Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage
Abstract
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Feb 08, 2022
- Source ID
- 10.1038/s41598-022-06088-9
Entities
People
- Anh T. P. Ngo
- Fehmi Civitci
- Hyun Ji Kim
- Matthew J. Rames
- Mayu Morita
- Owen J. T. Mccarty
- Randall Armstrong
- Samuel Tassi Yunga
- Terry K. Morgan
- Thuy T. M. Ngo
Organizations
- National Institutes of Health
- Oregon Health & Science University
- Susan G. Komen for the Cure
- United States Department of Defense