Transferring a Quantitative Molecular Diagnostic Test to Multiple Real-Time Quantitative PCR Platforms

Publication date: July 2018Source: The Journal of Molecular Diagnostics, Volume 20, Issue 4Author(s): Claudia Gürtler, Mark Laible, Wolfgang Schwabe, Heike Steinhäuser, Xingmin Li, Shujin Liu, Kornelia Schlombs, Ugur SahinQuantitative 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 real-time quantitative PCR assays across real-time quantitative PCR platforms without compromising analytical and clinical validity. The feasibility of our approach was tested on MammaTyper, an in vitro diagnostic assay that quantifies breast cancer biomarkers and dichotomizes results according to cutoff points. CFX96, Applied Biosystems 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 they were tested initially for equivalence via Bland-Altman and Deming regression analyses. A method comparison approach was adapted to adjust cutoffs for the new systems and the cross-platform agreement was evaluated. 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 tests successfully prequalified instruments, preventing constant and proporti...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research