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,Noam Shomron

doi:10.1371/journal.pone.0208508

M Rohan Fernando, Chao Jiang + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0208508

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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.

Highlights

The presence of fetal cell-free DNA in maternal blood was discovered in 1997 by Lo and colleagues [1]
It is critical that assay providers and new assay developers take appropriate measures to preserve the original proportion of cell-free fetal DNA (cffDNA) in maternal blood during the pre-analytical phase of the assay
The other possibility is an increase in maternal background cell-free DNA (cfDNA) concentration during the pre-analytical phase of the assay which contributes towards the reduction of the original proportion cffDNA

Summary

Introduction

The presence of fetal cell-free DNA (cffDNA) in maternal blood was discovered in 1997 by Lo and colleagues [1]. After this discovery, cffDNA in maternal blood has been used as genetic material for noninvasive prenatal diagnostic and screening tests in clinical practice [2, 3, 4, 5]. Utility cffDNA for noninvasive prenatal testing is challenging because cffDNA proportion in maternal blood is very low compared to background maternal cell-free DNA (cfDNA) proportion. The median cffDNA percentage in maternal blood is 10% (range 7.8–13%) and this value further decreases with increased maternal weight due to a dilution effect caused by increased maternal background cfDNA [6].

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