A Standardized Protocol for Donor-Derived Cell-Free DNA Quantification in the Diagnosis of Allograft Injury

Purpose There is an increasing interest in the use of donor-derived cfDNA (dd-cfDNA) in transplantation for non-invasive diagnosis of allograft injury. We hereby describe a non-commercial routine procedure for dd-cfDNA detection and quantification using digital droplet PCR and target-specific preamplification of a SNP-panel of 35 SNP-assays. The method is based on the difference in genotype between recipients and donors. After establishing our protocol, it has been used in a prospective study following recipients for 12 months after heart transplantation (HTx), and we here briefly describe our protocol. Methods A total of 728 samples from 67 HTx patients have been retrieved and analyzed. The recipients were genotyped preoperatively with respect to the SNP-panel. Upon transplantation, the donor genotype was interrogated with respect to the homozygous alleles found in the recipient. After HTx, blood samples were collected in cell free DNA collection tubes and delivered within the same day to the hospital laboratory. Plasma was separated by centrifugation at 2000g and 16000g, respectively. Isolated plasma was stored at -80C or used immediately for cfDNA extraction. Quality controls were performed by the 4200 Tape Station. The same primers were used for preamplification and downstream PCR. Non-template controls were included in all preamplification steps. The amount of dd-cfDNA was calculated as a fraction of the total cfDNA. Results The variation of extracted cfDNA yield was 10-130 ng/ml plasma. The 50-800 bp sized cfDNA constitutes 90% of the extracted samples, indicating a low background. An average of 2-5 informative SNPS assays for each transplant recipient were used for dd-cfDNA quantification with a detection rate down to 0.01% dd-cfDNA. Elevation of dd-cfDNA was seen not only in conjunction with cellular rejection but also during infection and in episodes of heart failure Conclusion Samples from HTx recipients demonstrated low levels of dd-cfDNA in consecutive samples during optimal treatment in non-eventful cases. Elevation of dd-cfDNA was seen not only during rejection but also in other conditions. This non-invasive method is relatively cheap, enables large sample yields by preamplification, has a fast turn-around time (48 hours) and is thus useful for clinical monitoring of HTx patients. However, it needs to be carefully related to clinical events.