The Evolution and Innovation of Donor-Derived Cell-Free DNA Testing in Transplantation

Background: Improvements to the donor-derived cell-free DNA measurement were implemented in the AlloSure assay to achieve faster turnaround time, streamlined laboratory workflow, and a lower limit of detection. Analytical validation established the performance characteristics. Clinical samples were used to demonstrate equivalence to the original assay. Materials and Methods: AlloSure 3.0 was implemented with a single multiplex library preparation and single read sequencing to streamline the workflow and decrease processing time. The analytical performance of the assay was characterized according to CLSI methods. Results: AlloSure 3.0 performance is improved relative to the performance of the original AlloSure assay. The reportable range is from 0.12% to 16% donor-derived cell-free DNA (dd-cfDNA). The precision has also improved, with run-to-run coefficient of variation of 2.7%. Following these improvements to the assay, we established 99% concordance to the original version of AlloSure for clinical samples. Conclusion: Ongoing improvements to the donor-derived cell-free DNA methods resulted in a larger reportable range, decreased turnaround time, and improved precision, while maintaining the same accuracy as demonstrated with the initial release of AlloSure. The improved performance enables greater range for evaluation of dynamic changes that have been demonstrated to inform on low-grade T cell mediated rejectionl.