Effects of preservative and temperature on ctDNA analysis

Purpose:Analysis of genomic alterations in cell-free DNA (cfDNA) is evolving as an approach to detect, monitor and genotype malignancies. Methods to separate the liquid from the cellular fraction of whole blood for circulating tumor DNA (ctDNA) analyses have been largely unstudied although these may be a critical consideration for assay performance. Experimental Design:To evaluate the influence of blood processing on cfDNA and ctDNA quality and yield, we compared the cfDNA levels in serum to those in plasma. Given the limitations of serum for ctDNA analyses, we evaluated the effects of two plasma processing approaches, K2EDTA and Cell-Free DNA™ BCT (BCT) tubes, on cfDNA and ctDNA recovery. A total of 45 samples from 9 cancer patients were collected in both tube types. Once collected, blood was processed into plasma immediately or kept at room temperature and processed into plasma at 1, 3, 5, or 7 days. Results:As early as 24 hours after collection, plasma isolated from blood collected in K2EDTA tubes contained an elevated level of cfDNA that increased over time compared with BCT tubes where no significant increase in cfDNA levels was observed. When samples from an additional six cancer patients, collected in the same manner, were stored at 4°C in K2EDTA tubes over the course of 3 days, total cfDNA and ctDNA levels were comparable between samples collected in BCT tubes.

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