Department of Paediatric Nephrology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK.
Paediatric Nephrology, University Hospital Southampton NHS Foundation Trust, Southampton, UK.
Clin Chem. 2020 Oct 1;66(10):1300-1309. doi: 10.1093/clinchem/hvaa173.
The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low fraction of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide polymorphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients.
We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was validated using genomic DNA (gDNA), cfDNA and donor samples where available.
The R "genotype-free" method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quantification 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients.
We have developed and validated a new assay to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients.
无细胞 DNA（cfDNA）作为一种非侵入性生物标志物来检测移植物损伤正在迅速普及。然而，量化供体来源的 cfDNA（ddcfDNA）的低分数具有挑战性，需要一种高度敏感的技术。通过独特的供体单核苷酸多态性（SNP）检测 ddcfDNA 是一种新的方法，但在儿科实体器官移植（SOT）受者中数据有限。
我们开发了一种使用 61 个 SNP 组合的检测方法，使用自定义 R 脚本准确地定量 ddcfDNA，该脚本需要对患者和供体基因型进行建模，只需从移植物受者中获得一个样本。使用基因组 DNA（gDNA）、cfDNA 和供体样本对检测方法的性能进行了验证。
R“无基因型”方法给出的结果与使用已知供体基因型时相当。适用于相关和不相关的配对，并且可以可靠地测量 ddcfDNA（空白限，低于 0.12%；检测限，高于 0.25%；定量限 0.5%，准确率为 84%）。对 159 名儿科 SOT 受者（肾、心和肺）进行了检测，无需供体基因分型。从 82 名患者中获得了连续采样。
我们开发并验证了一种新的检测方法来测量儿科 SOT 受者血浆中 ddcfDNA 的分数。我们的方法可以适用于任何供体-受者对，无需供体基因分型，并且可以在 48 小时内以低成本提供结果。需要进一步的前瞻性研究来证明其在大量儿科 SOT 受者中的临床有效性。
