Calprotectin Testing: A Methods Comparison Study

Abstract Background In conventional laboratory-based Enzyme Linked Immunosorbant Assays (ELISA) for calprotectin measurement, raw stool is returned to and manually processed by testing laboratories. The OCS-Pledia calprotectin test uses the same stool collection device as the OCS-Pledia FIT test used in the UK Bowel cancer screening programme. In brief, individuals sample their stool with a picker and transfer to a preprepared buffered diluent, which is then returned to the laboratory for fully automated analysis. We have previously shown that a stool concentration of >100ug/g measured using the IDK calprotectin ELISA was the optimal cut-off to distinguish IBS from IBD (sensitivity 86%, specificity 90%, positive predictive value 39%, negative predictive value 99%). We undertook a methods comparison study to report the stability, linearity and potential bias of the OCS-Pledia calprotectin assay compared with IDK ELISA. Methods 32 stool samples were collected from people with GI pathology, and homogenised. Samples were prepared for calprotectin testing using IDK and OCS-Pledia less than 4 hours from excretion (mean2.6 [SD]1.3 hrs). Samples were left at room temperature and further extracts taken at 6, 24, 48, 72, and 168 hours. Pairwise analysis of samples across the reporting range was undertaken and linearity and bias assayed by Pearson’s correlation and Bland-Altmann analysis, respectively. Results Calprotectin was stable over one week in both the OCS-Pledia buffered and the IDK ELISA unbuffered stool samples. However, OCS-Pledia results showed lower variance than that of the IDK ELISA, especially at later time points. There was a linear relationship between calprotectin measured with the OCS-Pledia and IDK-ELISA assays (R2=0.85; p<0.0001). The OCS-Pledia calprotectin assay has a positive bias across the reporting range. This led to 40 samples reading elevated calprotectin levels on OCS-Pledia but within normal range on IDK-ELISA. All 40 had colonic inflammation and therefore were false negative IDK-ELISA. Calprotectin concentrations of 600ug/g and 400ug/g measured using the OCS-Pledia assay were equivalent to concentrations of about 250ug/g and 100ug/g measured using the IDK ELISA. Conclusion OCS- Pledia assays for the measurement of calprotectin are likely to superseded conventional ELISAs because they are cheaper, easier to automate and do not involve laboratory staff pre-processing stool samples. Like conventional ELISA assays calprotectin is stable for at least one week. Clinicians will need to be aware, however, of the positive bias across the reporting range of calprotectin and adjust their positivity thresholds.