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Biomarkers are defined as parameters of biological processes that can be objectively measured and used in diagnosis, prognosis, and in monitoring response to therapy. Acute phase proteins (APPs) are plasma proteins whose serum concentration changes by at least 25% as part of a nonspecific systemic immune response. In cats, α1-acid glycoprotein (AGP), serum amyloid A (SAA), and haptoglobin (hp) are recognized as acute phase reactants. According to their kinetics in this species, AGP and SAA are classified as major, and hp as a moderate APP. To establish these proteins as biomarkers in cats, the first aim of this study was to objectively validate a sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of feline hp. The second aim was to examine and evaluate the use of AGP, SAA, and hp as diagnostic indicators and parameters for monitoring in cats with an obstructive urinary tract disease (obstructive FLUTD) and diabetes mellitus (DM).
Serum samples were obtained from 56 cats from June 2012 until January 2014 at the Clinic of Small Animals. The validation study of the ELISA for measurement of hp included an assessment of precision and accuracy (n = 6 cats), detection limit, method comparison with the spectrophotometric assay (n = 39), and evaluation of the overlap performance (n = 47). Statistical analysis was performed using descriptive statistics, Spearman's rank correlation (rs), Passing-Bablok regression, Bland-Altman difference plot and Kruskal-Wallis analysis. The clinical study comprised the establishment of reference intervals for AGP, SAA, and hp in healthy cats (n = 27) as well as measurements of these 3 APPs in diseased cats diagnosed with obstructive FLUTD (n = 16) and DM (n = 10). SAA and hp concentrations were measured using ELISA, AGP was determined by radial immunodiffusion. Samples from diseased cats were collected on the day of initial presentation and on follow-ups. Cats with obstructive FLUTD were subdivided based on the diagnosis of concurrent disorders during treatment (FLUTDuncomplicated, n = 7; FLUTDcomplicated, n = 9). Cats with DM were classified according to the diagnosis of a severe ketosis or diabetic ketoacidosis (DMuncomplicated, n = 4; DMcomplicated, n = 6). Statistical analysis included descriptive statistics, the calculation of 1- and 2-sided 95% reference intervals using a robust method, and analysis of the APP-concentrations of the diseased cats on the day of initial presentation (Kruskal-Wallis analysis, diagnostic sensitivity and specificity with 95% confidence intervals, Spearman's rank correlation rs) and of serial measurements (generalized linear models). Variables with p values ≤ 0.05 were considered statistically significant.
The results of the validation study revealed coefficients of variation from 2.5 to 4.7% for intra-assay variability and from 7.1 to 11.6% for inter-assay variability. The ratio of observed to expected dilutional parallelism of 4 serum samples was 108.1 to 118.4%. The ratio of observed to expected spike recovery of 4 serum samples was 90.8 to 94.0%. The detection limit was 0.19 mg/ml. Method comparison revealed a strong positive correlation (rs = 0.95, p < 0.0001) and a proportional bias between the methods of -38.9%. Agreement between the methods was not clinically acceptable. Overlap performance of the ELISA was deemed satisfactory. The commercially available sandwich ELISA measures feline haptoglobin with an analytical and overlap performance acceptable for clinical purposes. Given the observed bias, the ELISA cannot be used interchangeably with the spectrophotometric assay.
In the clinical study, the upper limit of a 1-sided 95% reference interval for AGP was calculated as 380.2 μg/ml and for SAA as 8.3 μg/ml. For hp, the upper limit of a 2-sided 95% reference interval was 2.05 mg/ml. A lower limit could not be determined due to hp-concentrations below the detection limit. The evaluation of the APP-concentrations in healthy and diseased cats revealed several significant differences. On the day of first presentation, the levels of AGP and SAA of the groups FLUTDuncomplicated, FLUTDcomplicated, and DMcomplicated were significantly elevated in comparison to concentrations measured in healthy cats. For hp, a significant elevation in concentration in comparison to the healthy cats was detected for FLUTDcomplicated, DMuncomplicated, and DMcomplicated. The highest increases in concentrations of all 3 APPs on the day of first presentation were noted in cats of the group DMcomplicated. SAA was revealed as the APP with the highest increase in concentration in cats of FLUTDuncomplicated, FLUTDcomplicated, and DMcomplicated. In consideration of the limited study population, hp seems to be a better diagnostic biomarker than AGP and SAA at detecting cats of the group FLUTDcomplicated on the day of first presentation. In the diabetic cats, SAA was deemed to be an excellent diagnostic indicator at detecting cats with a complicated DM. A strong correlation was revealed between the concentrations of AGP and hp in the diseased cats on the day of first presentation (rs = 0.77; p < 0.0001). Medium correlations were determined between SAA and hp and between AGP and SAA (rs = 0.62, p = 0.002; and rs = 0.61; p = 0.002, respectively).
The interpretation of the follow-up measurements of each patient showed a good agreement between the response to treatment and the corresponding APP-concentration profiles over time. For a correct evaluation, it was necessary to consider the nonspecific nature of an induced acute phase reaction, the latent period from an immunological stimulus to a measurable increase in concentration, and to include concentration profiles of at least 2 APPs. The small study population was deemed as an important limitation, which was taken into account when interpreting the results of this study.
In summary, AGP, SAA, and hp can be used as diagnostic and monitoring biomarkers in cats with obstructive FLUTD and DM. The results of the study confirm the recommendations of performing serial measurements and APP-profiles consisting of at least one major and one moderate APP. The current study contributes in establishing AGP, SAA, and hp as biomarkers in cats. Future studies regarding the development and validation of rapid assay kits and the clinical applicability are necessary to realize the potential of APPs in veterinary practice.