Assay-Dependent Variability in RBP4 and Adiponectin Quantification Affects Associations with Coronary Disease Severity and Doppler Echocardiographic Indices: A Comparative Study of Immunoturbidimetry and ELISA

Background: Differences among analytical platforms may contribute to inconsistent associations between circulating adipokines and coronary artery disease severity. This study compared the analytical robustness and clinical performance of the immunoturbidimetric assay (ITA) and enzyme-linked immunosorbent assay (ELISA) for measuring retinol-binding protein 4 (RBP4) and adiponectin (APN). Methods: A total of 128 patients with angiographically confirmed coronary artery disease were prospectively enrolled. Serum RBP4 and APN concentrations were measured in parallel using ITA and ELISA. Analytical performance was evaluated by precision, linearity, and recovery testing. In an additional cohort of 20 healthy volunteers, the effects of room-temperature storage, repeated freeze-thaw cycles, centrifugation conditions, and anticoagulant type were assessed. Coronary lesion severity was quantified using the Gensini score, and cardiac function was evaluated by Doppler echocardiography. Correlation, receiver operating characteristic, Bland-Altman, and multivariable regression analyses were performed. Results: Both assays demonstrated acceptable linearity and recovery, but ITA showed superior within-run and between-run precision. ELISA-derived measurements were more susceptible to prolonged room-temperature exposure, repeated freeze-thaw cycles, and lithium-heparin anticoagulation. RBP4 concentrations increased and APN concentrations decreased with increasing coronary lesion severity (all P<0.001). ITA-derived measurements showed stronger associations with Gensini score and selected echocardiographic indices than ELISA-derived measurements. ITA-derived RBP4 and APN demonstrated higher diagnostic discrimination for severe coronary artery disease, with areas under the curve of 0.872 and 0.851, respectively. Bland-Altman analysis showed that ELISA produced a positive systematic bias relative to ITA, with increasing intermethod variability at higher RBP4 concentrations. Conclusion: Assay-dependent analytical variability affects the magnitude of biomarker-disease associations and the classification of coronary disease severity. ELISA-derived RBP4 measurements exhibited a positive systematic bias relative to ITA; therefore, adipokine concentrations and proposed diagnostic thresholds should be interpreted with consideration of the analytical platform used. Assay harmonization and standardized preanalytical procedures are necessary before RBP4- and APN-based risk stratification can be applied reliably across laboratories.