Validation and method comparison for a point-of-care lateral flow assay measuring equine whole blood insulin concentrations

The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from 695 pmol/L (100 mu IU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139-278 pmol/L; 20-40 mu IU/mL), intermediate (278-417 pmol/L; 40-60 mu IU/mL), and high (> 417 pmol / L ; > 60 mu IU/mL) concentrations, respectively. For 10 replicates at each level (3 assay lots), inter-assay CVs were 15.9%, 11.0%, and 11.7%, respectively. In the weighted linear regression of 5 measured insulin concentrations against expected concentrations, R-2 = 0.98, slope = 1.02, and y-intercept = 14.4 pmol/L (2.08 mu IU/mL). The Spearman correlation coefficient (r(s)) was 0.90 (95% CI: 0.85-0.94) between the WRT and RIA; the WRT = f(RIA) Passing-Bablok regression yielded the fit, y = 1.005x + 24.3 pmol/L (3.50 mu IU/mL). The WRT result averaged 10.4% higher than the RIA result, with targeted bias of 25.9, 26.1, and 26.7 pmol/L (3.74, 3.76, and 3.84 mu IU/mL) for cutoffs used to diagnose insulin dysregulation of 312, 347, and 451 pmol/L (45, 50, and 65 mu IU/mL). Assay clinical sensitivities, specificities, and accuracies determined at the 3 selected clinical cutoffs and using the RIA as gold standard were 87-95%, 92-96%, and 91-95%, respectively (n = 99 samples). Observed total error was 28.4-30.4%. The WRT had acceptable precision, excellent linearity, and good association with the RIA.

Automated summary

This study evaluated the accuracy and reliability of the Wellness Ready Test (WRT) in measuring equine insulin and compared it with the radioimmunoassay (RIA). The results showed that the WRT had acceptable precision, excellent linearity, and a good association with the RIA.