*Purpose: Rejection is a major complication in solid organ transplant recipients and diagnosis often depends on periodic invasive biopsies. Thus there is a need for non-invasive and accurate tests to detect rejection. Elevated donor-derived cell-free DNA (dd cfDNA) in plasma has been shown to correlate with an increased incidence of organ rejection.

*Methods: Following analytical validation, 77 EDTA plasma biorepository samples from 25 kidney transplant recipients were assayed. Rejection status at sample collection was established by renal biopsy. Extraction was performed by the QIAamp Circulating Nucleic Acid extraction kit with analysis by the Qubit 1X dsDNA HS Assay Kit to determine dsDNA concentrations. Library preparation was performed by the NEBNext Ultra II kit with a final input of 0.5 ng/reaction. Pooled 4nM libraries were denatured, diluted to a final concentration of 1.2pM (with PhiX control library at 1% of final concentration), and sequenced by NextSeq 500/550 using mid-output flow cells and 2×75 cycle paired-end protocol. Sequence data were mapped to a human reference genome and, along with recipient genotype data generated using Illumina Omni2.5-8 beadchip arrays, were analyzed using a bioinformatics pipeline to calculate the percentage of dd cfDNA present.

*Results: Of the 77 samples tested, 55 were collected during a period free of acute rejection (AR); 3 samples were collected during borderline rejection and grouped with those characterized as free of AR. A total of 15 samples were collected during a period of biopsy-proven AR (humoral and/or cellular); 4 samples were collected during a period of BKV associated nephropathy and were grouped with those characterized as AR. Receiver operator characteristic (ROC) curve analysis was performed, demonstrating an AUC of 0.85 (95% CI 0.77 – 0.93, P < 0.001). Using the ROC results, a cutoff of >0.69% for positivity was selected. At this cutoff, the assay sensitivity, specificity, positive predictive value and negative predictive value were 57.9%, 84.5%, 55%, and 86%, respectively. Comparison of results for additional samples to an independent dd cfDNA assay (AlloSure, CareDx) demonstrated comparable performance (R2=0.97, slope=0.81, y-intercept=-0.15).

*Conclusions: The clinical performance of Viracor’s dd cfDNA was demonstrated to be equivalent to other recently published assays, suggesting that measurement of dd cfDNA has clinical utility as rejection biomarker.

« Back to 2020 American Transplant Congress