Abstract
Transplanted organs release cell-free DNA into the bloodstream of the recipient. This graft-derived cell-free DNA (GcfDNA) is a sensitive biomarker for organ health, since higher GcfDNA levels are indicative of increased cell-death in the graft. This protocol describes a method to measure relative GcfDNA concentrations by ddPCR assays. The method uses a set of preselected SNP assays from which the informative SNPs for each recipient–donor combination are selected in a straightforward two-step procedure that requires only one blood draw. Sampling of donor tissue and separate genotyping is not required, rendering the technique applicable also to patients, whose transplantation was not recent. In these patients there will be mostly no access to donor DNA anymore.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gielis EM, Ledeganck KJ, De Winter BY, Del Favero J, Bosmans JL, Claas FH, Abramowicz D, Eikmans M (2015) Cell-Free DNA: an upcoming biomarker in transplantation. Am J Transplant. https://doi.org/10.1111/ajt.13387
Zhang J, Tong KL, Li PK, Chan AY, Yeung CK, Pang CC, Wong TY, Lee KC, Lo YM (1999) Presence of donor- and recipient-derived DNA in cell-free urine samples of renal transplantation recipients: urinary DNA chimerism. Clin Chem 45(10):1741–1746
Gadi VK, Nelson JL, Boespflug ND, Guthrie KA, Kuhr CS (2006) Soluble donor DNA concentrations in recipient serum correlate with pancreas-kidney rejection. Clin Chem 52(3):379–382. https://doi.org/10.1373/clinchem.2005.058974
Lo YM (2011) Transplantation monitoring by plasma DNA sequencing. Clin Chem 57(7):941–942. https://doi.org/10.1373/clinchem.2011.166686
De Vlaminck I, Valantine HA, Snyder TM, Strehl C, Cohen G, Luikart H, Neff NF, Okamoto J, Bernstein D, Weisshaar D, Quake SR, Khush KK (2014) Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci Transl Med 6(241):241ra277. https://doi.org/10.1126/scitranslmed.3007803
Snyder TM, Khush KK, Valantine HA, Quake SR (2011) Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci U S A 108(15):6229–6234. https://doi.org/10.1073/pnas.1013924108
Beck J, Bierau S, Balzer S, Andag R, Kanzow P, Schmitz J, Gaedcke J, Moerer O, Slotta JE, Walson P, Kollmar O, Oellerich M, Schutz E (2013) 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 59(12):1732–1741. https://doi.org/10.1373/clinchem.2013.210328
Beck J, Oellerich M, Schulz U, Schauerte V, Reinhard L, Fuchs U, Knabbe C, Zittermann A, Olbricht C, Gummert JF, Shipkova M, Birschmann I, Wieland E, Schutz E (2015) Donor-derived cell-free DNA is a novel universal biomarker for allograft rejection in solid organ transplantation. Transplant Proc 47(8):2400–2403. https://doi.org/10.1016/j.transproceed.2015.08.035
Oellerich M, Schutz E, Kanzow P, Schmitz J, Beck J, Kollmar O, Streit F, Walson PD (2014) Use of graft-derived cell-free DNA as an organ integrity biomarker to reexamine effective tacrolimus trough concentrations after liver transplantation. Ther Drug Monit 36(2):136–140. https://doi.org/10.1097/FTD.0000000000000044
Kanzow P, Kollmar O, Schutz E, Oellerich M, Schmitz J, Beck J, Walson PD, Slotta JE (2014) Graft-derived cell-free DNA as an early organ integrity biomarker after transplantation of a marginal HELLP syndrome donor liver. Transplantation 98(5):e43–e45. https://doi.org/10.1097/TP.0000000000000303
Schütz E, Fischer A, Beck J, Harden M, Koch M, Wuensch T, Stockmann M, Nashan B, Kollmar O, Matthaei J, Kanzow P, Walson PD, Brockmöller J, Oellerich M, Singal A (2017) Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: a prospective, observational, multicenter cohort study. PLoS Med 14(4):e1002286
von Ahsen N, Wittwer CT, Schutz E (2001) Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg2+, deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. Clin Chem 47(11):1956–1961
Schütz E, von Ahsen N (2009) Influencing factors of dsDNA dye (high-resolution) melting curves and improved genotype call based on thermodynamic considerations. Anal Biochem 385(1):143–152
Acknowledgments
The authors thank Sarah Bierau and Stefan Balzer for their excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Beck, J., Oellerich, M., Schütz, E. (2018). A Universal Droplet Digital PCR Approach for Monitoring of Graft Health After Transplantation Using a Preselected SNP Set. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7778-9_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7776-5
Online ISBN: 978-1-4939-7778-9
eBook Packages: Springer Protocols