Clinical Case Report: 
Introduction: Donor derived cell-free DNA (dd-cfDNA) is a validated biomarker for allograft rejection. Donor fraction (dd-cfDNA%) has been routinely used to measure dd-cfDNA levels in plasma. However, evidence suggests that the estimated dd-cfDNA amount, measured as copies/ml (i.e., dd-cfDNA score [ddCFS]) may enhance interpretation of assay results, as total cell-free DNA (t-cfDNA) can confound the calculation of dd-cfDNA%. This case study explores the observed levels of dd-cfDNA% and ddCFS in a teenaged pediatric kidney transplant (KT) patient with a postoperative course complicated by delayed graft function, non-compliance, rejection, and multiple bacterial infections.
Case Description: A 19-year-old female KT recipient with a history of pauci-immune glomerulonephritis had 16 dd-cfDNA tests (the ProsperaTM test) performed over a 1.5 year period to monitor for rejection risk. dd-cfDNA test results were elevated above the rejection threshold (i.e., 1% and 78 cp/mL, respectively) on day 206 post-transplant (dd-cfDNA%=22.6%, ddCFS=1344 cp/mL), consistent with mixed rejection (TCMR/ABMR) observed from a contemporaneous biopsy. Following rituximab administration on day 269, dd-cfDNA% (2.5%) and ddCFS (245 cp/mL) decreased but remained above the threshold for rejection. This was accompanied by elevated t-cfDNA (9636 cp/mL). Between day 296-387, dd-cfDNA levels were below the rejection threshold. At day 443, dd-cfDNA% increased above the threshold (1.2%). Interestingly, this increase was accompanied by a reduction in t-cfDNA (1680 cp/mL). ddCFS remained low (21 cp/mL). A similar pattern of results for these measurements was seen over the next 4 surveillance tests. 
Discussion: The observed trends suggest that use of dd-cfDNA testing as a surveillance tool may provide insight into rejection events in KT patients. Our observation of prolonged elevation of dd-cfDNA levels after initiation of treatment for rejection may be explained by sustained injury from rejection or from treatment for ABMR. The increase in dd-cfDNA% after treatment was likely driven by a decrease in t-cfDNA leading to an inflated dd-cfDNA fraction estimate. The clinical significance of discordant dd-cfDNA measurements and the influence of t-cfDNA on these discrepancies requires further exploration.