Quantitative PCR of INDEL s to measure donor‐derived cell‐free DNA —a potential method to detect acute rejection in kidney transplantation: a pilot study

Abstract

Summary The quantification of donor‐derived cell‐free DNA (ddcfDNA) in recipient's plasma is a novel, but technically challenging noninvasive method to assist the diagnosis of acute rejection (AR). A quantitative real‐time PCR (qPCR) approach targeting insertion/deletion polymorphisms (INDEL) was adapted to measure ddcfNA in plasma samples from 29 kidney transplant recipients obtained at time of clinically indicated biopsies (eight patients with a histologically verified AR, nine with borderline rejection and 12 without evidence of rejection). Measured ddcfDNA levels of smaller INDEL amplicon targets differed significantly (P = 0.016, Kruskal–Wallis H test) between recipients with biopsy‐proven AR (median 5.24%; range 1.00–9.03), patients without (1.50%; 0.41–6.50) and patients with borderline AR (1.91%; 0.58–5.38). Similarly, pairwise testing by Mann–Whitney U‐tests revealed significant differences between recipients with AR and without AR (P = 0.012) as well as patients with AR and borderline histology (P = 0.015). Receiver operating characteristic (ROC) analysis revealed an area under the ROC curve for discriminating AR and non‐AR biopsies of 0.84 (95% CI: 0.66–1.00). The determined cutoff value of 2.7% ddcfDNA showed a sensitivity of 0.88 (95% CI: 0.63–1.00) and specificity of 0.81 (95% CI: 0.64–0.98). INDEL qPCR represents a novel method to quantify ddcfDNA on standard qPCR instruments within 6–8 h with high sensitivity and specificity to detect AR.

Donor‐derived cell‐free DNA as a biomarker for rejection after kidney transplantation: a systematic review and meta‐analysis

“…All studies were observational studies published during the last decade, including eight prospective cohort studies [6,8,9,12,19,22–24], one retrospective cross‐sectional study [7], four prospective cross‐sectional studies [10,13,18,25] and one study in which the data collection method was not described because of privacy or ethical restrictions, resulting in an unknown study design [20]. Nine studies used Cell‐Free DNA Blood Collection Tubes (BCT ® ) [6,8,10,12,13,19,22–24], two studies used EDTA tubes [9,25], and the three remaining studies did not specify the method of blood collection [7,18,20]. Single nucleotide polymorphism (SNP) based quantification techniques were used in most studies, either sequencing technology [6–8,12,13,18–20,22–24] or digital droplet PCR [9,10].…”

“…Single nucleotide polymorphism (SNP) based quantification techniques were used in most studies, either sequencing technology [6–8,12,13,18–20,22–24] or digital droplet PCR [9,10]. One study applied the quantitative real‐time PCR targeting insertion/deletion polymorphism (INDEL) technique [25].…”

Donor-Derived Cell-Free DNA in Kidney Transplantation as a Potential Rejection Biomarker: A Systematic Literature Review

“…Dauber et al [ 5 ] conducted a study in which they determined the dd-cfDNA levels in serum using quantitative RT-PCR, which analysed InDels in patients after KTx. In patients with AR, the dd-cfDNA level was 5.24% of the total cfDNA in the recipient’s plasma, while in the remaining patients it was 3.74% lower.…”

“…The indication for KTx is end-stage kidney disease, which diabetes, high blood pressure, glomerulonephritis, polycystic kidney disease, and other diseases can lead to [ 2 , 3 , 4 , 5 ].…”

“…There is no official Food and Drug Administration (FDA)-approved method of identifying the quantity of cell-free DNA (cfDNA). cfDNA studies have been performed in organs such as kidney, liver, heart and lung [ 5 ]. We believe that our review will inspire researchers to conduct further research.…”