Analytical Validation of a Single-Nucleotide Polymorphism-Based Donor-Derived Cell-Free DNA Assay for Detecting Rejection in Kidney Transplant Patients.

Background. Early detection of rejection in kidney transplant recipients holds the promise to improve clinical outcomes. Development and implementation of more accurate, noninvasive methods to detect allograft rejection remain an ongoing challenge. The limitations of existing allograft surveillance methods present an opportunity for donor-derived cell-free DNA (dd-cfDNA), which can accurately and rapidly differentiate patients with allograft rejection from patients with stable organ function. Methods. This study evaluated the analytical performance of a massively multiplexed polymerase chain reaction assay that targets 13 962 single-nucleotide polymorphisms, characterized and validated using 66 unique samples with 1064 replicates, including cell line-derived reference samples, plasma-derived mixtures, and transplant patient samples. The dd-cfDNA fraction was quantified in both related and unrelated donor-recipient pairs. Results. The dd-cfDNA assay showed a limit of blank of 0.11%, a limit of detection and limit of quantitation of 0.15% for unrelated donors, and limit of blank of 0.23%, a limit of detection and limit of quantitation of 0.29% for related donors. All other metrics (linearity, accuracy, and precision) were observed to be equivalent between unrelated and related donors. The measurement precision of coefficient of variation was 1.8% (repeatability, 0.6% dd-cfDNA) and was <5% for all the different reproducibility measures. Conclusions. This study validates the performance of a single-nucleotide polymorphism-based massively multiplexed polymerase chain reaction assay to detect the dd-cfDNA fraction with improved precision over currently available tests, regardless of donor-recipient relationships.