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J Heart Lung Transplant. 2017 Sep;36(9):1004-1012. doi: 10.1016/j.healun.2017.05.026. Epub 2017 May 20.

Applying rigor and reproducibility standards to assay donor-derived cell-free DNA as a non-invasive method for detection of acute rejection and graft injury after heart transplantation.

Author information

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.

KEYWORDS:

allograft rejection; automated; cell-free DNA; heart transplantation; reproducibility

PMID:
28624139
DOI:
10.1016/j.healun.2017.05.026
[Indexed for MEDLINE]

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