A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time

M Rohan Fernando; Chao Jiang; Gary D Krzyzanowski; Tifany Somer-Shely; Wayne L Ryan

doi:10.1371/journal.pone.0208508

A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time

PLoS One. 2018 Dec 6;13(12):e0208508. doi: 10.1371/journal.pone.0208508. eCollection 2018.

Authors

M Rohan Fernando^{1

2}, Chao Jiang¹, Gary D Krzyzanowski^{1

2}, Tifany Somer-Shely³, Wayne L Ryan²

Affiliations

¹ Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha NE, United States of America.
² Department of Research and Development, CFGenome Omaha NE, United States of America.
³ Methodist Physicians Clinic Women's Center, Elkhorn, NE, United States of America.

Abstract

This study was undertaken to evaluate a novel method for stabilizing and preserving the original proportion of cell-free fetal DNA (cffDNA) in maternal blood for extended periods of time without using crosslinking agents, such as formaldehyde, which compromise DNA integrity and extraction efficiency. Blood was drawn from pregnant donors into K3EDTA and Blood Exo DNA ProTeck® (ProTeck) tubes. Blood drawn into both tubes were aliquoted and stored at three different temperatures. At indicated times sample aliquots were processed for cell-free DNA (cfDNA) extraction. Plasma cfDNA and cffDNA quantified by droplet digital PCR (ddPCR) assay which amplify RASSF1A gene promoter region. ProTeck reagent is formaldehyde free and inhibits blood cell metabolism in blood samples during storage. Cell-free DNA concentration increased over time in blood plasma stored in K3EDTA tubes at 4, 22 and 30°C. Blood stored in ProTeck tubes, cfDNA concentration was stable at 4, 22 and 30°C for 21, 28 and 7 days, respectively. In K3EDTA tubes cffDNA proportion decreases steadily over time whereas in ProTeck tubes cffDNA proportion remained stable. This novel technology stabilizes cffDNA proportion in maternal blood samples at 4, 22 and 30°C for 21, 28 and 7 days, respectively.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blood Specimen Collection / instrumentation*
Blood Specimen Collection / methods
Cell-Free Nucleic Acids / blood*
Cell-Free Nucleic Acids / chemistry*
Female
Humans
Molecular Diagnostic Techniques
Polymerase Chain Reaction
Pregnancy
Pregnancy Trimester, First / blood
Pregnancy Trimester, First / genetics*
Promoter Regions, Genetic
Tumor Suppressor Proteins / genetics

Substances

Cell-Free Nucleic Acids
RASSF1 protein, human
Tumor Suppressor Proteins

Grants and funding

Funding for this research was provided by University of Nebraska Foundation, Obstetrics and Gynecology Research Fund # 1712.