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Optimization of blood handling for plasma extracellular vesicle isolation

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Abstract

Extracellular vesicles (EVs) are emerging targets for biomarker discovery. However, suitable clinical collection and storage methods for blood plasma-derived EVs have not been fully described. We investigated the long- and short-term effects of temperature and time on the plasma-derived EV concentration, size, protein, and RNA content. EVs isolated from freshly collected human blood stored for various periods of time at either room temperature (RT) or 4°C were analyzed for concentration, size, and protein content. In addition, EVs isolated from plasma stored at −80°C were compared to frozen EV pellets. We found that the number, size, protein, and RNA content of EVs were similar between blood samples stored at RT for 24h compared to samples processed shortly after collection or stored at 4°C vs. −80°C. It is therefore feasible to isolate and analyze comparable, stable EVs from blood plasma stored for varying periods of time and at various temperatures.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This work was supported by Loma Linda University’s Grants to Promote Collaborative and Translational GCAT Research program (NRW, MS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Author notes

  1. Maheswari Senthil and Nathan R. Wall contributed equally to this work.

Authors and Affiliations

  1. Department of Basic Science, Division of Biochemistry, Loma Linda University, Loma Linda, USA

    Janviere Kabagwira, Amber Gonda, Paul Vallejos, William H. R. Langridge & Nathan R. Wall

  2. Division of Surgical Oncology, Loma Linda University Health, Loma Linda, CA, USA

    Blake Babcock

  3. Division of Surgical Oncology, University of California- Irvine, 101, City Drive South, Building 55, Orange, CA, 92868, USA

    Maheswari Senthil

  4. Center for Health Disparities & Molecular Medicine, Loma Linda University, 11085 Campus Street, Mortensen Hall 160, Loma Linda, CA, 92350, USA

    Nathan R. Wall

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  1. Janviere Kabagwira
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Contributions

Janvier Kabagwira: conceptualization, methodology, writing original draft. Amber Gonda: conceptualization, methodology, supervision, validation, writing review, and editing. Paul Vallejos: software, analysis of data, writing review, and editing. Blake Babcock: conceptualization, methodology. William Langridge: conceptualization, supervision, writing review, and editing. Maheswari Senthil: conceptualization, supervision, methodology, project administration, funding, writing review, and editing. Nathan Wall: conceptualization, data curation, funding, methodology, project administration, resources, software, supervision, writing original draft, review, and editing. *Authors contributed equally to this manuscript.

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Supplementary information

Supplementary 1.

Blood storage at 4°C or 37°C does not alter EVs size and concentration over 24 hours. EVs were isolated from blood that was stored at either room temperature (A&C) or ice/ 4°C (B&D) from healthy control patients. The storage duration for the blood was 1, 2, 4 and 24 hours. Size (A&B) and concentration (C&D) of EVs was conducted using a Nanosight NS300 at each time point. Normalized EV protein was also compared (E). Data is representative of multiple replicates (n=5) performed for each sample. Non-significance is indicated by ns. (PNG 421 kb)

High resolution image (TIFF 857 kb)

Supplementary 2.

Blood storage at 4°C or 37°C does not alter EVs RNA yield over 24 hours. EVs were isolated from blood that was stored at either room temperature (A) or ice/ 4°C (B) from healthy control patients. The storage duration for the blood was 1, 2, 4 and 24 hours. Normalized RNA yield was compared with no significant difference observed (C) as a result of temperature at any time point. Data is representative of multiple replicates (n=5) performed for each sample. Non-significance is indicated by ns. (PNG 384 kb)

High resolution image (TIFF 857 kb)

Supplementary 3.

Long-term freezing of EVs does not affect EVs protein markers. Whole blood was collected and spun at 2000 rpm for 10 minutes to obtain plasma which was divided into two parts. One portion was frozen at−80 °C for 16 months and from the other portion EVs were isolated using ExoQuick measured and frozen−80 °C for 16 months. After 16 months, EVs were isolated from frozen plasma. Proteins from each sample were run on 12% SDS PAGE and probed with antibodies to EPCAM, CD81, CD63, and CD9. Data is representative of multiple replicates (n=2) performed for each sample. (PNG 372 kb)

High resolution image (TIFF 857 kb)

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Kabagwira, J., Gonda, A., Vallejos, P. et al. Optimization of blood handling for plasma extracellular vesicle isolation. J Nanopart Res 23, 65 (2021). https://doi.org/10.1007/s11051-021-05169-6

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