Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients

Authors

  • André Hoerning,

    1. Department of Pediatrics II, Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Children’s Hospital, University of Duisburg-Essen, Essen
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  • Halime Kalkavan,

    1. Department of Pediatrics II, Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Children’s Hospital, University of Duisburg-Essen, Essen
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  • Christian Rehme,

    1. Department of Pediatrics II, Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Children’s Hospital, University of Duisburg-Essen, Essen
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  • Julia Menke,

    1. Department of Nephrology, University Hospital Mainz, Johannes Gutenberg University Mainz, Mainz
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  • Karl Worm,

    1. Institute of Pathology and Neuropathology, University Hospital Essen, University Duisburg-Essen, Essen
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  • Henk S. P. Garritsen,

    1. Institute for Clinical Transfusion Medicine, Staedtisches Klinikum Braunschweig, Braunschweig, Germany
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  • Rainer Büscher,

    1. Department of Pediatrics II, Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Children’s Hospital, University of Duisburg-Essen, Essen
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  • Peter F. Hoyer

    1. Department of Pediatrics II, Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Children’s Hospital, University of Duisburg-Essen, Essen
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  • This work was supported in part by a research grant of the University Duisburg-Essen Medical School to A.H. (IFORES).

André Hoerning, Department of Pediatrics II, Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Children’s Hospital, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
Tel.: +49 201 723 3350
Fax: +49 201 723 5394
E-mail: a.hoerning@gmx.de

Abstract

Hoerning A, Kalkavan H, Rehme C, Menke J, Worm K, Garritsen HSP, Büscher R, Hoyer PF. Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients.
Pediatr Transplantation 2011: 15: 809–818. © 2011 John Wiley & Sons A/S.

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.

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