Skip to main content
Log in

Standardization of Blood Collection and Processing for the Diagnostic Use of Extracellular Vesicles

Current Pathobiology Reports

Abstract

Purpose of Review

Extracellular vesicles (EVs) are lipid membrane vesicles released by many types of cells in both health and disease. EVs can be found in most body fluids, carrying a plethora of biomolecules, including proteins, RNA, and DNA, that reflect the biomolecular composition of the tissue of origin. Parenchymal and stromal cells actively release EVs in the extracellular milieu and in circulation, providing valuable information that may be exploited for diagnostic applications. However, isolation of these EV subpopulations in circulation is extremely challenging as they are diluted within more abundant EV subpopulations derived from blood cells (red blood cells, platelets, and white blood cells).

Recent Findings

A number of preanalytical variables during blood collection and processing greatly impact the levels of blood-derived EVs, thus affecting sample quality. So far, lack of standard protocols for blood collection and processing as well as quality control metrics has limited the clinical validation and adoption of EV-based diagnostic assays.

Summary

In this review, we describe the preanalytical variables that affect sample quality and suitability for EV-based diagnostic approaches. Furthermore, we suggest biochemical and molecular quality control (QC) metrics to minimize intra- and interstudy variability and improve data robustness and reproducibility.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

References

Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance

  1. van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213–28.

    Article  CAS  PubMed  Google Scholar 

  2. Zhang H, Freitas D, Kim HS, Fabijanic K, Li Z, Chen H, et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol. 2018;20(3):332–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. •• Li Q, Wang X, Li X, He X, Wan Q, Yin J, et al. Obtaining high-quality blood specimens for downstream applications: a review of current knowledge and best practices. Biopreserv biobank. 2018. A review that summarizes the current best practices for blood specimen collection and handling.

  4. •• Witwer KW, Buzás EI, Bemis LT, Bora A, Lässer C, Lötvall J, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013;27:2 This review underlines the needs of standardization of biospecimen handling for downstream EV isolation and analysis.

    Google Scholar 

  5. Anfossi S, Babayan A, Pantel K, Calin GA. Clinical utility of circulating non-coding RNAs - an update. Nat Rev Clin Oncol. 2018;15(9):541–63.

    Article  PubMed  Google Scholar 

  6. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. György B, Pálóczi K, Kovács A, Barabás E, Bekő G, Várnai K, et al. Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube. Thromb Res. 2014;133(2):285–92.

    Article  CAS  PubMed  Google Scholar 

  8. Cvjetkovic A, Lötvall J, Lässer C. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles. 2014;25:3.

    Google Scholar 

  9. •• Ramirez MI, Amorim MG, Gadelha C, Milic I, Welsh JA, Freitas VM, et al. Technical challenges of working with extracellular vesicles. Nanoscale. 2018;10(3):881–906 Review that underlines the role of EVs in liquid biopsy and the variables to be considered for EVs studies during preanalytical and analytical phases.

    Article  CAS  PubMed  Google Scholar 

  10. •• Coumans FAW, Brisson AR, Buzas EI, Dignat-George F, Drees EEE, El-Andaloussi S, et al. Methodological guidelines to study extracellular vesicles. Circ Res. 2017;120(10):1632–48 This review suggests guidelines for sample collection and processing in EV studies.

    Article  CAS  PubMed  Google Scholar 

  11. EV-TRACK Consortium, Van Deun J, Mestdagh P, Agostinis P, et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat Methods. 2017;14(3):228–32.

    Article  CAS  Google Scholar 

  12. Mora EM, Álvarez-Cubela S, Oltra E. Biobanking of exosomes in the era of precision medicine: are we there yet? Int J Mol Sci. 2015;17(1).

  13. • Vila-Liante V, Sánchez-López V, Martínez-Sales V, Ramón-Nuñez LA, Arellano-Orden E, Cano-Ruiz A, et al. Impact of sample processing on the measurement of circulating microparticles: storage and centrifugation parameters. Clin Chem Lab Med. 2016;54(11):1759–67 This publication shows the influence of different sample processing and storage conditions on the microparticle count in plasma.

    Article  CAS  PubMed  Google Scholar 

  14. Štukelj R, Schara K, Bedina-Zavec A, Šuštar V, Pajnič M, Pađen L, et al. Effect of shear stress in the flow through the sampling needle on concentration of nanovesicles isolated from blood. Eur J Pharm Sci. 2017;98:17–29.

    Article  CAS  PubMed  Google Scholar 

  15. Clayton A, Buschmann D, Byrd JB, Carter DRF, Cheng L, Compton C, et al. Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017. J Extracell Vesicles. 2018;7:1 1473707.

    Article  CAS  Google Scholar 

  16. Tuck MK, Chan DW, Chia D, Godwin AK, Grizzle WE, Krueger KE, et al. Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group. J Proteome Res. 2009;8(1):113–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Betsou F and ISBER Biospecimen Science Working Group. Assays for qualification and quality stratification of clinical biospecimens used in research. Biopreserv Biobanking. 2016;14:398–409.

    Article  Google Scholar 

  18. •• Simundic A, Bölenius K, Cadamuro J, et al. Recommendation for venous blood sampling. EFML paper (European Federation of Clinical Chemistry and laboratory medicine), v. 1.1 2018. Joint EFLM-COLABIOCLI recommendations for blood sampling and post sampling procedures.

  19. • Magnette A, Chatelain M, Chatelain B, Ten Cate H, Mullier F. Pre-analytical issues in the haemostasis laboratory: guidance for the clinical laboratories. Thromb J. 2016;14:49 A systematic review about the preanalytical variables affecting sample quality.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. •• World Health Organization. WHO guidelines on drawing blood: best practices in phlebotomy. 2010 ISBN 978 92 4 159922 1 (NLM classification: WB 381). Summary of the best practices for venous blood sampling.

  21. Yuana Y, Bertina RM, Osanto S. Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb Haemost. 2011;105(3):396–408.

    Article  CAS  PubMed  Google Scholar 

  22. Hernandes VV, Barbas C, Dudzik D. A review of blood sample handling and pre-processing for metabolomics studies. Electrophoresis. 2017;38(18):2232–41.

    Article  CAS  PubMed  Google Scholar 

  23. Yin P, Lehmann R, Xu G. Effects of pre-analytical processes on blood samples used in metabolomics studies. Anal Bioanal Chem. 2015;407(17):4879–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. El Messaoudi S, Rolet F, Mouliere F, Thierry AR. Circulating cell free DNA: preanalytical considerations. Clin Chim Acta. 2013;424:222–30.

    Article  CAS  PubMed  Google Scholar 

  25. Wong KH, Sandlin RD, Carey TR, Miller KL, Shank AT, et al. The role of physical stabilization in whole blood preservation. Sci Rep. 2016;6:21023.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cheng HH, Yi HS, Kim Y, Kroh EM, Chien JW, Eaton KD, et al. Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS One. 2013;8(6):e64795.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Greening DW, Glenister KM, Sparrow RL, Simpson RJ. International blood collection and storage: clinical use of blood products. J Proteome. 2010;73(3):386–95.

    Article  CAS  Google Scholar 

  28. Trezzi JP, Bulla A, Bellora C, Rose M, Lescuyer P, Kiehntopf M, et al. LacaScore: a novel plasma sample quality control tool based on ascorbic acid and lactic acid levels. Metabolomics. 2016;12:96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jeyaram A, Jay SM. Preservation and storage stability of extracellular vesicles for therapeutic applications. AAPS J. 2017;20(1):1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Glinge C, Clauss S, Boddum K, Jabbari R, Jabbari J, Risgaard B, et al. Stability of circulating blood-based microRNAs - pre-analytic methodological considerations. PLoS One. 2017;12(2):e0167969.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bosch S, de Beaurepaire L, Allard M, Mosser M, Heichette C, Chrétien D, et al. Trehalose prevents aggregation of exosomes and cryodamage. Sci Rep. 2016;6:36162.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Onódi Z, Pelyhe C, Terézia Nagy C, Brenner GB, Almási L, Kittel Á, et al. Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma. Front Physiol. 2018;9:1479.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kuo WP, Tigges JC, Toxavidis V, Ghiran I. Red blood cells: a source of extracellular vesicles. Methods in molecular biology. Extracellular vesicles - methods and protocols 2017. ISBN 9781493972531.

  34. Tao SC, Guo SC, Zhang CQ. Platelet-derived extracellular vesicles: an emerging therapeutic approach. Int J Biol Sci. 2017;13(7):828–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yuana Y, Sturk A, Nieuwland R. Extracellular vesicles in physiological and pathological conditions. Blood Rev. 2013;27(1):31–9.

    Article  CAS  PubMed  Google Scholar 

  36. Danesh A, Inglis HC, Abdel-Mohsen M, Deng X, Adelman A, Schechtman KB, et al. Granulocyte-derived extracellular vesicles activate monocytes and are associated with mortality in intensive care unit patients. Front Immunol. 2018;9:956.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Díaz-Varela M, de Menezes-Neto A, Perez-Zsolt D, Gámez-Valero A, Seguí-Barber J, Izquierdo-Useros N, et al. Proteomics study of human cord blood reticulocyte-derived exosomes. Sci Rep. 2018;8(1):14046.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Juzenas S, Venkatesh G, Hübenthal M, Hoeppner MP, Du ZG, Paulsen M, et al. A comprehensive, cell specific microRNA catalogue of human peripheral blood. Nucleic Acids Res. 2017;45(16):9290–301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Momose F, Seo N, Akahori Y, Sawada S, Harada N, Ogura T, et al. Guanine-rich sequences are a dominant feature of exosomal microRNAs across the mammalian species and cell types. PLoS One. 2016;11(4):e0154134.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. •• Teruel-Montoya R, Kong X, Abraham S, Ma L, Kunapuli SP, Holinstat M, et al. MicroRNA expression differences in human hematopoietic cell lineages enable regulated transgene expression. PLoS One. 2014;9(7):e102259 This publication shows the contribution of hematopoietic cells to the miRNA content found in blood from healthy donors.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Merkerova M, Belickova M, Bruchova H. Differential expression of microRNAs in hematopoietic cell lineages. Eur J Haematol. 2008;81(4):304–10.

    Article  CAS  PubMed  Google Scholar 

  42. Pordzik J, Pisarz K, De Rosa S, Jones AD, Eyileten C, Indolfi C. The potential role of platelet-related microRNAs in the development of cardiovascular events in high-risk populations, including diabetic patients: a review. Front Endocrinol (Lausanne). 2018;9:74.

    Article  Google Scholar 

  43. Dempsey E, Dervin F, Maguire PB. Platelet derived exosomes are enriched for specific microRNAs which regulate WNT signalling in endothelial cells. Blood. 2014;124:2760.

    Article  Google Scholar 

  44. • Pritchard CC, Kroh E, Wood B, Arroyo JD, Dougherty KJ, Miyaji MM. Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies. Cancer Prev Res (Phila). 2012;5(3):492–7 This article shows evidence that blood cells are major contributors to miRNA levels in circulation, thus confounding RNA analyses for cancer biomarker discovery.

    Article  CAS  Google Scholar 

  45. Liu T, Zhang Q, Zhang J, Li C, Miao YR, Lei Q, et al. EVmiRNA: a database of miRNA profiling in extracellular vesicles. Nucleic Acids Res. 2018 Oct;18.

  46. Kuchen S, Resch W, Yamane A, Kuo N, Li Z, Chakraborty T, et al. Regulation of microRNA expression and abundance during lymphopoiesis. Immunity. 2010;32(6):828–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Choi JL, Li S, Han JY. Platelet function tests: a review of progresses in clinical application. Biomed Res Int. 2014;2014:456569.

    PubMed  PubMed Central  Google Scholar 

  48. Torri A, Carpi D, Bulgheroni E, Crosti MC, Moro M, Gruarin P, et al. Extracellular microRNA signature of human helper T cell subsets in health and autoimmunity. J Biol Chem. 2017;292(7):2903–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Fernández-Messina L, Gutiérrez-Vázquez C, Rivas-García E, Sánchez-Madrid F, de la Fuente H. Immunomodulatory role of microRNAs transferred by extracellular vesicles. Biol Cell. 2015;107(3):61–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ismail N, Wang Y, Dakhlallah D, Moldovan L, Agarwal K, Batte K, et al. Macrophage microvesicles induce macrophage differentiation and miR-223 transfer. Blood. 2013;121(6):984–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Sastre B, Cañas JA, Rodrigo-Muñoz JM, Del Pozo V. Novel modulators of asthma and allergy: exosomes and microRNAs. Front Immunol. 2017;8:826.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zocco D, Zarovni N. Extraction and analysis of extracellular vesicle-associated miRNAs following antibody-based extracellular vesicle capture from plasma samples. Methods Mol Biol. 1660;2017:269–85.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Davide Zocco.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by any of the authors.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This article is part of the Topical Collection on Biospecimens Science and Evidence-Based Standards for Precision Medicine

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Venturella, M., Carpi, F.M. & Zocco, D. Standardization of Blood Collection and Processing for the Diagnostic Use of Extracellular Vesicles. Curr Pathobiol Rep 7, 1–8 (2019). https://doi.org/10.1007/s40139-019-00189-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40139-019-00189-3

Keywords

Navigation