Digital PCR and copy number variants enable absolute and relative quantification of donor-derived cell-free DNA for post-transplant organ monitoring
About the session
Allograft cells undergoing apoptosis or necrosis release cell-free DNA that is genetically different from cfDNA released from recipient cells. We developed digital PCR-based assays to discriminate donor and recipient cfDNA for absolute quantification without sequencing, and to evaluate analytical performance.
45 CNV assays were developed with median zero copy allelic frequency 0.52 (IQR 0.43–0.59), one copy allelic frequency 0.41 (0.36–0.43), and two copy allelic frequency 0.08 (0.05–0.13). The assays performed linearly across the range <6–1280 copies/mL. LOB was 0 copies/mL, LOD was 6 copies/mL, and LOQ was 8 copies/mL.
This panel permits quantification of donor and recipient targets in cfDNA post-transplant. The CNV allelic frequencies maximise informativity and permit quantification against a negative background, unlike SNP-based approaches. In contrast to sequencing, dPCR permits rapid, direct quantification of the absolute concentration (copies/mL) of donor-derived cfDNA and total cell-free DNA, from which the relative measure of donor fraction (%) can be calculated.