Analytical and Clinical Validation of a Molecular Diagnostic Signature in Kidney Transplant Recipients

Context: The TruGraf test is a blood-based assay that provides non-invasive, accurate assessment of adequacy of immunosuppression in kidney transplant recipients. TruGraf relies on gene-expression “signatures” that differentiate a state of Transplant eXcellence (TX, indicating adequately immunosuppressed) from not-TX. Objective: To evaluate the performance of the TruGraf test. Design: Analytical performance studies to characterize stability of RNA in blood during collection and shipment, analytical sensitivity (input RNA concentration), analytical specificity (interfering substances) and assay performance (clinical validity, and intra-assay, inter-assay, inter-laboratory reproducibility). Results: Total RNA extracted from whole blood specimens collected in PAXgene Blood RNA tubes was stable up to 3 days at room temperature (stable RNA yield). Under routine ambient shipping conditions, storage and shipping temperatures did not affect results. However, specimen shipments exposed to temperatures u003e400°C or to ambient temperatures for u003e3 days were unacceptable for processing. Analytical sensitivity studies demonstrated tolerance to variation in RNA input (50 to 400 ng per 3’ IVT (in vitro transcript] labeling reaction). Specificity studies using genomic DNA spiked into 3 ’IVT reactions at 10-20% demonstrated negligible assay interference. The test was reproducible across operators, runs, reagent lots, and laboratories. External validation demonstrated that the TruGraf blood test accurately classified patients in 72% of 295 samples. Conclusions: Analytical sensitivity, analytical specificity, robustness, quality control, and clinical validity of the TruGraf blood test were successfully verified, indicating its suitability for clinical use.