Preanalytical validation of an in-house radioimmunoassay for measuring calprotectin in feline specimens.

ABSTRACT

BACKGROUND: Calprotectin is a marker of inflammatory disorders in people, and serum and fecal calprotectin were shown to be increased in dogs with gastrointestinal inflammation. Biomarkers of gastrointestinal inflammation are currently lacking in cats. OBJECTIVES: The purpose of the study was to analytically validate the canine calprotectin radioimmunoassay for quantification of calprotectin in feline specimens. METHODS: The immunoassay was analytically validated by determining assay working range, dilutional parallelism, spiking recovery, and intra- and inter-assay variability. Reference intervals for fecal calprotectin were established from healthy cats, and the influence of age, sex, and housing condition on fecal calprotectin was determined. RESULTS: The working range of the assay was 1.5-346.2 µg/g of feces and 11.2-8654.4 µg/L of serum. Observed-to-expected ratios (O/E) for serial dilutions of fecal extracts ranged from 77.3% to 112.0% (mean 99.2%) and from 95.7% to 161.4% (mean 118.5%) for spiking recovery. Intra- and inter-assay coefficients of variation for fecal samples were ≤ 11.0% and ≤ 12.8%, respectively. Fecal calprotectin concentrations ranged 1.5-66.5 µg/g (3-day sample mean) and 1.5-126.1 µg/g (3-day sample maximum). Housing conditions, sex, or age did not affect fecal calprotectin (all P > .05). For serial dilutions of serum samples, O/E ranged from 96.0% to 152.0% (mean 115.7%). Serum calprotectin concentrations in healthy cats ranged from 108.8 to 255.3 µg/L (median 158.2 µg/L). CONCLUSIONS: The canine radioimmunoassay for the measurement of calprotectin is analytically sensitive, linear, reproducible, accurate, and sufficiently precise (CVA  ≤ 43.2%) for use with feline feces (with a loss of accuracy at high calprotectin concentrations). The RIs for feline fecal calprotectin are comparable to those established for dogs. Independence of fecal calprotectin from age and sex agrees with findings in dogs.