Absolute Quantification of Donor-Derived Cell-Free DNA Following Pediatric and Adult Heart Transplantation.

Abstract

OBJECTIVE Traditional rejection surveillance after heart transplantation (HTx) is based on endomyocardial biopsies (EMB), which are invasive, expensive and associated with complications. Monitoring using cell-free DNA (cfDNA) is promising, but most studies report only on the donor fraction (DF) as the percentage of donor derived-cfDNA (dd-cfDNA) relative to total-cfDNA. We evaluated the performance of absolute as well as relative levels of dd-cfDNA to detect rejection. METHODS HTx patients were prospectively enrolled in a multicenter study, and blood samples collected concurrently with EMB. Dd-cfDNA was quantified using droplet digital PCR (ddPCR). Rejection was defined by EMB-results and compared to non-rejection EMB. Patients with symptomatic rejection were studied as a subgroup and test performance was determined using ROC-analysis. RESULTS We included 94 patients (70 adults and 24 children) undergoing rejection surveillance during the first year after HTx, which resulted in 1007 EMB and blood samples. In 19 patients, there were 32 rejection episodes > 14 days past HTx, with 15 of them being symptomatic. In ROC analysis, dd-cfDNA and DF could discriminate quiescence from rejection with an AUC of 0.68 and 0.65, respectively. Dd-cDNA at a threshold of 25 copies/ml showed an AUC of 0.87 to detect symptomatic rejection, significantly better than DF (AUC of 0.75). CONCLUSIONS dd-cfDNA found good discrimination between cardiac recipients with and without rejection. Absolute quantification of dd-cfDNA with ddPCR is a fast and effective method to monitor graft health. Analyzing absolute dd-cfDNA levels helps identify other factors, besides rejection, that may influence cfDNA levels, potentially reducing the need for EMB.