期刊
SCIENTIFIC REPORTS
卷 12, 期 1, 页码 -出版社
NATURE PORTFOLIO
DOI: 10.1038/s41598-022-06088-9
关键词
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资金
- Cancer Early Detection Advanced Research (CEDAR) center at Oregon Health and Science University's Knight Cancer Institute
- Cancer Research UK/OHSU Project Award [C63763/A27122]
- Kuni Foundation
- Susan G. Komen Foundation [CCR21663959]
- Department of Defense [W81XWH2110853]
- National Institutes of Health [R01HL101972, R21HD16-037, R01GM116184, R01HL047014]
This study examined the impact of differential centrifugation and freeze/thaw on the profiles of extracellular vesicles (EVs) and cell-free messenger RNA (cf-mRNA) in plasma using flow cytometric analysis. The results revealed that the differential centrifugation and post-freeze/thaw processing affected the EV subpopulations and cf-mRNA levels. Freezing plasma containing residual platelets resulted in irreversible ex vivo generation of EV subpopulations and cf-mRNA transcripts.
The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Despite growing interest in studying extracellular vesicles (EVs) and cell-free messenger RNA (cf-mRNA) as potential biomarkers, how blood processing and freeze/thaw impacts the profiles of these analytes in plasma was not thoroughly understood. We utilized flow cytometric analysis to examine the effect of differential centrifugation and a freeze/thaw cycle on EV profiles. Utilizing flow cytometry postacquisition analysis software (FCMpass) to calibrate light scattering and fluorescence, we revealed how differential centrifugation and post-freeze/thaw processing removes and retains EV subpopulations. Additionally, cf-mRNA levels measured by RT-qPCR profiles from a panel of housekeeping, platelet, and tissue-specific genes were preferentially affected by differential centrifugation and post-freeze/thaw processing. Critically, freezing plasma containing residual platelets yielded irreversible ex vivo generation of EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze/thaw. Our findings suggest the importance of minimizing confounding variation attributed to plasma processing and platelet contamination.
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