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Protocol for Measuring Concentrations of Extracellular Vesicles in Human Blood Plasma with Flow Cytometry

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Book cover Extracellular Vesicles in Diagnosis and Therapy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2504))

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Abstract

Extracellular vesicles (EVs) are lipid membrane enclosed particles that are released from cells into body fluids, such as blood. EVs offer potential new biomarkers of diseases, because the cellular origin, composition, concentration, and function of EVs change in health and disease. The concentration of EVs from specific cell types in blood can be determined with flow cytometry. A flow cytometer measures fluorescence and light scattering signals from single EVs, but only if these signals are sufficiently bright to be detected. Measured concentrations of EVs are therefore only reproducible and comparable if the detection ranges are known and reported in standard units, such as molecules of equivalent soluble fluorophore (MESF) for fluorescence signals and the diameter in nm for scatter signals. The goal of this protocol is to discuss all steps needed to derive the concentration of cell-type specific EVs within a known diameter range and fluorescence range. More specifically, this protocol describes how to determine the concentration of CD61+ (Integrin beta-3, platelet marker), CD235a+ (Glycophorin A, erythrocyte marker), and CD45+ (leukocyte common antigen) EVs in human blood plasma with an Apogee A60-Micro flow cytometer using scatter-based triggering. The principles behind this protocol could lay a firm basis for the design of a protocol suitable for other flow cytometers and body fluids.

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References

  1. Théry C, Witwer KW, Aikawa E et al (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7:1535750. https://doi.org/10.1080/20013078.2018.1535750

    Article  PubMed  PubMed Central  Google Scholar 

  2. van der Pol E, Böing AN, Harrison P et al (2012) Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64:676–705. https://doi.org/10.1124/pr.112.005983

    Article  CAS  PubMed  Google Scholar 

  3. Kuiper M, van de Nes A, Nieuwland R et al (2020) Reliable measurements of extracellular vesicles by clinical flow cytometry. Am J Reprod Immunol 85:e13350. https://doi.org/10.1111/aji.13350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. van der Pol E, Coumans FAW, Grootemaat AE et al (2014) Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 12:1182–1192. https://doi.org/10.1111/jth.12602

    Article  PubMed  Google Scholar 

  5. Welsh JA, van der Pol E, Arkesteijn GJA et al (2020) MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. J Extracell Vesicles 9:1713526. https://doi.org/10.1080/20013078.2020.1713526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. de Rond L, Coumans FAW, Nieuwland R et al (2018) Deriving extracellular vesicle size from scatter intensities measured by flow cytometry. Curr Protoc Cytom 86:e43. https://doi.org/10.1002/cpcy.43

    Article  CAS  PubMed  Google Scholar 

  7. Coumans FAW, Brisson AR, Buzas EI et al (2017) Methodological guidelines to study extracellular vesicles. Circ Res 120:1632–1648. https://doi.org/10.1161/CIRCRESAHA.117.309417

    Article  CAS  PubMed  Google Scholar 

  8. Lippi G, Salvagno GL, Montagnana M et al (2006) Influence of the needle bore size on platelet count and routine coagulation testing. Blood Coagul Fibrinolysis 17:557–561. https://doi.org/10.1097/01.mbc.0000245300.10387.ca

    Article  PubMed  Google Scholar 

  9. Piccin A, Murphy WG, Smith OP (2007) Circulating microparticles: pathophysiology and clinical implications. Blood Rev 21:157–171. https://doi.org/10.1016/j.blre.2006.09.001

    Article  CAS  PubMed  Google Scholar 

  10. Lippi G, Salvagno GL, Montagnana M et al (2006) Venous stasis and routine hematologic testing. Clin Lab Haematol 28:332–337. https://doi.org/10.1111/j.1365-2257.2006.00818.x

    Article  CAS  PubMed  Google Scholar 

  11. Hefler L, Grimm C, Leodolter S, Tempfer C (2004) To butterfly or to needle: the pilot phase. Ann Intern Med 140:935–936. https://doi.org/10.7326/0003-4819-140-11-200406010-00027

    Article  PubMed  Google Scholar 

  12. Van Ierssel SH, Van Craenenbroeck EM, Conraads VM et al (2010) Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied. Thromb Res 125:332–339. https://doi.org/10.1016/j.thromres.2009.12.019

    Article  CAS  PubMed  Google Scholar 

  13. Tuck MK, Chan DW, Chia D et al (2009) Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group. J Proteome Res 8:113–117. https://doi.org/10.1021/pr800545q

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pearson L, Thom J, Adams M et al (2009) A rapid flow cytometric technique for the detection of platelet-monocyte complexes, activated platelets and platelet-derived microparticles. Int J Lab Hematol 31:430–439. https://doi.org/10.1111/j.1751-553X.2008.01059.x

    Article  PubMed  Google Scholar 

  15. Lacroix R, Judicone C, Poncelet P et al (2012) Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. J Thromb Haemost 10:437–446. https://doi.org/10.1111/j.1538-7836.2011.04610.x

    Article  CAS  PubMed  Google Scholar 

  16. Mody M, Lazarus AH, Semple JW, Freedman J (1999) Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant. Transfus Med 9:147–154. https://doi.org/10.1046/j.1365-3148.1999.00188.x

    Article  CAS  PubMed  Google Scholar 

  17. Keuren JFW, Magdeleyns EJP, Govers-Riemslag JWP et al (2006) Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation. Br J Haematol 134:307–313. https://doi.org/10.1111/j.1365-2141.2006.06167.x

    Article  CAS  PubMed  Google Scholar 

  18. Lacroix R, Judicone C, Mooberry M et al (2013) Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost 11:1190–1193. https://doi.org/10.1111/jth.12207

    Article  Google Scholar 

  19. Rikkert LG, van der Pol E, van Leeuwen TG et al (2018) Centrifugation affects the purity of liquid biopsy-based tumor biomarkers. Cytometry A 93:1207–1212. https://doi.org/10.1002/cyto.a.23641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yuana Y, Bertina RM, Osanto S (2011) Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb Haemost 105:396–408. https://doi.org/10.1160/TH10-09-0595

    Article  CAS  PubMed  Google Scholar 

  21. Biro E, Sturk-Maquelin KN, Vogel GMT et al (2003) Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner. J Thromb Haemost 1:2561–2568. https://doi.org/10.1046/j.1538-7836.2003.00456.x

    Article  CAS  PubMed  Google Scholar 

  22. Simak J, Gelderman MP (2006) Cell membrane microparticles in blood and blood products: potentially pathogenic agents and diagnostic markers. Transfus Med Rev 20:1–26. https://doi.org/10.1016/j.tmrv.2005.08.001

    Article  PubMed  Google Scholar 

  23. van der Pol E, van Gemert MJC, Sturk A et al (2012) Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 10:919–930. https://doi.org/10.1111/j.1538-7836.2012.04683.x

    Article  CAS  PubMed  Google Scholar 

  24. Buntsma NC, Gąsecka A, Roos YBWEM, van Leeuwen TG, van der Pol E, Nieuwland R (2021) EDTA stabilizes the concentration of platelet-derived extracellular vesicles during blood collection and handling. Platelets:1–8. https://doi.org/10.1080/09537104.2021.1991569

  25. Cossarizza A, Chang H-D, Radbruch A et al (2019) Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol 49:1457–1973. https://doi.org/10.1002/eji.201970107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shapiro HM, Leif RC (2003) Practical flow cytometry, 4th edn. Wiley, Hoboken

    Book  Google Scholar 

  27. Maecker HT, Trotter J (2006) Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037–1042. https://doi.org/10.1002/cyto.a.20333

    Article  PubMed  Google Scholar 

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Acknowledgments

We would like to acknowledge Dr. Aleksandra Gąsecka for commenting on the sections about blood collection, plasma preparation , and plasma storage and Naomi Buntsma, M.Sc., for the data on CD45-APC titrations. E. van der Pol acknowledges funding from the Netherlands Organisation for Scientific Research - Domain Applied and Engineering Sciences (NWO-TTW), research programmes VENI 15924, and from the European Association of National Metrology Institutes, Grant/Award Number: 18HLT01 METVES II.

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Authors and Affiliations

  1. Laboratory Experimental Clinical Chemistry, Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands

    Najat Hajji, Chi M. Hau, Rienk Nieuwland & Edwin van der Pol

  2. Vesicle Observation Center, Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands

    Najat Hajji, Chi M. Hau, Rienk Nieuwland & Edwin van der Pol

  3. Biomedical Engineering and Physics, Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands

    Edwin van der Pol

Authors
  1. Najat Hajji
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  2. Chi M. Hau
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  3. Rienk Nieuwland
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  4. Edwin van der Pol
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Corresponding author

Correspondence to Edwin van der Pol .

Editor information

Editors and Affiliations

  1. National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy

    Maurizio Federico

  2. National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy

    Barbara Ridolfi

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Hajji, N., Hau, C.M., Nieuwland, R., van der Pol, E. (2022). Protocol for Measuring Concentrations of Extracellular Vesicles in Human Blood Plasma with Flow Cytometry. In: Federico, M., Ridolfi, B. (eds) Extracellular Vesicles in Diagnosis and Therapy. Methods in Molecular Biology, vol 2504. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2341-1_5

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  • DOI: https://doi.org/10.1007/978-1-0716-2341-1_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2340-4

  • Online ISBN: 978-1-0716-2341-1

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