Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage

Hyun Ji Kim; Matthew J Rames; Samuel Tassi Yunga; Randall Armstrong; Mayu Morita; Anh T P Ngo; Owen J T McCarty; Fehmi Civitci; Terry K Morgan; Thuy T M Ngo

doi:10.1038/s41598-022-06088-9

Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage

Sci Rep. 2022 Feb 8;12(1):2099. doi: 10.1038/s41598-022-06088-9.

Authors

Hyun Ji Kim^{1

2}, Matthew J Rames^{1

2}, Samuel Tassi Yunga^{1

2}, Randall Armstrong¹, Mayu Morita³, Anh T P Ngo², Owen J T McCarty², Fehmi Civitci¹, Terry K Morgan^{1

2

3}, Thuy T M Ngo^{4

5

6}

Affiliations

¹ Cancer Early Detection Advanced Research Center, Knight Cancer Institute (CEDAR), Oregon Health and Science University, 2720 SW Moody Ave, KR-CEDR, Portland, OR, 97201, USA.
² Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR, USA.
³ Department of Pathology, Oregon Health and Science University, Portland, OR, USA.
⁴ Cancer Early Detection Advanced Research Center, Knight Cancer Institute (CEDAR), Oregon Health and Science University, 2720 SW Moody Ave, KR-CEDR, Portland, OR, 97201, USA. ngth@ohsu.edu.
⁵ Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR, USA. ngth@ohsu.edu.
⁶ Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA. ngth@ohsu.edu.

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Blood*
Cell-Free Nucleic Acids*
Cryopreservation*
Extracellular Vesicles*
Flow Cytometry
Humans
RNA, Messenger*

Substances

Cell-Free Nucleic Acids
RNA, Messenger

Abstract

Publication types

MeSH terms

Substances

Grants and funding