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 (EMBs), 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 dd-cfDNA to detect rejection.

Methods: HTx patients were prospectively enrolled in a multicenter study, and blood samples were collected concurrently with EMB. Dd-cfDNA was quantified using droplet digital PCR (ddPCR). Rejection was defined by EMB results and compared to nonrejection EMB. Patients with symptomatic rejection were studied as a subgroup, and test performance was determined using receiver operation characteristic analysis.

Results: We included 94 patients (70 adults and 24 children), 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 receiver operation characteristic analysis, dd-cfDNA and DF could discriminate quiescence from rejection with an area under the curve (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.

Keywords: absolute quantification; cell-free DNA; droplet digital PCR; heart transplantation; pediatric; rejection.