Comparison of methods for donor-derived cell-free DNA quantification in plasma and urine from solid organ transplant recipients

In allograft monitoring of solid organ transplant recipients, liquid biopsy has emerged as a novel minimally invasive approach using quantification of cell-free DNA in plasma originating from the allograft, so-called donor-derived cfDNA (dd-cfDNA). Despite early clinical implementation and analytical validation of techniques, direct head-to-head comparisons of dd-cfDNA quantification methods are lacking. Furthermore, data on dd-cfDNA in urine is scarce and so far, no high-throughput sequencing-based method has been adapted to perform absolute dd-cfDNA quantification. In this publication, we present a comprehensive comparison of different analytical methods for the absolute and relative quantification of dd-cfDNA in urine and plasma from kidney and liver transplant recipients. Given the potential benefit of simultaneous dd-cfDNA quantification both as relative and absolute amounts, we also present for the first time a high-throughput sequencing method, which enables direct absolute dd-cfDNA quantification as copies/ml. Moreover, we investigated the potential of urinary-derived dd-cfDNA as a truly non-invasive biomarker to monitor solid organ transplant recipients and the results highlight the need for absolute dd-cfDNA quantification for its utilization. With these insights, this work thus substantially contributes to a safe translation of this novel biomarker in the area of transplantation medicine.