Evaluation of Storage Tubes for Combined Analysis of Circulating Nucleic Acids in Liquid Biopsies

Abstract

In the last decade, circulating nucleic acids such as microRNAs (miRNAs) and cell-free DNA (cfDNA) have become increasingly important in serving as potential novel biomarkers for a variety of human diseases. If cell-free nucleic acids are to become routinely used in diagnostics, the difference in plasma miRNA and cfDNA levels between healthy and diseased subjects must exceed pre-analytical and analytical variability. Until now, few studies have addressed the time limitations of pre-processing or explored the potential use of long-term blood storage tubes, which might need to be implemented in real-life diagnostics. In this study, we analyzed the stability of four breast cancer-associated miRNAs and two cancer-associated genes under various storage conditions, to test their limitations for potential application in clinical diagnostics. In two consecutive experiments, we tested the limits of conventional EDTA tubes, as well as long-term storage blood collection tubes (BCTs) from four different manufacturers. We found that circulating miRNAs are relatively stable when stored in EDTA monovettes for up to 12 h before processing. When stored in BCTs, circulating miRNAs and cfDNA are stable for up to 7 days, depending on the manufacturer. Norgen tubes were superior for cfDNA yield, while Streck tubes performed the worst in our study with hemolysis induction. In conclusion, plasma prepared from whole blood is suitable for the quantification of both cf-miRNAs and cfDNA simultaneously.

Pre‐analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients

“…ctDNA quantity and quality as well as VAF analysis, however, remain largely unaffected by the choice of tube if processed immediately. Nonetheless, in a clinical setting quick processing is not always possible; therefore, Streck tubes represent a good alternative as these tubes have been shown to conserve ctDNA, CTCs, and miRNA over longer time periods compared to EDTA tubes (Alidousty et al , 2017; Diaz et al , 2016; Qin et al , 2014; Ward Gahlawat et al , 2019). Taken together, ctDNA, CTC, and miRNA can be analyzed from a single blood collection tube, but the choice of the tube affects the outcome of the analysis, underlining the importance of pre‐analytical factors.…”

Liquid biopsy in pancreatic ductal adenocarcinoma: current status of circulating tumor cells and circulating tumor DNA

“…Antibodies can also be attached to microposts and other surfaces for CTC capture. Microfluidic devices have been developed based on the technology controlling the fluid flow, which offers advantages for CTC research such as improved capture efficiency and high purity (Warkiani et al, 2016). The geometrically enhanced differential immunocapture (GEDI) device uses geometrically enhanced microstructures and combines positive enrichment with hydrodynamic chromatography, which additionally enables cell sizebased separation.…”

“…If specimen processing can be performed within 6 h from collection, standard K2EDTA collection tubes are suitable for blood sampling. However, when the processing is delayed by up to 48 h, specialized cell-stabilizing blood collection tubes should be used to reduce contamination by genomic DNA released from leukocyte lysis (Alidousty et al, 2017;Medina Diaz et al, 2016;Merker et al, 2018;Risberg et al, 2018;Ward Gahlawat et al, 2019;Warton et al, 2017). Current evidence recommends that isolated plasma, not whole blood, can be stored frozen up to 9 months or up to a few years, depending on analytical goals (van Dessel et al, 2017;Meddeb et al, 2019).…”

The Growing Impact of Micro/Nanomaterial‐Based Systems in Precision Oncology: Translating “Multiomics” Technologies

“…[47] Conversely, Luk and co-workers reported significantly lower detection of CTC-associated mRNA by digital droplet PCR for samples collected in sBCTs relative to those collected in EDTA or acid-citrate and dextrose. [48] In the context of circulating miRNA, Ward Gahlawat et al compared the performance of four different long-term BCTs for both miRNA and cfDNA analysis. [48] This study reported substantial differences in the miRNA profile between the four tubes after seven days, which was hypothesized to be due to different rates of cell lysis.…”

“…[48] In the context of circulating miRNA, Ward Gahlawat et al compared the performance of four different long-term BCTs for both miRNA and cfDNA analysis. [48] This study reported substantial differences in the miRNA profile between the four tubes after seven days, which was hypothesized to be due to different rates of cell lysis. In contrast, the cfDNA profile was comparatively stable across all four tubes.…”