The effect of preservative and temperature on the analysis of circulating tumor DNA

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 with 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 nine patients with cancer 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 patients with cancer, collected in the same manner, were stored at 4°C in K2 EDTA tubes over the course of 3 days, total cfDNA and ctDNA levels were comparable between samples collected in BCT tubes. At day 3, there was a trend toward a decrease in ctDNA levels in both tubes that was more pronounced when measuring the mutant allele fraction for cases stored at 4°C in K₂EDTA tubes. Conclusions: In summary, methods of blood processing have a strong influence on cfDNA and ctDNA levels and should be a consideration when evaluating ctDNA in peripheral circulation.