A fast and simple method for detecting and quantifying donor-derived cell-free DNA in sera of solid organ transplant recipients as a biomarker for graft function

Martina Adamek 1 , Gerhard Opelz 1 , Katrin Klein 2 , Christian Morath 2 , and Thuong Hien Tran 1
  • 1 Institute of Immunology, Transplantation Immunology, University of Heidelberg, Heidelberg, Germany
  • 2 Department of Nephrology, University Hospital Heidelberg, Heidelberg, Germany
Martina Adamek, Gerhard Opelz, Katrin Klein, Christian Morath and Thuong Hien Tran

Abstract

Background: Timely detection of graft rejection is an important issue in the follow-up care after solid organ transplantation. Until now, biopsy has been considered the “gold standard” in the diagnosis of graft rejection. However, non-invasive tests such as monitoring the levels of cell-free DNA (cfDNA) as a sensitive biomarker for graft integrity have attracted increasing interest. The rationale of this approach is that a rejected organ will lead to a significant release of donor-derived cfDNA, which can be detected in the serum of the transplant recipient.

Methods: We have developed a novel quantitative real-time PCR (qPCR) approach for detecting an increase of donor-derived cfDNA in the recipient’s serum. Common insertion/deletion (InDel) genetic polymorphisms, which differ between donor and recipient, are targeted in our qPCR assay. In contrast to some other strategies, no specific donor/recipient constellations such as certain gender combinations or human leukocyte antigen (HLA) discrepancies are required for the application of our test.

Results: The method was first validated with serial dilutions of serum mixtures obtained from healthy blood donors and then used to determine donor-derived cfDNA levels in patients’ sera within the first 3 days after their kidney transplantation had been performed.

Conclusions: Our method represents a universally applicable, simple and cost-effective tool which can potentially be used to detect graft dysfunction in transplant recipients.

    • Supplementary material
  • 1.

    Pascual J, Perez-Saez MJ, Mir M, Crespo M. Chronic renal allograft injury: early detection, accurate diagnosis and management. Transplant Rev 2012;26:280–90.

    • Crossref
    • Export Citation
  • 2.

    Wagner J. Free DNA–new potential analyte in clinical laboratory diagnostics? Biochem Med 2012;22:24–38.

  • 3.

    Lo YM, Tein MS, Pang CC, Yeung CK, Tong KL, Hjelm NM. Presence of donor-specific DNA in plasma of kidney and liver-transplant recipients. Lancet 1998;351:1329–30.

    • Crossref
    • PubMed
    • Export Citation
  • 4.

    Araujo MB, Leonardi LS, Boin IF, Leonardi MI, Magna LA, Donadi EA, et al. Development of donor-specific microchimerism in liver transplant recipient with HLA-DRB1 and -DQB1 mismatch related to rejection episodes. Transplant P 2004;36:953–5.

    • Crossref
    • Export Citation
  • 5.

    Araujo MB, Leonardi LS, Boin IF, Leonardi MI, Meirelles L, Magna LA, et al. Molecular mechanisms associated with donor-specific microchimerism in peripheral blood of Brazilian patients after liver transplantation. Transplant P 2006;38:1411–7.

    • Crossref
    • Export Citation
  • 6.

    Crespo-Leiro MG, Hermida-Prieto M, Rodriguez JA, Muniz J, Barral S, Paniagua MJ, et al. Microchimerism after heart transplantation: prevalence, predisposing factors, natural history, and prognosis. Transplant P 2002;34:161–3.

    • Crossref
    • Export Citation
  • 7.

    Fu YW, Wang WG, Zhou HL, Cai L. Presence of donor-and-recipient-derived DNA microchimerism in the cell-free blood samples of renal transplantation recipients associates with the acceptance of transplanted kidneys. Asian J Androl 2006;8:477–82.

    • Crossref
    • PubMed
    • Export Citation
  • 8.

    Araujo MB, Leonardi LS, Leonardi MI, Boin IF, Magna LA, Donadi EA, et al. Prospective analysis between the therapy of immunosuppressive medication and allogeneic microchimerism after liver transplantation. Transpl Immunol 2009;20:195–8.

    • Crossref
    • PubMed
    • Export Citation
  • 9.

    Ayala R, Grande S, Albizua E, Crooke A, Meneu JC, Moreno A, et al. Long-term follow-up of donor chimerism and tolerance after human liver transplantation. Liver Transpl 2009;15:581–91.

    • Crossref
    • PubMed
    • Export Citation
  • 10.

    Gadi VK, Nelson JL, Boespflug ND, Guthrie KA, Kuhr CS. Soluble donor DNA concentrations in recipient serum correlate with pancreas-kidney rejection. Clin Chem 2006;52:379–82.

    • Crossref
    • PubMed
    • Export Citation
  • 11.

    Garcia Moreira V, Prieto Garcia B, Baltar Martin JM, Ortega Suarez F, Alvarez FV. Cell-free DNA as a noninvasive acute rejection marker in renal transplantation. Clin Chem 2009;55:1958–66.

    • Crossref
    • PubMed
    • Export Citation
  • 12.

    Lui YY, Woo KS, Wang AY, Yeung CK, Li PK, Chau E, et al. Origin of plasma cell-free DNA after solid organ transplantation. Clin Chem 2003;49:495–6.

    • Crossref
    • PubMed
    • Export Citation
  • 13.

    Macher HC, Suarez-Artacho G, Guerrero JM, Gomez-Bravo MA, Alvarez-Gomez S, Bernal-Bellido C, et al. Monitoring of transplanted liver health by quantification of organ-specific genomic marker in circulating DNA from receptor. PloS One 2014;9:e113987.

    • Crossref
    • PubMed
    • Export Citation
  • 14.

    Rutkowska J, Interewiczi B, Rydzewski A, Swietek M, Dominiak A, Durlik M, et al. Donor DNA is detected in recipient blood for years after kidney transplantation using sensitive forensic medicine methods. Ann Transplant 2007;12:12–4.

    • PubMed
    • Export Citation
  • 15.

    De Vlaminck I, Valantine HA, Snyder TM, Strehl C, Cohen G, Luikart H, et al. Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci Transl Med 2014;6:241ra77.

    • Crossref
    • PubMed
    • Export Citation
  • 16.

    Snyder TM, Khush KK, Valantine HA, Quake SR. Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci USA 2011;108:6229–34.

    • Crossref
    • Export Citation
  • 17.

    Beck J, Bierau S, Balzer S, Andag R, Kanzow P, Schmitz J, et al. Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury. Clin Chem 2013;59:1732–41.

    • Crossref
    • PubMed
    • Export Citation
  • 18.

    Kanzow P, Kollmar O, Schutz E, Oellerich M, Schmitz J, Beck J, et al. Graft-derived cell-free DNA as an early organ integrity biomarker after transplantation of a marginal HELLP syndrome donor liver. Transplantation 2014;98:e43–5.

    • Crossref
    • PubMed
    • Export Citation
  • 19.

    Oellerich M, Schutz E, Kanzow P, Schmitz J, Beck J, Kollmar O, et al. Use of graft-derived cell-free DNA as an organ integrity biomarker to reexamine effective tacrolimus trough concentrations after liver transplantation. Ther Drug Monit 2014;36:136–40.

    • Crossref
    • PubMed
    • Export Citation
  • 20.

    Hinds DA, Kloek AP, Jen M, Chen X, Frazer KA. Common deletions and SNPs are in linkage disequilibrium in the human genome. Nat Genet 2006;38:82–5.

    • Crossref
    • PubMed
    • Export Citation
  • 21.

    Sigdel TK, Vitalone MJ, Tran TQ, Dai H, Hsieh SC, Salvatierra O, et al. A rapid noninvasive assay for the detection of renal transplant injury. Transplantation 2013;96:97–101.

    • Crossref
    • PubMed
    • Export Citation
  • 22.

    Garcia Moreira V, de la Cera Martinez T, Gago Gonzalez E, Prieto Garcia B, Alvarez Menendez FV. Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR. Clin Chem Lab Med 2006;44:1410–5.

    • PubMed
    • Export Citation
  • 23.

    Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nuc Acids Res 1988;16:1215.

    • Crossref
    • Export Citation
  • 24.

    Zheng YW, Chan KC, Sun H, Jiang P, Su X, Chen EZ, et al. Nonhematopoietically derived DNA is shorter than hematopoietically derived DNA in plasma: a transplantation model. Clin Chem 2012;58:549–58.

    • Crossref
    • PubMed
    • Export Citation
  • 25.

    Lion T, Watzinger F, Preuner S, Kreyenberg H, Tilanus M, de Weger R, et al. The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation. Leukemia 2012;26:1821–8.

    • Crossref
    • PubMed
    • Export Citation
  • 26.

    Alizadeh M, Bernard M, Danic B, Dauriac C, Birebent B, Lapart C, et al. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002;99:4618–25.

    • Crossref
    • PubMed
    • Export Citation
  • 27.

    Willasch A, Schneider G, Reincke BS, Shayegi N, Kreyenberg H, Kuci S, et al. Sequence polymorphism systems for quantitative real-time polymerase chain reaction to characterize hematopoietic chimerism-high informativity and sensitivity as well as excellent reproducibility and precision of measurement. Lab Hematol 2007;13:73–84.

    • Crossref
    • PubMed
    • Export Citation
  • 28.

    Burns M, Valdivia H. Modelling the limit of detection in real-time quantitative PCR. Eur Food Res Technol 2008;226:1513–24.

    • Crossref
    • Export Citation
  • 29.

    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611–22.

    • Crossref
    • PubMed
    • Export Citation
  • 30.

    Bruno DL, Ganesamoorthy D, Thorne NP, Ling L, Bahlo M, Forrest S, et al. Use of copy number deletion polymorphisms to assess DNA chimerism. Clin Chem 2014;60:1105–14.

    • Crossref
    • PubMed
    • Export Citation
  • 31.

    Li Y, Hahn D, Zhong XY, Thomson PD, Holzgreve W, Hahn S. Detection of donor-specific DNA polymorphisms in the urine of renal transplant recipients. Clin Chem 2003;49:655–8.

    • Crossref
    • PubMed
    • Export Citation
Purchase article
Get instant unlimited access to the article.
$42.00
Log in
Already have access? Please log in.


or
Log in with your institution

Journal + Issues

Search