Sean Agbor-Enoh1, Ilker Tunc2, Iwijn De Vlaminck3, Ulgen Fideli4, Andrew Davis4, Karen Cuttin4, Kenneth Bhatti4, Argit Marishta4, Michael A Solomon5, Annette Jackson6, Grace Graninger7, Bonnie Harper7, Helen Luikart8, Jennifer Wylie8, Xujing Wang2, Gerald Berry8, Charles Marboe9, Kiran Khush9, Jun Zhu2, Hannah Valantine10. 1. Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland; Pulmonary and Critical Care Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland; Laboratory of Transplantation Genomics, National Heart, Lung, and Blood Institute, Bethesda, Maryland. 2. Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland. 3. Department of Bioengineering, Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York. 4. Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland; Laboratory of Transplantation Genomics, National Heart, Lung, and Blood Institute, Bethesda, Maryland. 5. Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland; Clinical Center, National Institutes of Health, Bethesda, Maryland. 6. Pulmonary and Critical Care Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland; Laboratory of Transplantation Genomics, National Heart, Lung, and Blood Institute, Bethesda, Maryland. 7. Clinical Center, National Institutes of Health, Bethesda, Maryland. 8. Department of Medicine, Stanford University School of Medicine, Palo Alto, California. 9. Department of Medicine, New York Presbyterian University Hospital of Cornell and Columbia, New York, New York. 10. Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland; Laboratory of Transplantation Genomics, National Heart, Lung, and Blood Institute, Bethesda, Maryland. Electronic address: hannah.valantine@nih.gov.
Abstract
BACKGROUND: Use of new genomic techniques in clinical settings requires that such methods are rigorous and reproducible. Previous studies have shown that quantitation of donor-derived cell-free DNA (%ddcfDNA) by unbiased shotgun sequencing is a sensitive, non-invasive marker of acute rejection after heart transplantation. The primary goal of this study was to assess the reproducibility of %ddcfDNA measurements across technical replicates, manual vs automated platforms, and rejection phenotypes in distinct patient cohorts. METHODS: After developing and validating the %ddcfDNA assay, we subjected the method to a rigorous test of its reproducibility. We measured %ddcfDNA in technical replicates performed by 2 independent laboratories and verified the reproducibility of %ddcfDNA patterns of 2 rejection phenotypes: acute cellular rejection and antibody-mediated rejection in distinct patient cohorts. RESULTS: We observed strong concordance of technical-replicate %ddcfDNA measurements across 2 independent laboratories (slope = 1.02, R2 > 0.99, p < 10-6), as well as across manual and automated platforms (slope = 0.80, R2 = 0.92, p < 0.001). The %ddcfDNA measurements in distinct heart transplant cohorts had similar baselines and error rates. The %ddcfDNA temporal patterns associated with rejection phenotypes were similar in both patient cohorts; however, the quantity of ddcfDNA was significantly higher in samples with severe vs mild histologic rejection grade (2.73% vs 0.14%, respectively; p < 0.001). CONCLUSIONS: The %ddcfDNA assay is precise and reproducible across laboratories and in samples from 2 distinct types of heart transplant rejection. These findings pave the way for larger studies to assess the clinical utility of %ddcfDNA as a marker of acute rejection after heart transplantation.
BACKGROUND: Use of new genomic techniques in clinical settings requires that such methods are rigorous and reproducible. Previous studies have shown that quantitation of donor-derived cell-free DNA (%ddcfDNA) by unbiased shotgun sequencing is a sensitive, non-invasive marker of acute rejection after heart transplantation. The primary goal of this study was to assess the reproducibility of %ddcfDNA measurements across technical replicates, manual vs automated platforms, and rejection phenotypes in distinct patient cohorts. METHODS: After developing and validating the %ddcfDNA assay, we subjected the method to a rigorous test of its reproducibility. We measured %ddcfDNA in technical replicates performed by 2 independent laboratories and verified the reproducibility of %ddcfDNA patterns of 2 rejection phenotypes: acute cellular rejection and antibody-mediated rejection in distinct patient cohorts. RESULTS: We observed strong concordance of technical-replicate %ddcfDNA measurements across 2 independent laboratories (slope = 1.02, R2 > 0.99, p < 10-6), as well as across manual and automated platforms (slope = 0.80, R2 = 0.92, p < 0.001). The %ddcfDNA measurements in distinct heart transplant cohorts had similar baselines and error rates. The %ddcfDNA temporal patterns associated with rejection phenotypes were similar in both patient cohorts; however, the quantity of ddcfDNA was significantly higher in samples with severe vs mild histologic rejection grade (2.73% vs 0.14%, respectively; p < 0.001). CONCLUSIONS: The %ddcfDNA assay is precise and reproducible across laboratories and in samples from 2 distinct types of heart transplant rejection. These findings pave the way for larger studies to assess the clinical utility of %ddcfDNA as a marker of acute rejection after heart transplantation.
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