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Abstract Background Calprotectin is a sensitive biomarker for inflammation and inflammatory response to infections. It can be measured in human plasma and serum samples using GCAL®, a turbidimetric particle-enhanced immunoassay (PETIA), which provides rapid and accurate results, facilitating timely clinical decision-making. The assay is available as an open-channel assay on multiple instrument platforms, including those from Roche, Siemens, Abbott, and Beckman Coulter.This study aimed to validate the Gentian GCAL® Calprotectin Immunoassay on the QuidelOrtho Vitros 5600 chemistry analyzer. Methods The GCAL® assay detects calprotectin levels by measuring the turbidity generated from antibody-antigen complex formation at 660 nm. Linearity was assessed according to the CLSI EP06 guideline using a dilution series of two lithium-heparin plasma samples with high and low calprotectin concentrations. Measuring range and antigen excess were evaluated following CLSI EP34. A high-concentration Li-hep plasma sample was tested to determine the security zone, ensuring either accurate measurements or appropriate reporting of values above the highest calibrator. For samples exceeding the measuring range, the system automatically triggers a 1:10 dilution to provide an accurate result. Precision was assessed according to CLSI EP05 in a three-day study, analyzing one serum sample, two Li-hep plasma samples, and two controls, each measured in five replicates, five times per day. Analytical sensitivity was tested using CLSI EP17, analyzing three serum samples over three days in ten replicates per day to determine the limit of quantification (LoQ), defined as the lowest analyte concentration meeting accuracy requirements (coefficient of variation (CV) and bias). Interference from haemoglobin, bilirubin, and intralipids was evaluated using CLSI EP07, where interferents were added to two serum samples with different calprotectin concentrations. Results The linearity was demonstrated in the measuring range from 0.41 mg/L to 19.85 mg/L, with no antigen excess observed up to 77.70 mg/L. Samples exceeding the highest calibrator concentration are automatically diluted 1:10 and reanalyzed. Precision: The total CV was 6.61% for samples with concentrations <1.00 mg/L and =3.23% for concentrations >1.00 mg/L. LoQ was established at 0.30 mg/L, with a CV of 18.2% and a bias of 0.03 mg/L. No clinically significant interference was observed for haemoglobin (2.5 g/L), bilirubin (0.6 g/L), or intralipids (10 g/L). Conclusion The Gentian Calprotectin Immunoassay, GCAL® is a robust turbidimetric assay which can be used for detection and assessment of inflammation in diverse inflammatory conditions. Validation on the Vitros 5600 analyzer confirmed its broad measuring range, linearity, and low imprecision, with no antigen excess or significant interference. The assay is compatible with high-throughput platforms from various manufacturers, offering fast, cost-effective, and reliable results for clinical applications.