Aim: Donor-derived cell free DNA (dd-cfDNA) has emerged as a promising biomarker for monitoring solid organ transplanted patients to discriminate between active and no rejection. To explore the analytical performance of Devysers new, next generation sequencing IVDR-certified Accept cfDNA kit based on 50 InDels we compared it to the established CareDx AlloSeq cfDNA kit based on 202 SNPs.
Methods: We assessed sonicated binary plasma mixes (BPM) to mimic cfDNA (~167 base pairs) at decreasing donor and cfDNA concentrations together with cfDNA samples from kidney allograft patients in the immediate post-transplant period. BPM samples were included to assess the 0.1% and 0.23% dd-cfDNA limit of detection (LOD) established by Devyser and CareDx, respectively and the 10 ng input concentration as required by both kits. Total cfDNA was assessed for purity by TapeStation analysis and concentration was confirmed by Qubit fluorometry. BPM samples were tested both at a constant 10 ng input cfDNA and six dilutions between 0.01% to 10% dd-cfDNA, and at a constant 1% dd-cfDNA and six dilutions between 0.5 ng to 50 ng input cfDNA.
Results: For both BPM and patient samples there was consensus between the percent dd-cfDNA detected in both kits (19% and 18%, respectively) and the BPM correlation between the two kits was R2>0.99. With 33 informative Devyser BPM markers identified the LOD was 0.05% dd-cfDNA and 1 ng input cfDNA, and interassay CV was 17% at 0.1% dd-cfDNA, the company-defined LOD. In our patient cohort (n=12) dd-cfDNA varied as expected in the immediate post-transplant period (Devyser 0.1-17.9%; CareDx 0.2-18.5%) which correlates with published data. Finally, hands-on time and total time from sample to result was comparable between the two assays and the user interface of the respective analysis software was straightforward for both options.
Conclusion: In conclusion, direct comparison of two NGS-based dd-cfDNA quantification methods demonstrated that the new Devyser kit has a slightly lower LOD and overall is a robust assay en par with the established CareDx assay for monitoring dd-cfDNA as an adjunct biomarker in organ transplanted patients.
