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Effects of collection and processing procedures on plasma circulating cell-free DNA from cancer patients

Risberg, B; Tsui, DW; Biggs, H; Ruiz-Valdepenas Martin de Almagro, A; Dawson, S-J; Hodgkin, C; Jones, L; ... Gale, D; + view all (2018) Effects of collection and processing procedures on plasma circulating cell-free DNA from cancer patients. The Journal of Molecular Diagnostics 10.1016/j.jmoldx.2018.07.005. (In press). Green open access

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Abstract

Circulating tumor DNA (ctDNA) offers new opportunities for non-invasive cancer management. Detecting ctDNA in plasma is challenging since it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the i) delay in processing, ii) storage temperatures, iii) different blood collection tubes, iv) centrifugation protocols, and v) sample shipment on cfDNA levels. Peripheral blood (n=231) from cancer patients (n=62) were collected into K3EDTA or Cell-free DNA BCT® (BCT) tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome (sWGS) sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0h, 6h, 24h, 48h, 96h, and 1 week at room temperature or 4 °C, or using different centrifugation protocols. Digital PCR showed that cfDNA levels increased gradually with time in K3EDTA tubes, but were stable in BCT tubes. K3EDTA samples stored at 4 °C showed less variation than room temperature storage, but levels were elevated compared to BCT. A second centrifugation at 3000g gave similar cfDNA yields compared to higher speed centrifugation. Next-generation sequencing showed negligible differences in background error or copy number changes between K3EDTA and BCT, or following shipment in BCT. This study provides insights into the effects of sample processing on ctDNA analysis.

Type: Article
Title: Effects of collection and processing procedures on plasma circulating cell-free DNA from cancer patients
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.jmoldx.2018.07.005
Publisher version: http://doi.org/10.1016/j.jmoldx.2018.07.005
Language: English
Additional information: Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0).
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Genetics, Evolution and Environment
URI: http://discovery.ucl.ac.uk/id/eprint/10056097
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