized by the allele-specific forward primer of the PCR-SSP approach, which could explain why the PCR amplification of the HPA-2a allele was hampered and leaded to a false negative result. Summary: PCR-SSP is a commonly used technique for platelet genotyping. The reliability of the typing results obtained with this method depends on the absence of a genomic mutation localized in the sequence of the PCR primers. A previous report already described one such mutation in the ITGB3 gene, which interfered with HPA-1 genotyping. As in that case, the mutation described in this report, has led to an incorrect HPA-typing result, with potential consequences in the diagnosis of maternal platelet alloimmunization. Autoimmune hemolytic anemias (AIHA) may occur when specific autoantibodies are developed against red blood cell (RBC) antigens. Anemia, signs of hemolysis, and detectable autoantibodies to RBCs are classical findings. The autoantibodies preferentially react at 37°C (warm autoantibodies). The majority of these autoantibodies are of the IgG class and less commonly of IgM or IgA warm classes. Roughly 50% of the patients have an associated disease (symptomatic or secondary AIHA), particularly lymphoproliferative, autoimmune disorders in adults, or viral infections in children. The cause of autoimmunization remains obscure in almost all other patients (idiopathic AIHA). Corticosteroids are the mainstay of therapy, but most patients with warm antibody AIHA require additional treatment with azathioprine, cyclophosphamide or other drugs. The most effective treatment in cases with life-threatening anemia is the blood transfusion. Cold agglutinins are the cause of hemolysis in <10% of patients with AIHA. These antibodies exhibit reactivity at temperatures <37°C. The most effective treatment is the consisten avoidance of exposure to cold. Rituxmimab is indicated only in isolated cases with cold agglutinins. Mixed type AIHA (cold agglutinines plus warm autoantibodies) is extremely rare. These patients may require a treatment similar to that used by AIHA of warm type. Donath-Landsteiner antibodies (paroxysmal cold hemoglobinuria) predominantly occur in children with a history of recent viral infection. In these cases, the hemolysis is usually acute and disappears within a few days. Drug-induced hemolysis occurs in about 10% of cases. The causative antibodies react with RBCs either in the presence and absence of the precipitating drug (drug-independent autoantibodies) or only in the presence of the drug and/or its metabolites (drug-dependent antibodies). The former antibodies are of the IgG class and predominantly cause extravascular hemolysis without complement activation, whereas the latter antibodies are of IgG and/or IgM classes that almost invariably cause intravascular hemolysis due to complement activation. Many patients have both types of antibodies. These cases are often confused with AIHA of the warm type. In some cases drug-dependent antibodies react in the presence of drug-metabolites, but not in the presence of the native drug.

The ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology currently acknowledges 308 blood group antigens, 270 of which belong to one of 30 blood group systems. The others are classified into collections (the 200 series) or the low-(700 series) and high-(901 series) frequency antigen series due to lacking molecular and genetic evidence. Thanks to rapid developments in molecular and cell biology, the genetic bases for all but one of the systems have been established. This knowledge has allowed new directions in immunohaematology including nucleic-acid-based blood typing and recombinant production of antigens as potential tools in various serological applications. In this context, any blood group antigen, the molecular genetic basis of which is not known, constitutes a challenge. If the molecule that carries the blood group antigen in question is unknown, then the corresponding gene cannot easily be identified and the underlying polymorphism not utilised in genotyping assays. Therefore, a focus of many immunohaematological research laboratories is to look for emerging new antigens or go back and revisit the orphan blood groups, i.e. antigens without a family (blood group system) or a home (genetic locus). Currently, 12, 18 and 8 antigens are categorized into the 200, 700 and 901 series, respectively, and specified by 6-digit identities. The 200 series contains six collections harbouring 1-3 antigens each. Of these, P k (209002) in the GLOB collection is known to be encoded by the A4GALT gene whilst the other 11 antigens have not been linked to genes yet. It has been debated whether the P k antigen should form a new system of its own or join the P1 antigen (003001) in the P blood group system, for which no genetic basis was known. Recent evidence linking the P 1 /P 2 phenotypes to a SNP in intron 1 of the A4GALT gene calls for a change. Thus, it is hereby suggested that P k is moved to join P1 in system 003, the name of which may simultaneously be changed from P to a name that reflects better the antigens belonging to it. Currently, the P antigen (028001) resides in the GLOB blood group system and is encoded by another glycosyltransferase gene, B3GALNT1. Therefore, the proposal is that system 003 changes its name, possibly to P1PK. Accordingly, the P1 antigen remains 003001 and P k becomes 003002. Eighteen low-frequency antigens remain to resolve although this is less crucial for clinical purposes. No information is yet available regarding their molecular/genetic homes. Several of the eight high-frequency antigens in the 901 series are clinically important (e.g. Vel, Lan, At a ) and all await assignment to either existing or new systems. Two of these antigens are known to be carbohydrates: Sda (901011) and MAM (901016). Additionally, new blood groups are waiting to be acknowledged and a couple of antigens presented at this meeting will join existing systems. New candidate antigens that comply with ISBT definitions are also emerging. Many of these are glycoconjugates likely to become officially accepted blood groups in the future. Background: Antigens of the Diego blood group system reside on the red cell membrane protein AE1 (band 3). The system currently comprises 21 antigens with two sets of antithetical antigens, Di a /Di b and Wr a /Wr b , of low and high incidence, respectively. The 17 remaining antigens are all of low incidence and no antithetical high incidence antigens are described. Many of these antigens reside on loop 3 of the band 3 protein which has a chymotrypsin cleavage site. All Diego antigens arise from a single nucleotide polymorphism in the SLC4A1 (DI) gene. We describe here a novel high incidence antigen which is antithetical to the low incidence Wu (DI9) antigen. Case study and methods: Blood samples from a healthy, untransfused, 34 year old primigravida (SK) were investigated for identification of an antibody detected following a miscarriage at 12 weeks gestation. Standard serological methods were used. The antiglobulin test (IAT) was carried out by LISS tube and DiaMed gel methods. DNA was extracted and genomic DNA sequencing was performed for exon 14 of SLC4A1, which encodes the third extracellular loop of the band 3 protein.

Results: SK plasma contained a very strong alloantibody reacting with all red cells tested by direct agglutination at 18°C and 37°C and marginally weaker by IAT. The cells of her brother were more weakly reactive than random control cells. Following extensive investigation and exclusion of all known antibodies to high incidence antigens we concluded that a novel antigen/antibody was involved. Testing against a range of proteolytic enzyme treated and chemically modified cells showed the determinant to be resistant to papain, trypsin and AET and sensitive to chymotrypsin. Serological testing for low incidence Diego antigens revealed a positive reaction with one multispecfic antiserum containing, amongst other specificities, anti-Wu. A homozygous G>C mutation was identified in exon 14 of SLC4A1 at position 1694 for SK. This results in a glycine to alanine change at position 565 of the band 3 protein (G565A) and corresponds to homozygosity for the allele encoding the Wu antigen. The 1694 G>C mutation was found in the heterozygous state in SK's brother. Summary/conclusions: We conclude that SK lacks a novel high incidence Diego antigen which is antithetical to Wu. We propose to call the antigen DISK (DI22). SK's Diego phenotype is DI:9,-22 and that of her brother DI:9,22. Wu and DISK represent the third pair of alleles in the Diego system. SK's antibody (anti-DISK) is extremely potent and has all the characteristics of a naturally occurring antibody, with similarities to anti-P,P1,PK made in pp individuals . It shows dosage in being more strongly reactive with DI:-9,22 cells than DI:9,22 cells. The serological characteristics suggest a highly clinically significant antibody but it is not known whether it played any part in her early miscarriage. Autologous units of SK are being frozen down should they be needed in the future. Background: Antigen D is still the leading cause of alloimmunization in D negative individuals and of hemolytic disease of the fetus and newborn. In 1953 anti-D was also detected in D positives; this seeming contradiction was attributed to partial defects of the D antigen. More than 200 RHD alleles are known, approximately one third of these are partial D, which may allow anti-D immunization. Aim: Since 1996 guidelines in Germany mandate the use of two monoclonal anti-D antibodies that do not recognize DVI for D typing in recipients. In 1998 the Rhesus Immunization Registry was launched within the framework of the German Society of Transfusion Medicine and Immunohematology (DGTI) as quality assurance for the then newly introduced D typing strategy. Methods: Cases of D positive individuals with anti-D are collected, analyzed and registered in an internet-based survey (www.uni-ulm.de/~flegel/RH/ RIR/). A questionnaire accompanies each sample documenting the gender of the patient, the transfusion history, pregnancies and the putative date of immunization. The samples are analyzed serologically by a standardized protocol including antibody differentiation, determination of antibody titers, performance of the direct antiglobulin test and antibody elution. Aberrant RHD alleles are identified by PCR and sequencing of genomic DNA. Results: Between 1998 and 2008, 123 samples were received. 109 were from Europe, nine from USA, three from Canada, one each from China and New Zealand. 74 were partial D: anti-D was observed in 12 cases with DVI, 12 with DIV, 11 with R0HAR, nine with DNB, six with DVII, five with DIII, five with weak D type 4.2 (DAR), two with DV. In addition 13 partial D were encountered with one observation each; among these were weak D type 11 (M295I) and weak D type 15. After 1997, three immunizations of DVI carriers were reported from Germany, one case involved massive transfusion, the other cases transfusions deviant from effective guidelines. Twelve individuals harbored DIV: two carried DIV type 1 and were of African decent; 10 carried DIV type 3 to type 5 and were of European origin. All individuals with DVII were from Europe. R0HAR carriers were all from Germany and predominantly from the state Nordrhein-Westfalen. 79% of the partial D carriers with anti-D were female; in about half of these pregnancy was a plausible immunization cause. Further samples with anti-D were from individuals who all had red blood cell bound anti-D and carried weak D type 1 to type 5, weak D type 33 or normal D. Conclusions: Besides DVI, other D variants like DIV are most frequently involved in Anti-D immunization events. Therefore, we propose an im-proved D typing strategy: a set of 2 monoclonal anti-D antibodies could be used with one not recognizing DVI, and another not recognizing other clinically relevant D variants. The combination of antibodies chosen may take account of prevalent alleles, such as DVI, DIV and R0HAR in Central Europe. Serologic typing should be combined with blood group genotyping. Especially pregnant women and their children would profit from this modified D typing strategy. Lund University, Lund, Sweden 2 University and Regional Laboratories, Lund, Sweden

Background: The A4GALT gene encodes galactosyltransferase that synthesizes the terminal Galactose-a1-4-Galactose binding in the P k glycosphingolipid, also known as Gb3/CD77, important in transfusion medicine, obstetrics and host-pathogen interactions. This locus has 3 exons with the entire coding region in the last. Curiously, crucial mutations in exon 3 abolish both the formation of P k and also another Gala1-4Gal-defined antigen, P1, the single member of the only blood group system for which a responsible gene has still not been established. Lack of P1 despite the presence of varying levels of P k , <ie. the P 2 phenotype, has been hypothesized to depend on polymorphisms in A4GALT, but neither found in the coding region nor the recently defined promoter. Aim: To elucidate the genetic basis of the P 1 /P 2 histo-blood group phenotypes. Methods: Transcripts from human bone-marrow-derived CD34+ cells in erythroid culture were subjected to 5¢/3¢RACE, cloning, RT-PCR, sequencing and quantitative real-time PCR. The recently reported promoter of A4GALT was cloned and deletion mutants made for use in luciferase reporter assays. Blood samples from 201 donors were analyzed by serological P1 typing and three newly-designed P1/P2-genotyping tests. Transcripts from A4GALT and 18S were semiquantified by TaqMan analysis. P k and P1 expression was evaluated by FACS in 15 samples (five of each genotype: P1P1, P1P2 or P2P2). Results: The 5¢/3¢-ends of A4GALT-mRNA were characterized and revealed a novel transcript containing only the non-coding exon 1 and a sequence from intron 1, here denoted exon 2a. This transcript included 5'cap, poly-A tail and 3 polymorphic sites, one of which correlated with P 1 / P 2 status in 50 samples by sequencing. A C>T substitution in exon 2a introduces a start codon that results in a potential open reading frame (ORF) in P2 alleles. Based on this, genotype screening assays for P 1 /P 2 status confirmed concordance in 200 of 201 samples. Luciferase experiments with P1 vs P2-related variants of the proximal promoter indicated that polymorphisms here do not affect transcription. Interestingly, P2/P2 samples demonstrated very low A4GALT transcript levels, whilst P1/P1 and heterozygotes had 28 and 4 times more, respectively. FACS and serology showed that P1 antigen expression was lower in heterozygotes than P1 homozygotes. Database searches identified multiple amino-acid sequences homologous to the new ORF. This may be because exon 2a consists mainly of a~300-bp Alu sequence, abundant in human genomes and found only in primates. However, Alu sequences are not thought to be translated. We found no other Alu elements with the C>T substitution in human database entries but, interestingly, an almost identical ORF in macaques. Conclusions: We observed striking variations in A4GALT transcript levels between P 1 and P 2 individuals but found no explanation for this among polymorphisms in the promoter or known exons. Instead, an alternative transcript based on a newly-discovered exon revealed a SNP that allows prediction of P 1 /P 2 status. Zygosity for the P1 allele correlated with transcript/antigen levels and appears to explain the known interindividual variation in P1 antigen strength. Current investigations focus on how the Alu-related sequence and/or ORF downregulate A4GALT transcription in P2 haplotypes.

Wednesday: Parallel Sessions 5A-S31 -Quality management in blood establishments 5A-S31-02

Quality has long been recognized as a necessary tool in the surveillance of complex processes in transfusion medicine. Dynamic development of transfusion medicine and continuing demand for upgrading the safety and quality of transfusion therapy have modified the perception of quality and its importance. Over decades, it has transformed from the basic quality control elements through individual and integrated quality management systems (QMS) to costumer focusing, holistic approach to quality and its achievement, and overall performance excellence. The critical role of quality and safety of transfusion therapy has been recognized in EU legislation. The ever wider implementation of quality system has entailed the need of a skilful professional to work on the issues related to quality management. Although the role and scope of the tasks covered by quality manager are generally dictated by the type of institution, they all share some basic responsibilities. Quality manager has a key role in QMS development, implementation, maintaining and improvement. He collaborates with the institution management in quality planning and setting quality goals, steers activities related to product and service quality in transfusion establishment, and promotes the philosophy of continuous quality improvement. Analyzing relevant data collected from the processes, he assesses conformity of the product and service quality with quality goals and costumer demands. Quality manager has an active role in stimulating quality communication and partnership with the costumers, assessment of their needs, and testing their satisfaction with the products and services provided. He is also responsible for promoting ethical standards and creating positive atmosphere, personnel motivation and correct management of human resources, in critical situations in particular. He is generally in charge of monitoring the change control system, assessment of supplier quality system, assistance and collaboration on inspection, certification and audit, and personnel education on all QMS aspects. Quality manager is responsible for the establishment of internal quality assessment program, where he designs annual internal audits plan, coordinates and supervises its performance, and monitors the efficiency of corrective measures. He takes active part in the development and management of the institution quality documentation. He is responsible for quality reporting at all levels. Besides due knowledge, quality manager should possess a number of skills to perform his function and tasks efficiently. In addition to formal authority and rational approach to management, a successful manager should possess leadership skills, especially to create such a working atmosphere where people believe in continuous improvement. The abilities of planning, proper organization and priority selection are necessary to manage the working tasks efficiently. Quality manager should be familiar with team work specificities and stimulate collaboration among different departments and professionals. By position, he has to closely collaborate with a great number of individuals, where good interpersonal skills, verbal and written communication skills are of utmost importance. Along with appropriate intellectual functioning, knowledge and professionalism, successful performance of quality manager is greatly influenced by emotional intelligence, which has direct impact on the process of communication, the ability of solving conflict situations, team work efficiency, motivation of coworkers, and leadership.

Who delivers Quality in an organisation? Think back to the professional workman of years gone by manufacturing highly valued bespoke goods. They might make furniture, jewellery or fine clothes and in doing this they would exert total control of the process using knowledge acquired through trail and error. They would listen to their customers understand their success in getting repeat business and gradually improve. In doing this they had no need for formal written specifications, they would source their material from suppliers they knew and understood, choosing what they knew would be valued by their customers and hand crafting it into a finished product, they had little need for systems but many, if they could write, would have kept notes about their suppliers, designs, successful outcomes so they could refer back to aid their memory. However whilst this would provide a living, except for the most valued artisan, it would only provide a living wage, rather than luxury. Slowly people came to realise that the way to earn money was through employing people to work on behalf of the craftsman, perhaps engaging apprentices who would learn the craftsman's skills and share the workload or employ assistants to carry out stages in the production process. From this point on ''manufacturing'' became more about control of people than the skills of the individual craftsmen allowing people to specialise in specific areas of work. The jobs became compartmentalised. This was of course seen in a variety of manufacturing industries particularly car manufacturing and defence, leading to some of the first principles of quality assurance which are part and parcel of what we do today. Quality Management systems aim to ensure that individual specialists work together to provide the best possible products and services. Clearly Blood Services have additional challenges. Collecting donations which are intrinsically dangerous, ensuring they are only given to compatible recipients and that components which might carry transfusion transmitted infection are effectively removed from the supply chain. In doing this we need work consistently, really understand how we can or do go wrong, and improve by implementing effective preventative action. Who drives that improvement? The quality professional, the clinician or the operational staff. In my experience this has been best achieved through adhering to and applying rigorously the principles of EU Good Pharmaceutical Manufacturing Process (EU GMP), and applying the increasingly sophisticated tools that evolving experience in its use EU GMP recommends. For example using risk management to assess the level of control a particular process requires. In a properly designed quality management system they will all have their part to play. Through some well chosen examples I will illustrate how applying simple GMP principles can assist in bringing about significant improvements which will ultimately benefit our donors and patients. Background: For more than a decade since 2000, through attaining certification and accreditation, the BTS has successfully established an integrated management system (IMS) in accordance with ISO9001, ISO14001, ISO15189 and GMP standard requirements. In line with global trend in sustainability development in blood industry, the BTS also recognizes its corporate social responsibility to provide a ''risk free'' work environment by minimizing incidents to its work force and the interested parties. OH-SAS18001 has been widely accepted as the basis for certification of Health and Safety Management System (HSMS) since launch. In view of the greatly improved compatibility with other management system requirements, the BTS has in early 2008 adopted the new OHSAS18001:2007 standard to strengthen its HSMS. Aim: (i)To develop and implement an HSMS meeting OHSAS18001:2007 requirements and to attain OHSAS18001 certification. (ii) To provide confidence to top management and other interested parties regarding the BTS's ability to comply with legal requirements and to achieve its health and safety and associated economic objectives. (iii) To integrate the HSMS into existing IMS by leveraging current resources and capitalizing existing practice and personnel. Methods: Phase 1: Adopt a risk-based approach to manage the health and safety aspects throughout manufacturing processes, where appropriate, and perform hazard identification and risk assessment as follows: 1. to identify, prioritize and document the risks associated with its activities, 2. to apply controls to eliminate or minimize these, as appropriate, 3. to relate the identified risks to relevant legal requirements, 4. to define health and safety policy, set appropriate objectives, preventive measures and their implementation program 5. to update IMS manual and compile HSMS procedures Phase 2: Apply ''Plan-Do-Check-Act''methodology to ensure effective implementation, verification, and improvement of BTS's HSMS, and prepared for certification. Results: The BTS attained OHSAS18001:2007 certification in 2009; meanwhile succeeded up-grading the established Quality-Environmental Management System to a total IMS. The BTS has established a comprehensive methodology for minimizing risk levels in work processes. The IMS holistic approach helps identify various aspects of each process, including quality, safety, environmental technical and social aspects, for integrated and effective monitoring, and continual improvement. Conclusions: In addressing health and safety problems throughout the blood manufacturing life cycles, the HSMS helps increase productivity by reducing additional unproductive costs for handling incidents and injury on duty, improving resource efficiency and building up employee morale, as well as creating a brand image of social responsibility to the BTS. The BTS is the first blood centre in the world to have successfully integrated quality, environmental and health and safety management systems resulting in one cohesive IMS that exhibits holistic approach results in balanced performance.

For implementation of good clinical transfusion practices it is necessary to establish hospital transfusion committees (HTC) all over the country. National Blood Transfusion Service initiated action to implement hospital transfusion committees in the year 2005 according to the WHO recommendations guided by the local and foreign experts. Initially it had been scheduled to be established only in all the teaching hospitals in Sri Lanka, but the success of this programme is debatable as the evidence recovered from records available revealed that it could not achieve the expected target. The number of hospitals where HTCs were established from 2005 to 2009 was <10 which will be shown in a table below. National Blood transfusion service is now being organized to achieve its aim of applying quality assurance to clinical interface of blood transfusion through hospital transfusion committees in the year 2010 by strengthening already established HTCs and taking steps to establish new hospital transfusion committees in all hospitals island wide. As the inaugural step a team is formulated by the national blood transfusion service in order to achieve this task. The target of this team is to establish HTCs in 15 regional blood centres initially and then expand it to 74 blood banks island wide. When establishing HTCs in a resource poor country like Sri Lanka strategies adapted by the NBTC were 1. Educate the medical officers in charge of each regional transfusion centre or blood bank. 2. Empower them to organize the HTC by annually or quarterly by providing necessary documents guidance and feedback by the NBTC. 3. Participation of a NBTC representative was made compulsory for each meeting (Consultant Transfusion Physician or Post graduate trainee in MD Transfusion Medicine) 4. All administrative problems arising in the meeting will be solved by the Director NBTS. 5. Recommendations to upgrade the blood bank performance as well as bed side transfusion practices will be sent to relevant authorities after each committee meeting by the National Blood Centre. 6. Guidance and resource persons will be provided for all awareness programmes required by the end users of blood and blood components. As there is a need to address the quality to be continued once the blood leaves the blood banks for use in wards and operation theatres, HTC is the key for it. There is no question that every one in each level of health care system can make a difference to clinical transfusion practices in their own hospital. Hence improving communication between blood bank and clinical staff is also important for fulfilling this task. By establishing hospital transfusion committees they can be given responsibility and authority to monitor and resolve any problems identified regarding transfusion practices. In the past three decades, the production of standard blood components from whole blood donations and from apheresis collections has reached a widely accepted high technical standard, paralleled by semi-automated methodology to safeguard the stability of the production process, and an internationally harmonized pharmaceutical quality. More recently followed methodological advances include pathogen inactivation as well as novel separation methods, which are challenging the previous achievements. This review aims (i) to summarize the current status of implementation of novel techniques into the routine preparation process of blood components, (ii) to discuss upcoming approaches in the area of component preparation, (iii) to identify clinical needs which justify novel investigations into blood component quality, and (iv) to pojnt out the status on the production of standard blood components by in vitro differentiation from stem and progenitor cells in vitro.

Hancock VJ, Cardigan RA, Thomas S NHSBT, Brentwood, United Kingdom Introduction: Red Cell Concentrates (RCC) may encounter temperatures outside those required for their normal storage (+2-+6°C) during their storage or supply. Although some temperature deviations are permitted in the UK Guidelines and previous studies have shown RCC can withstand these deviations (10°C for both 5 and 12 h) without adverse affects on their quality, it is not clear to what extent RCC storage temperature can be varied before quality is affected. Aim: The aim of this study was to evaluate the quality of RCC exposed to warm (22 ± 2°C) and cold (-2°C) deviations for 3 and/or 5 h at various and repeated points during their storage and supply. Methods: Blood was collected and stored overnight at 22 ± 2°C before being processed to RCC in SAGM. Subsequently, three sets of 10 RCC were pooled and split into three sets of nine test units. Eight of these units were exposed to single and multiple periods of warm (22 ± 2°C) and cold (-2°C) temperatures on days 3, 8, 15, 29, 36 and 43 of storage for 3 and/or 5 h before in vitro quality testing was carried out, alongside one control unit stored at 2-6°C for the 43 day storage period. One-way ANOVA was used to detect any differences in data from test and control units at the end of storage. Results: No significant differences were seen between control units or units exposed to any of the temperature deviations, when assayed for haemolysis, supernatant potassium, deformability, ATP and 2,3-DPG at day 43. In all units, haemolysis was below the 0.8% UK limit at the end of storage, although there was a trend towards lower haemolysis in packs incubated in cold rather than warm temperatures. Supernatant potassium gradually rose to a maximum of 6.1 mmol/unit at day 43. All 27 RCC units had ATP levels above 2.3 lmol/gHb at day 36, the day after the current NHSBT shelf life of RCC-SAGM. However, ATP levels in 6 units exposed to warm (22 ± 2°C) temperature deviations dropped below 2.3 lmol/gHb by day 43. All control units and units subjected to a deviation in temperature had levels of 2,3 DPG below 3 lmol/gHb from day 3. Deformability declined gradually during storage, as expected, as did RCC pH which was below 7.0 from the start of the study and gradually declined to a minimum of 6.35. Summary: These results suggest that multiple exposures of up to 3 · 3 h and 1 · 5 h to warm (22 ± 2°C) and cold ()2°C) temperatures did not significantly affect the in vitro quality of RCC in SAGM throughout their 43 day shelf life. Background: In transfusion medicine, red blood cells (RBCs) are liquid stored at 4°C up to a maximum of 5-6 weeks before being discarded. Alternatively, cryopreservation enables storage of RBCs for years. Cryopreservation is currently a valuable approach for long term storage of RBCs from donors with rare blood groups and for military deployment. Moreover, stockpiling frozen RBCs can be beneficial in emergency or clinical situations, where the demand exceeds the normal supply of RBCs. Currently RBCs are frozen and stored according to the high glycerol method (i.e. with a final concentration of 40% glycerol at )80°C). After thawing the RBCs need to be deglycerolized to prevent hemolytic transfusion reactions and renal failure after infusion. Once deglycerolized the shelf life of RBCs is limited to 2 days when stored in SAGM media. Little is known however, about the in vitro rheological properties (i.e. aggregability and deformability) of deglycerolized RBCs. The RBC aggregability and deformability are important determinants of the blood flow and hence the oxygenation of the tissues. Transfusion of rheologically impaired RBCs (i.e. enhanced aggregability and reduced deformablity) may hinder or obstruct the microcirculation, leading to reduced tissue perfusion, thrombus formation, ischemia or infarction. Aim: To assess the rheological properties of thawed deglycerolized RBCs and to compare the results with the rheological properties of liquid stored and fresh RBCs. Methods: Fresh RBCs were obtained from healthy volunteers. Leukoreduced liquid stored and thawed deglycerolized RBC units were obtained from the Sanquin blood bank. RBCs were in vitro tested for aggregability, deformability, osmotic fragility and various hematological variables such as hemolysis, mean cell volume (MCV), mean cell hemoglobin concentration (MCHC) and adenosine triphosphate (ATP) content. Results: The AI of cryopreserved RBCs was significantly reduced compared to fresh and liquid stored RBCs (P < 0.05). The time necessary to induce aggregation was slightly prolonged at day 0 post thaw as compared to liquid stored RBCs. The deformability of stored RBCs was significantly enhanced over a shear stress range of 2.0-26.4 Pa compared to fresh RBCs (P < 0.05). No significant differences in deformability between cryopreserved and 21 or 35 day liquid stored RBCs however were observed. The osmotic fragility, hemolysis, MCV and MCHC of cryopreserved RBCs were markedly altered compared to fresh or liquid stored RBCs (P < 0.05). The ATP content of cryopreserved RBCs was similar to 3 or 21 day liquid stored and fresh RBCs. Conclusions: Cryopreserved RBCs were more fragile during post-thaw storage than liquid stored and fresh RBC. The freeze-thaw wash process however, did not adversely affect the aggregability and deformability or the ATP content of deglycerolized RBCs. From the rheological point of view it is concluded that cryopreserved RBCs are a valuable alternative to liquid stored RBCs for usage in transfusion medicine.

Aim: The present study was performed to get data on the effect of late irradiation on day +28 or day +35 on RBCs that have been leukoreduced before subsequent storage. Currently, an irradiation later than on day +14 is not allowed by the Council of Europe recommendations. However, it is unknown if this restriction remains to be reasonable after the introduction of general prestorage leukoreduction. Methods: We studied 160 RBC units that were leukoreduced on the collection day and stored subsequently in the additive solution saline-adenine-glucose-mannitol (SAG-M). Forty components were irradiated with 30 Gy on Day +14, 40 on Day +28, and 40 on Day +35, and 40 served as nonirradiated controls. In vitro evaluation of both irradiated and nonirradiated RBC units was performed before and after irradiation on Days +3, +7, +14, +21, +28, +35, and +42 from the collection day. Results: Gamma irradiation induced enhanced leakage of potassium ions and lactate dehydrogenase and an enhanced in vitro hemolysis rate in the irradiated components. The enhancement of hemolysis and leakage was first detectable one week after irradiation. The mean in vitro hemolysis rate of both nonirradiated and irradiated components was remarkably lower than 0.8 percent, and the preservation of adenosine triphosphate over 42 days was satisfying. Particularly, at the end of shelf life the quality of RBCs, that were irradiated late, was superior to the quality of units that had been irradiated on Day +14. Conclusions: This study demonstrates that the restriction of the RBC storage period prior to gamma irradiation is no longer reasonable after the introduction of general prestorage leukoreduction. The Council of Europe recommendations should be revised in this regard.

Background: Platelet transfusions are an essential part of modern medical practice in the treatment or prophylaxis of bleeding due to qualitative or quantitative platelet defects. Platelet preparations as blood derivatives carry the same risk of transfusion as other blood components. Aim: Most blood banks prepare random donor platelets from whole blood donations by the PRP method (Platelet Rich Plasma) or the BC (Buffy Coat) method. The Buffy Coat method is still not popular in India. Use of prestorage leucodepletion and pooling of platelets is also not yet popular . Hence the following study was undertaken to develop best practices in the BC platelet preparation with prestorage leucodepletion and pooling. It also aimed to assess the quality of the platelet content of the pooled buffy coat. Modern health care depends to a large extent on blood and blood products. A small percentage (3-8%) of the eligible population contributes to the blood supply by donating blood. Since the task of recruiting new blood donors still poses a major challenge, the act of retaining blood donors is of critical importance to blood organizations Donating blood can be described as a form of pro-social behaviour, a free gift of blood to unnamed strangers. To donate blood implies an altruistic motive, ''I give blood to help others.'' Other motivational factors mentioned in the literature, that bear importance on the decision to give blood, include awareness of the need for blood, moral obligation, and convenience (opening hours, donation facility). In addition, studies have shown strong indications that donors partly donate for their own personal benefit. Reciprocal ability ''I give blood now, because I might need it myself in the future'', incentives (medical testing), and personal needs for recognition and self-esteem are important. Giving blood is partly selfish and ''might be more an act of benevolence rather than altruism'' (Ferguson, 2008) . Not only the recipient profits from the altruistic gift, but also the donor, i.e. feeling good about giving. Furthermore, it has been shown that the donation experience itself strongly impacts donor return. Having a bad donation experience, like being temporarily deferred, experiencing a donation reaction, or encountering unfriendly staff, is detrimental for making repeated donations. Negative experiences generate donor loss. Knowledge about the reasons underlying the blood donation decision is important for donor retention practices. Altruism is often mentioned as a major reason for donating, but it is debatable whether it is the most important element in the decision to start donating blood. No donor can be characterized by ''complete, disinterested, spontaneous altruism'' (Titmuss, 1972) . Individuals are multiply motivated, different motivational factors impact on the decision to donate. The distinction between more altruistic-based motives and self-regarding motives is valuable. Especially when taking into account the negative effect of bad donation experiences on donor return. Donating blood is not a mere altruistic deed. In one way or another, donors profit from their gift, by increased self-esteem and self-satisfaction. There is a longstanding tradition that recruitment appeals bear strong altruistic messages by conveying the need for blood. However, for retention practices, it may be fruitful to address self-regarding motives. It is important to stress the importance of the donor; ''you are of vital importance to us''. This may be especially relevant for those donors with negative donation experiences. Preventing all temporary deferrals and bad donation experiences is impossible. However, when focusing on donor retention strategies, blood organizations should take advantage of the fact that donors need to feel needed. Donors know about the need for blood and subscribe to giving for free, but they need to feel that they are still in the game, when they were not able to make a donation. This feeling strongly influences the delicate balance between altruistic and self-regarding motives, and thereby the good habit of continued blood donation. Background: In Europe, donor management faces several challenges, some of them with considerable political weight. 1. Migration: Migration throughout Europe has enormous effects on blood product supply and demand. Migrant populations show different disease patterns with different demands, while available data suggests that migrants tend not to be a blood donor in their new country. 2. Commercial activities: Commercial blood establishments now operate in 20% of the European countries. Commercial organizations are active in these countries that collect and process plasma. Non-remuneration of blood donors still is the written basic principle handled throughout Europe. However, in the plasma derived pharmaceutical business, paid donors do occur throughout the world, including the Americas and Europe. 3. Competition in the blood product supply chain: The debate on introducing competition in the field of blood supply for direct use in patients is growing throughout Europe. Pricing of blood components and access to donors are the major arguments for starting this debate. Competition and donor management: In case competition occurs, some aspects of donor management need special attention. Requested/potential donor base: The product range and market share of the blood establishment in question are the determinants for the qualitative and quantitative requirements of the donor base needed. For example, if a blood establishment decides to produce only apheresis plasma and only non-sub typed ABO-red cell and platelet concentrates for the lowest possible price, then the donor base will be essentially different from the donor base needed to produce the full range of blood products and blood components. Quality/Safety balance: Minimum quality and safety standards are laid down in European Directives and must be met by each blood establishment. However, a rise in quality assurance inevitably has its price. The precautionary principle confronts blood establishments with possibly large costs. Therefore, in straightened economic circumstances, the precautionary principle will, likely, be the first to be attacked with a subsequent potential rise in patient risk. Additional products and services: To remain attractive business partners for hospitals, a blood establishment could decide to offer additional services, such as 1. Additional and sub typed products: tissues, red cells and platelets; rare blood groups 2. Stem cells, including cord blood units 3. Product advice; consulting To remain attractive to donors, a blood establishment could offer them services, such as 4. Lifestyle advice 5. Periodic health check 6. Conditional sale (e.g. reduction in insurance premiums) Concluding remarks: 1. Migrational effects require adjusted recruitment/ retention strategies throughout Europe. 2. Blood establishments must anticipate fluctuations in both their client base (shopping hospitals) and donor base (shopping donors) in case competition and commercialisation in the blood product supply chain emerges. 3. The introduction of competition in donor management may influence the supply of blood products qualitatively and quantitatively.

Background: The American Red Cross instituted a donor hemovigilance program (HVP) in its 35 regional blood centers in 2003, to document adverse events occurring at the time of donation or reported later. The aims are to describe and quantitate rare events using standard definitions, define risk factors, design specific interventions and to assess outcomes. In the1990's most U.S. blood centers converted to 500 ml whole blood (WB) collection bags to maximize blood collection. The AABB instituted a standard (#5.4.1.A) to permit a maximum blood loss of 10.5 ml/kg and minimum donor weight of 50 kg (110 lb), allowing 525 ml of blood to be removed from the smallest donors. In the last decade, the majority of States have enacted legislation to allow 16 year old donors to donate with parental consent Methods: Standardized adverse event definitions and reporting were instituted in 2003 and adverse event data were collated and analyzed at the National Medical Office. Standard database and statistical analytical methods were utilized. Estimated blood volume was calculated based on height, weight and gender, using the method of Nadler et al. Results: Analysis of HVP data from 2002-2006 highlighted that young, first time, female donors were at risk for vasovagal reactions, injuries related to falls and the need for outside medical care. Multivariate analysis revealed young age, low estimated blood volume (<3.5 l) (EBV) and firsttime donation status as major independent risk factors. Calculation of EBV revealed that current AABB standards allow, and CFR limits do not prevent, 15-20% blood loss of EBV from short, low weight female donors. Interventions to reduce vasovagal reactions were piloted, starting in 2008, including: (i) a controlled environment through standardized work guidance, physical setup and staffing matrices; (ii) improved donor and parent educational material prior to donation; (iii) enhanced donor monitoring post donation; (iv) fluid intake encouraged within 30 min of donation; (v) distraction and muscle tension techniques; (vi) use of apheresis double red cell collection in eligible donors; (vii) enhanced post donation instructions to donors and patients; (viii) implementation of selective height and weight restrictions in donors <19 years old to avoid collection from donors with EBV <3.5 l. Conclusions: A national HVP has highlighted the issue of vasovagal syncope-like reactions in young first-time donors with low estimated blood volume leading to multiple interventions designed to reduce risk. Outcomes are being assessed and monitored. Current AABB standards do not adequately protect young donors with low height/weight from excess blood loss in the United States. Depletion of body iron stores is a frequent and significant side effect of blood donation. It may occur in whole blood as well as in apheresis donors. On the one hand, iron overload is associated with multiple organ damage. On the other hand, there is evidence that low body iron stores may decrease the risk of cardiovascular disease and cancer. If the latter is true, it would be desirable to tolerate mild iron deficiencies in blood donors. However, a number of trials have demonstrated that mild iron deficiency may lead to impaired cognitive function, fatigue and decreased physical endurance, even in the absence of anemia. Based on these trials, it can be concluded that lowering body iron stores below the limits of iron deficiency might be harmful for blood donors. Another aspect to consider is that donors who have been deferred due to low hemoglobin concentrations may potentially not return. Therefore, it appears quite appropriate to prevent iron depletion following blood donation in at least donors who are at a high risk of developing iron deficiency, i.e. female donors of childbearing age and male donors with a higher donation frequency. Major obstacles of iron supplementation include the risk of deterioration of undiagnosed hemochromatosis or masking of gastrointestinal bleeding. In order to avoid these complications and to improve compliance, an intermittent iron supplementation in a dose range which exclusively replaces iron loss from blood donation appears to be the most suitable solution. In a recent trial, a daily supplementation of 20 mg iron combined with 400 mg ascorbic acid over 30 days resulted in an adequate compensation of iron loss. Since intestinal iron absorption is inversely correlated with body iron stores, donors with iron deficiency were observed to benefit more from iron supplementation than donors with normal iron stores. In conclusion, blood donation centers should be aware of the potential harmful effects of iron deficiency in blood donors and must take responsibility in maintaining the donor health. Due to their multi/pluripotency and immunosuppressive properties Mesenchymal Stem/Stromal Cells (MSC) are important tools for treatment of immune disorders and tissue repair. The increasing uses of MSC lead to the development of production processes that need to be in accordance with good manufacturing practices (GMP). In cellular therapy, safety remains one of the main concerns, of these the risk of transformation is a major one and to avoid side effects accurate production controls have to be implemented. At this time, there are conflicting data regarding the genomic stability of culture expanded MSCs, and the risk of transformation. Conducted on immortalized human bone marrow MSCs, the first studies showed that transformation in MSCs was a long and multistep process involving genetic and epigenetic changes in cell experiencing more than 100 population doublings. Moreover, alterations of key genes, as p16ink4a, were mandatory for transformation. The first evidence of human MSC transformation during culture was reported for adipose tissue MSCs subjected to long term culture. If there were karyotype abnormalities, the transformation only appeared after p16 deletion, over-expression of cmyc and re-expression of human telomerase (hTERT). This transformation process could be linked to a mesenchymal-epithelial transition. One study on bone marrow MSCs reported that transformation could be a frequent and fast process. On the contrary, using karyotyping, array-comparative genomic hybridization (array-CGH) and fluorescent in situ hybridization (FISH), different studies on clinical-grade cultured MSC did not show any evidence of genetic instability and transformation. Recently, in two different clinical trials, we showed that aneuploidy could appear whatever the culture process was. However, presenting or not aneuploidy, all cultivated MSC reached senescence, and they did not exhibit any transforming events. In conclusion, if genetic stability of clinical grade expanded MSC has to be tested, karyotyping does not seem a relevant control. Focusing on the molecular events involved in transformation, a new approach of controls is needed. Background: Advanced Therapy Medicinal Products (ATMPs), especially the cell based medicinal products, have to be tested for sterility following pharmacopoeial regulations including membrane filtration or direct inoculation. The main disadvantage of the pharmacopoeial sterility test is the long incubation time of 14 days up to result. This fact impacts both patient and manufacturer because many of these ATMPs show a very short shelflife (often <2 days) from cell harvest until administration to the patient. Therefore, the sterility testing can be improved if the detection takes place in <14 days with a rapid detection method that does not rely upon high bacterial cell density for visualisation. Aim: The general aim of this study was to evaluate the use of the Milliflex Rapid Microbiology Detection and Enumeration System (Millipore, Molsheim, France) by using selected PEI Blood-Bacteria-Standards (BBS) and to demonstrate the suitability for bacterial monitoring of cell based products. This system is based on membrane filtration and ATP-bioluminescence technology and is widely used for bacterial monitoring of e.g. pharmaceutical water, bioburden and in food industry. Methods: All together 14 PEI BBS (13 bacterial species and 1 yeast) which are defined in identity and count were involved in the study. Exemplary artificially contaminated Chinese hamster ovary cells (CHO, 4 · 10 6 cells/ ml) and samples of physiological saline, both containing 20-80 CFU per sample, were filtered through a Milliflex funnel, incubated onto agar media, applicated with reagents on the Auto Spray Station and microcolonies were enumerated on the Detection Tower. CHO-samples were contaminated with three model organisms and pre-treated with a selective mammalian cell lysis solution and apyrase. Furthermore the influence of different incubation temperatures and agar media was investigated. Results: The vast majority (13 out of 14 microorganisms) could be detected between 4 and 14 h including the three organisms inoculated in CHO-cells. The anaerobic slow-grower Propionibacterium acnes was detectable after 38 h. The loss of CFU on the membrane was depended on the bacterial species and final sampling time of incubation. The agar media as well as the incubation temperature showed no significant influence on the recovering rate of the organisms on the membrane. Conclusions: ATMPs represent new dimensions in microbial safety of drugs since significant precautions in pharmaceutical industry are not applicable. The Milliflex is a unique system to detect and enumerate viable microorganisms in one-fourth to one-fifth the time of traditional microbiology methods. In this study, it is shown that the detection time necessary for reliable bacteria detection can be reduced if macroscopic observation of turbidity is replaced by the use of the Milliflex system. The results demonstrated the suitability of the Milliflex system for rapid bacterial monitoring of cell based products but there is a need for further evaluation of the principle in the use for ATMPs. Background: Platelets, the fundamental component of primary hemostasis, are also known as reservoirs of many growth factors (GFs) in their agranules. Platelet-rich plasma (PRP) has been used an autologous source of GFs for various field of tissue regeneration, but the benefit for bone repair is still controversial. Aim: The aim of this study was to confirm the effects of PRP on proliferation and osteogenic differentiation of human mesenchymal stem cells (HMSCs) in vitro, and to investigate which GFs in PRP affect proliferation and osteogenic differentiation. Methods: PRP was obtained by double centrifugation. The concentration of platelet-derived growth factor-AA (PDGF-AA), PDGF-AB, PDGF-BB, transforming growth factor-b1 (TGF-b1), fibroblast growth factor-basic (FGF-b), insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) were measured using ELISA. HMSCs were cultured in DMEM with 1%, 3%, 10% and 30% PRP or platelet-poor plasma (PPP) from three donors. After a 4-day and 12-day cultivation, DNA content and alkaline phosphatase (ALP) activity were measured. To evaluate the relationship between GF concentration and HMSC proliferation or osteogenic differentiation, HMSCs were cultured with 10% PRP from 39 donors for 12 days, and were then analyzed using multiple linear regression. Results: Both PRP and PPP had stimulatory effects on the proliferation of HMSCs with a 10% concentration after 12 day. However, the stimulatory effect of PRP was more significant as compared to PPP (P = 0.006). The ALP activity of cultured HMSCs decreased with the addition of PRP, but the addition of PPP did not affect the ALP activity of HMSCs. The concentrations of the GFs except for IGF-1 were significantly higher in PRPs as compared to PPPs. The amount of DNA was positively correlated with the concentration of TGF-b1 (P = 0.000) and negatively correlated with the age of donors (P = 0.017). ALP activity of cells was negatively correlated with the concentration of PDGF-BB (P = 0.008). Conclusions: PRP could stimulate the proliferation, but suppressed the osteogenic differentiation of HMSCs. The inter-individual variation of the concentration of GFs could lead to different effects of PRP. The positive effect on proliferation of HMSCs was mainly affected by TGF-b1, and the suppressive effect on osteogenic differentiation was mainly affected by PDGF-BB in PRP. Mesenchymal stem cells (MSC) are multipotent progenitor cells and can be isolated from various sources such as bone marrow, adipose tissue and placenta among others. It has been demonstrated that MSC elicit no alloreaction when co-cultured with allogeneic peripheral blood mononuclear cells (PBMC) in vitro and are able to suppress proliferation of stimulated PBMC. Thus they are promising candidates for tissue engineering and the treatment of graft versus host disease or autoimmune diseases. Furthermore, it has been shown that cell contact as well as soluble factors play a role in MSC mediated immunosuppression. Also, fully differentiated primary cells such as human renal tubular epithelial cells (RTEC) have been reported to modulate T-cell proliferation in vitro. Thus, the aim of this study was to compare two MSC populations: human adipose derived stem cells (ASC) and human amniotic mesenchymal stromal cells (HAMSC) to RTEC regarding their antiproliferative properties on stimulated PBMC and T-cells. With regard to a possible cell-contact driven mechanism, cell surface expression profiles of MSC and RTEC were compared under normal and inflammatory culture conditions and the effect of interferon-gamma (IFN-c) pre-treated MSC and RTEC on immune cell proliferation was observed. Concerning a cell-contact independent mechanism, the antiproliferative effect of MSC and RTEC on PBMC and Tcells was detected in an indirect co-culture setting (Transwell Ò ). Cell proliferation in all co-cultures was measured by bromodeoxyuridine (BrdU) ELISA. In this work, we demonstrate that pre-treatment of MSC and RTEC with IFN-c leads to expression of major histocompatibility complex class-II molecules (MHC-II) and the immunoregulatory surface molecules PD-L1 and PD-L2. A high number of HAMSC already constitutively expressed PD-L1 and PD-L2 on their surface. IFN-c stimulated MSC even show enhanced antiproliferative properties in direct co-culture with stimulated PBMC. This beneficial effect was only moderately present in RTEC co-cultures. Furthermore, MSC as well as RTEC have an antiproliferative effect on PBMC and T-cells in a cell-contact independent setting. Experimental data demonstrate that RTEC exert similar properties as MSC regarding their effect on immune cells in vitro, although the extent of their antiproliferative properties is less pronounced than observed with MSC. Regarding direct co-cultures, it still has to be determined whether the immunosuppressive effect of MSC and RTEC is mediated by PD-1/PD-L1/ PD-L2 pathway, other receptor/ligand interactions or an interplay between surface and cytokine signalling. Additionally, cytokine analysis of coculture supernatants will provide further details regarding a cell-contact independent mechanism and possible differences in the action of MSC and RTEC. The constant increase of population movements related to emigration, business or tourism, leads to the spread of infections until now restricted to endemic areas. In the case of Central and South Americas, Chagas disease has become a matter of concern during the last decade in countries which receive Latino American immigrants. This disease is caused by a protozoan parasite, Trypanosoma cruzi, transmitted in endemic areas predominantly by a Triatomine vector. The probable routes of infection in nonendemic countries, where the vector is nonexistent, are therefore blood transfusion, organ transplantation and vertical transmission. In Europe, Spain is the country that receives most part of immigrants proceeding from Central and South Americas. In Spanish blood banks, anti-T. cruzi screening is mandatory since September 2,005 in donors at-risk for Chagas disease. From this date, this new selective screening allowed to identify 110 infected blood donors in the Catalonian Blood Bank. A large part of positive donors was from Bolivia, country with the highest seroprevalence of infection. Other positive donors were from Argentina, Paraguay, Ecuador etc. but also from Spain (mother born in endemic area, or donor born in endemic area or who had resided in endemic area). At the same time, look-back procedures were initiated in those donors who had donated before and whose blood products had been transfused. Screening for T. cruzi infection is generally selective, only done in at-risk donors, although the USA blood banks perform a universal screening since 2007. The selective screening strategy is finally discussed. Besides Chagas disease, malaria is also a parasitic infection caused by Plasmodium and transmitted by the bite of Anopheles mosquito in endemic areas. Two of the five Plasmodium species, P. falciparum and P. vivax, are most often found in Central and South Americas. Cases of transfusiontransmitted malaria are mostly associated to P. falciparum, although cases of transmission of P. malariae, P. vivax or P. ovale have also been described. The growing number of donors who travel to malaria endemic areas, or who were born in an endemic region increases the probability to fail to identify an at-risk donor and to transfuse a contaminated blood component. In non-endemic countries, the measures taken to prevent malaria transmission by transfusion generally consist in deferral of at-risk donors, based on questionnaire reporting travels to or residence in malaria areas. Some blood bank use a screening test to detect anti-Plasmodium antibodies, but have then to face the problem of confirmation of an initially reactive result and of donor's counselling. To opt for a screening or for a deferral strategy could depend once again on the epidemiological profile of blood donors. Background: Testing for T.cruzi antibody (the protozoan responsible for Chagas' disease) has been carried out in the USA since 2007. As an interim measure until testing of ''at risk''donors could be started, Canadian Blood Services, which manages the blood supply for all other provinces and territories in Canada except for the province of Quebec, implemented risk questions in February 2009 and ceased making platelets and transfusible plasma from donors identified as having risk. Aim: To evaluate the effectiveness of these screening questions in identifying ''at risk''donors and to estimate the number of positive donations that may be missed with a selective testing approach. Methods: Donor responses to the screening questions were monitored from the date of implementation (February 9, 2009) until December 31, 2009. A survey of 4,006 donors (of whom 2677, 67% responded) who had denied all risk factors on screening were invited to participate in a telephone interview to re-evaluate risk. Based on the survey results and the previously estimated prevalence of donors with Chagas' risk factors, an estimate for the number of donors with risk missed by the questions that may be positive for T.cruzi antibody was calculated. Results: In total 457,749 donors were screened of whom 7,243 (1.58%) indicated that they had risk. The most common risk factor was birth in Mexico, Central or South America (0.82%), with the rest being individuals whose mother or grandmother was born in one of these regions or who had travelled there for a continuous 6 month period or more. There were 233,004 donors who had more than one donation. The vast majority of these had consistent responses to the risk questions (98.68%), but a small proportion switched from ''risk'' to ''no risk'' (406, 0.17%) or from ''no risk'' to ''risk'' (519, 0.22%). However, all donors who are assessed as having risk are flagged for testing in the system irrespective of their current response to the questions. Of the 2,677 donors in the survey who claimed to have no risk on screening, 7 (0.26%) had been born in a risk country, and an additional 15 (0.56%) had spent time there, or their mother or maternal grandmother was born there. It was estimated that of about 420,000 Canadian donors per year, in the first year of testing a mean of 0 (95% CI 0 -5) donors who failed to acknowledge risk could be positive for T.cruzi antibody. Conclusions: Screening questions for Chagas' disease risk identify most donors, although a few will fail to answer correctly and may be missed by a selective strategy. These missed donors may pose a very small risk of transfusing a T.cruzi positive product to a recipient. Background: Malaria is a potentially serious disease caused by a parasite. At present recognised as an emerging disease, is one of commons transfusion transmitted infections, since infected asymptomatic donor with low level parasitaemia (antibody positive) can be parasite reservoirs. In Portugal, the incidence rate of malaria (1993-2002) was 0.74/100000 per year, all cases relating to imported malaria, mostly from Portuguese speaking African countries.The number of donors deferred, because of potential malaria risk is increasing as donors travel to malarial endemic areas, and populations move from endemic to non-endemic areas. Aim: To evaluate the Newmarket malarial antibody enzyme-linked immunoassay (EIA) performance in screening of blood donors with history of clinical malaria and/or potentially exposed to malaria. Methods: We used the Newmarket malaria EIA (Newmarket Laboratories Ltd, Newmarket, UK, distributed in Portugal by Bio-Rad, Portugal), programmed in accordance with the manufacturer's recommended protocol into the Tecan robotic microplate processor (model Genesis 200).This assay is a sandwich EIA incorporating four recombinant antigens for P. falciparum and P. vivax, detecting specific immunoglobulin G, immunoglobulin M and immunoglobulin A.We studied a panel of 12 positive samples for malaria, from Reference Centers (National Blood Service, London, UK and Centro de Transfusions de la Comunidad Valenciana, Spain). Between October 2008 and December 2009, we studied a total of 566 serum samples from blood donors, 386 from donors with a previous history of clinical malaria, 173 from donors potentially exposed to malaria (malarial area visitors and residents), at least four months after their last exposure, and seven samples from donors without any risk of malaria exposure. Results: The reference malaria positive samples were all reactive with the Newmarket malaria EIA. Of the 386 with a history of clinical malaria, 198 (51.3%) were negative and 188(48.7%) were reactive for antibodies to plasmodium sp. Of the remaining 173 donors potentially exposed to malaria, 149 (86.1%) were negative and 24 (13.9%) were reactive. The seven samples from donors without risk of malaria exposure were all negative. Conclusions: The detection of anti-plasmodium sp antibodies by this enzyme immunoassay appears to be a valid method of screening to be implemented in blood banks. Should be considered the test of malaria to all donors who have been in endemic area, regardless of whether or not they have had malaria crises, allowing to recover a significant number of donors with a history of clinical malaria, and to reduce deferral period of donors potentially exposed to disease. Background: Considering the high infectivity and wide-spread distribution all over the world, infection with Plasmodium (P.) sp. poses a risk of transfusion transmitted disease. According to the Croatian transfusion service regulations, stay in areas endemic for malaria necessitates deferral of blood donation for the next two years. With the introduction of high sensitivity testing such as real-time PCR for detection of malaria parasite, the status of blood donors upon their return from those areas can be resolved earlier.

Aim: The aim of the study was introduction and evaluation of a commercial diagnostic test, Malaria Parasites Real Time PCR kit (CE, IVD, Shanghai ZJ Bio-Tech Co., China) on an AB 7500 Real Time PCR System (Applied Biosystems, USA). The kit is intended for detection of malaria DNA (P. falciparum, P. malariae and P. ovale). Methods: Testing was performed by use of the 1st WHO International Standard for P. falciparum DNA Nucleic Acid Amplification Techniques. The standard consists of freeze-dried whole blood preparation collected from patient by exchange transfusion. Standard samples for evaluation were prepared without dilution (10E+9 IU/ml) and at dilutions of 1:10E-3, 1:10E-4, 1:10E-5 and 1:10E-7. The QIAamp DNA Mini Blood kit (Qiagen, Germany) was used on nucleic acid isolation. Real time PCR testing was done in duplicate to test the result intra-run reproducibility. The kit negative and positive control and negative human plasma were included in the sample series. A calibration curve with known titer standards (10E+4, 10E+5, 10E+6 and 10E+7 cop/ml) was plotted to test the amplification quality and efficacy. According to the manufacturer's instructions, standards were prepared by dilution of the kit positive control of defined titer (10E+7 cop/ml). The IU/ml translation to cop/ml was done by the conversion factor of 1 IU/ml = 5.6 cop/ml. Results: Ct value for the kit positive control was within the range defined by the manufacturer (19.85). Negative human plasma and negative control from the kit yielded negative result with positive internal standard signal, confirming the absence of inhibition. All the WHO standard dilutions tested yielded positive signals (Ct 14.01-36.00), with good intra-run reproducibility. Calibration curve showed good amplification efficacy (R2 0.999664; curve slope -4.0235). Methods: US blood donor screening for T. cruzi was implemented 29/01/ 07. Confirmed positive donors were invited to provide follow-up samples for hemoculture testing to determine parasitemia. Participating donors provided blood samples (heparin) that were centrifuged, plasma removed, added to LIT media and incubated at 27°C. Positive hemocultures were expanded in LDNT media and parasites isolated for lineage determination. Culture isolates were mixed with GE lysis buffer, DNA extracted, purified and run on six separate traditional PCRs with primer sets amplifying the small subunit rRNA, mini-exon genes and sequence characterized amplified region markers. DNA bands were analyzed for characteristic lineage markers and classified as TcI or TcIIa-e. Results: A total of 155 confirmed positive donors were tested by hemoculture and 15 (9.7%) were parasitemic. The country of birth for parasitemic donors was: Bolivia (7), Argentina (2), US (2), Chile (1), Colombia (1), El Salvador (1), and Paraguay (1). Rates of hemoculture positive donors was significantly higher (P < 0.0001) for donors with a TcII origin (11 of 24; 45.8%) compared to donors with a TcI origin (2 of 90; 2.2%). Thirty-four donors born in the US were tested and 2 (5.9%) were hemoculture positive, presumably representing autochthonous infections. Seven donors of other/ unknown origin were tested by hemoculture; all negative. Of the 15 hemoculture positives, 14 were successfully expanded and typed: 12 typed as TcIIb,d or e and 2 typed as TcI (Colombia and El Salvador origins). The US autochthonous isolates typed as TcII2d and 2e. Conclusions: Seropositive donors with a southern South American origin, consistent with TcII, were more likely to be parasitemic than donors with a TcI origin (i.e. Mexico, Central America, northern South America). Seropositive donors identified through blood screening in the US are predominately from Central America and Mexico, but as clearly demonstrated in this study are less likely to be parasitemic than those from southern South America. Thus, the relatively low rate of transmission observed in recipients of US blood products may be related to the low proportion of TcII donors in the US. In contrast, Spain has perhaps seen relatively more transfusion cases because their seropositive donors have primarily southern South America origins. Originating from the hematopoietic stem cells, red and white blood progenitor cells depend on internal and external cues for their differentiation, the most prominent ones being provided by lineage (specific) cytokines and transcription factors. It has come to a surprise that primary genetic defects in very basal cellular functions, here exemplified in secretory cargo transport and mitochondrial energy provision, may result in blood lineage specific deficiencies. Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDAII is the most frequent CDA. CDAII patients show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. CDAII is characterized by ineffective erythropoiesis and by the presence of bi-/ multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red cells are protein and lipid dysglycosylation and ER doublemembrane remnants. Development of other haematopoietic lineages is normal. By reverse genetics we elucidated that the secretory COPII component SEC23B is mutated in CDAII patients. In some cases a reduction in SEC23B expression occurs, which in erythrocytes may not be sufficiently compensated for by the highly conserved paralogue SEC23A and thus may result in erythrocyte specific defective COPII-mediated ER-export, ultimately leading to the clinical and cellular phenotype. Reticular dysgenesis (RD) or aleukocytosis (MIM: % 267500) is the most severe human primary combined immunodeficiency and accounts for <2% of SCID. It is characterized by the absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood, hypoplasia of the thymus and secondary lymphoid organs, and the lack of innate and adaptive humoral and cellular immune functions, leading to fatal septicemia within days after birth. In RD bone marrow, myeloid differentiation is blocked at the promyelocytic stage, while erythro-/megakaryocytic maturation appear generally normal. These features exclude a defect in hematopoietic stem cells but point to a unique aberration of the myelolymphoid lineages. We identified that the mitochondrial energy metabolism enzyme, adenylate kinase 2 (AK2), is mutated in RD patients. The results provide in vivo evidence for AK2 selectivity in leukocyte differentiation and we suggest that a human immunodeficiency syndrome is causally linked to energy metabolism and therefore can be classified as a mitochondriopathy.

Following these examples of blood cell specific diseases, apparently in cases of some inborn genetic defects the survival of blood progenitor cells is under less stringently redundant surveillance of cellular pathways as are other body cells. Regenerative potential of stem and progenitor cells has been under intense investigation. Endothelial progenitor cells (EPCs) have been isolated from the peripheral blood of adult individuals, cultured in-vitro and committed into an endothelial lineage in a specific condition. Based on initial EPC biology studies, researchers then pursued the notion of ''therapeutic vasculogenesis'', whereby systemic delivery of EPCs may augment neovascularization. Following preclinical studies, a phase I/ IIa clinical trial regarding transplantation of autologous CD34+ cells, the EPCenriched fraction, was performed in no-option patients with atherosclerotic peripheral artery disease (PAD) or Buerger's disease representing critical limb ischemia (CLI) for 5 years in our institutes. Our collaborators in USA performed the Phase III trial of transplantation into chronic severe myocardial ischemia (CMI) patients for 3 years. The outcomes of both prospective clinical studies indicate safety and feasibility of CD34+ cell therapy in patients with CLI and CMI. However, despite lots of research development on EPC biology for 10 years following the isolation of EPC, EPC biology is still controversial among researchers without the definitive concept of EPC identification and differentiation hierarchy. I introduce the development of basic and translational researches regarding ''What is EPC?''and ''How do we use EPCs?''in these days. Furthermore, recent basic and preclinical studies indicate optional role of EPCs for organ reconstruction, including the tissue preparation for regeneration and trigger signals for organ differentiation. The vasculogenesis effect on organogenesis seems essential for any organ recovery from ischemic disease or other pathological disorder. This issue will be discussed for the future direction of regenerative medicine.

Adverse events related to insertional mutagenesis or ectopic transgene expression may compromise the therapeutic prospects of gene therapy in the hematopoietic system. Over the past years, major progress has been made in the understanding of the underlying mechanisms, opening rational approaches to increase the therapeutic index. We have established sensitive nonclinical models, in transplanted C57BL/6J mice or using cultured cells, to reveal the transforming capacity of insertional activation of cellular proto-oncogenes such as Evi1 or Prdm16, and developed integrating gene vectors designed to reduce the risk of insertional transformation by altering the insertion pattern and the expression cassette of the transgene. In a murine model of Mpl deficiency, major adverse events related to ectopic protein expression could be controlled by transcriptionally targeted vectors. Two important principles emerge to ensure undisturbed long-term hematopoiesis from gene-modified cells. The first is the establishment of polyclonal hematopoiesis using vectors with an untargeted integration profile and ''physiological'' transgene cassettes. The second is the generation of clonally defined hematopoiesis derived from induced pluripotent cells with genetic modification in bona fide safe harbours. Blood banks first implemented serologic testing of donors for syphilis in the 1950s, employing non-specific assays for antibodies to cardiolipin using the VDRL or RPR versions of the original Wasserman test. The original assays for HBsAg, developed in 1970, employed simple gel diffusion, a technique too insensitive and slow for routine blood bank use. Second generation tests, which used counterelectrophoresis to accelerate precipitate formation, were for use in donor screening by FDA in 1972. Third generation HBsAg tests used a solid phase radioimmunoassay, which were replaced by an enzyme-labeled techniques (EIAs) in the 1980s and chemiluminescence-based assays (ChIAs) now currently in use, which can detect~1000 HBV virions/ml. Additional protection for prevention of posttransfusion hepatitis was provided in 1986 EIAs for antibody to the HBV-Core Antigen (anti-HBc) were implemented in the US and other countries, in parallel with ALT screening to interdict non-A, non-B hepatitis. The AIDS epidemic in 1981 and reports of transfusion AIDS in 1983, led to rapid development/implementation of first generation HIV antibody (Ab) EIAs in 1985. Although a major advance, these early tests could not detect HIV antibodies until~3 months post-infection, and also failed to detect some non-B clades of HIV-1 and HIV-2. Advances over then next decade included use of immunodominant recombinant HIV-1/2 antigens, and 3rd generation antigen-sandwich-format EIAs and ChIAs that detect antibodies within 2-3 weeks of acute viremia. EIAs for HTLV-I and then HTLV-I/II were developed in the late 1980s implemented for donor screening in many countries, later replaced by ChIAs. The discovery of HCV in 1990 led to rapid implementation of first-generation HCV EIAs with limited antigen representation; although a major advance these assays missed~20% of chronic infections and failed to detect seroconversion for 2-3 months following infectious viremia. HCV antibody assays were enhanced over the subsequent decade through addition of immunodominant, multi-cladereactive recombinant and peptide antigens, resulting in detection of virtually all HCV infections (except rare serosilent carriers) within 6-8 weeks of acute viremia. Increasing concern over residual transfusiontransmission due to pre-seroconversion window-phase viremia led to the parallel development of nucleic acid amplification technologies (NAT), separate HIV and HCV antigen (Ag) EIAs, and 4th generation Ag/Ab combi-assays employing EIA and ChIA technologies. These 4th generation tests, which detect 40-60% of window phase donations detected by NAT tests, are increasingly employed in donor screening in developing countries (where NAT is not feasible/affordable) as well as in diagnostic settings. Other recent advances include development of immunoassays for additional transfusion-transmitted agents such as T cruzi, malaria and dengue, although use of these assays is controversial, with recent development of regional or selective testing strategies based on geographic/temporal exposure or one-time donor screening. There is ongoing work to develop multiplexed screening tests capable of detection of Ags and Abs for multiple TT-pathogens using novel point-of-care rapid and microarray technologies. Finally there have been advances in serological assays for confirmation and typing of infections that yield information of use in counseling donors and allow reinstatement of non-infected deferred donors.

The description of the ABO blood group system by Landsteiner and coworkers was a big milestone in making blood transfusions feasible and safe for a broad range of indications. Nevertheless with an increase of blood transfusions side effects like transfusion transmitted infections (TTIs) became more and more important. A big challenge in transfusion medicine was and is to develop screening assays with maximum analytical sensitivity and analytical specificity in order to reduce the diagnostic window period as much as possible. Until the late 1990s, blood screening for TTIs depended entirely on serological assays. Except for HBV, where the antigen can be detected using HBs-antigen assays, tests for detection of other TTIs relied almost exclusively on antibody detection. These tests, however, are associated with a relatively long diagnostic window period, since they detect the response of the immune system to an infection. In the mid 1990ies the residual risk of transfusion associated HCV infection was estimated at less than 1:5,000. New upcoming molecular technologies like the polymerase chain reaction (PCR) were examined in order to investigate how these methods could be implemented into blood donor screening to reduce this risk. This review reports on the development of nucleic acid amplification tests and describes the current state of technology. Functional principles of the different nucleic amplification technologies (NAT) are depicted and blood donor screening by NAT for different viruses is described. Additionally the special situation of bacterial detection by NAT is discussed. Blood donor screening by NAT was started using in-house methods. Over the last decade these systems were significantly improved and certified by the FDA or the EU. Currently three fully automated and barcode controlled NAT systems are available for blood donor screening (detection in individual donations or in mini-pools up to 96 samples per pool). In most developed countries NAT screening for HCV-RNA and HIV-1-RNA is performed. Depending on the screening strategy blood donor testing by NAT is able to reduce the diagnostic window period for TTIs like HCV to a minimum of 4-6 days. This led to a residual risk of TTIs of less than 1:1 million for HCV and HIV-1 in countries using NAT and underlines the efficiency of these methods. However, in some cases NAT detection may fail due to mutations in the genome of the pathogen. Several cases of TTIs were reported in the literature in which mutations in primer and probe binding regions were the major cause for a reduced analytical sensitivity and for screening failures. Amplification in at least two conserved genomic regions is a promising approach to overcome this risk. Generic bacterial detection can also be done by NAT, but there are a few drawbacks. Serological combo assays may be economic alternatives to NAT, but are associated with a longer diagnostic window period compared to NAT systems. Pathogen-inactivation methods are feasible for platelets and plasma products, but general inactivation methods for all three blood products are still eagerly awaited. The early days of the AIDS epidemic brought tragic consequences for blood recipients but also brought attention and resources to the prevention of transmission of infectious agents by transfusion of blood. Sensitive and specific assays discussed in the two other presentations of this session are the most effective ''layer of safety''for the selection of safe donors. While regulatory oversight assures compliance with regulations and good manufacturing practices, history questions have lower sensitivity and poor predictive value (e.g. history of travel or hepatitis) and deferral files have a minor role in an age of computers and reproducible tests. Thus, test selection deserves attention from those involved in the preparation of blood for transfusion. Despite public demand for unattainable ''zero risk'', investments in transfusion safety also need to be balanced with other healthcare priorities appropriate for each environment. Different technologies address different issues. Serological assays detect antibodies against the infectious agent (signal amplification results from the immune response) while molecular assays detect the nucleic acid sequences, RNA or DNA through amplification that takes place in the test tube. Serological assays are effective for detection of antibodies in infected individuals in a population, or prevalent cases. Molecular assays (nucleic acid amplification tests or NAT) are effective for the detection of recent infections, or incident cases. Improvements in technology have brought sensitivity of 4th generation serological assays closer to that of NAT. In some instances, like detection of parasitic infections, serological tests are more effective because the few parasites present in a component may be sufficient to transmit infection but may be missed in the small specimen used for testing (Poisson distribution). Test selection must also consider regional epidemiology. Screening for hepatitis B (HBV) in a low prevalence area may be based on a combination of assays that include HBV surface antigen or HBsAg, antibodies to core of HBV (HBcAb) and NAT for HBV, depending on availability and resources. Selection is more difficult in high prevalence areas because a large segment of the population has been exposed to HBV and is positive for HBcAb, and often resources for the implementation of NAT are not available; thus, screening must rely on high sensitivity HBsAg assays. Regional epidemiology has also been a factor in the selective approach chosen for T. cruzi in the U.S. and HTLV-I/II in Europe. Serological assays are not effective for Dengue, West Nile Virus and probably other arboviruses because they are only transmitted when there is viremia which gradually wanes after the appearance of antibodies. There has been great progress in the development of assays based on new technologies, including microarray chips and nanotechnology, with high sensitivity and specificity which allow simultaneous screening for multiple agents in a cost effective manner. However, we cannot continue to add screening assays for every newly recognized pathogen transmissible by transfusion. Hopefully, in the foreseeable future, safe and effective pathogen inactivation technologies will allow us to limit the number of screening assays needed to ensure the safety of the blood supply. Three vital gases ''oxygen, nitrogen, and carbon dioxide'' intersect at the level of the human red blood cell. The delivery of oxygen to all tissues by red cells is essential to human life. Evolution has created a complex molecule, hemoglobin, designed for efficient uptake and off-loading of oxygen. Iron rests at the center of the heme moiety and is critical for oxygen exchange. Although studied for over a century, some details of oxygen transport by the red cell remain uncertain. Recent research has focused on the interaction of hemoglobin with a complex of cellmembrane proteins centered on band 3. In addition, there is renewed interest in the question of whether or not stored red cells deliver oxygen to tissues as well as fresh red cells. Nitric oxide (NO) physiology is directly related to oxygen delivery by the red cell. NO serves a local vasodilator to increase blood flow to hypoxic tissue beds. NO binds strongly to hemoglobin which serves as an NO sink. Under normal conditions, plasma NO synthesized by endothelial cells is not consumed by red cell hemoglobin due to a diffusion blockade across the red cell membrane that results from membrane structures not fully identified. Under conditions of red cell lysis, free hemoglobin scavenges NO reducing local vasodilation. Scavenging of NO is now recognized as an important component of the physiologic response to chronic hemolysis and is very likely to play an important role in the renal lesion of acute hemolysis. The interactions of CO2 with the red cell have drawn far less research attention. Plasma CO2 released by tissues serves as an essential trigger for oxygen release by hemoglobin via the Bohr effect. CO2 transport depends up conversion of CO2 to bicarbonate via red cell carbonic anhydrase in conjunction with chloride exchange across the red cell membrane. There is very little research on the effect of blood storage on CO2 excretion although this aspect of respiratory physiology is every bit as important as oxygen delivery. Oxygen, nitrogen, and carbon dioxide are also the three principal gases of the Earth's atmosphere. While CO2 is the least abundant of the three by far, it is also likely to be the most critical to the future survival of life on Earthbecause small further increases in the concentration of CO2 will result in continued climate change and large increases will be deadly. Thus, the management of three vital gases by the collection of red cells found within us has broad similarities to the collective management of our atmosphere. Just as the survival of individual tissue cells depends upon proper balance of these three respiratory gases, so too will their proper balance be the key to survival of life on Earth. And as the results from running RCTs will still take years to become available, policy will have to be based on the available observational data. Due to publication bias and the heterogeneity of the studies it is impossible to perform a valid metaanalysis on the observational studies. Looking more closely at the design and analyses of the individual published studies, it is however possible to explain a large part of the differences in the reported conclusions. A major issue concerning the design and analyses of the studies is the comparison of patient groups that differ in more aspects than storage time alone. The most frequently encountered imbalance is in the number of RBC transfused to patients. As shown by van de Watering et al, there is a strong association between the total number of RBC transfused to a patient and storage time variables (see figure) . As a result of this association, using storage time to define patients groups may lead to a significant bias in total number of transfusions. In literature, some authors have recognised this imbalance and controlled it's biasby matching, stratification, or multivariate analyses. Within these studies the vast majority of the initially significant adverse associations of prolonged storage disappeared after controlling for number of RBC. However, lots of reports on adverse effects are based on crude observational data without controlling for imbalances in number of RBC transfused. Another important issue is the fact that adverse effects are predominantly reported from North America, and hardly ever from Europe. Whether this is based on differences in blood processing (hard/soft spin; buffy-coat removal), storage solution (AS1, CPD, SAGM), plasticisers (DEHP) and/or publication policy remains a question. Given the fact that the running RCTs on this topic are all performed in North America, we're not assured they will provide us with an legitimate answer for Europe. The collection of European data is imperative to prevent a looming scenario where ''European'' RBC, clinically still good enough after storage, will nevertheless have their maximum storage time reduced as a precautionary measure (introducing serious logistic problems) based solely on North-American RCT data.

quality of the RBCs, however, continuously decreases during hypothermic storage. Transfusion of hypothermically stored RBCs has been associated with proinflammatory and immunomodulatory effects, an increased length of stay in the hospital, and ultimately, increased morbidity and mortality in transfused patients. Cell membranes are one of the primary sites of injury during hypothermic storage. Cold storage of RBCs leads to a cascade of damaging events including phase changes and phase separation of lipids, lipid loss through microvesiculation, and ultimately, hemolysis. Our study investigates whether small unilamellar liposomes (100-200 nm) composed of specific natural phospholipids stabilize RBCs during hypothermic storage. Methods: A variety of liposomes composed of different types of saturated and unsaturated lipids (dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC)) have been incubated with RBCs to investigate the effect of liposomes on RBC membranes and vice versa that of RBCs on liposomal membranes. FTIR-Spectroscopy and MALDI-TOF mass spectrometry were used to study the interaction between liposomes and RBCs. The effect of various liposome compositions on RBC deformability and hemolysis during hypothermic storage was investigated using ektacytometry and spectrophotometric Drabkin's analysis. Results: FTIR and MALDI-TOF analysis showed that liposomes composed of DOPC or DMPC exhibited signs of lipid and cholesterol transfer to RBCs, whereas DPPC lipids did not. FTIR analysis showed that the Tm of DOPC liposomes shifted to higher temperatures after incubation (T G 10°C). The Tm of DMPC increased after incubation, whereas the Tm of DPPC lipsomes was not affected after incubation with RBCs. FTIR analysis on ghosts prepared from liposome treated RBCs revealed that liposomes composed of unsaturated lipids have a fluidizing effect on RBC membranes. RBC deformability was impaired during hypothermic storage duration in all cases, however in DPPC-liposome treated RBCs this impairment was slightly reversed. In addition, treatment and storage of RBCs with DPPC liposomes depressed hemolysis as compared to untreated RBCs stored under the same conditions. In contrast, RBCs stored with liposomes composed of DOPC and DMPC lipids showed increased levels of hemolysis and a decreased ability to deform.

The data indicate transfer of lipid components between RBCs and liposomes during incubation. In addition, exposure to liposomes improved the deformability of hypothermically stored RBC. The mechanism by which liposomes confer protection to RBCs is currently under investigation. The findings of this study suggest that liposomes composed of long-chain saturated phospholipids may confer protection to RBC membranes during hypothermic storage. Background and aim: Concerns have recently been raised about the safety and efficacy of transfusing stored red blood cells. Increased risk of posttransfusion complications has been reported with increasing ''age'' of blood 1 . The liver is a target for injury in low flow states associated with trauma and hemorrhage 2 . The aim of the present study was to evaluate the effect of the age of transfused blood on liver injury and to explore possible mechanisms in a rat model. Methods: Anesthetized rats were randomized to either control group (CG; sham), hemorrhagic shock group (HSG) [controlled bleeding to a mean arterial pressure (MAP) of 25 mmHg] or resuscitation groups (10 min following controlled hemorrhage as in HSG, resuscitation with fresh blood (BRG-d0) or blood stored for 4 or 7 days (7 ml, 1 ml/min). Liver injury was evaluated using fMRI combined with hypercapnia and hyperoxia for perfusion analysis (DS maps) as described 2,3 , liver enzymes, apoptosis and liver histology. To explore possible mechanisms linking adverse liver outcome with increased duration of blood storage, we evaluated 2,3 DPG, reactive oxygen species, IL-1b, and TNFa levels in the transfused blood, as well as red blood cell deformability, aggregation and adhesion to the endothelium. Results: Liver outcome following blood transfusion: Resuscitation with fresh blood significantly attenuated liver injury induced by hemorrhagic shock as reflected by the significantly lower liver enzymes levels in serum, reduced liver injury on histology examination and significant reduction in apoptosis. Both fMRI and liver enzymes showed significant differences between resuscitation with fresh blood (BRG-d0) versus blood stored for both 4 and 7 days (BRG-d4 and BRG-d7). However, significant aggravation of liver injury on histological examination and increased apoptosis were only observed following transfusion of blood stored for 7 days. Analysis of parameters of the stored blood: Analysis of blood samples stored for 0, 1, 4, 7, 11 and 14 days showed that 2,3-DPG levels were significantly reduced already at 4 days of blood storage, in good agreement with fMRI results. Blood levels of TNFa and reactive oxygen species did not change with time of storage, while IL-1b levels were significantly elevated already after 1 day of storage. Red blood cell deformability was considerably reduced only at 7 days into the storage period inducing a five-fold increase in the number of rigid, undeformable cells, whereas RBC/endothelial cells adherence was not significantly affected by the increased storage time up to 7 days. Conclusions: In rats with acute bleeding, transfusion of blood stored longer than 4 days increased liver injury. This was associated with significant changes in deformability of the stored erythrocytes and probably unrelated the TNFa, IL-1b, reactive oxygen species, 2,3-DPG levels in the stored blood or the RBC/endothelial cells adherence properties. As transfusion of fresh stored blood is not an available option owing to blood shortages and current blood banking practices, our findings suggest a potential treatment target to reduce transfusion-related injury. The retention of previous donors and the recruitment of new donors is a serious challenge for many blood donation services in their effort to prevent blood shortages. More and more services make use of some sort of donation incentives. However, the use of (material) incentives to motivate blood donors is fiercely controversial and there is a longstanding (ethical) debate about whether it should be allowed that donors receive material rewards. Interestingly, this debate is dealt with in almost complete absence of systematic empirical evidence on the effectiveness of material incentives in encouraging people to donate. In this paper, we argue that the discussion on what is ethical in motivating blood donors should be enriched with empirical evidence based on field experiments. We confront the Titmuss controversy with recent results from an experiment administering lottery tickets as a motivation device. Moreover, we take up a neglected phenomenon in the study of blood donors: many non-donors are not principally against donating blood they have just never made up their mind about becoming active blood donors. We propose active decisions as a mechanism to transform latent prosocial preferences into actual prosocial behavior. The regulation of cells and tissues in the United States can be divided into two major categories. If the cells/tissues are minimally manipulated (their composition is not altered) then these are considered ''361'' products and the Good Tissue Practice (GTP) regulations apply (21 CFR 1271). The primary focus of the GTP regulations is the prevention of the spread of communicable diseases and therefore focuses heavily on the screening of the donor and possible infectious disease risks the donor may transmit to the patient. Examples of these types of products include autologous and allogeneic (first and second degree relative) hematopoietic progenitor cells, CD34 selected cells and density gradient enriched mononuclear cells. The Food and Drug Agency (FDA) require that facilities involved in GTP manufacturing must be registered with the agency. If the cells/tissue are more than minimally manipulated (composition may be altered by either culture, gene modification, etc) then these are considered ''351'' products and the Good Manufacturing Practice (GMP) regulations apply (21CFR 200 and 600 series). The GMP regulations focus more on the reproducible manufacturing of a cellular or tissue products and the stringent testing of the product before release. Examples of this type of product include hematopoietic cells from unrelated donors, ex vivo expanded HPC and gene modified cells. The FDA intends that facilities involved in manufacturing of 351 products will eventually apply for a Biological License Application (BLA) for the manufacturing of these products. Today's discussion will focus on both the similarities and the difference between the GTP and GMP regulations. In addition, we will discuss the recent Guidance document for Cord Blood Licensure And finally we will discuss some of the recent findings of the agency when inspecting establishments that are manufacturing cellular and tissue products. (1), entered into force on December 30, 2008. ATMP include gene therapy medicinal products, somatic cell therapy medicinal products and engineered tissues. In the USA, MSCs are considered in the context of human cells, tissues, or cellular and tissue-based products (HCT/Ps) . Therefore they must comply with Current Good Tissue Practice (CGTP) requirements, under Code of Federal Regulations Title 21, Part 1271, whose essential requirements relate to the prevention of the transmission of communicable diseases. Translation of research-based protocols into good manufacturing practice (GMP) compliant procedures for large scale production of clinical-grade MSC requires careful analysis of risks and benefits with the aim to identify and control all critical aspects. In particular, for MSC as for any other cellular products obtained in culture, aseptic conditions (defined in Annex 1 of GMP) must be validated. The availability of automated and closed devices can therefore help to maintain asepsis, reduce the number of in process controls and facilitate large scale expansion of MSC. Moreover, for large scale production of MSC, it should be considered that after 12-15 population doublings, the proliferation rate slows down and MSCs partly loose their multi-potency (2) . For that reason, in order to maintain phenotypic and genotypic stability of MSC during multiple passages, the culture condition should be optimized for MSC GMP production. State-of-the art culture conditions for research grade MSC expansion require FCS supplemented media, but recently also human serum or plasma (3, 4) and growth factors have been proposed as alternatives to FCS. In fact, FCS shows large batch-to-batch variability, which may negatively affect production efficiency. Moreover, the risk of transmission of infectious diseases and the possible occurrence of sensitization against FCS proteins in the recipients (5) must be considered in view of clinical applications. The replacement of FCS with human or humanized components and its regulation is therefore one of the most challenging aspect of GMP translation in MSC production.

In the member states of the European Union, the field of cell based medicine and tissue preparations is determined by the law of the European Community (EC). The implementation of the EC directives is mandatory and makes Community law binding in the EU Member States. The Tissues and Cells Act of July 20, 2007 transposed the Directive 2004/23/EC, which is setting standards for human tissues and cells, into German law. This Tissue Act is not a law on its own, but makes significant amendments of the Medicinal Products Act, the Transplantation Act, and the Transfusion Act. According to the Amsterdam Treaty (1997) and the Directive 2004/23/EC itself, the national legislators may stipulate stricter provisions, thus exceeding the minimum requirements specified in this EC directive. Accordingly, a real harmonization of the standards for human tissues and cells within the European Union is not intended and not possible. The Directive 2004/23/EC includes minimum standards to ensure high quality and safety margins for human tissues and cell preparations released for clinical application in humans. The standards cover the donation, procurement, testing, processing, preservation, storage, and distribution of human tissues and cells. The German Tissue Act defines tissues and cell preparations as pharmaceutical drugs governed by the German Drug Act. The need for high standards in quality and safety is highlighted, most of the loopholes that might otherwise allow operation outside national drug legislation in the field of local tissue banking and tissue engineering have been closed. As a consequence, donated tissues and stem cell preparations are subjected to strict regulations which mainly aim to prevent the most serious adverse effects of allogeneic tissue transplants, the transmission of infectious pathogens. Whenever possible, a validated inactivation procedure should be included in the manufacturing process. Tissues and stem cell preparations now usually require national approval, a procedure which in principle is comparable, although somewhat simplified, to the wellestablished national licensing procedure for blood components seeking postmarketing approval from the national authorities (Paul Ehrlich Institute). According to the highly positive experiences obtained with the standardization and licensing of blood components combined with a local and national system for surveillance, it can be expected that the national approval and local and national surveillance of stem cell and tissue preparations will contribute to a significantly improved quality and higher safety profile for the patients. However, the bureaucratic efforts which are inevitable consequences of the new regulations are still a matter of some dispute.

Takamoto S Aichi Medical University School of Medicine, Nagakute, Japan

The production and trade of all medical instruments and medicines in Japan, including blood products, is regulated by the Pharmaceutical Affairs Law, implemented in 1960 and revised many times thereafter. The recent initiation of cellular therapy worldwide has forced several amendments that apply this law to cell based medicine. In 1999, notification on securing the quality and safety of medical instruments and medicines using cells or tissues was published by the Japanese Ministry of Health, Labour and Welfare (MHLW), and effectively adds a ''securing''step between the preclinical and clinical study stages in the application process for the approval of any new medicines. This is to ensure the quality and safety of new medicines which use cells or tissues derived from humans or animals. After many twists and turns, ''artificial skin''has finally become the only cell based medicine approved to date. Conversely, medical treatment is regulated by the Medical Practitioners Law and the Medical Care Law, which were enacted in 1948, also revised many times, and which control the licence and business of medical doctors and hospitals and clinics. These laws permit any Institutional Review Board (IRB)-approved medical treatments, including advanced treatment of clinical study, to be carried out under the responsibility of medical doctors. Regarding clinical research, MHLW created the Ethical Guidelines for Clinical Research in 2003. It includes basic concepts, responsibilities of researchers, IRBs, informed consent, and by-laws. Therefore, clinical research is regulated by these rules. Furthermore, the Guideline for Clinical Research using Human Stem Cells was enacted by the MHLW in 2006. Although Embryonic Stem (ES) cells were excluded, the rest of cell based medicine and clinical research using human cells is now obliged to follow it. Thus, cellular therapy, on the whole, is executable as long as the treatment follows the laws and guidelines described above, including approval by the IRB and implementation by responsible medical doctors belonging to the institute. However, obtaining cell products for cellular therapy from other hospitals or medical institutes, as well as from companies, is forbidden. The committee on regenerative and cellular therapy at the medical facilities of the MHLW is now preparing a proposal on those subjects, including making biological products available inter-institutionally. It is likely to refer to (i) basic concepts; (ii) general considerations which cover (a) the provision of regenerative and cellular therapies, (b) application of therapy and procurement of cells and tissues, (c) management systems for processing and quality control including Cell Processing Center (CPC) facilities, (d) transplantation and administration, (e) data and records management, and f) assessment of the effects and safety of the therapy; (iii) requirements for multiple institutes to perform therapies in collaboration, (a) mutual system for providing therapies, and (b) conveying; (iv) roles expected of the relevant academic societies. Regulation of cell based medicine in Japan is still underway, and systems governing ES cells and iPS cells should be consolidated in the near future.

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they emerge -instead of only reacting to them. This requires intensified research into the ecology of viruses and their reservoirs. Bats have come into focus as potential reservoirs of several pathogenic RNA viruses. Our recent research has yielded a range of bat-borne viruses sharing surprisingly recent common ancestors with human pathogens. Hipposideros cf ruber in Ghana harbours a virus of common ancestry with human coronavirus (hCoV)-229E, a ubiquitous common cold agent of humans. About 200 years ago, a bat virus may have spread globally in humans, potentially associated with a SARS-like epidemic. Rhinolophus blasii and R. euryale in Bulgaria carry a close relative of SARS-Coronavirus. Reverse genetic studies on one of the major Interferon antagonists of SARS-Coronavirus suggest that this virus might be even more efficient in circumventing the human interferon response, and thus, might be more pathogenic if switching hosts into humans. However, pseudotyping of the spike protein of this virus into reporter gene-expressing vesicular stomatitis virus suggests no spike-dependent entry of the Bulgarian SARS-like CoV into human cells. In Ghanaian Eidolon helvum, we have detected Henipa-and other paraxmyxoviruses. For the Henipavirus genus that is associated with severe encephalitis in humans, sequence diversity suggests an African rather than Asian origin. Finally, the same bat species shows a considerable detection rate of prototype members of the GB viruses, which define the genus Hepacivirus together with hepatitis C virus, one of the most important pathogens in humans.

Cserti-Gazdewich M University Health Network/University of Toronto, Toronto, Canada Plasmodium falciparum malaria, as ancient as hominid evolution itself, has provoked more change within the human genome than any other pathogen. JBS Haldane observed the overlapping distributions for thalassemia and malaria endemicity, and proposed ''balanced polymorphisms''as advantageous heterozygous mutant states. We now appreciate the wider range of hemoglobinopathies, membranopathies, and enzymopathies as distinct evolutionary adjustments to the erythrocyte, the very compartment which P falciparum hijacks to sicken the host. Unlike other Plasmodium species, P falciparum's power over the erythrocyte consists of its limitless red cell infectivity, and its capacity to render the infected red blood cell (iRBC) adhesive enough to arrest in the circulation. This latter cytoadhesivity is achieved by sticky knobs (known as ''Plasmodium falciparum erythrocyte membrane protein-1''[PfEMP-1]), which are trafficked to the red cell exterior from the parasite within. PfEMP-1 is designed to latch onto endothelial cells of the post-capillary venules (''sequestration''), as well as onto other uninfected red blood cells and platelets (''rosetting''). In so stalling flow towards the spleen, the iRBC doubly harms the host by resisting the first defence of reticuloendothelial clearance, and congesting the host's microvasculature. The youngest, most malaria-naïve suffer malaria's highest case fatality rate, revealing just how critical this innate (pre-adaptive) immune control of parasitaemia is. The biochemical means by which PfEMP-1 achieves its cytoadhesive promiscuity is in part through one particular lectin-like domain, DBL1a. This domain binds not only to heparan sulphate-like glycosaminoglycans, but to two blood group antigens expressed densely on erythrocytes: the group A carbohydrate in the ABO system, and antigens (including those of the Knops system) on CR1 (CD35). If indeed these ligands are critical in the molecular pathogenesis of malaria fatalities, then we might expect to observe non-adhesive variants ascending to higher prevalence in the most malaria-endemic parts of the world. The cytoadhesivity of wildtype group A hosts is theoretically, and in vitro, demonstrably mitigated by what we now know are the mutant phenotypes which define the polymorphisms of the ABO system. These include the group O or B alleles, the weaker A types, and the genetics influencing the quantity of secreted (competitive) free A antigen in group A hosts. Each of these phenotypes is observed at higher frequencies in malaria-endemic areas. Certain CR1 polymorphisms are also more frequently found in these parts of the world.

The assembly of in-vitro, geographic, and clinical evidence weighs heavily towards ABO evolution being a highly specific response to P falciparum. Rather than bypassing invasion or enhancing clearance, these mutations are special because they highlight the importance of escape from cytoadhesion. Forthcoming are the results of the first prospective study powered to confirm the impact of ABO on malaria mortality (NCT 00707200, www.clinicaltrials.gov). Should the results of emerging studies confirm a survival advantage among group O individuals, the basic strategy for transfusion support in malaria may shift to greater use of group O red cells. The clinical value of this approach, more immediately available than any new drug or vaccine development, will need to be tested in clinical trials. Background: Xenotropic Murine Leukemia Virus-related Retrovirus (XMRV) is a human gammaretrovirus recently discovered in familial prostate cancer tissue. XMRV was subsequently identified in lymphocytes of patients with chronic fatigue syndrome and shown to exhibit cell-free (plasma) and cell-associated transmission from activated lymphocytes. Aim: The goals of the present study were to examine viral replication kinetics, tissue tropism and the host immune response to XMRV. Development of serologic assays to detect XMRV-specific antibodies would facilitate screening for individuals infected with XMRV and provide the foundation for epidemiologic studies to establish the etiologic role of XMRV infection in human disease. Methods: Five rhesus macaques were inoculated intravenously with XMRV. Blood was collected throughout the course of infection, and tissue from multiple organs was harvested at necropsy. Two macaques were necropsied at day 6 or 7 and 1 at day 144 post infection. The remaining two animals were re-inoculated with XMRV on day 158 and necropsied on day 291. XMRV-specific immunoreactivity was monitored by Western Blot (WB) using viral lysate. Prototype serologic assays to detect antibodies to env gp70, p15E and gag p30 were developed on the high-throughput automated ARCHITECT instrument system (Abbott Diagnostics). Results: XMRV inoculation resulted in low transient plasma viremia although proviral DNA persisted in circulating PBMCs for several weeks. Of interest, the earliest leukocyte targets were CD4+ T cells and NK cells; CD14+ monocytes were negative. Animals sacrificed at the acute stage showed evidence of viral replication in spleen, lung, lymph nodes, liver and reproductive organs. Two animals were reinoculated with XMRV at 5 months and showed transient proviral DNA in blood cells. However, following immunization with adjuvanted XMRV proteins, low level plasma viremia was detected with greater dissemination of XMRV in various organs including the GI, reproductive and urinary tract as well as in vaginal tissue of the one female. By WB analysis, all three chronically infected macaques developed antibody responses to env and gag proteins. The serologic assays demonstrated 100% sensitivity by detecting all WB positive serial bleeds from the XMRV-infected macaques. Preliminary results showed evidence of detectable reactivity to multiple XMRV antigens in a low proportion (~0.1%) of U.S. blood donors. Conclusions: These data suggest that lymphocytes are a primary target for replication of XMRV. Acute infection may lead to detectable plasma viremia and there is potential for reactivation of viral replication after immune activation. This study identified specific serological markers useful for detection of antibodies elicited by XMRV infection. The prototype antibody assays will be useful for large-scale epidemiological studies. and 10 (6%) into genotype 4, both of which were assumed to be Japanindigenous strains. Of the 118 donors responding to the questionnaire, 84 (69%) had a history of eating the animal viscera such as intestine and/or liver. Look-back survey found that three of previous donations were also positive for HEV RNA, none of which caused TTI. Of the 48 donors who could be followed-up at least twice a month after the donation, 27 (56%) showed transient elevations of ALT higher than 45 IU/l. Conclusions: The present study revealed that Japan-indigenous HEV strains were consistently circulating in blood donors by zoonotic foodborne route in Hokkaido. Most of the donors positive for HEV RNA were asymptomatic but about half of them showed ALT elevation. HEV RNA is much more useful marker for identification of HEV-positive donors than HEV antibodies. The prevalence rate of HEV infection was roughly half of that of HCV infection in blood donors in Hokkaido and therefore HEV infection is not negligible for blood safety in Hokkaido.

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Sachs UJ Justus Liebig University, Giessen, Germany

The use of blood products is not without hazards, and side effects are seen in a relevant numbers of transfusions. Side effects are defined as problems that occur in addition to the desired therapeutic effect; or that occur when treatment goes beyond the desired effect. On the one hand, side effects can be based on the presence of wanted or unwanted (contaminating) substances in the blood product. As an example, red blood cells may lead to alloimmunization; contaminating viruses may lead to infection. On the other hand, side effects can occur as a consequence of the incorrect use of blood components, in which ''incorrect''can be obvious and avoidable or masked and not (or not always) avoidable. As an example, red blood cells may lead to acute haemolysis in ABO-incompatible transfusion; the transfusion of two units of compatible red blood cells may lead to volume overload and lung oedema. Over the past decade, concern regarding side effects associated with the transfusion of blood products has shifted from infectious disease transmission to non-infectious side effects. In industrialized countries, a patient is 1000-fold more likely to experience a noninfectious side effect than an infectious complication of transfusion. Many of these non-infectious side effects are currently not or not completely avoidable. These side-effects will be in the focus of this overview. Some will present as acute transfusion reactions within minutes or hours after transfusion including, transfusion-related acute lung injury (TRALI), transfusion-associated overload (TACO), transfusion-associated sepsis (TAS), and allergic/anaphylactic reactions. These acute transfusion reactions can be difficult to evaluate. The fact that pulmonary manifestations may dominate the clinical features further impedes the differential diagnosis. In contrast, non-ABO HTR, transfusion-associated graft-versus-host disease (TA-GVHD), and post-transfusion purpura (PTP) do usually not manifest before days or weeks after transfusion and present with more or less distinct clinical manifestations that will allow for a definite diagnosis. In addition, blood transfusion appears to carry a not well-defined risk of increased mortality (transfusion-associated mortality, TAM) that cannot be attributed to any of these side effects. TAM is been suspected to be associated with red blood cell storage lesions and with transfusion-related immunomodulation potentially promoting cancer recurrence and perioperative infections. The suspected pathophysiologic mechanisms, currently available strategies for diagnosis and treatment and current efforts to prevent these noninfectious side effects will be presented. Our laboratory studies the role of platelets in various biological processes using mice as a model system. Platelet depletion with anti-platelet antibody causes profound thrombocytopenia that can be maintained for several days. Platelets can be replenished by transfusion if they are genetically altered so as not to be recognized by the depleting antibody. In 2006, we reported that platelets support experimental angiogenesis and that nascent vessels bleed in thrombocytopenia (Kisucka J et al., PNAS, 2006) . Since angiogenesis is accompanied by recruitment of inflammatory cells, we proceeded to study mature vessels at sites of local inflammation in the presence of low or normal platelet count. To our surprise, we observed that, within minutes of the onset of thrombocytopenia, postcapillary venules of the inflamed skin began to bleed. We could observe this in real time by intravital microscopy. Similarly, other organs subjected to LPS (lung) or ischemia/reperfusion induced inflammation (brain) began to hemorrhage in thrombocytopenia (Goerge T et al., Blood, 2008) . Using platelet transfusion, we showed that platelets deficient in major platelet adhesion receptors could rescue the phenotype and prevent bleeding. These receptors are all crucial in the hemostatic process indicating that it is a special function of the platelets, different from platelet plug formation, that maintains vasculature during inflammation. Both angiogenesis and inflammation are involved in tumor progression in cancer. In experimental cancer models, we showed that platelet depletion causes almost immediate bleeding of tumor vessels and no bleeding elsewhere in the animal (Ho-Tin-Noe B et al., Cancer Res., 2008). Again, platelet transfusion could prevent the tumor hemorrhage. This protective function of platelets was linked to platelet granule release, as platelets activated to degranulate prior to transfusion could not rescue the tumor from bleeding. More recently, we found that it is the leukocyte recruitment at the site of inflammation or tumor rather than increased vascular permeability that causes the hemorrhaging in thrombocytopenia (Ho-Tin-Noe B et al., Am. J. Path., 2009). Exactly how platelets counteract these negative effects of inflammatory cells on the vasculature is not known. We are currently investigating whether induction of tumor bleeding by thrombocytopenia may make the tumor more susceptible to chemotherapy (Melanie Demers, manuscript in preparation) and what could be the platelet granule component(s) that prevent hemorrhage in inflammation.

MANAGEMENT OF ALLOIMMUNIZED PATIENTS Nance SJ American Red Cross, Philadelphia, United States of America Alloimmunization in the transfused patient is relatively common, especially in the chronically transfused population. There are certain populations of patients for whom alloimmunization has extreme consequences, pregnant women and sickle cell disease (SCD) patients. For these, consequences can be fetal demise or a clinical state of untransfusability. Determination of the clinical significance of antibodies is important in cases with multiple antibodies or an antibody to a high prevalence antigen making compatible red cell products difficult to find. Some contributing factors are temperature of reactivity, IgG subclass, and reactivity in the monocyte monolayer assay. Patient specific factors that may contribute to alloimmunization potential are less well known. SCD patients present challenges in the frequency of alloimmunization and the nature of the antibodies formed. People of Black African descent more commonly have partial RH antigens (partial e and D). Testing for these antigens has become more accurate with the advent of molecular testing. There are facilities who match these patients with partial antigen expression with compatible type donors. This approach may be valuable in decreasing alloimmunization. For some patients, sensitization to even the easiest blood group antigen (like K) is devastating. The pregnant patient with an alloantibody to an antigen the fetus possesses is cause for alarm. Some forward thinking countries have established that females receive K and c negative blood until beyond childbearing years. Through unmatched transfusion, K and c can cause hemolytic disease of the newborn. Most countries match for D. An important point in the management of the alloimmunized patient is the prevention of alloimmunization especially in the two groups previously mentioned. The ''side effect''(allo-antibody) of an unmatched transfusion that results in the patient being less able to receive treatment (red cell transfusion) that other patients can receive should be considered in the selection of blood used for transfusion. Molecular and racial matching should be evaluated in the sickle cell patient. Transfusion of blood negative for c and K should be required for females until their childbearing years have ended There is some thought that other patient populations (like those with solid malignancies, previous allogeneic hematopoietic stem cell transplant and history of diabetes) may have increased risk of alloimmunization. The number of transfusion exposure plays a role in the rate of alloimmunization. There have also been a number of studies evaluating the immune response and the role of various mediators. Avoiding all red cell alloimmunization is not possible. Patients with clinically significant antibodies should receive antigen negative blood. Sometimes blood is not available locally, or regionally or even nationally. Programs should be in place or started to provide antigen negative blood. Often these are termed Rare Donor Programs. Some countries have national programs; others have regional programs that collaborate. If there is no blood in the country, the WHO International Donor Program, operated by the International Blood Group Reference Laboratory in Bristol UK, is a resource to find rare blood all over the world. The ISBT Working Party for Rare Donors is also an international resource. Background: Fetal and neonatal thrombocytopenia (FNAIT) is caused by maternal antibodies directed against human platelet antigens (HPA) on fetal platelets. In 85% of FNAIT cases antibodies are directed against the HPA1a antigen. FNAIT is the platelet equivalent of hemolytic disease of the fetus and the newborn (HDFN) due to red cell (rhesus) alloimmunization. In several countries screening of pregnant women for anti-HPA1a antibodies is therefore considered. In pregnant women with anti-HPA1a antibodies non-invasive fetal genotyping of HPA1a would offer a safe method to determine whether a fetus is at risk of FNAIT. However, a non-invasive fetal HPA1a genotyping assay has not been described yet. Aim: To develop a reliable assay for non-invasive fetal genotyping of human platelet antigen HPA1a in maternal plasma. Methods: The HPA1a allele differs one single nucleotide (176 T > C) from the HPA1b allele. The T > C substitution in the HPA1b allele creates an Msp1 endonuclease restriction site. Pre-PCR processing of cell-free DNA with Msp1 will digest the HPA1b allele, while leaving the fetal HPA1a allele intact. We analyzed samples of 34 HPA1a negative pregnant women with anti-HPA1a antibodies (plasma n = 26; serum n = 8) using real-time PCR on Msp1 digested cell-free DNA. Mean gestational age was 31 + 2/ 7 weeks (range 12-41 weeks). Results: Conclusive results were obtained in 30 out of 34 samples. In four cases incomplete digestion of an HPA1bb control donor was observed and results were considered invalid. In 23 women the fetus was found to be HPA1a positive. In seven women the fetus was HPA1a negative. All results were concordant with serological typing of platelets or genotyping by amniocentesis. The difference in cycle threshold (Ct) values between the blood samples of women carrying an HPA1a positive fetus (mean Ct value 34.4 ± 1.8 SD) and of those carrying an HPA1a negative fetus (mean 44.4 ± 2.0 SD) was 10.0 (95% CI 9.24-10.81). Conclusions: We have developed a reliable non-invasive fetal HPA1a genotyping assay, which, similar to fetal RHD genotyping assays, can be used in high risk pregnancies as well as in screening programs aimed to prevent severe FNAIT. NAIT occurs about once in every 1000 live births. A specific serologic diagnosis should always be established because subsequent pregnancies may be (more) severely affected, and prenatal therapy is available. Aim: The first child of a woman with a history of previous miscarriages was born on term with a platelet count of 25 · 109/l and petechial haemorrhages. Serological cross-matching with maternal serum and paternal platelets revealed a strong alloimmunization against GP IIb/IIIa. There were no apparent discrepancies in HPA-1, -3, -6, and -9 genotypes between mother and child. The maternal serum was non-reactive with a panel of 12 different platelet suspensions. Extended genotyping ruled out the presence of additional rare alloantigens on paternal ITGB3 (GPIIIa). Additional studies were performed to identify the molecular basis of the suspected new alloantigen (Sec). Methods: Paternal and maternal platelets were immunoblotted with maternal serum. ITGB3 was amplified by PCR and sequenced. ITGB3 and ITGA2B (GPIIb) cDNA were cloned into mammalian expression vectors. ITGB3 was modified by site-directed mutagenesis to generate the Sec mutation. Chinese hamster ovary (CHO) cells were transfected with ITGA2B and either wild-type or mutant ITGB3 to express wild-type and mutant (Sec) GP IIb/IIIa, respectively. Transfected CHO cells were analyzed for their reactivity with Anti-HPA-1a and Anti-Sec in flow cytometry and immunoprecipitation. To analyze a possible functional impact of the Sec mutation, CHO cells were analyzed for their capability to adhere to fibrinogen and to bind LIBS (after addition of RGDW) and PAC-1 (after DTT activation). Adhesion of paternal (Sec) platelets to fibrinogen was also analyzed. Finally, 300 individuals and all available members of the family Sec were screened for the presence of the Sec mutation by real-time PCR. Results: Maternal serum reacted with GPIIIa from paternal platelets in immunoblotting. Sequence analysis revealed a single nucleotide exchange in exon 10 of ITGB3 (G331T), changing Lys580 (wild-type) to Asn580 (Sec) in the EGF4 domain. CHO cells transfected with the Sec variant of GP IIb/ IIIa, but not wild-type GP IIb/IIIa, showed binding of Anti-Sec in flow cytometry. Immunoprecipitation studies corroborated this finding. Adhesion of transfected CHO cells to fibrinogen showed reduced binding of the Sec variant compared to wild-type GPIIb/IIIa at low fibrinogen concentrations. LIBS binding was reduced after addition of RGDW to mutant, but not wild-type, CHO cells. PAC-1 binding to DTT-activated mutant cells was affected even more clearly. Adhesion of paternal (Sec) platelets to fibrinogen was not altered when compared to wild-type platelets, most likely because of heterozygous expression. Four members of the family Sec were positive for the G331T mutation, but none of 300 unrelated blood donors. Summary/conclusions: In the analyzed case, NAIT was caused by maternal alloimmunization against a previously unreported, low-frequency polymorphism on GPIIIa (G331T = Lys580Asn), termed Sec. This is the first low-frequency polymorphism on GPIIIa which influences the receptor's function, most likely because of the mutation's position within the EGF4 domain which participates in fibrinogen binding. International standards are necessary to ensure that the diagnostic tests used meet a minimum standard of diagnostic performance. International standard test methods set baseline analytical and diagnostic performance requirements for new reagents or methodologies. The tests that are used to qualify blood and biologics for international movement must provide a degree of confidence that those biologicals that give negative test results are free of a particular infectious disease agent. The WHO Expert Committee on Biological Standardization (ECBS), formed in 1947, is responsible for establishing WHO International Biological Reference Preparations that create global norms and standards to help define products of assured quality. Members of the Expert Committee are scientists from national control agencies, academia, research institutes, public health bodies and the pharmaceutical industry acting as individual experts and not as representatives of their respective organizations. ECBS reviews data from qualified laboratories and endorses standards and reference preparations and reference panels. International Standards are used extensively to calibrate secondary standards and working reagents that are used in assay validation. For example, The 1st International Standard for HCV RNA was established by ECBS in 1997 and has been pivotal in the implementation of HCV RNA NAT screening of plasma used in fractionation for the manufacture of plasma-derived products. The availability of the standard enabled laboratories to ensure that assays are of sufficient sensitivity and comply with regulatory requirements for limits of detection of HCV RNA. Their use has extended to clinical laboratories and kit manufacturers, where HCV RNA loads are expressed in IU/ml. International standard sera serve as primary reference standards and as both reference materials for the calibration of test methods and reagents and prototypes for the production of national and working standards. Without the development of standards for diagnostic tests, true international harmonization cannot be achieved.

Heiden SM, Seitz R Paul-Ehrlich-Institut, Langen, Germany

First pathogen inactivated blood products became available already in the early 80th. The main goal was to overcome haemophilia treatment associated hepatitis transmissions but it helped also to avoid transmissions of HIV not yet known at that time. Since then several virus inactivation and reduction procedures had been implemented in the manufacturing process of plasma derivatives, each of them with a specific inactivation profile and its specific side effect profile. Nevertheless, owing to a tight regulatory control nowadays these blood products are invariably characterized by a high quality and safety. The risk of spreading infectious diseases by products made of large plasma pools is much higher than that by blood components. However, many publications report on new emerging infectious agents, some of them with impact on the transfusion safety, and there are many arguments that merely testing for pathogens may not be sufficient to sustain a save blood supply in the future. In consequence, efforts have been made by many manufacturers to implement pathogen inactivation into the transfusion chain, starting more than 20 years ago with methylene blue/light treatment of fresh frozen plasma, followed later by S-303 (Helinx) treatment of red blood cells, whose development was interrupted due to an observed antigenicity. Recognizing the current worldwide epidemiological situation and the changed perception of blood safety, regulators should support those developments and escort them until access on the market. What kind of support by regulators is needed to achieve quality and safety standards comparable to that of plasma derivatives? First of all, the requirements for a market access should be transparent. Basic requirements with regard to the demonstration of non-toxicity are well described by ICH guidelines; the requirements for validation of virus inactivation capacity are also clearly defined. Minimal requirements to describe the component's quality are missing. The experience with plasma derivatives shows that a certain loss in activity following pathogen inactivation is to expect, which may become evident e.g. as a diminished coagulation capacity of plasma, an impaired platelet response to different stimuli, and an increase in red cell storage lesions. But, especially for cellular components it is impossible to define precise quality standards as there is no single strong correlation known between laboratory parameters and clinical efficacy of a given component. That means any information on quality and efficacy has to be confirmed by clinical data. The minimal requirements for the market access of pathogen inactivated blood components therefore should include demonstration of non-toxicity, description of the pathogen inactivation capacity against the relevant pathogens, description of the well defined manufacturing procedure, description of the product quality and functionality by laboratory parameters, and confirmation by clinical studies according to principles of Good Clinical Practice that efficacy and tolerability are not inferior compared to the native component. Special attention should be given to a consistent component preparation as small deviations can result in a remarkably impaired product quality. Finally, as for plasma derivatives a permanent tight regulatory control will guaranty a continuously high product quality and safety. The clinical effects of natural killer (NK) cell biology are evident in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). Our understanding of how NK killer Ig-like receptor (KIR) interaction with MHC class I ligands drives NK function has clarified the molecular mechanisms underlying previously described clinical correlations. Meanwhile, studies correlating KIR immunogenetics with transplant outcomes continue to provide additional insights to the impact of activating NK receptors, about which comparatively little is known and whose ligands remain largely obscure. HLA-mismatched allogeneic HSCT has served as a useful model to examine the clinical effects of ''missing self'' by licensed or educated NK cells, whereby recipients lacking class I ligands present in the donor experience lower relapse and higher survival. Interestingly, beneficial NK effects can also seen in HLA-matched HSCT where donors possess inhibitory KIR receptors for which neither the donor nor the recipient has the relevant class I ligand. In HLA-matched HSCT, comparison of survival and relapse rates between patients with and without ligand for donor inhibitory KIR revealed that acute myelogenous leukemia patients with ''missing KIR ligand'' have significantly improved overall and disease-free survival secondary to lower relapse. We have recently extended these findings to the autologous HSCT setting for neuroblastoma, where patients lacking HLA class I molecules for autologous inhibitory KIR experience improved progression-free survival. Studies correlating donor KIR genotype with HSCT outcomes may ultimately help to guide donor selection. Acute myelogenous leukemia patients with donors exhibiting genotypes with more activating KIR experience higher relapse-free survival. In addition, patients with donors exhibiting the activating KIR3DS1 experience less graft-versus-host disease. Taken together, these findings indicate that NK cells play an important role in controlling malignant disease, supporting their use in adoptive cell therapy for the treatment of specific cancers. Minor histocompatibility antigens (mHags) play a major role in the induction of detrimental graft-versus-host disease (GvHD) and the development of beneficial graft-versus-leukemia (GvL) effect in HLA-matched hematopoietic stem cell transplantation (HSCT). These antigens are defined as immunogenic peptides derived from polymorphic proteins and can be recognized by allogeneic cytotoxic T cells (CTLs) in the context of HLA molecules. The tissue distribution of mHags and the HLA molecules by which they can be presented play a significant role in the clinical outcome of T-cell responses against these antigens. In part, differential recognition by T cells of mHags specifically expressed in hematopoietic cells, including the malignant cells from the recipient, may result in GvL reactivity without concurrent GvHD. Furthermore, T-cell responses against proteins solely expressed in hematopoietic cell lineages from which the malignancy is derived may be appropriate mediators of GvL reactivity without GvHD induction. Therefore, following HLA-identical HSCT, mHags with hematopoiesis-restricted expression may serve as the primary target structures for T-cell-mediated GvL and anti-tumor reactivity (graft-versus-tumor reactivity, GvT). Targeting these haematopoietic mHags is an attractive strategy to induce specific GvL/GvT effects without increasing the risk of GvHD. To date, experimental and bioinformatics tools were utilized to identify more than 30 mHags at their molecular level, but the number of immunotherapeutically used mHags has reached only 13. The contribution of these mHags in GvL/GvT has been evaluated mainly by T cell-to-antigen-based approaches by testing the capacity of CD8+ CTLs specific for HLA class I-restricted mHags to recognize and lyse the malignant cells and/or their clonogenic precursors derived from patients with different types of leukaemia or myeloma. Because the identified mHags can only target a small fraction of haematological malignancies, focused efforts are still necessary to identify mHags that display balanced allele frequencies and are presented by prevalent HLA alleles. Additionally, experimental studies have revealed that mHag-specific CD4+ T cells not only provide proper help for the induction and expansion of CD8+ T cells, but also kill the malignant cells directly. To provide CD4+ T cell help for the induction and expansion of mHag-specific CD8+ effector T lymphocytes the identification of more HLA class II-restricted haematopoietic mHags is important. Unfortunately, initial attempts to clinically implement mHag-specific therapeutic approaches were not very successful. Therefore the development of novel techniques, such as the T-cell receptor (TCR) transfer approach and the emergence of novel insights in the in vivo survival of adoptively transferred CD4+ and CD8+ T cells directed at haematopoietic mHags raise hope to increase the efficacy and to realize the mHag-specific adoptive immunotherapy significantly. This presentation will summarize the current efforts to identify therapeutically relevant haematopoietic mHags and outline the strategies to apply mHag-based cellular immunotherapy to treat recurrent hematologic malignancies after allogeneic SCT. Background: Allogeneic stem cell transplantation (SCT) may trigger NK cell mediated graft versus leukemia (GvL) effects leading to improved outcome for patients with malignant haematologic diseases. GvL effects of donor-derived NK cells are caused by a repertoire of alloreactive cells expressing inhibitory killer cell immunoglobulin-like receptors (KIR) for HLA-ligands missing in the recipient (KIR ligand mismatch). However, until now, no general conclusion can be drawn from the genetically predicted NK cell receptor repertoire for the beneficial effect of the NK cell alloreactivity. For a better prediction of the GvL effect mediated by NK cells, an extensive phenotypic and functional characterization of the alloreactive cells of the donor is needed. Aim: The aim of the study is to establish a method to phenotypicaly and functionally characterize alloreactive donor-NK cells in the context of KIR ligand mismatch between donor and patient and to use these cells as effectors against leukaemic blasts. Methods: HLA and KIR genotyping was performed by PCR-SSO/SSP. Sequence based typing was used for high resolution typing of HLA-Cw. KIR phenotyping was performed by flow cytometry with antibodies against KIR2DL1/2DL2/3/3DL1 and NKG2A. To determine the frequency of alloreactive donor-NK cells for theoretical patients with a KIR ligand mismatch, combinations of these antibodies were used. To analyze the NK cell mediated cytotoxicity, B-LCL-stimulator target cells expressing HLA-C group C1, C2 or no HLA-C ligand were incubated with NK cells. In addition, CML blasts were isolated from an individual with HLA-C1 group. For cell discrimination immunostaining with CD19 and CD56 was performed and dead cells were excluded by staining with 7-AAD. NK cell activity was calculated as the percentage decrease of the absolute amount of vital target cells relative to the culture of target cells alone. Additionally, the reactivity of donor-derived NK cell populations expressing certain KIRs was evaluated by CD107 degranulation assay. Results: KIR phenotyping showed extensive individual variations in NK cell populations expressing certain inhibitory KIRs. With respect to particular KIR genes, we observed an inter-individual NK cell frequency up to twofold between individuals that were tested positive for the respective KIR by genotyping (KIR2DL1+:14-26%, n = 6; KIR2DL2,3+:17-29%, n = 4; KIR2DL3+:0-26%, n = 5). Experiments setting-up theoretical patient/donor pairs for a KIR2DL2, -2DL3/HLA-C group 1 mismatch showed a variation of 8% vs 15% in the alloreactive NK cell frequency. In addition, coincubation of NK cells with the L721. Background: Unrelated-cord blood transplantation (UCBT) represents for some patients the best chance for cure and surviving. The success of this procedure is based principally, on the grade of HLA-matching, but other factors, such as stage of the disease, the preparative regimen and cell dose also play an important role. Due to the immaturity of lymphocytes at birth this kind of transplant allow up to three donor-recipients HLA-allele mismatched. The higher disparity on HLA-allele however, correlates directly in most cases, with the severity of Graft versus Host Disease (GVHD) and success of transplant. Aim: To determine the frequency of HLA-A, B and DRB1 recipient-donor allele matched in a Mexican Cord Blood Bank and its relation with the incidence of GVHD. Methods: We reviewed retrospectively the HLA allele-matching and outcomes of a total of 172 patients underwent UCBT with cord blood units released from the Banco de Sangre de Cordó n Umbilical del Centro Nacional de la Transfusió n Sanguinea between May 2004 and December 2009. The typification of HLA-allele Class I (A and B) and Class II (DRB1) was performed in our laboratory using a low-resolution technique. Results: From the total of transplanted patients 18 (10%) were HLAmatched, 70 (40%) had one HLA difference, 81 (47%) were 4/6 HLA-allele matched and 3 (2%) had three HLA differences with the donor. The most disparities (63%) were observed among the HLA class I. With regard to the severity of GVHD there was not significant difference between patients transplantated with a HLA-matched cord blood unit and patients receiving a cord blood transplant with one or two HLA-allele mismatched since the incidence in both groups was 80% and 76% respectively. In the group with more than two HLA-allele disparities GVHD was documented in all of them. Conclusions: At the present it is still difficult to find a 6/6 HLA-allele donor-recipient matched in our population. The Mexican ethnic diversity influences undoubtedly these findings. The most of patients are transplantated with a 4/6 HLA-compatibility. Despite the HLA disparities there is no evidence of an increased rate on the severity of GVHD between 6/6 and 4/6 HLA, so that the transplantation with a 4/6 HLA matched is still valid. Additionally it is not strictly necessary to perform a high-resolution typing for HLA-A, -B and -DRB1 in cord blood transplantation. Background: Exosomes are small membrane vesicles, 30-100 nm in diameter, produced from different cell types. Exosomes are formed by invagination and budding from late endosomal membranes. They are then secreted to the extracellular environment by the fusion of late endocytic compartments with the plasma membrane. In recent years, exosomes also have been shown to be present in physiological fluids such as blood, urine, breast milk. Exosomes provide a novel mode of cell-cell communication and play important roles in immune activity and tolerance. In vitro exosomes derived from mature DC can activate lymphocytes while tumorderived exosomes can inhibit proliferation of lymphocytes and enhance the suppressive function of Treg cells. We previously reported that exosomes with immunity-associated molecules present in human plasma and induce CD4+T cell apoptosis in vitro. But the effect of plasma exosomes on other cells and the mechanism of immunotolerance are unknown. In blood and immune cells, WNT signalling controls the proliferation of progenitor cells and might affect the cell-fate decisions of stem cells. The number of studies on WNT signalling on immunity system has increased sharply during the past few years. Recent studies indicate that WNT proteins also regulate effector T cells development, regulatory T cells activation and dendritic cell maturation. When canonical WNT pathway was activated, b-catenin phosphorylation was inhibited and b-catenin translocates to the nucleus to form an active transcription factor complex. Aim: In the present study we investigated exosomes from human plasma might induce T cells tolerance and the mechanism of regulation function. Methods: We previously reported that exosomal-like vesicles isolated from human plasma by differential ultracentrifugation and ultrafilter display exosomal shape, with 30-100 nm in diameter. They also expressed exosomal marker protein and immunity-associated molecules such as Wnt3a, Wnt5a. Here, we purified exosomes from human plasma as before. CD4+ T cells and CD4+ CD25+ Foxp3+ Treg cells were purified using human Magnetic cell sorting kit. We determined the expression of Frizzled (Fzd, Wnt receptor) in CD4+ T cells and CD4+ CD25+ Foxp3+ Treg cells by RT-PCR. Then we examined the changes of b-catenin phosphorlation in CD4+ T cells and Treg cells by flow cytometry after stimulation with plasma exosomes. At last, after incubating with plasma exosomes, the apoptosis of Treg was measured and the expression of apoptosis gene in CD4+ T cells and Treg cells were examined. Results: After magnetic cell sorting, we could obtain yielded over 95% CD4+ T cells and CD4+ CD25+ T cells with high expression of Foxp3. The CD4+ T cells and CD4+ CD25+Foxp3+ Treg cells expressed mRNA encoding Frizled1, Frizled2, Frizled3 and Frizled4. We found that after incubating 2 days with plasma exosomes, the level of b-catenin phosphorlation in CD4+ T cells and Treg cells decreased, the canonical WNT pathway was activated. Furthermore, apoptosis assay show that exosomes from plasma maintain survival of Treg cells and don't enhance expression of apoptotic gene (Bax, c-myc) in Treg. And exosomes induced apoptosis by increasing expression of apoptotic gene (Bax, c-myc) in CD4+ T cells. Conclusions: exosomes from human plasma can induce CD4+ T cells apoptosis and promote Treg cells survival via Wnt signalling to influence immune responses. Risk modeling studies in blood safety play an important but occasionally misunderstood role. These studies are intended to quantify and contrast risks and benefits. This information is critical for policy development and intervention decision making. The limitations of risk modeling should be considered alongside the results obtained. The goal of this manuscript and presentation is to review current risk modeling techniques used in blood safety and to discuss the pros and cons of using this information in the decision making process. The types of questions that can be answered include: the extent of a risk or threat; implications of action or inaction; identification of effective strategies for risk management; or whether to adopt specific interventions. These analyses can be focused on a risk alone, but are often combined with economic information in order to gain an understanding of feasible risk interventions given budgetary or other monetary considerations. Thus, analyses that include risk modeling provide insights along multiple policy lines. As important, the analyses also provide information on what is not known or uncertain about a potential hazard and how much that uncertainty may influence the decision-making process. Specific examples of the range of risk analyses the author has participated in will be reviewed and include ongoing process improvement in testing laboratories such as error identification/eradication, estimating the risk of malaria exposure based on the specific locations of travel, evaluation of blood supply and demand during an influenza pandemic, cost-utility analyses of screening interventions for infectious diseases in countries with different human development indices, and insurance against emerging pathogen risk. Each of these analyses has a different purpose and seeks to answer different questions, but all rely on similar methods. The tool kit for risk analysis is broad and varied, but does have limitations. The chief limitation of risk modeling is that risk analyses are not scientific experiments or otherwise controlled studies. Consequently the analyses are more apt to be influenced by assumptions. These assumptions may be necessary in order to structure a problem in a way that will allow the question of interest to be answered or may result from incomplete or missing information. Another potential limitation is that commissioners of such studies, those who undertake them, and the intended audience, such as regulatory agencies, may have distinct and differing interpretations of the results. Risk modeling is a set of techniques that can be used to inform and support decision making at all levels in transfusion medicine. Advances in risk modeling techniques allow for continued expansion in the scope of possible questions that can be analyzed. Expanded use also improves the acceptance of the utility of these studies in blood safety and transfusion medicine. Background: In France, men who have sex with men (MSM) are permanently excluded from blood donation. This policy is felt to be discriminatory by MSM activists who request a change to a deferral based on sexual behaviour and not only on orientation. In addition, the deferral policy is not fully respected since some MSM do not report their sexual orientation before donating. Aim: We estimated the excess risk of HIV associated with the lack of compliance of MSM with the current policy and assessed the number of additional HIV transfusion associated infections of a new strategy in which MSM would donate blood and be deferred if they report more than one male sexual partner in the last 12 months in comparison with the current strategy. Methods: The study-period included 3 years, from 2006 to 2008. First, we estimated the fraction of the current HIV residual risk attributed to MSM, since some MSM are regularly found HIV positive when donating blood despite the deferral policy. We then constructed a model based on data obtained from behavioural and epidemiological surveys among MSM to assess the impact of the new strategy. Results: Thirty-one HIV seroconversions were observed among blood donors who had made at least two donations during the study-period, accounting for an incidence of 1.3 per 100,000 person-years. On the basis of this incidence rate, the current HIV residual risk was estimated at 0.41 per million donations or one in 2,400,000 donations (95% confidence interval: 0-1 in 700,000). Among the 31 HIV incident cases observed between 2006 and 2008, 16 (52%) were MSM (information obtained during post-donation medical interview). If all MSM had abstained from donating blood during the study period, the risk would have been 1 in 4,700,000 donations, a twofold reduction in the current risk. The new strategy would result in an overall HIV residual risk of 1 in 3,750,000 to 1 in 800,000 donations which corresponds to a maximum of two additional donations potentially infected with HIV each year in France in comparison with the current risk (one donation infected with HIV each year). Conclusions: Changing the current MSM deferral policy would probably increase the risk of transfusion-transmission of HIV. However, it does not take into account a likely change in compliance of MSM linked to a modification of the deferral policy. As some MSM currently consider ''lifetime deferral''discriminatory, they give blood wittingly (more than half of HIV incident cases among blood donors turn out to be MSM). A less stringent policy would be perceived to be more equitable and should enhance responsibility. In this perspective, further qualitative assessment is needed in addition to our quantitative analysis. Background: Screening of syphilis is one of the WHO recommended strategies for reducing transfusion transmitted infections. It has however been suggested by some experts in the developed world that screening should be discontinued. Transfusion transmitted syphilis (TTS) is now a rare occurrence and has not been reported in the international literature for >25 years. Storage of blood in temperatures at 2-80°C, which is lethal to Treponema pallidum, largely accounts for the elimination of TTS. In developing countries, however, where blood is in high demand and blood supply is largely by replacement donation, blood units are kept in the fridge for only a short time. In such conditions, it is possible that TTS still occurs. Aim: This aim of this study was to determine if syphilis sero-conversion occurs in transfusion recipients in a hospital where screening of blood units for syphilis is not routinely performed. Methodology: This study was conducted in Medicine, Obstetrics and Gynaecology and Paediatrics departments of the Komfo Anokye Teaching Hospital (KATH) in Kumasi Ghana. KATH does not screen its donated blood for syphilis. After obtaining informed consent, a pre-transfusion sample from the patient and another sample from the unit of blood being transfused were obtained. Plasma was aliquoted, stored in a -20 freezer and later tested in batches. Samples were screened for syphilis initially using an enzyme immunoassay (EIA, Bioelisa Syphilis 3.0, Biokit). Confirmation of positive EIA results was by the Treponema pallidum haemagglutination assay (TPHA, Biokit Syphagen TPHA). A rapid plasma regain test (RPR, Biokit RPR Redirect) was used to determine disease activity in EIA/TPHA positive samples. Recipients who were sero-negative prior to transfusion and who received a syphilis positive blood unit were recalled for follow up testing with EIA, TPHA and RPR to determine if sero-conversion had occurred. Patients with positive serology results prior to transfusion and those who received blood with positive serology results were given appropriate counseling and treatment. Results: Voluntary donors comprised 87% of donations. 34.3% of the donors were female. 200 transfusion recipients were recruited into the study; results are currently available for 150. 7.3% of donated blood units and 13.3% of transfusion recipients were EIA/TPHA positive. 36.6% and 30% respectively were also RPR positive. Two recipients were initially EIA/ TPHA positive and went on to receive EIA/TPHA positive blood. Two patients who received EIA/TPHA positive blood died before subsequent samples could be obtained to test for sero-conversion. One patient became EIA/TPHA positive one month after receiving syphilis positive blood. The blood had been transfused after 1 day's storage in the blood bank fridge. Serum has been stored for further confirmatory tests. This donated blood came from a walk in voluntary donor. Conclusions: This study shows that transfusion transmitted syphilis could potentially be a problem in our hospital. Further patient follow up and testing for sero-conversion is planned. Discussions are ongoing with the hospital management and transfusion committee to institute syphilis screening of all donated blood. Methods: Donors were screened for HIV Ab by an HIV-1/2 EIA and confirmed by western blot or IFA. HIV-1 RNA was detected by TMA in pools of 16 with reactive pools resolved to the individual donation; HIV-1 RNA confirmation of Ab-donors was performed using PCR. The drug resistance profile of the protease and RT genes was determined using the ViroSeq HIV-1 Genotyping System (v2.0, Celera Diagnostics). HIV genetic subtype was determined by phylogenetic analysis of the protease-RT viral sequence.

Results: Drug resistance profiles were obtained for 203 donor specimens; 9.9% (20 of 203) had mutations that confer resistance to antiretroviral drugs. Ten show resistance to a single drug class; one to nucleoside RT inhibitors (NRTI) and nine to non-nucleoside RT inhibitors (NNRTI). Eight show two drug class resistance; 5 NRTI + NNRTI, 2 NRTI + protease inhibitors (PI), and 1 NNRTI + PI. Two show three drug class resistance (NRTI+NNRTI+PI). Non-B strains were identified in 2.5% (5 of 203) of donors and consisted of subtypes A and D, CRF02_AG, CRF43_02G, and URF_BF. Drug resistance and non-B subtypes were only found in the established infection group. Conclusions: Combining the data from this study with the previous study shows that antiretroviral drug resistant HIV-1 is present in 9.1% (24 of 265) of HIV infected blood donors. While drug resistance was higher in established infections (9.3%; 22 of 236), resistant strains were present in 6.9% ( . HBV-DNA in seroconversion panels was determined by Siemens bDNA 3.0 assay. Detection limits of HBsAg assays were estimated in IU/ml from results obtained in an international proficiency programme in which lyophilised HBsAg standard dilutions equivalent to the WHO standard are used. HBsAg cut off crossing points in dilutions were determined by regression analysis on logit S/CO ratios and compared to 50% and 95% HBsAg detection limits in probit analysis. The 95% and 50% detection limits of Ultrio Plus (Gen-Probe/Novartis) were determined on HBV-DNA genotype A standard dilutions calibrated in copies/ml in the bDNA 3.0 assay and compared to the ones previously obtained with the first generation tiplex Ultrio assay on the same reference panels (Assal A et al. Transfusion 2009;49:289-300). The pre-seroconversion WPs were estimated according to refined formulas of Weusten J. et al (Transfusion, submitted) assuming a HBV doubling time of 2.56 days and a minimum infectious dose of 1 DNA copy/20 ml plasma. Results: A consistent correlation of 1 IU of HBsAg to~100,000 HBV-DNA copies was found between the two HBsAg assays and the bDNA 3.0 assay in the five seroconversion panels. This corresponds to a~1:1000 ratio of HBV to HBsAg particles. The table compares 95% and 50% detection limits and estimated pre-seroconversion WPs of NAT and HBsAg assays.

The parallel and consistent correlation between viral and subviral particles in the ramp up phase proves that detection limits on well calibrated HBV-DNA or HBsAg standard dilution panels can be used in WP risk models. The modeling shows that Ultrio and Ultrio Plus assays in ID-NAT configuration reduce the infectious WPs by 16.7-25.8 days as compared to HBsAg assays. Purpose: ''The rules governing medicinal products in the European Union (EU)'' contain in Volume 4 ''Good Manufacturing Practice (GMP) Guidelines''the following requirements: (4.1) Records provide a history of each batch of product; (4.8) Records should be made or completed at the time each action is taken and in such a way that all significant activities concerning the manufacture of medicinal products are traceable; (4.9) Data may be recorded by electronic data processing systems[3DOTS] if documentation is handled by electronic data processing methods, only authorised persons should be able to enter or modify data in the computer and there should be a record of changes and deletions; access should be restricted by passwords or other means and the result of entry of critical data should be independently checked; and (5.8) Checks on yields, and reconciliation of quantities, should be carried out as necessary to ensure that there are no discrepancies outside acceptable limits. The German Red Cross Blood Donation Service East collects 350,000 whole blood units per year. A new center in Dresden was constructed and validated between the years 2002 and 2006 with the objective to consolidate the manufacturing from five former sites to one location. The goal related to documentation was to replace the existing process on paper sheets with electronic data records. Methods: First, existing manufacturing documentation in the blood bank management system BAS/400 was identified, thus the outcome of products was registered, however the different steps were not documented. In the next step, documentation and technical conditions needed to fulfil the requirements above were defined and the inadequate documentation for centrifugation and freezing garnered the highest priority. Results: The manufacturer of the centrifuge offers a software solution with data interface and the manufacturer of the freezer offers to create a solution, but both are too expensive. Thus, solutions were created with less external support. Further, records for other steps were improved or newly created. Today all needed product, processes and user information are recorded. Data will be checked in BAS/400 and critical information will be evaluated. Products out of specification are listed on protocols for evaluation by the Qualified Person. Conclusions: Data management solutions, in connection with Auto-Identification methods, enable the staff to capture easily detailed product, manufacturing and user information. The automated safety checks to predefined requirements and the enlisting of products ''out of specification''result in a clear overview of the manufacturing process for the Qualified Person and the data checks permit an automatic blocking of conspicuous products for further use until evaluation by the Qualified Person. Further the actual product status and position could be tracked and staff involved in the manufacturing could be identified. Thus EU GMPrequirements could be completely satisfied in larger production sites. The presentation deals with the EU requirements, the implementation and validation process as well as the final solutions. [2] . Collaboration between the ISBT Standing Committee on Education coordinating the ISBT e-learning program has been started to develop educational material based on the Guideline. 2. RFID Task Force reviewed the current state of RFID development and has recommended guidelines for use of RFID in transfusion medicine. The Guideline considers use of RFID throughout the blood supply chain from donation through transfusion, including involvement and participation by blood bag manufacturers. It also addresses standardization details. The Guideline is expected to be published during the spring 2010. In addition to these major undertakings the Interface Task Force has been focusing on the equipment and information management system interfaces to provide an enhanced level of standardization that would compliment the relevant existing standards HL7 and LIS2. The aim is to provide a degree of standardization that will ensure critical data fields are transmitted in a tightly defined format allowing a generic interface to correctly receive and interpret required information. A pilot project aimed as a proof-of-concept with blood collection weigher/shaker instruments is awaiting final commitments and a poster introducing the standard concept will be presented during the ISBT/DGTI Congress 2010. In the future the Task Force will also be looking into the system-to-system interface challenges. The future activities will involve Traceability Task Force, a joint effort between the WPIT and the WP on Hemovigilance, which is planned to be activated during the ISBT/DGTI Congress 2010. Other new topics awaiting consideration and possible activation will be discussed in this presentation. reporting tool allows organisations to access registration and completion data for monitoring uptake and meeting accreditation requirements. Approximately 4% of users have encountered a problem. These include completion certificates not being received, lack of an email address required for registration, typographic errors by users, and inadequate technology (software and hardware) to access this. Solutions and support have been developed to address these challenges. Future Directions: With uptake and endorsement by all states and territories of Australia a successful submission was made for national funding to provide access and support across all of Australia. AUD$1.2 million has been provided over 3 years for a program management office, additional staff, ongoing support, further development including new content, and a promotional strategy and resources. A comprehensive evaluation of the effectiveness of this tool will also be undertaken during the funding term. Correspondence: david.peterson@health.sa.gov.au Blood is an essential adjunct to modern clinical practice across the EU however, there is increasing awareness that the blood supply has been steadily declining. Unnecessary transfusion of a blood component exposes patients to unnecessary risk and wastes the donor's gift. Over the last 20 years it has been known there are differences between countries and between different hospitals within countries, in the use of blood in similar surgical operations. During this time haemovigilance schemes have demonstrated that errors in prescribing, and in the clinical transfusion process are the cause of significant risks to patients. As part of the 2006 EU Public Health Programme, the Scottish National Blood Transfusion Service (SNBTS) has led a 3 year project to promote the optimal use of blood. Twenty project partners representing 16 EU member states have developed a manual, which aims to provide practical guidance for those seeking to improve the safety of the clinical transfusion process, and the effectiveness of the use of blood components. A 1 day symposium will launch the Manual, which is supported by a website. The symposium will explore the importance of improving safety and optimising this scarce resource by looking at experiences in different EU countries and also from the international aspect. The meeting is suitable for medical and nursing staff involved in blood transfusion, staff within the blood services and hospital blood banks who are interested in quality improvement. The enthusiasm demonstrated by the project participants has encouraged us to believe that sharing information and best practice on the optimal use of blood is the best way to protect patients from the known and unknown risks of transfusion. There is interest in building a network of committed transfusion professionals and the symposium will facilitate wider discussion on how this can be taken forward. Background: Research in epidemiology, virology, and immunology is important to improving blood safety in low and middle income countries. However such research has been limited by a lack of trained clinical research personnel. The number of candidates who can complete long-term training in Europe of the USA is limited by high cost and long absence from primary job responsibilities. We reasoned that an in-country short course could help to develop methodological skills on study design and data analysis relevant to blood safety. Aim: Develop and implement a 2-week research training course with the following objectives: (i) provide a state-of-the-art review of research in blood donor selection and laboratory testing methodologies currently utilized to minimize the risk of TTI's; and (ii) provide practical training in epidemiology and clinical research methods which will allow the trainees to design and conduct studies to improve transfusion safety at their own blood centers. The ISBT currently recognises 308 red cell surface antigens, 270 of which belong to 1 of 30 blood group systems, although this number will almost certainly increase at the Berlin ISBT congress. Most of the systems represent a single gene, whereas three (Rh, Xg, Chido/Rodgers) have two genes and one (MNS) has three, making a total of 35 identified and sequenced blood group genes. The molecular bases of all clinically significant blood group polymorphisms have been defined, as have the molecular backgrounds to many blood group variants. This makes it possible to predict, with a high level of accuracy, most blood group phenotypes from a DNA sample, and this is becoming widely applied for a variety of purposes. Most blood group polymorphisms are associated with single nucleotide polymorphisms (SNPs), but a number of other genetic mechanisms are also involved, including gene deletion, mutations within the promoter sequence, recombination between homologous genes, and splice-site mutations. Protein-based blood group phenotypes can also be altered by changes to genes other than the ones encoding the blood group protein. For example, expression of Lutheran antigens and antigens in several other systems are depressed by mutations within the genes for the transcription factors EKLF and GATA1. Many proteins expressing blood group antigens are part of complexes within the red cell membrane. Changes in one protein of a complex may affect expression of antigens on one or more others. There appears to be at least two major complexes. There is substantial evidence for a complex consisting of band 3 (Diego), glycophorins A and B (MNS), Rh proteins and RhAG, ICAM-4 (LW), CD44 (Indian), and CD47, and linked to the cytoskeleton through protein 4.2 and ankyrin. This complex may be part of a metabolon involved in the transport and processing of respiratory gases. Another complex, so far only demonstrated in mice, contains band 3, glycophorin C (Gerbich), the Rh proteins, the Kell glycoprotein, Xk, and the Duffy glycoprotein, and is linked to the spectrin-actin junction of the cytoskeleton through protein 4.1R, p55, and adducin. Some blood group abnormalities are not inherited, but result from somatic mutations. One example of this is the reduced expression of the blood group antigens located on glycosylphosphatidylinositol-linked proteins in paroxysmal nocturnal haemoglobinuria. Somatic mutations an X-linked gene, PIGA, involved in the biosynthesis of the GPI anchor, is responsible for a proportion of the red cells lacking the GPI-linked proteins with the loss of antigens of the Cromer, Dombrock, Yt, and JMH systems. Another example is Tn polyagglutination, which results from somatic mutations in another X-linked gene, C1GALT1C1, which encodes a chaperone for Tsynthase. Without active T-synthase, the carbohydrate Tn cryptantigen is not converted to T and is expressed at the red cell surface. Methods: In the present study we included 38 samples with aberrant RHCE phenotypes that have been previously investigated for the entire RHCE coding region. No alteration of the RHCE sequence has been observed. A 1457 bp fragment including the promoter and the exon 1 region was PCRamplified using RHCE-specific primers and subsequently sequenced. Results: In 34 of the 38 samples (90%) the promoter region revealed a normal DNA sequence as expected from the C/c phenotype and genotype. In two cases with diminished e-antigen expression and cE/ce genotype the promoter sequence indicated heterozygous C/c alleles. This indicated a ce allele with C sequence characteristics in the promoter. One sample showed weak expression of the C antigen with a proposed Ce/ce genotype. In the promoter region the DNA sequence indicated homozygous c characteristics. Thus, the Ce allele is under control of a c-promoter and may cause a lower expression of the encoded antigens. Another sample with normal expression of C, c and e but low expression of E antigen (proposed genotype Ce/cE) revealed heterozygous C/c promoter sequence as expected. In addition, a heterozygosity was observed at the D-specific position )58C > G. Presumably, the mutation is located on the cE-allele and may cause lower expression of the encoded protein. However, only the E antigen showed altered expression whereas the c antigen was normal. Conclusions: Promoter variants can be observed among samples with aberrant RHCE phenotypes. Only 10% of the 38 selected samples showed an DNA sequence alteration in the RHCE promoter region. Other mechanisms such as DNA methylation of the promoter elements may also contribute to diminished antigen expression and needs to be investigated. Background: As the clinical problem of alloanti-D immunizations after transfusion of undetected weak D phenotypes has to be overcome, uncertain serological results are resolved by antigen prediction based on the genotype of the RHD locus. Sequence Based Typing (SBT) is used in-house to detect molecular alterations in the RHD gene. Due to the steadily rising number of testings, the establishment of new high throughput RHD genotyping methods is necessary. Aim: By this pilot study comprising 26 patient sequence data we intended to establish a new routine application and compare the validated Sanger sequencing with the methodical potency of the massively parallel sequencing approach regarding routine molecular screening of mutations in the RHD gene. Methods: Twenty-six EDTA blood samples were collected and typed as unsure weak D types by standard serological analysis. Sanger based SBT was performed for exon 1-10 of the RHD gene. In parallel all samples were sequenced by the Genome Sequencer (GS) FLX technology (454 Life Sciences, Bradford). Exon specific primer sequences for GS were identical with the amplification primers in the Sanger approach (except exon 2). Amplification of all 260 amplicons was performed with one temperature profile, followed by amplicon purification and quantification by PicoGreen (Invitrogen). Appropriate dilution and pooling of the amplicons resulted in 8 amplicon libraries each containing 3-4 RHD patients à 10 amplicons. GS-FLX sequencing procedures were performed according to manufacturer's instructions. Results: Twenty-one of 26 analysed RHD samples revealed molecular alterations (homo-and heterozygous single nucleotide exchanges and a 37 bp duplication) in Sanger based SBT. We detected 1-4 mutations per patient with mutations spread along all 10 exons, so that we characterized alleles of weak D types 1, 2, 3, 4, 4.3, 11, 20, 21, 33, 45, 66 and 70, D categories DIII type 6 and DVII, partial D DNB and DHMi as well as the alleles Del RHD(IVS3+1 G > A), D negative RHD(W16X) and RHDw. All mutations detected by Sanger SBT were correctly reconfirmed by GS-FLX Sequencing. Homozygous alterations were detected in a range of 82-100% of reads, heterozygous mutations in 35-55% of reads. While the heterozygous 37 bp duplication of RHDw leads to a signal overlay in the electropherograms in the Sanger sequencing approach causing difficulties in interpretation, GS-FLX sequencing enables a clear discrimination of the alleles due to the clonal sequencing technology. Conclusions: In this pilot study we could impressively show that high throughput sequencing by GS-FLX is capable of detecting mutations within the RHD blood group system in full concordance with standard Sanger sequencing approach. Main advantage of massively parallel pyrosequencing is the improvement of cost effectiveness in RHD genotyping by screening up to 50 patients in parallel. Alternatively sequencing runs can be amended by further amplicons covering other blood group systems for detailed genotype information. However, future methodical developments regarding read length and automatisation will be applied on the presented GS-FLX sequencing approach prior to validation and introduction of the system as a routine service. Background: The Rh system is complex with numerous different alleles of RHD and RHCE genes. These two genes are highly homologous and are potentially subject to many gene conversion events creating hybrid genes. Furthermore, various non-templated point mutations have also been described. With an increasing number of routinely RH-genotyped samples several new alleles are detected each year -especially those with rarer occurence and with minor serological differences from normal RhD. Aim: In 2009 more than 300 samples were genotyped in the Czech population by microarray or PCR-SSP. Discrepancies found between serological and genotyping results were further investigated. The aim was to characterize three potentially new genotypes that were identified by our screening. Material and methods: Routine and extended phenotyping was performed with CE-certified monoclonal reagents (Immucor; ID-Partial D Typing Set, DiaMed; D-Screen, Diagast). A cohort of 200 random blood donors was genotyped on microarray BloodChip v.2.0 and other 130 samples with serological suspicion of a weak or variant RhD type were genotyped with PCR-SSP kits (INNO-TRAIN). Selected cases were sequenced. Results: Case 1: RHD 48G > C point mutation /exon 1/. This blood donor was phenotyped as RhD+ (C+c+E-e+Cw-) and expression was apparently normal in extended typing with the monoclonal anti-D panels. The mutation was detected by the BloodChip and confirmed by sequencing and predicts an amino acid W16C change. This polymorphism has been observed together with other substitutions in the weak D type 4.1 (which was excluded by BloodChip) but has not been reported on its own. Case 2: RHD 787G > A point mutation /exon 5/. A patient with weak RhD expression in routine tests (C-c+E-e+Cw-) showed equivocal pattern in tests with panel of monoclonal anti-D. The molecular basis remained unresolved by PCR-SSP kits /although it appeared DV-like/. Sequencing of RHD exons 4 and 5 revealed the single mutation predicting G263 change. This polymorphism, although present in many D variants involving this part of RHD gene, has not been reported on its own. Case 3: RHD(1-8)-CE(9)-D(10) hybrid gene. A blood donor phenotyped as RhD+ (C+c+E-e+Cw-), also with apparently normal expression. Analysis by BloodChip revealed the absence of signal for all exon 9 SNPs, which was confirmed by exon-scanning PCR-SSP. This too, is an apparently new RHD-CE-D hybrid gene. Interestingly, the substitution by RHCE exon 9 does not appear to affect the normal expression. Conclusions: We describe three novel RHD alleles detected by discrepancies between routine phenotyping and genotyping. In these cases, changes in peripheral regions of RHD, e.g. the point mutation in exon 1 and the exon 9 exchange, show no apparent effect on RhD strenght and D epitope expression. The single point mutation in RHD exon 5 in case 2 results in phenotype corresponding to a novel weak type. Detailed serological and molecular studies are in progress to fully characterize these novel RHD alleles. Support: MZdCR 23736 pisacka@uhkt.cz Translational research is transforming how research is conceived and conducted, dynamically linking together healthcare systems with disciplines that study the molecular biology of humans and disease, and ensuring that blood and transfusion medicine continue to provide its patients and customers with innovative products and services. As a critical enabler of this transformation, information technology functions as a conduit in the bidirectional translation in this feedback loop, from bench to bedside and back. This presentation explores the role of information technology and data sharing as key facilitators of biomedical translational research. We differentiate between information technology and informatics as disciplines in the healthcare domain. The presentation also highlights a case study at BloodCenter of Wisconsin, USA where IT and Informatics are enabling translational research. The intended audience for this content includes: healthcare and healthcare IT executives, senior managers, clinicians, and researchers. In 2009 two new guidelines were approved for publication and WorkShops focusing on these Guidelines will be held during the ISBT/DGTI Congress 2010: 1. Validation Task Force completed a three-years comprehensive rewrite of The ISBT Guidelines for Validation of Automated Systems in Blood Establishments and the version two was published in December 2009 [2] . Collaboration between the ISBT Standing Committee on Education coordinating the ISBT e-learning program has been started to develop educational material based on the Guideline. 2. RFID Task Force reviewed the current state of RFID development and has recommended guidelines for use of RFID in transfusion medicine. The Guideline considers use of RFID throughout the blood supply chain from donation through transfusion, including involvement and participation by blood bag manufacturers. It also addresses standardization details. The Guideline is expected to be published during the spring 2010. In addition to these major undertakings the Interface Task Force has been focusing on the equipment and information management system interfaces to provide an enhanced level of standardization that would compliment the relevant existing standards HL7 and LIS2. The aim is to provide a degree of standardization that will ensure critical data fields are transmitted in a tightly defined format allowing a generic interface to correctly receive and interpret required information. A pilot project aimed as a proof-of-concept with blood collection weigher/shaker instruments is awaiting final commitments and a poster introducing the standard concept will be presented during the ISBT/DGTI Congress 2010. In the future the Task Force will also be looking into the system-to-system interface challenges. The future activities will involve Traceability Task Force, a joint effort between the WPIT and the WP on Hemovigilance, which is planned to be activated during the ISBT/DGTI Congress 2010. Other new topics awaiting consideration and possible activation will be discussed in this presentation. Background: ORBS is a web based ordering and receipting system that was designed, developed and implemented by the Queensland Blood Management Program (QBMP) in conjunction with key stakeholders including the Australian Red Cross Blood Service, Australian National Blood Authority, and private and public pathology organisations. It facilitates electronic ordering and receipting of blood and blood products supplied to both public and private sectors, and has replaced manual fax based systems. Aim: ORBS was developed to streamline the ordering process for blood products, provide traceability of products from the supplier to pathology laboratories, improve the quality and safety of the supply process, provide demand data in relation to blood products, and to support the reconciliation of invoices for blood products. ORBS is designed to interface with a range of other systems to facilitate data flows and operational efficiencies, and was developed with a view to national implementation should the concept prove effective. Methods: ORBS is a web-based system that was designed and developed ''in house''by the Queensland Blood Management Program after substantial consultation and business analysis with stakeholder organisations. Following 18 months of development and piloting, the system was implemented across the private and public sectors in Queensland over a 13 week period from 1 September to 3 December 2008. Training and on-site ''golive'' support was provided by two staff members of the QBMP. After 12 months of operation the success of ORBS was reviewed in Australia at a national level and the decision taken to pilot the system in two further jurisdictions to evaluate its suitability for national roll out. Results: ORBS is now operational in 66 laboratories spanning five private and public sector organisations in Queensland. Implementation in a further thirty facilities in other states of Australia is scheduled to commence in February 2010. In addition to process enhancements, the system has led to quality improvements in the distribution process and provides valuable data for understanding and managing the blood supply. Work is underway to further enhance the system through the development of interfaces to supplier and laboratory computer systems, and to a national data warehouse and reporting system. A high level of user acceptance has been demonstrated. Summary/conclusions: The presentation will outline the development process, lessons learned during implementation and benefits realised from the novel on-line ''Ordering and Receipting Blood System''. Results of a ''User Satisfaction Survey'' conducted 6 months post-implementation will be presented. Data sets and information now available to support the management of the blood supply, including ''real life'' examples of interest to transfusion laboratory personnel and users of transfusion services, will support discussion on the merits of the system.

EGYPTIAN EXPERIENCE OF NBTS STAFF IT LITERACY AND TRAINING ON COMPUTERIZED BLOOD TRANSFUSION SERVICES ACTIVITIES activities to implement an integrated system for automating NBTS activities. Consequently, training medical and paramedical staff in blood banks has become an important issue. Applying Blood Bank Management Software system will diminish errors that might be generated manually, such as keeping track of inventory stock level, keeping records of donors, patients and staff members, creating a billing system that handles financial issues concerning patients and hospitals, and facilitate the implementation of haemovigilance system. Computerization will improve the interaction and communication between different departments within the same transfusion center and with other transfusion centers within the service. Aim: Our objectives in this period include: 1. General training for preparing staff to become good users to achieve overall computer literacy. Besides, other courses cover essential knowledge of networking, data security and disaster recovery. 2. Specialized training is organized for every department as follows: 3. Training staff in donor care department to contact donors via web site to update their data and use it in recalling system. 4. Training help desk members know how to receive and answer questions and requests from patients and HBBs via web site. 5. Training data entry personnel to update donors' database, store test results, inform donors with positive results and mark them as deferred. 6. Training people in warehouse on how to monitor stock levels of empty blood bags and medical tools necessary for campaigns. 7. Training People in issuing department on how to update database of current blood stock to assign the patient to the nearest blood bank where the required blood components are available. Methods: To achieve the above mentioned aims, reference manuals were prepared; trainers were assigned at different courses 1. Training programs and material for all categories working in the National Blood Transfusion Service have been tailored to meet the target. 2. Installing Local Area Network connecting departments of the National Blood Transfusion Center which is the headquarter of the NBTS. 3. Training lab equipped with one server, one data show and 15 PCs connected to the main LAN is founded and dedicated for IT training.

Results: The following table shows the number of (489) staff in Egyptian NBTS who completed their training during the period from January 2008 to June 2009: Conclusions: IT training is quite important for everybody working in blood banks to be familiar with Blood Management Software Systems which facilitate their daily work. People working in blood drives perform much better when having laptops connected to the main server in the Headquarters. Accountants, HR specialists and other administrative staff are in need to do their job much better when they are well trained to deal with computer applications tailored for relevant activities. Generally, IT training improves the whole organization performance to unlimited extent. Cord Blood Tranplantation has been performed for the past 20 years. The advantages of using Cord Blood (CB) as a source of stem cells are less Graft vs Host Disease,and hence less stringent Histocompatability (HLA) matching requirements, availability of the unit (usually within 3-4 weeks as opposed to 8 weeks for bone marrow), ability to target ethnic minorities to increase the HLA donor pool of the bank, and currently the material is a waste product from the birthing process. However, while the concentration of stem cells in a CBU is extremely high, the small volumes results in a low absolute number of CD34+ cells after collection. Therefore, while readily available for children, there is limited use in adults. The two major strategies for increasing the availability to adults is (i) use of double cord blood units, (ii) expansion of the stem cell pool within the unit before infusion. Our laboratory has been very interested in the later method for the treatment of adult patients with malignant diseases. Our first clinical trial was a randomization of patient to receive either two unmanipulated cords or one unmanipulated cord and one cord that had been ex vivo expanded for 14 days in complete medium supplemented with G-CSF, TPO, Flt-3 and SCF. The second trial evaluated the benefit of culturing these cells on a mesenchymal progenitor cell layer (MPC). The MPCs provided a three dimensional milieu and potential growth factors that would enhance the proliferation of progenitor cells. The current laboratory and clinical results of these trials along with our future directions will be discussed during the presentation. Micro RNAs are emerging as key regulators of development, proliferation, differentiation and apoptosis. According to some data from micro RNA expression profiling during hematopoiesis, we investigate the role of miR-10a down regulation in megakaryocytic differentiation of UCB Cd133+ cells using anti miR-10a. Material and methods: CD133+ cells from three UCB were isolated by MACS method and expanded in cytokine cocktails (SCF, TPO and Flt3L) for a week, then were transfected by anti miR-10a (exiqon) along with TPO treatment (as positive control) and culturing without any cytokines (as negative control). Differentiation was followed by flow cytometric detection of surface markers, micro RNA real time and forming of colonies in mega cult (stem cell technology). Results: The mean ± SD of CD41 positive cells percentage was 7.2 ± 3.2 in untreated group, whereas in anti miR-10a transected cells was 31.1 ± 9.8, that showed significant difference(P-value < 0.05).This value in positive control cells was86.6 ± 7.4.Real time PCR results confirmed low expression of miR-10a in anti miR-10a transfected group. These cells formed three size colonies in Megacult after 2 weeks. Discussion: The results indicated that down regulation of miR10-a can induces differentiation of UCB CD133+ cells to megakaryocytic series with retaining of colonogenic capacity. According to data from micro RNA predicting targeting sites, Hox A1 has target sites in its 3¢UTR for miR-10a and because Hox A1 is up-regulated in megakaryocytic differentiation, it seems that down regulation of miR-10 affects increasing in HOXa1 expression and megakaryocytic differentiation. Background: Cord blood (CB) as a source of hematopoietic stem cells is an alternative to bone marrow or peripheral blood stem cells in clinical transplantation. Cryopreserved CB units may be stored for years before they are used for transplantation. However at present it is unclear how long CB units can be preserved without affecting their potential of CFU-GM formation. The Mannheim Cord Blood Bank was established in 1996 and collects CB at seven maternity clinics in the Southwest of Germany. Currently, about 1800 allogeneic CB units are stored for up to 145 months. Here we report our data of long-term stored CB units. Methods: Thirty-four volume-reduced CB units were analyzed after longterm storage (121 ± 16 months (mean ± SD), range 103-145 months) in the vapor phase of liquid nitrogen. The NC count of all CB exceeded 5 · 108 after preparation. The medium time from collection to freezing was 26.8 ± 10.2 h. Autologous CB plasma was used to prepare the cryoprotectant solution with a final dimethyl sulfoxide (DMSO) concentration of 5%. After thawing, we determined nucleated cell (NC) and CD34+ cell count, viability (colony-forming unit-granulocyte-macrophage, CFU-GM) as well as cell recovery. Results: The NC recovery after thawing averaged 38.7 ± 12.2% (range 17.8-69.2%), the CD34+ cell recovery showed wide variation: 92.5 ± 53.5% (range 22.8-213.0%, n = 31). In the viability assays, mean CFU-GM was 7.0 ± 5.1 · 105 (range 0.4-19.3 · 105). Interestingly we found no decline in NC recovery or CFU-GM in CB units £105 months vs 134-145 months: NC recovery 32.9 ± 9.9%, CFU-GM 5.4 ± 2.6 · 105 (103-105 months, n = 12) vs NC recovery 44.9 ± 13.4%, CFU-GM 8.4 ± 6.2 · 105 (134-145 months, n = 13). Conclusions: The present data show that the potential of CFU-GM formation of CB remains preserved after long-term storage up to 145 months. We found a considerable loss of cells after thawing, however values varied within a wide range. Further studies will have to focus on possible individual parameters impacting on this. As we observed no storage-time-dependent decrease of NC recovery and NC-viability, we conclude that even longer successful cryopreservation of allogeneic CB-units is possible. Background: Umbilical cord blood (UCB) contains a high quantity of haematopietic stem cells (HSC) that have demonstrated its use as an alternative source of HSC for haematopoietic stem cell transplantation. Aim: To maximize cost-effectiveness of public cord blood banking service provision in Hong Kong, this study attempted to determine the optimal inventory size of Hong Kong Red Cross Catherine Chow Cord Blood Bank (CBB), the public cord blood bank in the territory operated by the Hong Kong Red Cross Blood Transfusion Service.

We estimated the probability of finding one or more compatible UCB units for 464 previous local patients (107 children & 357 adults) for whom searches had been initiated as a function of three levels of HLA matching (4, 5 and 6 out of 6 loci by HLA-A, -B and -DRB1 low resolution HLA typing) according to various inventory levels that the CBB would be able to achieve in count of years. CBB of various inventory levels were constituted by simulation through random drawing of HLA typing results from donors in Hong Kong Bone Marrow Donor Registry which is also operated by the BTS. Results: With an inventory level of 5 000, 10 000, 15,000 and 20,000 UCB units, 21.1%, 27.6%, 32.1% and 34.9% respectively will have at least one unit available that is 6 out of 6 loci matched (median 2, 3, 3 and 4 donors per patient respectively); 66.2%, 92.0%, 95.3% and 95.5% respectively will have at least one unit available that is 5 out of 6 loci matched (median 6, 16, 21 and 28 donors per patient respectively); 99.1%, 99.4%, 100 and 100% respectively will have at least one unit available that is 4 out of 6 loci matched (median 93, 184, 265 and 354 donors per patient respectively). Conclusions: Ethnic Chinese accounts for 95% of the local population. Based on the observed HLA matching frequency among local patients and donors, an inventory level of 10,000 high-quality UCB units is considered optimal for Hong Kong. Doubling the inventory level to 20,000 UCB units will yield ‡1 compatible USB for additional 3.5% of patients at the 5 of 6 allele match level; the increase in chance of finding suitable units at this matching level for transplantation follows the law of diminishing return and is considered as marginal in the context of cost-effectiveness. Background: MSCs appear to be an appealing cell source for transplantation because of the ease of harvest and expansion ex vivo. During preparation of MSCs, They must undergo serum free and hypoxia conditions. On the other hand, the low cellular survival rate after transplantation, for example, into an infarcted heart within the first few days engenders only marginal functional improvement. Thus, it is necessary to reinforce MSCs against the arduous microenvironment incurred from ischemia, hypoxia, inflammatory response, and proapoptotic factors in order to improve the efficacy of cell therapy. Aim: In this study, in order to fulfill the above goals we manipulate MSCs with one of the potent cytoprotective factor i.e Hemoxygenase (HO-1). Methods: MSCs were exposed to UV irradiation in order to induce HO-1. Full length cDNA of HO-1cDNA was isolated with specific primersand was cloned into prokaryotic TOPO vector by TOPO cloning reaction The construct was ligated to gateway adapted adenovirus expression vector by LR recombination reaction. The Recombinant virus produced in appropriate mammalian cell line. The recombinant virus expressing HO-1 infected to MSCs. HO-1 engineered cells were exposed to hypoxic and oxidative stress condition followed by cell viability and apoptosis. Results: MSCs infected with recombinant adenovirus expressing HO-1 as determined by RTP-CR and Western Blot analysis. After exposure to hypoxia and oxidative stress condition cell viability and apoptosis were higher and lower in HO-1 engineered cells compared to the control. HO-1-MSCs retain their multidifferentiation potential into adipogenic, chondrogenic, and cardiomyocyte lineages Conclusions: Transient expression of HO-1 in MSCs increase cell viability against stresses that MSCs inevitably faced to them and may lead to improve the efficacy of cell therapy. Introduction: The entire African continents counties are classified as developing countries according to the World Bank criteria. It is ironic that poverty is a cause of endemic disease which in turn is a cause of poverty. It has been described in the 2008 UNAIDS report that sub-Saharan Africa remains the region most heavily effected by HIV, accounting for 67% of all people living with HIV and 75% of all AIDS deaths in 2007 and in this environment millions of units of blood are collected. This paper is based on a review of the literature as well as information provided by various transfusion services in Africa. Results: Table 1 shows the prevalence of disease in some transfusion services. There are various systems for the provision of blood, the hospital based systems which consist mainly of transfusion units attached to laboratories at the hospital, most of which use donor replacement schemes or the more centralized transfusion centers that usually have a system for voluntary non remunerated altruistic donors. Currently most countries have a hybrid of these two systems with 70% of the blood coming from donor replacement schemes. A range of screening strategies are used in Africa and it was estimated in 2004 that only 80% of the blood was screened for Transfusion Transmissible infections (TTI's). 155 countries reported to the WHO global database that 100% screening was performed but of these only 71 were performed in a quality assured manner. Various assay systems with differing sensitivities and specificities are used. A rapid assay is performed prior to donation in some settings which has advantages and disadvantages. Studies have shown that performing two different rapid assays in serial or parallel is more sensitive than as a single test but may not be feasible in resource limited settings. The majority of African countries test using an ELISA method which is the recommendation of the WHO. Many creative studies have been performed to try and make the screening as cost effective as possible without too much loss in sensitivity. One such study showed that if a serial testing algorithm was used that tested HBsAg first and then HIV on the seronegative donations and then syphilis on the subsequent seronegative donations and finally HCV the costs per annum in screening could be reduced by €90,860 per annum in Ghana. The final screening strategy that is used in some developing countries is Nucleic acid testing (NAT), this strategy is used in South Africa, Namibia, Egypt and Ghana. Although this strategy increases the safety of the blood supply it has been shown that in some settings it is not cost effective and should be clearly investigated prior to implementation. Conclusions: There are various screening strategies in Africa and due to a large amount of work performed by different organizations the plan is to have 100% voluntary blood donors and 100% screening by 2012. To do this a National blood screening programme and budgeting nationally for blood will be required. To compare results of serology screening using Enzyme Immunoassay (EIA) and NAT results over a period of 5 months at the NBTC. Methods: Routine ID-NAT was implemented at the Egyptian NBTC in the serology department in August 2009, using PROCLEIX ULTRIO Assay on the fully automated PROCLEIX TIGRIS System (Chiron -Novartis Gen-Probe), which is a single screening assay for multiple viruses in a multiplex format designed with Transcription mediated amplification (TMA) technology. NAT screening was done in parallel with EIA screening for HBsAg,HCV-Ab & HIV-Ag/Ab following the national screening algorithms. Initially Reactive (IR) samples by both EIA and NAT were marked as positive, we discarded the donations and did donors counseling. NAT negative EIA IR samples were extensively tested following the EIA screening algorithm while EIA negative, NAT IR samples were tested following NAT algorithm, which include repeat ultrio and Repeatedly Reactive (RR) samples will be tested using discriminatory assay. Results: Over a 5 months period (from 1st August till 31of December 2009) approximately 27,537 blood donations were screened both serologically and using ID-NAT, the following results were obtained: 794 (2.9%) were IR by both NAT and EIA, 11 samples were NAT RR and seronegative in the original screening, the discriminatory testing revealed that five samples were HCV RNA reactive and six samples were HBV DNA reactive (further characterization including viral load, genotyping, sequencing and extensive serological markers (anti-HBc, anti-HBs, HBeAg, anti-HBe) should be done to complete the image and identify the status of the donors (a window period donations or may be an occult HBV cases). Additionally 48 (0.17%) of blood donations tested were reactive for HBsAg, similarly 314 (1.14%) donations were reactive for HCV-Ab, the reactivity of these samples was confirmed by Chemiluminescent Immunoassay (CLIA, Architect, abbott diagnostics), however these samples were NAT negative These discrepancies between serology and NAT testing are possibly due to chronic HCV and HBV carriers with low viral loads undetectable by current NAT assays (quantification and viral load should be done). Conclusions: From our results, we concluded that it is mandatory to implement NAT as routine screening for donors, as there was a considerable number of yield cases. However, the increased incidence of non viremic,seropositive blood donors reinforce the notion that NAT technology cannot replace serology screening assay yet .NAT and serology assays are complementary to each other for the advantage of blood safety. Background: Although NAT testing is still not mandatory in Brazil, our service, representing a group of hospital blood banks in São Paulo state, voluntarily implemented minipool NAT testing for HCV and HIV-1 as a supplemental screening test in 1998 using an ''in-house''PCR test. . At the time, HBV was not included since the low viral load in the window-period required individual donation testing, which was costly and difficult to incorporate into the ''in-house''PCR test. However, a case of HBV transfusion-transmission at one of our hospitals made it necessary for us to introduce HBV-DNA testing in addition to HCV and HIV-1. In June, 2009 we replaced our ''in-house''PCR with the Roche cobas TaqScreen MPX / cobas s 201 technology (Roche, Brazil). Aim: To report overall performance and correlation to other HBV markers of the NAT system in use. Methods: The cobas s 201 is a fully automated system performing simultaneous detection of HBV, HCV, HIV-1 Group M, HIV-1 Group O and HIV-2, in addition to an internal control, in a multiplex format. Donations are tested in minipools of six donations and reactive pools are resolved in a follow-up algorithm, where the individual reactive donation is identified. Discrimination of the virus present in the sample is performed by an ''inhouse''real-time PCR method that detects HCV/HIV-1 and an internal control in a multiplex format with distinct fluorescent probes for each target. HBV DNA is detected in a separate assay. These confirmatory assays have 95% limits of detection of 18 IU/ml, 60 IU/ml and 91 IU/ml for HBV, HCV and HIV-1 respectively. Results: Since June 1st 2009, 33,113 donations were tested with the MPX test. Thirty-three HCV RNA reactive (0.1%), nine HIV-1 RNA reactive (0.03%) and 18 HBV-DNA reactive (0.06%) donations were identified. All donations were also positive for the respective antibody, except for one NAT HBV yield case. This donation was anti-HBc and HBsAg non-reactive by our standard screening (Biomeriéux) and also HBsAg non-reactive by two other more sensitive HBsAg tests (Abbot Axsym and Roche Chemiluminescence). Seroconversion to anti-HBs (16.4 mIU/ml) and anti-HBc IgM was observed in a follow-up sample obtained 46 days after the index donation, when HBV-DNA was no longer detectable. Noticeably, HBsAg was never detected in this donor; even when the most sensitive assay available (PRISM, Abbott) was used. Conclusions: In the first days of HBV DNA screening, the transmission of HBV to at least two recipients was prevented by NAT testing. Results from a larger number of donations will provide the full potential impact of HBV NAT on the Brazilian blood supply. The rate of so-called occult HBV infections (OBI) was unexpectedly low; HBV DNA was not detected in 319 anti-HBc reactive individuals.. This may be partially explained by the low prevalence of anti-HBc and HBsAg in this donor population of 1.02% and 0.12% respectively. This is rather low compared to the Brazilian blood donor population in general (5% and 0.5% respectively) where a rate of 1-3% of HBV-DNA carriers has been described among anti-HBc reactive donors. Background: Although highly sensitive hepatitis B surface antigen (HBsAg) EIA and 4th generation HCV and HIV antigen-antibody serologic screening assays were routinely performed, Shenzhen Blood Center decided to conduct nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) as a part of routine screening by using TMA (Novartis Diagnostics, USA), Roche PCR (Roche, USA), PCR-CHIP (Haoyuan, China), or real time fluorescence PCR (Kehua, China) since January 2006. NAT screening was undertaken on serology negative donations only. Aim: To evaluate the feasibility of NAT for routine blood screening in China, to compare different NAT technologies, to increase the safety of the blood supply, and to determine the rate of seronegative NAT yield donations. Methods: Seronegative donor samples were detected with one of five different NAT systems for the presence of HBV DNA, HCV RNA and HIV-1 RNA during difference period. NAT yield samples were confirmed by BCP (basic core promoter region) nested PCR. All positive donors were traced for serological markers and viral quantitative analysis using fluorescence molecular detection system (TaqMan PCR). Results: Of 143,234 seronegative donations tested, 29 were confirmed as HBV DNA positive by BCP nested PCR and donor follow-up testing. The rate of HBV DNA positive yield by difference methods ranged from 1:2010 to 1:18,521 (Table) . The HBV viral load in these yield samples ranged from 17.2 to 8.02-103 IU/ml. Twenty-one of these 29 donations were anti-HBc positive. Follow-up testing showed 8 of 17 donors seroconverted, four cases were proved to be window period donations, two donors recovered from HBV infection naturally (HBsAg and HBV DNA became undetectable,anti-HBs appeared), five donors had decreasing viral load and eventually became undetectable. A donor was confirmed as HCV RNA positive and donated during window period with a viral load of 2-105 IU/ml 3 months after the index donation.

Conclusions: Our data show that (i) implementation of highly sensitive HBV and HCV NAT for blood screening can enhance the safety of the blood supply; (ii) the main problem for the blood supply in China remains to be infectious blood donated by chronic HBV carriers with low viral loads but HBsAg seronegative; (iii) ID-NAT (such as TMA technology with high sensitivity) is recommended for use in high HBV prevalent area. Background: National Blood Service Zimbabwe (NBSZ) recruits donors nationally. All the donor information is stored in the Wide Area Network database (SafeNet). The testing of samples is centralised and results downloaded into the network system. The database was introduced in 1994 and it allows electronic follow-up of donors from date of first donation. Donors are lost due to various reasons including Transfusion Transmissible Infections (TTIs) (HIV, HBV, HCV and Syphilis) that leads to permanent deferrals. The longer the donor survive (not deferred) is a key indicator for a successful blood donor program. The study focuses on the survival patterns of the donors from date of first donation until permanently deferred after testing positive to TTI's markers. Aim: To estimate duration and blood donor survival patterns from date of first donation until permanently deferred due to TTIs seropositivity. Methods: An analysis of data from database of 2316 donors who were permanently deferred due to TTIs seropositive results. The demographic variables include gender, date of first donation, age and date of deferral, type of TTI, donor type and collection site. Kaplan-Meier functions are used to estimate survival patterns. Multivariate analyses examined the effect of age, and gender and other predictor variables on survival (in months). The survival time was calculated as the period from the date of first donation to the date of last donation/deferral. Results: The characteristics of 2316 donors are shown in Table 1 . The overall median survival time was 16.4 months (95% CI 15.9-17.5). Univariate analysis showed that survival times varied by gender (male 19.2 vs female 13.5) with a log-rank chi-square test statistic of 70. Eight (df = 1, P-value < 0.001), age group (df = 1, P < 0.001), no. Donations (df = 1, P < 0.001), year first donate(df = 1, P < 0.001), deferral reason (df = 3, P < 0.001), donor category(df = 1, P < 0.001), and by donation site (df = 4, P < 0.001). The above 20 years age group had higher survival time (43.7 vs 12). Similarly higher median survival times were observed for those with more than 10 donations (92.9 vs 14.3), started to donate before 2002 (67.3 vs 12.1) and are pledge 25 donors (32.4 vs 16.1). The HBV had the least median survival time of 12.2 months than HCV (20.8), Syphilis (21.2) and HIV (25). Thus, most donors are seroconverting to HBV than any other TTIs. It is interesting to note that HIV has the highest median survival time which may reflect that most donors may be paying much attention (in their lifestyles) to HIV (maybe it's the most known TTIs) and avoids possible exposure. A multivariate analysis using Cox Proportional Hazard Model eliminated gender (P = 0.575) and donations site (P = 0.140) as the possible predictor variables when the other five variables are included in the model. Summary/conclusions: The blood donor program can be strengthened by looking at predictor variables with higher survival times as the donors will be able to donate for a long time before they are permanently deferred due to TTIs seropositivty. In many clinical settings severe bleeding and massive transfusion may occur. Substantial morbidity and mortality is related to (sometimes excessive) bleeding and associated (poly)transfusion. In fact, bleeding is one of the leading causes of trauma-related mortality. A dilutional coagulopathy as a result of massive blood loss in combination with circumstancers such as acidosis and hypothermia may aggravate the bleeding tendency. The extent of the coagulopathy is often underestimated by conventional coagulation tests. Management of bleeding consists of local control (e.g. packing or surgical hemostasis), measures to retain adequate circulation, and proper transfusion procedures. In addition to these strategies, pro-hemostatic treatment may in some cases support the treatment of (severe) bleeding. Pro-hemostatic therapy aims at an improvement of hemostasis, which may be achieved by amelioration of primary hemostasis, stimulation of fibrin formation or inhibition of fibrinolysis. These treatment strategies may be applied to specifically correct a defect in one of the pathways of coagulation, but have in some situations also been shown to be effective in reducing bleeding in patients without a primary defect in coagulation. Besides the transfusion of platelets in case of thrombocytopenia or severe platelet disorders, a pharmacological improvement of primary hemostasis may be achieved by the administration of desmopressin. The administration of DDAVP results in a marked increase in the plasma concentration of Von Willebrand factor (and associated coagulation factor VIII) and (also by yet unexplained additional mechanisms) a remarkable potentiatiØon of primary hemostasis as a consequence. DDAVP is used for the prevention and treatment of bleeding in patients with von Willebrand disease or mild hemophilia A, and further in patients with an impaired function of primary hemostasis, such as in patients with uremia, liver cirrhosis or in patients with aspirin-associated bleeding. Based on the current insight that activation of coagulation in vivo predominantly proceeds by the tissue factor/factor VII(a) pathway, recombinant factor VIIa has been developed as a prohemostatic agent and has recently become available for clinical use. Indeed, in uncontrolled clinical studies this compound has been shown to exert a potent procoagulant activity and appeared to be highly effective in the prevention and treatment of bleeding, although most experience so far has been obtained in patients with severe and complicated coagulation defects. At present, a more general use of this agent for bleeding patients without an apparent coagulation defect is the subject of a number of ongoing clinical trials. Agents that exert anti-fibrinolytic activity are aprotinin and the group of lysine analogues. Aprotinin was shown to be effective in reducing blood loss in patients undergoing major surgery, however, has been associated with less faviourable outcomes, such as deterioration of kidney function and excess (cardiovascular) morbidity. Lysine analogues, however, are effective agents in reducing perioperative blood loss and appear to be safer, and therefore can be considered as clinically helpful pro-hemostatic agents.

Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model

Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM

No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients

Conclusions: After only five years, a short course in clinical and epidemiological research has already been instrumental in stimulating blood safety research in low and middle income countries of Africa and Latin America, and in promoting North-South collaborations. Future plans include obtaining funding for masters and doctoral level training of promising candidates, and starting a web-based system for networking and communication among former trainees. Supported by an ISBT Foundation Grant, Blood Systems Research Institute, UCSF Center for AIDS Research, an educational grant from Novartis and NIH grants K24-HL-075036, D43-TW000003 and D43-TW05799.

Thursday: Parallel Sessions 6B-S46 -How do I train best? How to safeguard good practice 6B-S46-01

Bein G University of Giessen, Giessen, Germany Regulatory T cells (Treg cells; CD25+CD4+Foxp3+) are present in the normal immune system and function as natural controllers of auto-reactive T cells. Depletion of Treg cells in mice produces autoimmune diseases. In the steady-state homeostasis, phagocytosis of endogenous apoptotic cells by quiescent antigen presenting cells, e.g. immature dendritic cells, leads to the generation of Treg cells maintaining peripheral self-tolerance. This finding led to the concept that systemic (i.v.) administration of apoptotic donor leucocytes could be used to induce donor-specific immunosuppression through induction of Treg cells controlling allo-reactive T cells. Apoptotic cell therapy by means of extracorporeal photopheresis has been demonstrated to be effective in the treatment of graft-versus-host disease in allogeneic bone marrow transplantation as well as in the treatment of organ transplant rejection. The perspectives of apoptotic cell therapy for the clinical induction of Treg cells in the field of allogeneic transplantation will be discussed. In 1999 the Scottish Government Health Department (SGHD) published a health service circular recommending that all staff involved in the transfusion process were supported by training and education (MEL1999(9)). A further health service circular launching the NHS Scotland (NHSS) Better Blood Transfusion (BBT) Programme in 2003 reinforced this recommendation (NHSHDL (2003)19)). To assist NHS Scotland hospitals implement the recommendations, the Scottish National Blood Transfusion Service (SNBTS) developed the Learnbloodtransfusion (LBT) continuing education programme. The aim of the LBT education programme is to assist all staff involved in the transfusion process provide high standards of care to patients, improve transfusion practice and minimize risk to patients and practitioners. The LBT programme is delivered using a variety of teaching methods including: face-to-face teaching programmes, written support and self-directed learning materials, and an interactive eLearning resource, www.learnbloodtransfusion.org.uk. The NHS Quality Improvement Scotland Clinical Transfusion Standards recommend the Module 1: Safe Transfusion Practice as mandatory for all staff involved in the transfusion process in Scotland (www.nhshealthquality.org). The Learnbloodtransfusion programme has been developed by UK subject experts and currently includes the following modules: A number of initiatives have been used to maximise user uptake in the hospitals including local training agreements owned by the hospital clinical managers, regular training reports disseminated to all NHS Hospital Transfusion Committees and clinical managers by the BBT Transfusion Practitioners (TPs) and review of individual training certificates as part of the practitioner's annual performance review meeting. In 2010, we have added new functionality to the eLearning Learner Management System (learnPro NHS TM ) that allows the BTT Transfusion Practitioners and clinical managers to identify staff with valid, elapsed (a defined grace period in which staff can renew their training), or expired training records, targeting learners via automated emails advising them their training is about to expire. The BBT programme has also committed to a promotional programme in 2010, developing a communication strategy targeting the promotion and revalidation of blood transfusion education. Promotional materials included individual messaging e.g. staff pay slips, revalidation memos, dissemination of promotional items (pens, pencils, mouse mats) and centrally produced posters and leaflets promoting the LBT web address and key message; staff must have current, valid training to participate in the transfusion process. Each hsopital is encouraged to use the promotional materials at promotional and regular training events. The NHSS Better Blood Transfusion remains committed to supporting all staff in Scotland achieve training appropriate to their role and to monitoring effectiveness. Background: Australia has some significant challenges in providing consistent, quality education to clinical staff to improve clinical transfusion practice. These include large distances between major centres and a large number of rural and remote hospitals and health centres with small numbers of staff administering transfusions on an occasional basis. In addition,as blood in Australia is supplied free of charge to patients, it is often perceived as a ''no-cost'' product with very few cost/price indicators given to clinicians that may assist with controlling usage. In late 2007 the BloodSafe transfusion safety and quality improvement program in South Australia released an e-learning education program (www.bloodsafelearning.org.au) for doctors, nurses, scientists and support staff involved in the transfusion chain. This aimed to overcome some of these challenges and assist hospitals with new accreditation requirements for transfusion training and credentialing. Results and uptake: Initially developed for a single state (South Australia) this resource has been adopted by all states and territories, with 36,601 users registered to December 31, 2009. A significant number of users come from rural and remote areas. Completion of this is mandatory in some health services and regions and it has been incorporated into university medical and nursing curricula. User testing with final year medical students (n = 98) has demonstrated that the content has provided increased understanding of the appropriate use of blood (83% of respondents), increased confidence and ability to organise a transfusion including appropriate consent (90%) and confidence in responding to a transfusion reaction (79%). Unsolicited feedback has been extremely positive with users commenting on the instructional design, use of video and interactivity to model best practice, and case studies that demonstrate how easy it is for problems to both occur, and also be prevented. An administrative and Background: The Rh blood group system, with 50 antigens, is the most complex system. Further complexity arises because some antigens, notably D, C, c, E, and e, have altered forms; the so-called partial antigens. The low prevalence antigen, Crawford (RH43), was first recognized as a separable antibody in a polyclonal anti-D reagent and is detected by a monoclonal anti-D (GAMA401). The molecular bases associated with its expression was elucidated (RHCE*ce 48G > C, 697C > G, and 733C > G; RHCE*ceCF) (Flegel, et al., Transfusion 2006; 46:1334) . We report four cases with the RHCE*ceCF allele, which collectively reveal that the encoded c and e are partial antigens and that Crawford is antithetical to the high prevalence antigen, CELO. Aim: The aim of this study was to determine the RHCE alleles in two patients whose serum contained an antibody to a high prevalence Rh antigen with apparent alloanti-Rh17 specificity, and the alleles in a c+ patient with alloanti-c, and a e+ patient with alloanti-e. Methods: Hemagglutination tests, DNA extraction, PCR-RFLP, AS-PCR, reticulocyte RNA isolation, Rh-cDNA analyses, cloning, and sequencing were performed by standard procedures. Results: RBCs from two patients typed D+, C-, E-, c+, e±, hr S + W /-, hr B -and their serum contained a strongly reactive (3+) alloantibody to a high prevalence antigen in the Rh system (alloanti-Rh17) in as much as the antibody reacted with RBCs of common Rh phenotype but did not react with RBCs with the Rh null or D-phenotype. In Case 1, a pregnant African American female, DNA analyses showed homozygosity for RHCE*ceCF. In Case 2, a pregnant Hispanic female, DNA analyses showed the presence of RHCE*ceCF in trans to a silenced RHCE*cE. Eluates (prepared following incubation of serum with antigen-positive RBCs) and RBCs from these two cases were mutually compatible. Serum from Case 1 did not agglutinate RBCs with the DC W -phenotype and serum from both patients did not agglutinate RBCs with the Dc(e) phenotype (encoded by RHCE*ceBP), which lack Arg229 (Chen, et al., Transfusion 2004;44:391). Adsorption and elution with serum from Case 1 demonstrates these Dc(e) RBCs express the antigen extremely weakly. Case 3 was an African American female whose serum contained alloanti-c but her RBCs typed C+c+. DNA analyses showed the presence of RHCE*Ce and RHCE*ceCF, thereby revealing that the c carried on the RhceCF protein is a partial antigen. Case 4 was an African American female whose serum contained alloanti-e but her RBCs typed E+e+. DNA analyses showed the presence of RHCE*cE, and RHCE*ceCF, indicating that the e carried on the RhceCF protein is a partial antigen. Conclusions: This study reveals the amino acid changes on RhceCF (Trp16Cys, Gln233Glu, and Leu245Val) encode partial c and partial e antigens, and that the protein does not express a high prevalence antigen, which we have named CELO (provisional ISBT 004058; RH58). CELO is antithetical to Rh43 (Crawford) and is also absent from RBCs with the Rh null , D-, or DC W -phenotype and is markedly depressed on RBCs with Dc(e) phenotype encoded by RHCE*ceBP. Recent advances have broadened the application of allogeneic hematopoietic stem cell transplantion and contributed to the continuosly increasing numbers of transplantations performed worldwide. These include (i) greater utilisation of reduced intensity conditioning and improvement of supportive care allowing transplantation of patients up to 70 years of age and with preexisting medical problems, and (ii) expansion of the acceptable stem cell donor pool to unrelated cord blood HSC. Thus, selection of the particular transplant procedure should be guided by patient characteristics such as type and stage of the disease, previous therapies, age and comorbidities. However, HLA-typing of patient and siblings at diagnosis is essential to allow the timely initiation of an unrelated donor search if needed.