title: Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients.
abstract: The presence of microchimerism in peripheral blood of solid organ transplant recipients has been postulated to be beneficial for allograft acceptance. Kinetics of donor cell trafficking and accumulation in pediatric allograft recipients are largely unknown. In this study, we implemented SNPs of the HVRs I and II of mitochondrial DNA to serve as molecular genetic markers to detect donor-specific cell chimerism after pediatric renal transplantation. Serial dilution of artificial chimeric DNA samples showed a linear correlation coefficient of R > 0.98 and a detection sensitivity of 0.01% with high reproducibility. Longitudinal semiquantitative analysis of donor-specific SNPs was then performed in peripheral blood mononuclear cells samples up to two yr post-transplant. Quantity of donor-specific cell chimerism in peripheral blood was highest in the early post-transplant period reaching values of ~10% after liver-kidney and 2.8% after renal transplantation. From one wk after transplantation, renal transplant patients exhibited an amount of donor-specific mtDNA ranging from 0.01% to 0.1%. We developed a highly accurate, sensitive, and rapid real-time quantitative PCR method using sequence-specific primers and fluorescent hydrolysis probes for the detection of at least 0.01% donor-specific cells in the recipient's peripheral blood after renal transplantation.
authors.affiliation: Department of Pediatrics II, Children's Hospital, University of Duisburg-Essen, Essen, Germany. a.hoerning@gmx.de
authors.forename: André
authors.initials: A
authors.lastname: Hoerning
authors.forename: Halime
authors.initials: H
authors.lastname: Kalkavan
authors.forename: Christian
authors.initials: C
authors.lastname: Rehme
authors.forename: Julia
authors.initials: J
authors.lastname: Menke
authors.forename: Karl
authors.initials: K
authors.lastname: Worm
authors.forename: Henk S P
authors.initials: HS
authors.lastname: Garritsen
authors.forename: Rainer
authors.initials: R
authors.lastname: Büscher
authors.forename: Peter F
authors.initials: PF
authors.lastname: Hoyer
mesh_terms: D004252:DNA Mutational Analysis; D004272:DNA, Mitochondrial / Q000235:genetics*; D003576:Electron Transport Complex IV / Q000235:genetics; D006801:Humans; D016030:Kidney Transplantation / Q000276:immunology*; D016031:Liver Transplantation / Q000276:immunology; D027381:Liver-Specific Organic Anion Transporter 1; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014019:Tissue Donors; D018183:Transplantation Chimera / Q000235:genetics*
chemical_list: D004272:DNA, Mitochondrial; D027381:Liver-Specific Organic Anion Transporter 1; D003576:Electron Transport Complex IV