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

Abstract

Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals. The assays are capable of accurate quantification of down to 0.1% minor allele content when analyzing 165 ng of human DNA. We found no significant differences in the yield of extracted cfDNA using the three different commercial kits tested. More cfDNA was extracted from the plasma of KTx patients than from healthy volunteers, especially early after transplantation. The median level of donor-derived minor alleles in KTx samples was 0.35%. We found that ddPCR using the evaluated assays within specific range is suitable for analysis of KTx patients' plasma but recommend prior genotyping of donor DNA and performing reliable preamplification of cfDNA.

Extracellular vesicle‐bound DNA in urine is indicative of kidney allograft injury

“…Monitoring kidney function using urinary evDNA is particularly useful in allogenic kidney transplants, as we can use the differences in genotype to specifically track the DNA released from the allograft (ddDNA) due to apoptosis, necrosis, or active release (Oellerich et al., 2021; Sigdel et al., 2013; Zhang et al., 1999). Here, we have successfully measured dd‐evDNA fraction and dd‐evDNA copy number in the urine of our kidney transplant patient cohort, using our recently developed ddPCR method for determining dd‐cfDNA in plasma (Jerič Kokelj et al., 2021). The ddDNA fraction of evDNA was highly variable, ranging from 2.7% to 99.7%, but no significant differences were detected among the three patient groups.…”

Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury

“…Thus, we can use the differences in genotype to specifically track the DNA released from the allograft (ddDNA) due to apoptosis, necrosis, or active release (Oellerich et al, 2021; Sigdel et al, 2013; Zhang et al, 1999). Here, we have successfully measured dd-evDNA fraction and dd-evDNA copy number in the urine of our kidney transplant patient cohort, using our recently developed ddPCR method for determining dd-cfDNA in plasma (Jerič Kokelj et al, 2021). Because a recent study reported a significant effect of variable water-load on the concentration of uEVs (Blijdorp et al, 2021), we adjusted the uEV and DNA values to urine creatinine to account for interpatient variability in urine concentration (Erdbrügger et al, 2021).…”

“…Custom TaqMan SNP Genotyping Assays (Thermo Fisher) used for genotyping of single nucleotide polymorphisms (SNPs; rs1707473, rs2691527, rs7687645, rs1420530, rs9289628, rs6070149) and quantification of ddDNA were described and validated previously on blood plasma (Jerič Kokelj et al, 2021). SNPs were chosen based on their potential to distinguish DNA of two individuals and are characterized with high minor allele frequency, low global fixation index, proximity to cfDNA fragment peaks and no overlap with repetitive elements.…”

White blood cell count affects fetal fraction and test failure rates in noninvasive prenatal screening

“…All blood samples were collected in EDTA tubes following pretest counseling. cfDNA was extracted from 200 μl of plasma using a QIAamp DSP DNA Blood Mini Kit (Qiagen) ( 14 ). Library construction and sequencing were performed using the BGISEQ-500 platform (MGI, China).…”