title: Probe-Free Digital PCR Quantitative Methodology to Measure Donor-Specific Cell-Free DNA after Solid-Organ Transplantation.
abstract: Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA can be costly and/or laborious. A probe-free droplet digital PCR (ddPCR) methodology using small deletion/insertion polymorphisms (DIPs) was developed to circumvent these limitations without compromising the quantification of dscfDNA. This method was called PHABRE-PCR (Primer toHybridize across anAllelicBREakpoint-PCR). The strategic placement of one primer to hybridize across an allelic breakpoint ensured highly specific PCR amplification, which then enabled the absolute quantification of donor-specific alleles by probe-free ddPCR.
  
  
  
  dscfDNA was serially measured in 3 liver transplant recipients. Donor and recipient genomic DNA was first genotyped against a panel of DIPs to identify donor-specific alleles. Alleles that differentiated donor-specific from recipient-specific DNA were then selected to quantify dscfDNA in the recipient plasma.
  
  
  
  Lack of amplification of nontargeted alleles confirmed that PHABRE-PCR was highly specific. In recipients who underwent transplantation, dscfDNA was increased at day 3, but decreased and plateaued at a low concentration by 2 weeks in the 2 recipients who did not develop any complications. In the third transplant recipient, a marked increase of dscfDNA coincided with an episode of graft rejection.
  
  
  
  PHABRE-PCR was able to quantify dscfDNA with high analytical specificity and sensitivity. The implementation of a DIP-based approach permits surveillance of dscfDNA as a potential measure of graft health after solid-organ transplantation.
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia.|Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia.
authors.forename: Su Kah
authors.initials: SK
authors.lastname: Goh
authors.affiliation: Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia.
authors.forename: Vijayaragavan
authors.initials: V
authors.lastname: Muralidharan
authors.affiliation: Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia.
authors.forename: Christopher
authors.initials: C
authors.lastname: Christophi
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia.|School of Cancer Medicine, La Trobe University, Bundoora, Victoria, Australia.|Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Hongdo
authors.initials: H
authors.lastname: Do
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia; alex.dobrovic@onjcri.org.au.|School of Cancer Medicine, La Trobe University, Bundoora, Victoria, Australia.|Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
mesh_terms: D004247:DNA / Q000235:genetics*; D006801:Humans; D016377:Organ Transplantation; D016133:Polymerase Chain Reaction; D014019:Tissue Donors
chemical_list: D004247:DNA