How to transfer a quantitative molecular diagnostic test to multiple qPCR platforms

Abstract Quantitative gene expression assays are increasingly used for diagnosis and research, but are often restricted to specific instrumentation. We propose a robust technical and statistical framework that enables transferring of established RT-qPCR assays across qPCR platforms without compromising analytical and clinical validity. The feasibility of our approach on MammaTyper®, an in vitro diagnostic assay which quantifies breast cancer biomarkersand dichotomizes results according to cut-offpoints, was tested. CFX96, ABI 7500 Fast, and Mx3000P were chosen as the candidate platforms whereas the LightCycler 480 II was used as a reference. Two instruments were used per platform, and tested initially for equivalence via Bland-Altman and Deming regressionanalyses. A method comparison approach was adapted to adjust cut-offsfor the new systems, and evaluated the cross-platformagreement. Finally precision was estimated for each platform. The performance on the candidate devices was highly comparable to the reference platform with a 7 log quantification range and amplification efficiencies of 97% to 103%. The equivalence testssuccessfully pre-qualified instruments, preventing constant and proportional errors, and enabling reliable adjustments of cut-offs, which resulted in cross-platformmarker and subtype agreements of 91% to 100% and Kappa values between 0.78 and 1.00. Provided that platform-specific adjustments are implemented, the described process can help expand the operability of quantitative diagnostic tests while maintaining assay performance characteristics.