Time-Dependent Apparent Increase in dd-cfDNA Percentage in Clinically Stable Patients Between One and Five Years Following Kidney Transplantation

Abstract

Background Donor-derived cell-free DNA (dd-cfDNA) is reportedly a valuable tool for graft surveillance following kidney transplantation (KTx). Possible changes in dd-cfDNA(%) reference values over time have not been evaluated. For long-term monitoring after KTx, changes in host cfDNA might represent a biasing factor in dd-cfDNA(%) determinations. Methods Plasma samples were obtained (n = 929) 12–60 months after engraftment in a cross-sectional cohort of 303 clinically stable KTx recipients. Total cfDNA(copies/mL), dd-cfDNA(%), and dd-cfDNA(copies/mL) were determined using droplet-digital PCR. Stability of threshold values in these stable KTx recipients over time was assessed by 80th, 85th, and 90th quantile regression. Results Upper percentiles of total cfDNA showed a significant decline of −1902, −3589, and −4753 cp/mL/log(month) (P = 0.014, <0.001, and 0.017, respectively), resulting in increasing dd-cfDNA(%) percentiles by 0.25, 0.46, and 0.72%/log(month) (P = 0.04, 0.001, and 0.002, respectively), with doubling of the 85th percentile value by 5 years. In contrast, dd-cfDNA(cp/mL) was stable during the observation period (P = 0.52, 0.29, and 0.39). In parallel increasing white blood cell counts and decreasing tacrolimus concentrations over time were observed. After 5 years, the median total cfDNA was still 1.6-fold (P < 0.001) higher in KTx recipients than in healthy controls (n = 135) and 1.4-fold (P < 0.001) higher than patients with other medical conditions (n = 364). Conclusions The time-dependent decrease of host cfDNA resulted in an apparent increase of dd-cfDNA fraction in stable KTx patients. For long-term surveillance, measurement of absolute dd-cfDNA concentrations appears to be superior to percentages to minimize false positive results.

Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients

“…The cfDNA extraction yield from plasma of our group of KTx patients was similar to that previously reported for KTx patients (21) but lower than that reported for liver transplanted patients (28). We observed a higher cfDNA extraction yield from plasma of KTx patients compared to plasma cfDNA from apparently healthy individuals, which is in agreement with Schütz et al who also reported a higher total cfDNA in a group of KTx patients compared to a healthy control group or a group with other medical conditions (29).…”

Absolute or Relative Quantification of Donor-derived Cell-free DNA in Kidney Transplant Recipients: Case Series

“…One patient exhibited very low total cfDNA below the fifth percentile of the reference population, leading to absolute quantification of 22 copies/mL, which is below the cutoff, whereas relative quantification was 1.42% and above the cutoff. 13 For this patient, a sound explanation for low total cfDNA was possible, being low leukocyte count due to chronic myeloid leukaemia (CML) and nilotinib treatment. Despite the fact that no repeated biopsy was performed, the clinical course suggested no ongoing activity of antibody-mediated rejection (AMR) in this patient at the time of dd-cfDNA measurement.…”

“…We suspect this in 1 case, where CML and nilotinib treatment led to a low leukocyte count of 4.6 × 10 9 /L, resulting in total cfDNA levels below the fifth percentile of the reference population. 13 …”

“…Reference ranges for total cfDNA in the posttransplant course were assessed in a cohort of 300 KTR, as described previously. 13 …”

“… 12 CNI seem to have a negative effect on cell stability. 13 Consequently, relative dd-cfDNA values are higher in patients in the late posttransplant period, presumably because of a decrease in apoptosis rate for white blood cells as immunosuppressant drug doses are tapered off.…”