Challenges in measuring serum calprotectin in autoinflammation: a comparative study of different immunoassays

BACKGROUND: Serum calprotectin (S100A8/A9 complex) is increasingly used in the clinical evaluation of autoinflammatory diseases (AID). However, systematic comparisons of commercially available assays remain limited. This study aimed to assess comparability, and clinical applicability of commercial calprotectin assays to support standardized biomarker-guided diagnostics. METHODS: Serum samples from 40 individuals-systemic juvenile idiopathic arthritis (sJIA, n = 10), adult-onset Still's disease (AOSD, n = 10), familial Mediterranean fever (FMF, n = 10), PAPA syndrome (n = 2), infections (n = 5), leukemia (n = 3), and healthy pediatric controls (n = 10)-were analyzed. Calprotectin concentrations were measured using four ELISAs (BÜHLMANN, R&D Systems, BioLegend, BMA), two PETIAs (sCAL turbo, GCAL), and one CLIA (QuantaFlash) and compared with a BÜHLMANN ELISA reference. ELISA assays were measured with a secondary 10-fold dilution, while PETIA and CLIA assays were performed on automated platforms with reflex dilution. Spiking experiments were conducted to evaluate recovery. RESULTS: All assays except BMA (r = 0.52) showed strong correlation with the reference (r ≥ 0.96). Median values in healthy controls varied markedly (0.29-3.22 µg/mL), with inter-assay differences exceeding one log scale. Several assays underestimated very high concentrations (> 50 µg/mL), likely due to Hook effects, and upper detection limits differed substantially. Spike recovery ranged from 79 to 268%, with the most consistent results observed for sCAL turbo. CONCLUSIONS: Commercial assays reliably quantify serum calprotectin in the normal to moderately elevated range but are not interchangeable. Variability in detection limits and susceptibility to Hook effects may lead to underestimation in severe inflammation. Assay-specific reference ranges and additional dilution steps are recommended, particularly when extreme elevations are suspected.