Analytical Validation of a Single-nucleotide Polymorphism-based Donor-derived Cell-free DNA Assay for Detecting Rejection in Kidney Transplant Patients

Abstract

Background: Early detection of rejection in kidney transplant recipients holds the promise to improve clinical outcomes. Development and implementation of more accurate, noninvasive methods to detect allograft rejection remain an ongoing challenge. The limitations of existing allograft surveillance methods present an opportunity for donor-derived cell-free DNA (dd-cfDNA), which can accurately and rapidly differentiate patients with allograft rejection from patients with stable organ function.

Methods: This study evaluated the analytical performance of a massively multiplexed polymerase chain reaction assay that targets 13 962 single-nucleotide polymorphisms, characterized and validated using 66 unique samples with 1064 replicates, including cell line-derived reference samples, plasma-derived mixtures, and transplant patient samples. The dd-cfDNA fraction was quantified in both related and unrelated donor-recipient pairs.

Results: The dd-cfDNA assay showed a limit of blank of 0.11%, a limit of detection and limit of quantitation of 0.15% for unrelated donors, and limit of blank of 0.23%, a limit of detection and limit of quantitation of 0.29% for related donors. All other metrics (linearity, accuracy, and precision) were observed to be equivalent between unrelated and related donors. The measurement precision of coefficient of variation was 1.8% (repeatability, 0.6% dd-cfDNA) and was <5% for all the different reproducibility measures.

Conclusions: This study validates the performance of a single-nucleotide polymorphism-based massively multiplexed polymerase chain reaction assay to detect the dd-cfDNA fraction with improved precision over currently available tests, regardless of donor-recipient relationships.

FIGURE 1.

Workflow of a clinical grade next-generation sequencing assay. Donor-derived cfDNA is released from renal allograft into circulation; blood is drawn and centrifuged, and plasma is isolated. cfDNA is extracted from plasma samples and used for library preparation followed by targeted PCR amplification of 13 926 SNPs, performed using mmPCR. Amplicons are sequenced on a next-generation sequencer, and sequencing data are analyzed using a maximum likelihood estimate method to give a dd-cfDNA fraction, which is reported to the physician. cfDNA, cell-free DNA; dd-cfDNA, donor-derived cell-free DNA; mmPCR, massively multiplexed polymerase chain reaction; PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism.

FIGURE 2.

Histograms of measured dd-cfDNA for LoB analysis. A, Related method, lot 1. B, Unrelated method, lot 1. C, Related method, lot 2. D, Unrelated method, lot 2. dd-cfDNA, donor-derived cell-free DNA; LoB, limit of blank.

FIGURE 3.

Measured CV values (%) as a function of the corresponding empirical means (%) for LoQ analysis. A, Related samples. B, Unrelated samples. CV, coefficient of variation; LoQ, limit of quantitation.

FIGURE 4.

Measured dd-cfDNA as a function of the corresponding attempted spike levels, along with the calculated linear fit, for linearity analysis. A, Related only. B, Unrelated only. C, Related and unrelated cases together. dd-cfDNA, donor-derived cell-free DNA.

FIGURE 5.

Measured dd-cfDNA as a function of the corresponding ddPCR values, along with the calculated linear fit for accuracy analysis. A, Related only. (b) Unrelated only. (c) Related and unrelated cases together. dd-cfDNA, donor-derived cell-free DNA; ddPCR, digital droplet polymerase chain reaction.

FIGURE 6.

Measured dd-cfDNA from lot 2 as a function of the values from lot 1. A, On linear scale, along with the calculated linear fit. B, On log-log scale. dd-cfDNA, donor-derived cell-free DNA.