Digital Droplet PCR for Rapid Quantification of Donor DNA in the Circulation of Transplant Recipients as a Potential Universal Biomarker of Graft Injury

Abstract

BACKGROUND: Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR).

Cell-Free DNA: An Upcoming Biomarker in Transplantation

“…While amplification of Y-chromosome genes in the recipient's plasma and urine has been widely used in gender-mismatched transplantations (21,22,30,31,(34)(35)(36)(37)(38)(39), this strategy is limited to about 25% of the transplant population. A more universal approach is the use of informative genetic polymorphisms whereby a particular allele differs or is absent in the donor genome compared to the recipient genome; in addition to the quantification of donor-specific human leukocyte antigen deoxyribonucleic acid (HLA DNA) (40) and copy number deletion polymorphisms (41), single nucleotide polymorphisms (SNPs) can be targeted to determine the percentage of ddcfDNA by counting donor and recipient's bases at informative SNP sites with digital droplet polymerase chain reaction (ddPCR) (42)(43)(44) or massive parallel shotgun sequencing (MPSS) (34,45).…”

“…The high ddcfDNA levels immediately after transplantation may be caused by ischemia-reperfusion injury (40,42,43). If this is the case, higher levels of ddcfDNA are expected in recipients with grafts of deceased donors.…”

“…qPCR of a housekeeping gene such as beta-globin (22,37,39,40) Donor-derived plasma/serum cfDNA Directly interrogates graft integrity (42) Increased in case of graft damage (22,(37)(38)(39)(40) Quantification:…”

“…Low fraction of total cfDNA (0.06-3.5%) (34,42,45) qPCR or ddPCR of Y-chromosome genes (22,34,(37)(38)(39) (56) cfDNA isolation:…”

Therapeutic monitoring of immunosuppressive drugs in pediatric patients: special considerations

“…Another promising new approach is determination of graftderived circulating cell-free DNA (GefDNA) [13][14][15]. As solid organ transplants are also genome transplants, GefDNA could be determined in plasma and used as a marker of allograft injury [9].…”