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.

Speakers

Doug Bost

JETA Molecular BV

Mr. Doug Bost is a Co-Founder and CEO of JETA Molecular, as well as a Board Member and Vice-President of Transplant Monitoring at Omixon Biocomputing in Budapest, Hungary. JETA is diagnostics company focused on commercialization of non-invasive reagent and software solutions intended for monitoring recipients of allogeneic stem cell transplants. Prior to founding JETA, Mr. Bost was Director of Human Genetic and Transplantation Diagnostics R&D at Celera/Quest Diagnostics Mr. Bost received his B.A. in Microbiology and Immunology from the University of California, Berkeley, and is also the primary inventor of multiple issued patents related to kits and methods of selective nucleic acid isolation.