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

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...

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Veröffentlicht in:The Journal of molecular diagnostics : JMD 2016-11, Vol.18 (6), p.890-902
Hauptverfasser: Grskovic, Marica, Hiller, David J, Eubank, Lane A, Sninsky, John J, Christopherson, Cindy, Collins, John P, Thompson, Kathryn, Song, Mindy, Wang, Yue S, Ross, David, Nelles, Mitchell J, Yee, James P, Wilber, Judith C, Crespo-Leiro, Maria G, Scott, Susan L, Woodward, Robert N
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container_issue 6
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container_title The Journal of molecular diagnostics : JMD
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creator Grskovic, Marica
Hiller, David J
Eubank, Lane A
Sninsky, John J
Christopherson, Cindy
Collins, John P
Thompson, Kathryn
Song, Mindy
Wang, Yue S
Ross, David
Nelles, Mitchell J
Yee, James P
Wilber, Judith C
Crespo-Leiro, Maria G
Scott, Susan L
Woodward, Robert N
description 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.
doi_str_mv 10.1016/j.jmoldx.2016.07.003
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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. 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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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>27727019</pmid><doi>10.1016/j.jmoldx.2016.07.003</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record>
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subjects Alleles
Cell Line
DNA - blood
DNA - genetics
Female
Gene Frequency
Genetic Markers
Genetic Testing - methods
Genetic Testing - standards
Genotype
Graft Rejection - diagnosis
Graft Rejection - genetics
Graft Rejection - immunology
High-Throughput Nucleotide Sequencing
Humans
Male
Organ Transplantation
Pathology
Polymorphism, Single Nucleotide
Reference Standards
Reproducibility of Results
Sensitivity and Specificity
Tissue Donors
Transplant Recipients
title Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients
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