Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients

Abstract

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

Biological Variation of Donor-Derived Cell-Free DNA in Renal Transplant Recipients: Clinical Implications

“…Robust statistics are preferred for nonnormal distributions to reduce the influence of outlier values. The low analytical variability [analytical CV (CV A ) = 6.8%] has been previously established by performing replicate measurements of reference materials mimicking transplant patients' cfDNA across a wide range of dd-cfDNA levels and replicated in patient samples (6). The index of individuality (II) was calculated as the CV I :CV G ratio.…”

“…We measured dd-cfDNA using AlloSure, a clinical-grade, targeted next-generation sequencing assay described in detail previously (6). Briefly, 266 SNPs were selected on the basis of high allele frequency, low error, and minimal linkage.…”

“…Target genomic regions containing these SNPs are amplified by PCR and sequenced to identify allele frequencies for each SNP. The allele frequencies are used to accurately quantify dd-cfDNA in transplant recipients without the need for separate genotyping of the recipient or the donor (6). The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs.…”

“…The ddcfDNA assay is precise across the linear quantifiable range (0.2%-16%) with a mean across-run analytical CV of 6.8%. Assay results of the clinical samples in this study were evaluated against established QC criteria described previously (6), and only passing results were used for the analysis. Samples that failed QC were repeated at the step where they failed or were repeated using plasma from the duplicate Streck Cell-Free DNA BCT tube collected at the same venipuncture as the first sample.…”

“…Observations of increased levels of ddcfDNA during acute rejection and correlation to severity of rejection have indicated the potential utility of dd-cfDNA as an early noninvasive indicator of allograft injury (2)(3)(4)(5)(6)(7)(8). Among the various strategies to measure dd-cfDNA, we have demonstrated the analytical validity of a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms (SNPs) to accurately quantify dd-cfDNA in the plasma of transplant recipients without the need for genotyping either the donor or the recipient (6).…”