Analytical and biological validation of a multiplex immunoassay for acute kidney injury biomarkers

Background: Acute kidney injury (AKI) is a dynamic process that can involve inflammatory, hypoxic, and structural changes to the kidney. We evaluated a multiplex panel of markers representing different AKI mechanisms as a tool to provide integrated assessment of AKI status in a single assay. Methods: Urinary cystatin C (CysC), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and interleukin-18 (IL-18) were measured by multiplex electrochemiluminescence immunoassay. Analytical performance was compared to the biological and pathological variation of these markers in human samples. Results: Linearity was established over a 3- to 4-log range for all markers, which spanned the reference ranges established from healthy donors. Imprecision was below 15%, comparing favorably with the observed biological variation of these markers. Control patients fell within donor-derived reference ranges for most markers, but a subset of patients showed CysC and KIM-1 elevations in the absence of documented AKI. Conclusion: The multiplex assay is reliable for simultaneous quantitation of CysC, IL-18, KIM-1 and NGAL in human urine, and performs at levels sufficient for clinical application. The observed differences in biological variability and baseline levels suggest that clinical strategies to detect AKI will need to vary depending upon the specific markers used.