A Novel High-throughput Droplet Digital PCR-based Indel Quantification Method for the Detection of Circulating Donor-derived Cell-free DNA After Kidney Transplantation

Abstract

Background. Donor-derived cell-free DNA (ddcfDNA) is a promising minimally invasive biomarker for acute rejection (AR) in kidney transplant recipients. To assess the diagnostic value of ddcfDNA as a marker for AR, ddcfDNA was quantified at multiple time points after kidney transplantation with a novel high-throughput droplet digital PCR indel method that allowed for the absolute quantification of ddcfDNA. Methods. In this study, ddcfDNA in plasma samples from 223 consecutive kidney transplant recipients was analyzed pretransplantation; at 3, 7, and 180 d after transplantation; and at time of for-cause biopsies obtained within the first 180 d after transplantation. Results. Median (interquartile range) ddcfDNA concentration was significantly higher on day 3 (58.3 [17.7-258.3] copies/mL) and day 7 (25.0 [10.4-70.8] copies/mL) than on day 180 after transplantation (4.2 [0.0-8.3] copies/mL; P < 0.001 and P < 0.001, respectively). At time of biopsy-proven AR (BPAR), between day 11 and day 180 after transplantation, ddcfDNA concentration was significantly higher (50.0 [25.0-108.3] copies/mL) than those when biopsies showed non-AR (0.0 [0.0-15.6] copies/mL; P < 0.05). ddcfDNA concentration within the first 10 d after transplantation showed no significant difference between recipients with BPAR and those with non-AR in their biopsy or between recipients with BPAR and ddcfDNA measured at day 3 and day 7. Conclusions. Unfortunately, ddcfDNA concentration is not a good biomarker to detect AR within the first 10 d after transplantation; however, BPAR occurring after 10 d after transplantation can be detected in kidney transplant recipients by ddcfDNA using a novel and unique high-throughput droplet digital PCR indel method.

Direct detection of circulating donor-derived extracellular vesicles in kidney transplant recipients

“…At any rate, the detection of dd-EVs can be associated with stable allograft function – which is in contrast to the detection of e.g. donor-derived cell-free DNA, which has been observed to increase in concentration as a consequence of allograft damage 40 .…”

Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury: The Impact of the Timing of the Liquid Biopsy

“…These fluctuations in recipient cfDNA occur both during physiological conditions ( 9 , 10 ), as well as pathological conditions, including infection and cancer ( 11 , 12 ), that occur frequently in heart transplant recipients ( 13 ). For this reason, using ddcfDNA concentration might be more accurate to avoid the variability of ddcfDNA% ( 14 ). Additionally, the EMB procedure might not only affect the level of donor cfDNA but also of the recipient cfDNA.…”

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients

“…2018-035). 13 PBMCs were thawed using RPMI medium with DNase (DNase I, Roche, Germany) and stained with anti-CD3-BV510, anti-CD19-PE-Cy7, anti-CD16-PE, anti-CD56-PerCP, anti-CD45-APC, anti-CD15-BV421, and anti-CD14-FITC. All antibodies were from Biolegend (San Diego, CA) except anti-CD14 (BD Bioscience, San Jose, CA).…”