Analytical and clinical validation of a donor-derived cell-free DNA (dd-cfDNA) assay for renal allograft rejection

Background: Recently published studies have demonstrated analytical and clinical validation of a single nucleotide polymorphism (SNP)-based dd-cfDNA assay using multiplexed PCR (mmPCR) technology for detection of renal allograft rejection. Here, we review the favorable performance of this assay compared to the standard of care based on estimated glomerular filtration rates (eGFR) and serum creatinine levels.

Materials and Methods: Analytical validity was measured in 66 unique samples with 1064 replicates from cell line-derived reference samples, plasma-derived mixtures, and transplant patient samples. Clinical validity was performed using 217 biopsy-matched plasma samples. Both validations applied a SNP-based mmPCR assay targeting over 13,000 SNPs, without the need for prior knowledge of donor or recipient genotypes.

Results: Analytical validation of the cfDNA assay showed a limit of detection (LoD) of 0.15% for unrelated and 0.29% for related donors. Precision measurements showed a coefficient of variation of <2%. At a predefined cutoff, clinical validation demonstrated significantly higher median dd-cfDNA in samples with biopsy-proven active (2.3%) vs non-active (0.47%) rejection samples. The dd-cfDNA assay showed improved performance over eGFR and SCr, with high sensitivity (88.7% vs. 67.7% vs. 51.6%), specificity (72.6% vs. 65.3% vs. 67.5%) and AUC (0.87 vs. 0.74 vs. 0.68).

Conclusions: This dd-cfDNA assay has shown analytical and clinical performance superior to the current standard of care for renal allograft rejection monitoring.