The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/µL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry.

BIOCEV 1st Faculty of Medicine Charles University 252 50 Vestec Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University 128 44 Prague Czech Republic

Department of Neurology and Clinical Neuroscience 1st Faculty of Medicine Charles University and General University Hospital 128 21 Prague Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University 128 00 Prague Czech Republic

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University 128 00 Prague Czech Republic

Zobrazit více v PubMed

Thompson A.J., Baranzini S.E., Geurts J., Hemmer B., Ciccarelli O. Multiple sclerosis. Lancet. 2018;391:1622–1636. doi: 10.1016/S0140-6736(18)30481-1. PubMed DOI

Saposnik G., Sempere A.P., Raptis R., Prefasi D., Selchen D., Maurino J. Decision making under uncertainty, therapeutic inertia, and physicians’ risk preferences in the management of multiple sclerosis (DIScUTIR MS) BMC Neurol. 2016;16:9. doi: 10.1186/s12883-016-0577-4. PubMed DOI PMC

Trapp B.D., Nave K.A. Multiple sclerosis: An immune or neurodegenerative disorder? Annu. Rev. Neurosci. 2008;31:247–269. doi: 10.1146/annurev.neuro.30.051606.094313. PubMed DOI

Nylander A., Hafler D.A. Multiple sclerosis. J. Clin. Investig. 2012;122:1180–1188. doi: 10.1172/JCI58649. PubMed DOI PMC

Raposo G., Stoorvogel W. Extracellular vesicles: Exosomes, microvesicles, and friends. J. Cell Biol. 2013;200:373–383. doi: 10.1083/jcb.201211138. PubMed DOI PMC

Porro C., Trotta T., Panaro M.A. Microvesicles in the brain: Biomarker, messenger or mediator? J. Neuroimmunol. 2015;288:70–78. doi: 10.1016/j.jneuroim.2015.09.006. PubMed DOI

Carandini T., Colombo F., Finardi A., Casella G., Garzetti L., Verderio C., Furlan R. Microvesicles: What is the role in multiple sclerosis? Front. Neurol. 2015;6:7. doi: 10.3389/fneur.2015.00111. PubMed DOI PMC

Akers J.C., Gonda D., Kim R., Carter B.S., Chen C.C. Biogenesis of extracellular vesicles (EV): Exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J. Neuro-Oncol. 2013;113:1–11. doi: 10.1007/s11060-013-1084-8. PubMed DOI PMC

Sáenz-Cuesta M., Osorio-Querejeta I., Otaegui D. Extracellular Vesicles in Multiple Sclerosis: What are They Telling Us? Front. Cell. Neurosci. 2014;8:100. doi: 10.3389/fncel.2014.00100. PubMed DOI PMC

Burnouf T., Chou M.-L., Goubran H., Cognasse F., Garraud O., Seghatchian J. An overview of the role of microparticles/microvesicles in blood components: Are they clinically beneficial or harmful? Transfus. Apher. Sci. 2015;53:137–145. doi: 10.1016/j.transci.2015.10.010. PubMed DOI

Ohno S., Ishikawa A., Kuroda M. Roles of exosomes and microvesicles in disease pathogenesis. Adv. Drug Deliv. Rev. 2013;65:398–401. doi: 10.1016/j.addr.2012.07.019. PubMed DOI

Julich H., Mims A., Lukacs-Kornek V., Kornek M. Extracellular vesicle profiling and their use as potential disease specific biomarker. Front. Immunol. 2014;5:6. doi: 10.3389/fimmu.2014.00413. PubMed DOI PMC

Słomka A., Urban S.K., Lukacs-Kornek V., Żekanowska E., Kornek M. Large Extracellular Vesicles: Have We Found the Holy Grail of Inflammation? Front. Immunol. 2018;9:2723. doi: 10.3389/fimmu.2018.02723. PubMed DOI PMC

Croese T., Furlan R. Extracellular vesicles in neurodegenerative diseases. Mol. Asp. Med. 2018;60:52–61. doi: 10.1016/j.mam.2017.11.006. PubMed DOI

Blonda M., Amoruso A., Martino T., Avolio C. New Insights Into Immune Cell-Derived Extracellular Vesicles in Multiple Sclerosis. Front. Neurol. 2018;9:8. doi: 10.3389/fneur.2018.00604. PubMed DOI PMC

Pieragostino D., Lanuti P., Cicalini I., Cufaro M.C., Ciccocioppo F., Ronci M., Simeone P., Onofrj M., van der Pol E., Fontana A., et al. Proteomics characterization of extracellular vesicles sorted by flow cytometry reveals a disease-specific molecular cross-talk from cerebrospinal fluid and tears in multiple sclerosis. J. Proteom. 2019;204:10. doi: 10.1016/j.jprot.2019.103403. PubMed DOI

Minagar A., Jy W., Jimenez J.J., Sheremata W.A., Mauro L.M., Mao W.W., Horstman L.L., Ahn Y.S. Elevated plasma endothelial microparticles in multiple sclerosis. Neurology. 2001;56:1319–1324. doi: 10.1212/WNL.56.10.1319. PubMed DOI

Sheremata W.A., Jy W., Delgado S., Minagar A., McLarty J., Ahn Y. Interferon-β1a reduces plasma CD31+ endothelial microparticles (CD31+EMP) in multiple sclerosis. J. Neuroinflamm. 2006;3:5. doi: 10.1186/1742-2094-3-23. PubMed DOI PMC

Lowery-Nordberg M., Eaton E., Gonzalez-Toledo E., Harris M.K., Chalamidas K., McGee-Brown J., Ganta C.V., Minagar A., Cousineau D., Alexander J.S. The effects of high dose interferon-beta 1a on plasma microparticles: Correlation with MRI parameters. J. Neuroinflamm. 2011;8:6. doi: 10.1186/1742-2094-8-43. PubMed DOI PMC

Marcos-Ramiro B., Nacarino P.O., Serrano-Pertierra E., Blanco-Gelaz M.A., Weksler B.B., Romero I.A., Couraud P.O., Tunon A., Lopez-Larrea C., Millan J., et al. Microparticles in multiple sclerosis and clinically isolated syndrome: Effect on endothelial barrier function. BMC Neurosci. 2014;15:13. doi: 10.1186/1471-2202-15-110. PubMed DOI PMC

Sheremata W.A., Jy W., Horstman L.L., Ahn Y.S., Alexander S., Minagar A. Evidence of platelet activation in multiple sclerosis. J. Neuroinflamm. 2008;5:6. doi: 10.1186/1742-2094-5-27. PubMed DOI PMC

Saenz-Cuesta M., Irizar H., Castillo-Trivino T., Munoz-Culla M., Osorio-Querejeta I., Prada A., Sepulveda L., Lopez-Mato M.P., de Munain A.L., Comabella M., et al. Circulating microparticles reflect treatment effects and clinical status in multiple sclerosis. Biomark. Med. 2014;8:653–661. doi: 10.2217/bmm.14.9. PubMed DOI

Alexander J.S., Chervenak R., Weinstock-Guainan B., Tsunoda I., Ramanathan M., Martinez N., Omura S., Sato F., Chaitanya G.V., Minagar A., et al. Blood circulating microparticle species in relapsing-remitting and secondary progressive multiple sclerosis. A case-control, cross sectional study with conventional MRI and advanced iron content imaging outcomes. J. Neurol. Sci. 2015;355:84–89. doi: 10.1016/j.jns.2015.05.027. PubMed DOI PMC

Momen-Heravi F., Balaj L., Alian S., Trachtenberg A.J., Hochberg F.H., Skog J., Kuo W.P. Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front. Physiol. 2012;3:6. doi: 10.3389/fphys.2012.00162. PubMed DOI PMC

Welsh J.A., Horak P., Wilkinson J.S., Ford V.J., Jones J.C., Smith D., Holloway J.A., Englyst N.A. FCMPASS Software Aids Extracellular Vesicle Light Scatter Standardization. Cytom. Part A. 2020;97:569–581. doi: 10.1002/cyto.a.23782. PubMed DOI PMC

Dubochet J., Adrian M., Chang J.J., Homo J.C., Lepault J., McDowall A.W., Schultz P. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 1988;21:129–228. doi: 10.1017/S0033583500004297. PubMed DOI

van der Pol E., van Gemert M.J.C., Sturk A., Nieuwland R., Van Leeuwen T.G. Single vs. swarm detection of microparticles and exosomes by flow cytometry. J. Thromb. Haemost. 2012;10:919–930. doi: 10.1111/j.1538-7836.2012.04683.x. PubMed DOI

Colombo E., Borgiani B., Verderio C., Furlan R. Microvesicles: Novel biomarkers for neurological disorders. Front. Physiol. 2012;3:6. doi: 10.3389/fphys.2012.00063. PubMed DOI PMC

Properzi F., Logozzi M., Fais S. Exosomes: The future of biomarkers in medicine. Biomark. Med. 2013;7:769–778. doi: 10.2217/bmm.13.63. PubMed DOI

Jy W., Minagar A., Jimenez J.J., Sheremata W.A., Mauro L.M., Horstman L.L., Bidot C., Ahn Y.S. Endothelial microparticles (EMP) bind and activate monocytes: Elevated empmonocyte conjugates in multiple sclerosis. Front. Biosci. Landmark. 2004;9:3137–3144. doi: 10.2741/1466. PubMed DOI

Simak J., Holada K., Risitano A.M., Zivny J.H., Young N.S., Vostal J.G. Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria. Br. J. Haematol. 2004;125:804–813. doi: 10.1111/j.1365-2141.2004.04974.x. PubMed DOI

Ojeda-Fernandez L., Recio-Poveda L., Aristorena M., Lastres P., Blanco F.J., Sanz-Rodriguez F., Gallardo-Vara E., de las Casas-Engel M., Corbi A., Arthur H.M., et al. Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response. PLoS Genet. 2016;12:24. doi: 10.1371/journal.pgen.1005935. PubMed DOI PMC

Rossi E., Pericacho M., Bachelot-Loza C., Pidard D., Gaussem P., Poirault-Chassac S., Blanco F.J., Langa C., Gonzalez-Manchon C., Novoa J.M.L., et al. Human endoglin as a potential new partner involved in platelet-endothelium interactions. Cell. Mol. Life Sci. 2018;75:1269–1284. doi: 10.1007/s00018-017-2694-7. PubMed DOI PMC

Suades R., Padro T., Vilahur G., Martin-Yuste V., Sabate M., Sans-Rosello J., Sionis A., Badimon L. Growing thrombi release increased levels of CD235a(+) microparticles and decreased levels of activated platelet-derived microparticles. Validation in ST-elevation myocardial infarction patients. J. Thromb. Haemost. 2015;13:1776–1786. doi: 10.1111/jth.13065. PubMed DOI

Ingersoll M.A., Spanbroek R., Lottaz C., Gautier E.L., Frankenberger M., Hoffmann R., Lang R., Haniffa M., Collin M., Tacke F., et al. Comparison of gene expression profiles between human and mouse monocyte subsets. Blood. 2010;115:E10–E19. doi: 10.1182/blood-2009-07-235028. PubMed DOI PMC

Swerlick R.A., Lee K.H., Wick T.M., Lawley T.J. Human Dermal Microvascular Endothelial but not Human Umbilical Vein Endothelial-Cells Express Cd36 in vivo and in vitro. J. Immunol. 1992;148:78–83. PubMed

Mitjavila-Garcia M.T., Cailleret M., Godin I., Nogueira M.M., Cohen-Solal K., Schiavon V., Lecluse Y., Le Pesteur F., Lagrue A.N., Vainchenker W. Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells. Development. 2002;129:2003–2013. doi: 10.1242/dev.129.8.2003. PubMed DOI

Rheinlander A., Schraven B., Bommhardt U. CD45 in human physiology and clinical medicine. Immunol. Lett. 2018;196:22–32. doi: 10.1016/j.imlet.2018.01.009. PubMed DOI

Wang K.M., Wei G.Q., Liu D.L. CD19: A biomarker for B cell development, lymphoma diagnosis and therapy. Exp. Hematol. Oncol. 2012;1:7. doi: 10.1186/2162-3619-1-36. PubMed DOI PMC

Vermes I., Haanen C., Reutelingsperger C. Flow cytometry of apoptotic cell death. J. Immunol. Methods. 2000;243:167–190. doi: 10.1016/S0022-1759(00)00233-7. PubMed DOI

Skotland T., Hessvik N.P., Sandvig K., Llorente A. Thematic Review Series: Exosomes and Microvesicles: Lipids as Key Components of their Biogenesis and Functions Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology. J. Lipid Res. 2019;60:9–18. doi: 10.1194/jlr.R084343. PubMed DOI PMC

Sodar B.W., Kittel A., Paloczi K., Vukman K.V., Osteikoetxea X., Szabo-Taylor K., Nemeth A., Sperlagh B., Baranyai T., Giricz Z., et al. Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci. Rep. 2016;6:12. doi: 10.1038/srep24316. PubMed DOI PMC

Vickers K.C., Palmisano B.T., Shoucri B.M., Shamburek R.D., Remaley A.T. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat. Cell Biol. 2011;13:423–433. doi: 10.1038/ncb2210. PubMed DOI PMC

Lima E.S., Maranhao R.C. Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma. Clin. Chem. 2004;50:1086–1088. doi: 10.1373/clinchem.2004.032383. PubMed DOI

Jamaly S., Ramberg C., Olsen R., Latysheva N., Webster P., Sovershaev T., Braekkan S.K., Hansen J.B. Impact of preanalytical conditions on plasma concentration and size distribution of extracellular vesicles using Nanoparticle Tracking Analysis. Sci. Rep. 2018;8:11. doi: 10.1038/s41598-018-35401-8. PubMed DOI PMC

Jayachandran M., Miller V.M., Heit J.A., Owen W.G. Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J. Immunol. Methods. 2012;375:207–214. doi: 10.1016/j.jim.2011.10.012. PubMed DOI PMC

Kong F.C., Zhang L.M., Wang H.X., Yuan G.L., Guo A.Y., Li Q.B., Chen Z.C. Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis. Exp. Ther. Med. 2015;10:2093–2101. doi: 10.3892/etm.2015.2780. PubMed DOI PMC

Hujacova A., Brozova T., Mosko T., Kostelanska M., Stranak Z., Holada K. Platelet Extracellular Vesicles in Cord Blood of Term and Preterm Newborns Assayed by Flow Cytometry: The Effect of Delay in Sample Preparation and of Sample Freezing. Folia Biol. 2020;66:204–211. PubMed

De Rond L., Libregts S., Rikkert L.G., Hau C.M., van der Pol E., Nieuwland R., van Leeuwen T.G., Coumans F.A.W. Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry. J. Extracell. Vesicles. 2019;8:10. doi: 10.1080/20013078.2019.1643671. PubMed DOI PMC

Erdbrugger U., Rudy C.K., Etter M.E., Dryden K.A., Yeager M., Klibanov A.L., Lannigan J. Imaging Flow Cytometry Elucidates Limitations of Microparticle Analysis by Conventional Flow Cytometry. Cytom. Part A. 2014;85:756–770. doi: 10.1002/cyto.a.22494. PubMed DOI

Lucchetti D., Battaglia A., Ricciardi-Tenore C., Colella F., Perelli L., de Maria R., Scambia G., Sgambato A., Fattorossi A. Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality? Int. J. Mol. Sci. 2020;21:15. doi: 10.3390/ijms21176257. PubMed DOI PMC

Holcar M., Kanduser M., Lenassi M. Blood Nanoparticles—Influence on Extracellular Vesicle Isolation and Characterization. Front. Pharmacol. 2021;12:20. doi: 10.3389/fphar.2021.773844. PubMed DOI PMC

Chandler W.L. Measurement of Microvesicle Levels in Human Blood Using Flow Cytometry. Cytom. Part B Clin. Cytom. 2016;90:326–336. doi: 10.1002/cyto.b.21343. PubMed DOI

Yuana Y., Koning R.I., Kuil M.E., Rensen P.C.N., Koster A.J., Bertina R.M., Osanto S. Cryo-electron microscopy of extracellular vesicles in fresh plasma. J. Extracell. Vesicles. 2013;2:7. doi: 10.3402/jev.v2i0.21494. PubMed DOI PMC

Masvekar R., Mizrahi J., Park J., Williamson P.R., Bielekova B. Quantifications of CSF Apoptotic Bodies Do not Provide Clinical Value in Multiple Sclerosis. Front. Neurol. 2019;10:11. doi: 10.3389/fneur.2019.01241. PubMed DOI PMC

de Rond L., van der Pol E., Bloemen P.R., Van Den Broeck T., Monheim L., Nieuwland R., van Leeuwen T.G., Coumans F.A.W. A Systematic Approach to Improve Scatter Sensitivity of a Flow Cytometer for Detection of Extracellular Vesicles. Cytom. Part A. 2020;97:582–591. doi: 10.1002/cyto.a.23974. PubMed DOI PMC

Thery C., Witwer K.W., Aikawa E., Alcaraz M.J., Anderson J.D., Andriantsitohaina R., Antoniou A., Arab T., Archer F., Atkin-Smith G.K., et al. 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. 2018;7:43. doi: 10.1080/20013078.2018.1535750. PubMed DOI PMC