Multi‐centre analytical performance verification of an IVD assay to quantify donor‐derived cell‐free DNA in solid organ transplant recipients

Abstract

Donor‐derived cell‐free DNA (dd‐cfDNA) has been widely studied as biomarker for non‐invasive allograft rejection monitoring. Earlier rejection detection enables more prompt diagnosis and intervention, ultimately improving patient treatment and outcomes. This multi‐centre study aims to verify analytical performance of a next‐generation sequencing‐based dd‐cfDNA assay at end‐user environments. Three independent laboratories received the same experimental design and 16 blinded samples to perform cfDNA extraction and the dd‐cfDNA assay workflow. dd‐cfDNA results were compared between sites and against manufacturer validation to evaluate concordance, reproducibility, repeatability and verify analytical performance. A total of 247 sample libraries were generated across 18 runs, with completion time of <24 h. A 96.0% first pass rate highlighted minimal failures. Overall observed versus expected dd‐cfDNA results demonstrated good concordance and a strong positive correlation with linear least squares regression r2 = 0.9989, and high repeatability and reproducibility within and between sites, respectively (p > 0.05). Manufacturer validation established limit of blank 0.18%, limit of detection 0.23% and limit of quantification 0.23%, and results from independent sites verified those limits. Parallel analyses illustrated no significant difference (p = 0.951) between dd‐cfDNA results with or without recipient genotype. The dd‐cfDNA assay evaluated here has been verified as a reliable method for efficient, reproducible dd‐cfDNA quantification in plasma from solid organ transplant recipients without requiring genotyping. Implementation of onsite dd‐cfDNA testing at clinical laboratories could facilitate earlier detection of allograft injury, bearing great potential for patient care.