Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients.

The Journal of Molecular Diagnostics : JMD
Bente RisbergDavina Gale

Abstract

Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because 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 delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into K3EDTA or Cell-free DNA BCT tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0, 6, 24, 48, 96 hours, 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 with BCT. A second centrifugation at 3000 × g gave similar cfDNA yields compared with higher-speed...Continue Reading

Citations

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Methods Mentioned

BETA
PCR
Amplicon Sequencing
Amplicon deep sequencing
DNA-seq
TAm-Seq
biopsy
blood collection

Software Mentioned

Rsamtools
Picard
BWA
SAMtools
Biostrings
MarkDuplicates
mem
R package CNAclinic
R
Tools

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