key: cord-0005427-1al2xtwu
authors: Hansen, A. E.; Schaap, M. K.; Kjelgaard-Hansen, M.
title: Evaluation of a Commercially Available Human Serum Amyloid A (SAA) Turbidimetric Immunoassay for Determination of Feline SAA Concentration
date: 2006
journal: Vet Res Commun
DOI: 10.1007/s11259-006-3373-6
sha: 73afef161640be5bcfb3b546ca09e67e182bc8ca
doc_id: 5427
cord_uid: 1al2xtwu

Serum amyloid A (SAA) is an acute-phase protein in cats likely to be useful for diagnosing and monitoring inflammatory diseases, especially if rapid, reliable and automated assays can be made available. A commercially available automated human SAA turbidimetric immunoassay (SAA-TIA) was evaluated for determination of SAA in cats. Intra-assay and inter-assay imprecisions were in the ranges 2.1–9.9% and 7.0–12.5%, respectively, and without significant inaccuracy. Eighty-eight cats were divided into groups according to (A) the presence or absence of an acute-phase response (APR) (n = 23 and 65, respectively) and (B) clinical diagnosis (clinically healthy cats, cats diagnosed with inflammatory/infectious diseases, endocrine/metabolic diseases, neoplastic diseases, and miscellaneous disorders (n=43, 13, 8, 4 and 20, respectively)). The observed SAA concentrations were, as expected, different for (A) cats with and without an APR and (B) cats with inflammatory/infectious diseases compared to other diagnostic groups, except neoplastic diseases. In conclusion, the SAA concentration in cats could be measured reliably using the commercially available TIA designed for measuring human SAA, which should facilitate implementation of the parameter for routine diagnostic purposes.

The acute-phase response is part of the initial response to inflammatory stimuli. Following the inflammatory insult, the hepatic synthesis of a heterogeneous group of acute-phase proteins is increased (Jensen and Whitehead, 1998) . Serum amyloid A (SAA) has been identified as an acute-phase marker in humans, with levels increasing 100-1000-fold in response to inflammatory stimulation (Steel and Whitehead, 1994; Gabay and Kushner, 1999; Uhlar and Whitehead, 1999) . Earlier studies have concluded that SAA, α 1 -acid glycoprotein (AGP) and haptoglobin are useful indicators of the acute-phase status in cats (Kajikawa et al., 1999; Sasaki et al., 2003; Giordano et al., 2004) .

Part of the study was presented at the 5th Annual International Colloquium on Animal Acute Phase Proteins, 14-15 March 2005, Dublin, Ireland. Concerning the dynamics of SAA following an inflammatory stimulus, it has been reported that SAA increased 8 h (Kajikawa et al., 1999) and 3-6 h (Sasaki et al., 2003) following an inflammatory stimulus, reaching a maximum 36-48 h (Kajikawa et al., 1999) and 21-24 h (Sasaki et al., 2003) after initial stimulation. In comparison, AGP exhibits increased serum concentration later than SAA following an inflammatory stimulus (Steel and Whitehead, 1994) , and concentrations also wane later (Kajikawa et al., 1999) .

The widespread use of acute-phase proteins as biomarkers for inflammatory status in human medicine has led to growing attention to their use in animal medicine (Eckersall, 2004) . Significant differences in feline SAA concentration between clinically healthy cats and cats with various inflammatory diseases and disorders have already been found by direct ELISA using feline SAA-specific monoclonal antibodies (Sasaki et al., 2003) . However, reliable, rapid, automated and readily available assays are needed if feline SAA measurements are to be implemented for routine diagnosis and monitoring. The aim of the present study was to evaluate the validity of a commercially available automated human SAA turbidimetric immunoassay (SAA-TIA) in detecting and measuring SAA concentrations in cats, by means of assessing imprecision, inaccuracy, detection limit and overlap performance.

Serum samples were obtained from 45 client-owned cats (client-owned patients) presented at the Small Animal Veterinary Teaching Hospital, The Royal Veterinary and Agricultural University of Copenhagen, Denmark, for various diagnostic, therapeutic or prophylactic measures. An additional 43 clinically healthy Maine coon cats (age and sex not registered) participating in a cardiomyopathy screening programme at the hospital were assigned to the study as a control group (healthy controls). All cats were subjected to clinical examination and standard haematological and biochemical profiles (Jensen et al., 2001) . Further diagnostic procedures on individual cats were conducted at the discretion of the individual clinician (e.g. radiography, cytology, histopathology, histological smears, endocrine testing).

All serum samples were obtained by centrifugation (1500g, 5 min) of blood samples after collection in plain vials containing clot activator and separation gel (Vacuette, Greiner bioone, Kremsmuenster, Austria). Samples were stored in plastic vials at −80 • C until analysis, never for longer than 4 months.

SAA concentrations were determined using a human turbidimetric immunoassay (SAA-TIA; LZ-SAA (lot no. 47007), Eiken Chemical Co., Tokyo, Japan) and analyses were performed on an automated analyser (ADVIA 1650 Chemistry System, Bayer, Newbury, UK) according to the manufacturer's recommendations. Calibration curves were created using a human SAA calibrator from the same manufacturer (Eiken Chemical Co.).

Analyses were performed using a feline-specific single radial immunodiffusion assay (Feline α1AG Plate, The Institute for Metabolic Ecosystem, Miyagi, Japan), following the directions supplied by the manufacturer.

The SAA content was determined for all 88 cats. Three pools with high, low and medium concentration of SAA were obtained by mixing serum samples with high, low and medium SAA concentration (range, number of samples mixed): high pool 47-150.6 mg/L, n = 3; medium pool 21.4-33.3 mg/L, n = 4; low pool 1.0-3.3 mg/L, n = 4). The intra-assay variation was determined by measuring SAA concentration within-run on alternately high, low and medium pools seven successive times. The inter-assay variation was assessed by SAA determination of 11 between-run measurements on the same three pools. The pools were frozen in separate identical vials for the between-run analyses and only vials needed were thawed, to prevent potential variation due to repetitive freeze-thaw cycles. Linearity during dilution using physiological saline was determined to indirectly estimate inaccuracy by duplicate within-run measurements on continuous dilutions (0%, 10%, 30%, 50%, 70%, 90% and 100%) of the high SAA concentration serum pool (Jensen, 2000) . Detection limit was calculated from data of 15 replicate measurements of sterile 0.9% sodium chloride (blanks).

Overlap performance of the assay was assessed in two ways: (A) ability to identify cats with an acute-phase response, and (B) ability to show expected differences in SAA concentration between different groups of diseased and healthy cats. In detail: (A) The individual cat was categorized according to serum concentration of AGP, with a high concentration indicating the presence of an acute-phase response (Kajikawa et al., 1999) . A cut-off value of 0.65 g/L was used (based on data reported by Selting et al., 2000) . (B) All cats were assigned retrospectively to five different diagnostic groups according to the final diagnosis of the attending clinician, with regard to clinical, haematological, clinical biochemical and pathological findings, i.e. clinically healthy cats, cats diagnosed with inflammatory/infectious diseases, endocrine/metabolic diseases, neoplastic diseases, and miscellaneous disorders (n = 43, 13, 8, 4 and 20, respectively). Cats with neoplastic diseases had tumours that were cytologically and/or histologically characterized as neoplastic. Cats with infection had a pathogen identified that could be related to the pathological findings. Cats with endocrine/metabolic disorders had a final diagnosis that could be related to hormone or metabolic malfunction/disorder. Cats with miscellaneous diseases had various diagnoses and no clinical findings to associate them with any other groups. The 43 healthy controls were assigned to the group of clinically healthy.

Overlap performance was assessed by comparing the distribution of SAA levels within the various groups in setting (A) and (B).

Standard deviations, arithmetic means, medians and intra-and inter-assay coefficients of variation (CV) were assessed using standard descriptive procedures (Box et al., 1978; Büttner et al., 1980) . Detection limit was estimated from the mean value of blanks plus three standard deviations (Jensen, 2000) .

Linearity during dilution was analysed by ordinary linear regression analysis. Runs test was performed to investigate whether data followed a straight line (Jensen, 2000) . The correlation of AGP and SAA was assessed by Spearman's correlation coefficient. The Mann-Whitney non-parametric test was used to investigate statistically significant differences between the SAA concentrations of cats in the groups with and without the presence of an acute-phase response according to AGP concentration (Siegel, 1998) .

The Kruskall-Wallis one-way analysis was used to detect differences of medians between the diagnostic groups. On an indication of different distributions, Dunn's multiple comparison test was applied to obtain additional information regarding the deviation (Siegel, 1998) . The level of significance in all tests was set at 0.05.

The observed intra-and inter-assay imprecisions were in the ranges 2.1-9.9% and 7.0-12.5%, respectively (Table I) . Linearity under dilution was observed, with no signs of deviation from a straight line model with slope of unity and intercept of zero (Table II) . Detection limit was 0.38 mg/L (mean 0.19 mg/L, SD 0.06 mg/L). 

Clinical characteristics, descriptive data, and serum AGP and SAA concentrations of the included cats are given in Table III (Kajikawa et al., 1999) and 10.2 ± 8.3 mg/L (Giordano et al., 2004) ). This variation between studies is most likely due to the use of different methodologies and the lack of available material for standardization of feline SAA assays. Detection limit was estimated as 0.38 mg/L, which was within the observed physiological range of SAA concentration of healthy cats. This should not impair clinical application, as the primary purpose of SAA measurements would be to identify ( Horizontal lines indicate median SAA concentration of the individual groups except those with neoplasia. This is in accordance with previous reports of the overlap performance of feline SAA (Kajikawa et al., 1999; Sasaki et al., 2003) . Furthermore, the differences appeared to be of a magnitude that would make the observed imprecision and detection limit acceptable for clinical use. The cut-off value for AGP used to differentiate between cats with and without an acutephase response was established independently of the present study (upper 95% confidence limit (Solberg, 1996) of the AGP levels in healthy cats; data including 51 healthy cats in which the AGP serum concentration was measured using the same method as in the present study (Selting et al., 2000) ). The previously reported kinetic differences between SAA and AGP (Kajikawa et al., 1999) could cause minor uncertainties regarding the parallel presence of both proteins during an acute-phase response, but the effect was considered to be negligible in the present study. This assumption was sustained by the observed significant correlation of the two acute-phase proteins.

No significant difference was found between the group of infectious/inflammatory disorders and the group of neoplastic diseases, presumably because the group of neoplastic disorders was very heterogeneous in acute-phase response, as also observed in another study (Selting et al., 2000) . Thus, the assay demonstrated an overlap performance expected for feline SAA. Variation due to the freezing and storage of samples in the present study should be negligible as SAA has been reported to be stable at −20 • C (McDonald et al., 1991) and AGP is reported to be unaffected by freezing (Ganz et al., 1983) . Previous reports are available on species-specific determination of feline SAA (DiBartola et al., 1989; Sasaki et al., 2003) and on heterologous determination by means of rabbit anti-canine SAA-antibodybased ELISA (Kajikawa et al., 1996 (Kajikawa et al., , 1999 and anti-human SAA-antibody-based ELISA (Giordano et al., 2004) . However, none of these assays seem to be sufficiently practicable for routine application in terms of availability and automation. To the authors' knowledge there are no previous reports that demonstrate sufficient reliability of an SAA-TIA based on anti-human SAA antibodies for heterologous determination of feline SAA concentration. Analogously to their use in human medicine, feline SAA measurements are suggested to be applicable for diagnostic and monitoring purposes (Kajikawa et al., 1999; Sasaki et al., 2003) . The availability of a fast, reliable, readily available and automated assay such as the SAA-TIA should facilitate measurements of feline SAA in diagnostic laboratories and thus routine applicability in feline medicine.

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