rxCOV: A metric for assessing immunoassay analyte fidelity

Abstract Immunoassay based bioanalytical measurements are widely used in a variety of biomedical research and clinical settings. In these settings they are assumed to faithfully represent the experimental conditions being tested and the sample groups being compared. Although significant technical advances have been made in improving sensitivity and quality of the measurements, currently no metrics exist that objectively quantify the fidelity of the measured analytes with respect to noise associated with the specific assay. Here we introduce ratio of cross-coefficient-of-variation (rxCOV), a fidelity metric for objectively assessing immunoassay analyte measurement quality when comparing its differential expression between different sample groups or experimental conditions. We derive the metric from first principles and establish its feasibility and applicability using simulated and experimental data. We show that rxCOV assesses fidelity independent of statistical significance, and importantly, identifies when latter is meaningful. We also discuss its importance in the context of averaging experimental replicates for increasing signal to noise ratio. Finally, we demonstrate its application in a Lynch Syndrome case study. We conclude by discussing its applicability to multiplexed immunoassays, and to paired and unpaired data. We anticipate rxCOV to be adopted as a simple and easy-to-use metric to help assess robustness of immunoassay results in real-time.