Abstract 4586: Evaluation of available blood collection tubes for use in stabilizing concentrations of extracellular vesicles / exosomes and associated cell-free RNA

Abstract

Abstract Introduction: Liquid Biopsy has revolutionized the detection and the monitoring of cancer disease states. In addition to the widely utilized circulating cell-free DNA (ccfDNA), cell-free RNAs (cfRNA) and the extracellular vesicles (EV's) / exosomes they are stored in, is drawing significant interest in cancer diagnostics. Analysis of disease-specific EV's / exosomes or cfRNA requires that collected samples maintain draw-time concentrations of these analytes in order to remain relevant. Blood components, such as platelets, red, and white blood cells, are unstable when stored in normal blood collection tubes (BCT's), thus leading to increases of EVs and associated cfRNA concentration. We undertook an analysis of currently available commercial BCT's to determine whether any of these sufficiently stabilize EV / exosome and cfRNA concentration. Materials and Methods: Healthy consenting adult donors were drawn into blood collection tubes per approved protocol. Samples were either processed immediately (Day-0) or allowed to incubate at room temperature for the indicated times. Cleared plasma was isolated and subsequently frozen at -80oC until use. Assay of extracellular vesicle concentration utilized the Nanosight-300 (Malvern Instruments) per manufactures protocol. Isolation of cell-free RNA utilized the Circulating Nucleic Acid Isolation Kit (Qiagen) per manufacturer's protocol with the exception of the lysis step; this was extended from 30 minutes to 60 minutes. Digital PCR (ddPCR) utilized the Bio-Rad workflow (QX200 Droplet Generator and Reader). Conclusion: Stabilization of EV's and cfRNA was assayed for blood samples drawn into a variety of Streck nucleic acid stabilizing BCT's, non-Streck nucleic acid stabilizing BCT's, and a traditional K3EDTA BCT. A majority of the assayed tubes led to time-dependent increases in EV concentration with the most robust increases occurring between Day-3 and Day-7. Time-dependent increases in cfRNA concentration mirrored the increases observed in EV's, and based on ddPCR analysis; the majority of this increase was likely the result of erythrocyte membrane shedding. One type of Streck BCT was found to stabilize both EV and cfRNA concentration as a function of blood storage time. Incorporation of this tube into the Liquid Biopsy workflow will maximize assay target sensitivity by preventing the deleterious effects of excess cfRNA from non-specific release of EV's / exosomes. Citation Format: Nicholas George. Evaluation of available blood collection tubes for use in stabilizing concentrations of extracellular vesicles / exosomes and associated cell-free RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4586.