Circulating calprotectin in rheumatoid arthritis: unravelling the impact of (pre-)analytical confounders on meta-analysis results

Background: Circulating calprotectin (cCLP) is a promising diagnostic and prognostic biomarker in neutrophil-related inflammation, with most evidence in rheumatoid arthritis (RA). In the current study we aimed to investigate the impact of pre-analytical and analytical confounders on the inter-study heterogeneity of recent publications (01/2000–02/2023). Methods: A systematic literature search was performed in the electronic databases Medline, Embase and Cochrane databases (March 23st, 2023). After removal of duplicates, article eligibility was assessed using following inclusion criteria: (I) English (II) full-texts, describing (III) unique study results obtained from (IV) clinically defined RA patients and healthy control (HC) cohort, based on (V) clearly specified sample types (serum/plasma) and (VI) analytical method, and (VII) providing quantitative cCLP data. MEDCALC version 22.021 (MedCalc Software Ltd., Belgium) was applied for meta-analysis, including the Egger regression test to evaluate the publication bias. Results: Our literature search resulted in a total of 646 titles: 44 titles were retrieved for full-text screening and 18 studies including 21 cCLP datasets were eligible for inclusion in the meta-analysis. Totally, cCLP was measured in 2,627 RA and 897 HC and cCLP levels were significantly higher in RA patients [estimated standard mean difference (SMD) =1.032; 95% confidence interval (CI): 0.767–1.297]. Significant (P<0.0001) heterogeneity was observed between the different studies (Cochran’s Q =171.04; I2 statistic =88.31%; 95% CI: 83.52–91.71%). cCLP analyses was tested using 14 different cCLP assays. Only in 9 out of the 18 included studies, adherence to pre-analytical conditions was specified. Subgroup analysis of the datasets (n=12) of these 9 studies, still showed significant heterogeneity (Cochran’s Q =96.107; I2 statistic =88.55%; 95% CI: 81.91–92.76%). Excluding the Bühlmann (n=4) datasets, the datasets compliant to pre-analytical recommendations (n=9), using eight different cCLP assays on samples of 856 RA patients and 425 HC, showed absence of heterogeneity (Q =4.84; I2 statistic =0.00; 95% CI: 0.00–42.28), whilst in the other subgroup (n=8) significant inter-study heterogeneity persisted (Q =66.03; I2 statistic =89.40%; 95% CI: 81.48–93.93%). cCLP concentrations remained significantly higher in RA patients compared to HC (estimated SMD =0.698; 95% CI: 0.551–0.844). Conclusions: The inter-study variability of cCLP in RA is, besides analytical issues, mainly caused by pre-analytical confounders. Adherence to pre-analytical recommendations, significantly reduced inter-study heterogeneity and provided a reliable SMD, accurately revealing higher cCLP levels in RA versus HC.