Background: Donor derived cell-free DNA (dd-cfDNA) is a validated biomarker for allograft rejection in adult kidney transplant (KT) recipients. Recently, improved performance in detecting rejection using a two-threshold algorithm incorporating dd-cfDNA fraction (dd-cfDNA%) and estimated dd-cfDNA amount measured in cp/mL (dd-cfDNA score [ddCFS]) was reported. Elevated levels of either  dd-cfDNA% or ddCFS indicate increased risk for allograft rejection. We detail the experience of a single pediatric transplant center using the two-threshold algorithm for KT rejection surveillance.
Methods:From Nov 2020-May 2022, a cohort of 11 KT recipients, (mean age: 16 yrs, range: 11-19, 54.5% male), underwent serial dd-cfDNA testing performed monthly for the first year and quarterly thereafter for the duration of the study (mean post-transplantation monitoring: 17 months), with for-cause biopsies performed as needed. 
Results: A total of 66 dd-cfDNA tests were performed with 12 resulting in high risk findings (i.e. dd-cfDNA% and/or ddCFS elevated above 1% and 78 cp/ml, respectively). Ten of these high risk results (5 with elevated dd-cfDNA% and dd-CFS, and 5 with elevated dd-cfDNA% only) came from a single patient with biopsy proven rejection (TCMR/ABMR) who experienced prolonged elevation of dd-cfDNA levels after initiation of treatment for rejection. A stable patient, 18 days post-transplant, had elevated dd-cfDNA% (1.84%) but not ddCFS (46 cp/mL). dd-cfDNA% fell below the threshold (0.47%) at the next surveillance test, while ddCFS remained low. One patient had elevated ddCFS (78 cp/mL) but not dd-cfDNA% (0.93%). This patient required subsequent admission for management of E.Coli Pyelonephritis and C. Diff colitis. Biopsy found no rejection in other patients.
Conclusions: In this cohort, the two-threshold algorithm identified rejection following KT. Interestingly one patient who would have been called as low-risk with dd-cfDNA% alone was called as high risk by ddCFS. This patient did not have rejection but did have a clinically significant transplant pyelonephritis. Our observation of a clinically stable patient with elevated dd-cfDNA% and normal ddCFS should be studied in larger cohorts investigating clinical utility of dd-cfDNA testing in pediatric populations.