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Licensed Unlicensed Requires Authentication Published by De Gruyter November 30, 2020

Effect of collection matrix, platelet depletion, and storage conditions on plasma extracellular vesicles and extracellular vesicle-associated miRNAs measurements

  • Martina Faraldi ORCID logo , Marta Gomarasca ORCID logo , Silvia Perego ORCID logo EMAIL logo , Veronica Sansoni ORCID logo , Giuseppe Banfi ORCID logo and Giovanni Lombardi ORCID logo

Abstract

Objectives

The interest around circulating extracellular vesicles and their cargo in diagnostics has greatly increased; however, several pre-analytical variables affect their determination. In this study, we investigated the effects of sample matrix, processing, and plasma storage delay and temperature on extracellular vesicles and their miRNA content.

Methods

Blood was collected from 10 male volunteers in dipotassium ethylendiaminotetraacetate-coated tubes (K2EDTA), either with plasma-preparation tube (PPT) or without (K2E) gel separator. A stepwise centrifugation was applied to K2E aliquots to obtain platelet-poor plasma (PPP). K2E, PPP and PPT plasma, stored under different conditions, were assayed for extracellular vesicles concentration and size distribution, through dynamic laser light scattering, and microRNAs content, by qPCR.

Results

PPP samples were characterized by the lowest extracellular vesicles count and miRNA detectability. Although having no effects on extracellular vesicles total concentration, storage conditions influenced microRNAs detectability, mainly in PPP and PPT samples. Extracellular vesicles-associated miRNAs levels in K2E were, in general, higher than in PPP and to a very limited extent to PPT. Storage temperature and delay did not affect their profile in K2E samples.

Conclusions

Extracellular vesicles count and extracellular vesicles miRNA profile changed under the analyzed pre-analytical variables, showing the greatest stability in K2E samples. Since pre-analytical variables differently affected extracellular vesicles and their miRNA content, they should be considered in each experimental setting and clinical routine.


Corresponding author: Silvia Perego, Ph.D., Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto Ortopedico Galeazzi, Via Riccardo Galeazzi, 4 – 20161, Milan, Italy, Phone: +39 0266214068, E-mail:

Funding source: Italian Ministry of Health

Acknowledgments

The authors are grateful to Prof. Giberto Chirico and Prof. Maddalena Collini (Department of Physics, National Institute of Nuclear Physics, Università degli Studi di Milano-Bicocca) for their help in designing, conducting and interpreting the DLS experiments.

  1. Research funding: This work has been funded by the Italian Ministry of Health.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by Comitato Etico Ospedale San Raffaele, Milano, Italia (SportMarker, ClinicalTrials.gov registration number NCT03386981).

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2020-1296).


Received: 2020-08-25
Accepted: 2020-11-17
Published Online: 2020-11-30
Published in Print: 2021-04-27

© 2020 Walter de Gruyter GmbH, Berlin/Boston

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