Donor-derived Cell-free DNA Investigated by Digital PCR After Targeted Pre-Amplification: A Prospective Clinical Study of Heart-transplant Patients

Donor-derived cell-free DNA (dd-cfDNA) has gained emerging interest as a highly specific marker of rejection after heart transplantation (HTx). However, the method is not established, in part due to technical issues: Sequencing techniques come with high cost and time delay, while PCR-based methods struggle with a low yield of cfDNA-harvest. Hitherto there has been a focus on either relative (dd-cfDNA versus all cfDNA, including recipient-derived cfDNA [rd-cfDNA] + dd-cfDNA) or absolute measurements of dd-cfDNA alone. We propose a novel approach involving a targeted pre-amplification step before digital droplet PCR using probe-based SNP-assays is performed, allowing for the measurement of both relative and absolute values of dd-cfDNA at the same time. This approach is currently being evaluated in an ongoing, prospective single-center study of HTx recipients.