The Effect of Preservative and Temperature on the Analysis of Circulating Tumor DNA

Abstract

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. 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, KEDTA 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. As early as 24 hours after collection, plasma isolated from blood collected in KEDTA 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 KEDTA 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 KEDTA tubes. 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. .

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

“…The tubes were handled and centrifuged within 14 days according to the manufacturer's instructions (double spin at 1,600 g and 16,000 g ). Previous research has shown that the cfDNA quantity from BTC and EDTA tubes was highly comparable, with cfDNA quantity from the BTC tubes remaining stable over time . cfDNA was isolated from 4 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Venlo, the Netherlands) according to the manufacturer's instructions.…”

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

“…For blood storage until analysis, the choice between commonly available EDTA tubes and cell stabilizing tubes should depend on the transportation time: for in‐house samples processed within 4–6 hours after collection, EDTA tubes are sufficient to extract cfDNA. 62–64 Longer periods of storage or transportation warrant the use of tubes containing cell‐stabilizing reagents . From these tubes, steady cfDNA quality can be obtained after up to 1 week of storage, independent of the temperatures tested in the analysis (4°C or room temperature) .…”

“…In a comparative study, cfDNA concentrations from identical artificially spiked plasma samples extracted at 56 different laboratories revealed wide variations in cfDNA yields ranging from 2.87 to 224.02 ng/mL with a median of 23.15 ng/mL of plasma . While it is still debated whether blood plasma or serum contains more and higher quality cfDNA, there is evidence that serum is prone to high‐molecular‐weight DNA contamination due to delayed processing . For cfDNA‐based analytics of primary and metastatic brain tumors it is important to note that cerebrospinal fluid harbors more ctDNA than blood as the blood‐brain barrier reduces molecular exchange with the peripheral circulation, total volume is comparably low and contamination by cellular DNA is limited .…”

Liquid biopsies

“…Commercially available cfDNA BCT blood collection tubes are claimed to stabilize nucleated blood cells, preventing the release of cellular DNA into plasma, and also inhibiting degradation of cfDNA . Commercially available cfRNA BCT blood collection tubes are also claimed to stabilize nucleated blood cells, preventing the release of cellular DNA into plasma, and also inhibiting degradation of cfRNA …”

“…The influence of preanalytical parameters on ctDNA and CTC analysis is crucial and could potentially explain the various discrepancies reported in the literature and the poor reproducibility and differences in handling procedures . Numerous studies have already evaluated parameters potentially affecting ctDNA concentration and fragmentation and have described the optimal preanalytical handling conditions for ctDNA analysis …”