2025 Report of the Sensitization in Transplantation: Assessment of Risk Working Group on donor-derived cell-free DNA: Establishing analytical validity as the basis for appropriate and effective clinical utilization

A working group under the Sensitization in Transplantation: Assessment of Risk initiative was established in 2023 to develop guidelines for analytical and clinical validity of lab-based testing for donor-derived cell-free DNA (dd-cfDNA). Measurement of dd-cfDNA as a minimally invasive marker of allograft injury has become more widely used over the last few years. To date, various technical and quantitation methods have hindered the standardization and interpretation of the results, leading to variability in understanding how to best utilize cell-free DNA in transplantation. Kits are being formulated for local laboratory testing, but we lack an organized framework for laboratory quality assurance. Further, threshold values and methods of measurement have changed over time, indicating that assay sensitivity and clinical relevance are still being refined. Harmonization and reproducibility will be critical as the field moves forward to local laboratory-based testing. The goal of this work group was to review and analyze technical and biological variables and clinical settings that could contribute to disparities in results, which will ultimately influence clinical validity and utility. High-quality, standardized decentralized dd-cfDNA testing is the essential prerequisite for conducting real-world evidence-generating multicenter studies to establish the appropriate context of use for this promising assay.