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

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.