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

Abstract

Circulating tumour 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 (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 analysed by digital PCR (dPCR), 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 (RT) or 4°C, or using different centrifugation protocols. dPCR 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 RT 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.