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

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


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.

Publication types

  • Comparative Study

MeSH terms

  • Acute Disease
  • Adult
  • Biomarkers / blood
  • Cell-Free Nucleic Acids / analysis*
  • Female
  • Graft Rejection / blood*
  • Heart Transplantation / adverse effects*
  • Heart Transplantation / methods
  • Humans
  • Linear Models
  • Male
  • Primary Graft Dysfunction / blood*
  • Primary Graft Dysfunction / physiopathology
  • Reproducibility of Results
  • Statistics, Nonparametric
  • Tissue Donors*


  • Biomarkers
  • Cell-Free Nucleic Acids