High Concordance between ddPCR- and NGS-Based Quantification of Donor-Derived Cell-Free DNA Percentage after Kidney Transplantation

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) enables early detection and monitoring of graft injury after transplantation. However, clinical adoption has been slowed by pricing and limited reimbursement. Commercially available assays based on different methodologies are used in practice, but their comparability remains uncertain. Demonstrating interchangeability may support more cost-effective use in transplant surveillance. METHODS: This study compared 2 dd-cfDNA assays in 254 samples: 134 samples from 97 patients undergoing indication biopsy and repeated dd-cfDNA testing, and 120 samples from 44 clinically stable patients. Both assays quantify dd-cfDNA via predefined single nucleotide polymorphisms (SNPs): AlloSeq (CareDx) detects 202 SNPs using next-generation sequencing, while GraftAssure (Insight Molecular Diagnostics) analyzes 45 SNPs using droplet digital PCR. RESULTS: dd-cfDNA values demonstrated excellent agreement between AlloSeq and GraftAssure (Pearson's r = 0.96). At a clinically relevant threshold of 0.5%, assay concordance reached 99.2%. The highest dd-cfDNA levels were observed in antibody-mediated rejection (mean ± standard deviation: AlloSeq 1.96 ± 1.06%, GraftAssure 2.08 ± 1.07%; P = 0.99). Among patients with stable graft function, dd-cfDNA levels were similarly low and consistent (AlloSeq 0.18 ± 0.11%, GraftAssure 0.17 ± 0.13%). Biological variability analysis using GraftAssure in 18 stable patients, each with at least 3 values above the quantitative detection limit, revealed an intraindividual coefficient of variation of 9.7% and a reference change value of 40.9% at a median dd-cfDNA level of 0.17%. CONCLUSIONS: This study provides the first comparison of 2 established dd-cfDNA testing methods and demonstrates excellent diagnostic agreement. These findings support flexibility in assay selection based on availability and cost, supporting the integration of dd-cfDNA testing into routine practice. GERMAN CLINICAL TRIALS REGISTRY: DRKS00023604.