A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR

Abstract

Objectives: To study the ability of a stabilizing reagent to prevent cellular DNA contamination of cell-free DNA (cfDNA) in plasma during whole blood sample storage and shipping.

Design and methods: Samples were drawn from healthy donors into K₃EDTA and Cell-Free DNA BCTs (BCT) and stored at room temperature (RT). Aliquots were removed at specified time points and cfDNA was purified from the plasma. A Droplet Digital PCR (ddPCR) assay that amplifies a short β-actin gene fragment (136 bp) was used to measure the total plasma cfDNA (pDNA) concentration while a longer β-actin fragment (420 bp) was used to quantify genomic DNA (gDNA). In a follow-up experiment, blood samples drawn into the same types of tubes were shipped round trip by overnight air before cfDNA was isolated and analyzed.

Results: Blood stored in K₃EDTA tubes at RT showed increases in pDNA and gDNA concentrations over time. However, both pDNA and gDNA levels remained stable in BCT for at least seven days. On day 14, there was a 4.5-fold increase in pDNA in BCT as compared to >200-fold increase in K₃EDTA tubes. Likewise, gDNA increased <2-fold on day 14 in BCT as opposed to a 456-fold increase in K₃EDTA tubes. Similar results were observed after samples were shipped.

Conclusions: Cell-Free DNA BCTs prevent gDNA contamination that may occur due to nucleated cell disruption during sample storage and shipping. This novel blood collection tube provides a method for obtaining stable cfDNA samples for rare target detection and accurate analysis while mitigating the threat of gDNA contamination.

Keywords: Blood specimen collection; Clinical laboratory techniques; DNA contamination; DNA in plasma; Investigative techniques.

Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.