Ekkehard Schütz

Chapter 7 – Graft-derived cell-free DNA as a marker of graft integrity after transplantation

Genome transplant dynamics is a particularly promising new approach for the detection of graft injury based on the determination of graft-derived circulating cell-free DNA (cfDNA) in the blood of the recipient. An increase of donor DNA is an early indication of organ damage. A novel potential routine assay for graft-derived circulating cfDNA quantification has been developed using droplet digital polymerase chain reaction for the determination of the donor/recipient circulating cfDNA ratio. This method is very cost-effective and provides results on the same day. Monitoring graft-derived cfDNA has the advantage that it directly interrogates the health of the donor organ, and it allows early detection of transplant injury (“liquid biopsy”). The detection of subclinical rejection would be desirable to allow early intervention. Undiagnosed chronic damage can result in chronic rejection. The determination of graft-derived circulating cfDNA may complement or possibly replace other approaches for post-transplant monitoring, and it may improve the chances of long-term graft survival. This method will be helpful to individualize immunosuppressive regimens. Personalized immunosuppression will in the future shift emphasis from reaction to prevention, which could make immunosuppressive drugs safer and more effective and also reduce the cost of health care.

A Universal Droplet Digital PCR Approach for Monitoring of Graft Health After Transplantation Using a Preselected SNP Set

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.