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Extracellular vesicles as biomarkers in liver diseases: A clinician's point of view

  • Sara Thietart
    Affiliations
    Université de Paris, Centre de recherche sur l'inflammation, Inserm, F-75018 Paris, France
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  • Pierre-Emmanuel Rautou
    Correspondence
    Corresponding author. Address: Service d'hépatologie, Hôpital Beaujon, 100, boulevard du Général Leclerc, 92100 Clichy, France. Tel.: +33.1.40. 87.52.83; Fax: +33.1. 40.87.55.30.
    Affiliations
    Université de Paris, Centre de recherche sur l'inflammation, Inserm, F-75018 Paris, France

    Service d'Hépatologie, DHU Unity, Pôle des Maladies de l'Appareil Digestif, Hôpital Beaujon, AP-HP, Clichy, France

    Centre de Référence des Maladies Vasculaires du Foie, French Network for Rare Liver Diseases (FILFOIE), European Reference Network (ERN) ‘Rare-Liver’
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      Summary

      Extracellular vesicles are membrane-bound vesicles containing proteins, lipids, RNAs and microRNAs. They can originate from both healthy and stressed cells, and provide a snapshot of the cell of origin in physiological and pathological circumstances. Various processes that may give rise to the release of extracellular vesicles occur in liver diseases, including hepatocyte apoptosis, hepatic stellate cell activation, liver innate immune system activation, systemic inflammation, and organelle dysfunction (mitochondrial dysfunction and endoplasmic reticulum stress). Numerous studies have therefore investigated the potential role of extracellular vesicles as biomarkers in liver diseases. This review provides an overview of the methods that can be used to measure extracellular vesicle concentrations in clinical settings, ranging from plasma preparation to extracellular vesicle measurement techniques, as well as looking at the challenges of using extracellular vesicles as biomarkers. We also provide a comprehensive review of studies that test extracellular vesicles as diagnostic, severity and prognostic biomarkers in various liver diseases, including non-alcoholic and alcoholic steatohepatitis, viral hepatitis B and C infections, cirrhosis, primary liver cancers, primary sclerosing cholangitis and acute liver failure. In particular, extracellular vesicles could be useful tools to evaluate activity and fibrosis in non-alcoholic fatty liver disease, predict risk of hepatitis B virus reactivation, predict complications and mortality in cirrhosis, detect early hepatocellular carcinoma, detect malignant transformation in primary sclerosing cholangitis and predict outcomes in acute liver failure. While most studies draw on data derived from pilot studies, which still require clinical validation, some extracellular vesicle subpopulations have already been evaluated in solid prospective studies.

      Keywords

      Linked Article

      What are extracellular vesicles?

      Extracellular vesicles form a heterogeneous group of membrane-bound vesicles that contain cell-derived biomolecules, such as proteins, lipids, RNAs and microRNAs (miRNAs).
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      In this review, we therefore use the terms “small”, and “larger” extracellular vesicles, using a size cut-off of 100 to 200 nm (the smallest size of extracellular vesicle that can be detected using flow cytometry).
      How to name extracellular vesicles?
      Once released into the extracellular compartment, extracellular vesicles can reach target cells for intercellular communication. The methods by which extracellular vesicles communicate are varied and include activation of surface receptors, transfer of vesicle content into the target cell by vesicle internalisation (phagocytosis, endocytosis) or membrane fusion.
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      The role of extracellular vesicles in cell-cell communication in liver diseases has been thoroughly reviewed elsewhere.
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      Circulating extracellular vesicle concentrations and composition thus vary with disease conditions, with some specificity for the ongoing pathological process. These characteristics make extracellular vesicles useful tools for personalised medicine, as they can be used to diagnose disorders, and evaluate prognosis, disease progression and response to treatment. Circulating extracellular vesicles have therefore emerged as attractive biomarkers in liver diseases, as discussed in this review.

      Measurement methods in clinical use

      This section provides the reader with an overview of the pre-analytical and analytical requirements for high-throughput measurement of extracellular vesicles in a clinical setting. Therefore, we do not discuss techniques for the analysis of extracellular vesicles in research laboratories (which are reviewed extensively elsewhere
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      Methodological guidelines to study extracellular vesicles.
      ).

      Patient requirements

      Like most routinely used blood tests, plasma extracellular vesicle concentrations can be influenced by factors such as age, sex, pregnancy, menopausal status, fasting, circadian variations, exercise, body mass index, diet, comorbidities, and medication.
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      It is therefore important to collect these data and use matched control groups in order to manage this variability.

      Pre-analytical requirements

      Several pre-analytical factors can influence extracellular vesicle measurement. Hence, standardisation of plasma sample collection and processing is necessary to ensure comparability among studies and, ultimately, to permit the correct interpretation of the results at an individual level.
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      Plasma sample collection, preparation, and storage

      Many factors can induce the release of extracellular vesicles by blood cells during or after blood draw.
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      Consequently, over the past few years, each step of sample collection and preparation has been standardised to limit this unwanted release of extracellular vesicles by blood cells in test tubes. It could be assumed that extracellular vesicles derived from non-circulating cells, such as hepatocytes, would be less likely to be subject to additional extracellular vesicle release in the test tube, since mother cells are not present in the tube. In any case, each step – from sample collection to plasma preparation and storage – should be meticulously described.
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      Researchers can upload experimental protocols from a crowd-sourcing knowledge base called EV-TRACK (http://evtrack.org).
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      The International Society on Thrombosis and Haemostasis
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      and the International Society for Advancement of Cytometry
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      Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants.
      recommend the use of citrate tubes (as opposed to ethylene diamine tetraacetic acid and heparin), as their use better prevents platelet activation and formation of extracellular vesicles.
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      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.
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      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      The American Heart Association's (AHA) 2017 guidelines
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      and the International Society for Extracellular Vesicles' (ISEV) 2017 position paper
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      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      recommend that the choice of anticoagulants be adapted to downstream analysis. For example, acid citrate dextrose is considered to be more suitable for RNA analysis.
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      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
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      Blood sample collection, platelet-free plasma preparation and storage, adapted from ISEV 2013, AHA 2017, ISAC 2016 and ISTH 2015 guidelines.
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      • Gool E.L.
      • Nieuwland R.
      Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Witwer K.W.
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      • Bemis L.T.
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      • Lässer C.
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      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ,
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      • Lamm C.
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      • Preißing F.
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      Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants.
      Tabled 1
      AHA, American Heart Society; EDTA, ethylene diamine tetraacetic acid; ISAC, International Society for Advancement of Cytometry; ISEV, International Society of Extracellular Vesicles; ISTH, International Society of Thrombosis and Hemostasis.
      Plasma is stored at −80°C, avoiding repeated freeze-thaw cycles and thawed at 37°C. However, a unique freeze-thaw cycle does not alter plasma extracellular vesicles.
      • Lacroix R.
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      • Key N.S.
      • Dignat-George F.
      • et al.
      Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop.
      It has been shown that small extracellular vesicle miRNAs could still be assessed from samples frozen for up to 8 years.
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      Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
      Extracellular vesicles are membrane-bound vesicles, containing cell-derived biomolecules, such as proteins, lipids and RNAs.

      Separation methods

      Most techniques for measurement of extracellular vesicle concentration require separation of extracellular vesicles from the rest of the plasma.
      Currently, no extracellular vesicle separation method can entirely separate extracellular vesicles from the remaining plasma. A balance must be found between maximising recovery of extracellular vesicles (to avoid loss of information) and minimising contaminants (soluble proteins, protein aggregates, lipoproteins, nucleic acids and viruses). Some authors use combinations of techniques such as ultrafiltration, density gradients or washing with extracellular vesicle-free buffer. This increases purity, but is more labour intensive.
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      The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray.
      ,
      • Mol E.A.
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      ,
      • Cheng L.
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      Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood.
      Separation methods can isolate non-vesicular circulating miRNAs or RNAs together with extracellular vesicles.
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      Reassessment of exosome composition.
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      The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.
      Since treatment of extracellular vesicles with deoxyribonuclease/ribonuclease (DNase/RNase) does not interfere with RNA analysis and degrades externally bound RNA, its use is recommended by the ISEV and AHA.
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      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
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      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Hill A.F.
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      Interpretation of data should take into account isolation techniques and use of ribonuclease treatment. For small cohorts, it is useful to verify the reliability of separation methods, by quantifying contaminants (measuring albumin, ApoB100, ApoA1 and ApoB48) and/or by characterising the aspect of separate extracellular vesicles (for example, by using transmission electron microscopy).
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      ,
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      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
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      • Buzás E.I.
      • Bemis L.T.
      • Bora A.
      • Lässer C.
      • Lötvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ,
      • Jayachandran M.
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      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ,
      • Andreu Z.
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      • Sanguino-Pascual A.
      • Lamana A.
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      • et al.
      Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
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      The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.
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      • Módos K.
      • Marton N.
      • et al.
      Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods.
      • Vergauwen G.
      • Dhondt B.
      • Van Deun J.
      • De Smedt E.
      • Berx G.
      • Timmerman E.
      • et al.
      Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.
      • Rautou P.-E.
      • Bresson J.
      • Sainte-Marie Y.
      • Vion A.-C.
      • Paradis V.
      • Renard J.-M.
      • et al.
      Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.
      • Payancé A.
      • Silva-Junior G.
      • Bissonnette J.
      • Tanguy M.
      • Pasquet B.
      • Levi C.
      • et al.
      Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis.
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • Roux O.
      • Payancé A.
      • Lebrec D.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.
      • Enderle D.
      • Spiel A.
      • Coticchia C.M.
      • Berghoff E.
      • Mueller R.
      • Schlumpberger M.
      • et al.
      Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel Spin column-based method.
      Some separation methods are unlikely to be suitable for use in clinical routines, including differential centrifugation, density gradient centrifugation and immunoaffinity capture. These methods are described in the supplementary text.
      • van der Pol E.
      • Böing A.N.
      • Gool E.L.
      • Nieuwland R.
      Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Witwer K.W.
      • Buzás E.I.
      • Bemis L.T.
      • Bora A.
      • Lässer C.
      • Lötvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ,
      • Van Deun J.
      • Mestdagh P.
      • Agostinis P.
      • Akay Ö.
      • Anand S.
      • Anckaert J.
      • et al.
      EV-TRACK Consortium
      EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.
      ,
      • Gardiner C.
      • Di Vizio D.
      • Sahoo S.
      • Théry C.
      • Witwer K.W.
      • Wauben M.
      • et al.
      Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey.
      ,
      • Momen-Heravi F.
      • Balaj L.
      • Alian S.
      • Mantel P.-Y.
      • Halleck A.E.
      • Trachtenberg A.J.
      • et al.
      Current methods for the isolation of extracellular vesicles.
      • Taylor D.D.
      • Shah S.
      Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.
      • Stranska R.
      • Gysbrechts L.
      • Wouters J.
      • Vermeersch P.
      • Bloch K.
      • Dierickx D.
      • et al.
      Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma.
      ,
      • Enderle D.
      • Spiel A.
      • Coticchia C.M.
      • Berghoff E.
      • Mueller R.
      • Schlumpberger M.
      • et al.
      Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel Spin column-based method.
      Other separation methods seem to be better adapted to clinical settings: (a) Size-exclusion chromatography separates extracellular vesicles from other components based on size.
      • Böing A.N.
      • van der Pol E.
      • Grootemaat A.E.
      • Coumans F.A.W.
      • Sturk A.
      • Nieuwland R.
      Single-step isolation of extracellular vesicles by size-exclusion chromatography.
      It uses a column containing porous beads: proteins are small enough to pass through the pores, but extracellular vesicles are not. Extracellular vesicles therefore migrate at a higher speed than soluble proteins (Fig. 1). However, if the molecule (such as a lipoprotein) is the same size as the extracellular vesicle, both will migrate at the same speed and can therefore be co-isolated.
      • Takov K.
      • Yellon D.M.
      • Davidson S.M.
      Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential.
      This technique has been shown to give intact and functional vesicles, as well as fewer protein and lipoprotein contaminants than other methods, although it achieves suboptimal purity.
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Mol E.A.
      • Goumans M.-J.
      • Doevendans P.A.
      • Sluijter J.P.G.
      • Vader P.
      Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation.
      ,
      • Takov K.
      • Yellon D.M.
      • Davidson S.M.
      Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential.
      ,
      • Stranska R.
      • Gysbrechts L.
      • Wouters J.
      • Vermeersch P.
      • Bloch K.
      • Dierickx D.
      • et al.
      Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma.
      ,
      • Baranyai T.
      • Herczeg K.
      • Onódi Z.
      • Voszka I.
      • Módos K.
      • Marton N.
      • et al.
      Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods.
      ,
      • Hong C.-S.
      • Funk S.
      • Muller L.
      • Boyiadzis M.
      • Whiteside T.L.
      Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.
      Automated acquisition methods have recently been developed, which make this technique less labour intensive and more useful in clinical settings. (b) Filtration can be used to separate smaller-sized soluble components (which can pass through pores) from extracellular vesicles which are retained on the filter. The filter reference number is important, as filter type and pore size have been shown to heavily influence recovery.
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Vergauwen G.
      • Dhondt B.
      • Van Deun J.
      • De Smedt E.
      • Berx G.
      • Timmerman E.
      • et al.
      Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.
      The effect on vesicle disruption of the forces applied to push samples through the filters is unknown.
      • Taylor D.D.
      • Shah S.
      Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.
      (c) Precipitation kits (such as, ExoQuick™, miRCURY™ Exosome Isolation Kit or Invitrogen™ Total Exosome Isolation Kit) recover large quantities of extracellular vesicles, but are associated with poor purity, as they also recover protein complexes and extracellular vesicle aggregates.
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Van Deun J.
      • Mestdagh P.
      • Sormunen R.
      • Cocquyt V.
      • Vermaelen K.
      • Vandesompele J.
      • et al.
      The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.
      Their application should be limited to samples known to be rich in small extracellular vesicles, for the study of RNA, or as a concentration method after using another separation method.
      • Witwer K.W.
      • Buzás E.I.
      • Bemis L.T.
      • Bora A.
      • Lässer C.
      • Lötvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ,
      • Andreu Z.
      • Rivas E.
      • Sanguino-Pascual A.
      • Lamana A.
      • Marazuela M.
      • González-Alvaro I.
      • et al.
      Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
      ,
      • Taylor D.D.
      • Shah S.
      Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.
      Thus, as a general rule, precipitation kits should be considered with caution in a biomarker setting.
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      Table 1Main separation methods of extracellular vesicles.
      MethodType of EV recoveredEV recoveryPurityCommentApplicability in clinical settingsRef.
      Methods unlikely to be used in clinical routine
       Differential centrifugationSmall & larger EVsIntermediate (2–80%): depends on size and density (i.e. EV cargo)Intermediate: co-recovery of protein aggregates and virusesHigh variability (depending on rotors, dilution method, sample viscosity)

      Risk of EV aggregation or damage
      No:

      Time consuming (2–9 h), laborious, low-throughput
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Witwer K.W.
      • Buzás E.I.
      • Bemis L.T.
      • Bora A.
      • Lässer C.
      • Lötvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ,
      • Jayachandran M.
      • Miller V.M.
      • Heit J.A.
      • Owen W.G.
      Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
      ,
      • Momen-Heravi F.
      • Balaj L.
      • Alian S.
      • Mantel P.-Y.
      • Halleck A.E.
      • Trachtenberg A.J.
      • et al.
      Current methods for the isolation of extracellular vesicles.
      ,
      • Momen-Heravi F.
      • Balaj L.
      • Alian S.
      • Trachtenberg A.J.
      • Hochberg F.H.
      • Skog J.
      • et al.
      Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles.
       Density gradient centrifugationSmall&larger EVsLow (10–50%).

      High for small EVs
      Intermediate: co-recovery with lipoproteins.

      High for small EVs
      High variability.

      Risk of damage and loss of biological activity.
      No: time consuming (6–48 h), laborious, low-throughput
      • van der Pol E.
      • Böing A.N.
      • Gool E.L.
      • Nieuwland R.
      Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Van Deun J.
      • Mestdagh P.
      • Sormunen R.
      • Cocquyt V.
      • Vermaelen K.
      • Vandesompele J.
      • et al.
      The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.
      ,
      • Taylor D.D.
      • Shah S.
      Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.
       Immunocapture assaysSmall & larger EVs

      Specific subpopulations
      IntermediateIntermediate, poor on plasmaVariability depending on antibody panel.

      Low hand-on time.
      Only for media with few protein contaminants.

      Not for plasma.

      High-throughput when using multiwell plates.
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Witwer K.W.
      • Buzás E.I.
      • Bemis L.T.
      • Bora A.
      • Lässer C.
      • Lötvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ,
      • Taylor D.D.
      • Shah S.
      Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.
      ,
      • Stranska R.
      • Gysbrechts L.
      • Wouters J.
      • Vermeersch P.
      • Bloch K.
      • Dierickx D.
      • et al.
      Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma.
      ,
      • Enderle D.
      • Spiel A.
      • Coticchia C.M.
      • Berghoff E.
      • Mueller R.
      • Schlumpberger M.
      • et al.
      Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel Spin column-based method.
      Methods adapted to clinical routine
       Size-exclusion chromatographySmall & larger EVsHigh

      Depends on pore size.
      High

      Depends on pore size and column height.
      Important dilution

      Commercialised columns available
      Maybe: easy and fast (30 min) but labour intensive
      • van der Pol E.
      • Böing A.N.
      • Gool E.L.
      • Nieuwland R.
      Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.
      ,
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Stranska R.
      • Gysbrechts L.
      • Wouters J.
      • Vermeersch P.
      • Bloch K.
      • Dierickx D.
      • et al.
      Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma.
      ,
      • Baranyai T.
      • Herczeg K.
      • Onódi Z.
      • Voszka I.
      • Módos K.
      • Marton N.
      • et al.
      Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods.
       FiltrationSmall & larger EVsVariable: Depends on membrane type and pore size.HighHigh variability depending on filter typeYes, for larger EVs: easy, fast (20 min) and reproducible if same filter is used.

      Not for small EVs.
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Andreu Z.
      • Rivas E.
      • Sanguino-Pascual A.
      • Lamana A.
      • Marazuela M.
      • González-Alvaro I.
      • et al.
      Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
      ,
      • Vergauwen G.
      • Dhondt B.
      • Van Deun J.
      • De Smedt E.
      • Berx G.
      • Timmerman E.
      • et al.
      Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.
      • Rautou P.-E.
      • Bresson J.
      • Sainte-Marie Y.
      • Vion A.-C.
      • Paradis V.
      • Renard J.-M.
      • et al.
      Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.
      • Payancé A.
      • Silva-Junior G.
      • Bissonnette J.
      • Tanguy M.
      • Pasquet B.
      • Levi C.
      • et al.
      Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis.
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • Roux O.
      • Payancé A.
      • Lebrec D.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.
       Precipitation kitsSmall EVsHigh (90%)Poor: co-recovery of protein complexes and non-EV particlesShould be used as an EV concentration method or for studying EV RNAsYes: inexpensive, fast, small sample volumes
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Andreu Z.
      • Rivas E.
      • Sanguino-Pascual A.
      • Lamana A.
      • Marazuela M.
      • González-Alvaro I.
      • et al.
      Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
      EV(s), extracellular vesicle(s).
      Figure thumbnail gr1
      Fig. 1Size-exclusion chromatography.
      Left. The column contains porous beads separating soluble proteins (which migrate at lower speed, as they are small enough to go through the pores) from extracellular vesicles. Right. As an example, plasma from a patient with Child-Pugh C cirrhosis goes through a size-exclusion chromatography column, progressively separating the extracellular vesicle compartment from the bilirubin-rich protein compartment. The chromatogram shows absorbance at 280 nm of each 2 ml fraction of plasma: extracellular vesicles are detected first, followed by soluble proteins.

      Other biofluid collection and separation methods

      In addition to plasma, extracellular vesicles have been detected in other biological fluids, including serum, urine, ascites and bile. Methods to collect these biofluids and separate extracellular vesicles are described in the supplementary text.
      • van der Pol E.
      • Böing A.N.
      • Gool E.L.
      • Nieuwland R.
      Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Witwer K.W.
      • Buzás E.I.
      • Bemis L.T.
      • Bora A.
      • Lässer C.
      • Lötvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ,
      • Wisgrill L.
      • Lamm C.
      • Hartmann J.
      • Preißing F.
      • Dragosits K.
      • Bee A.
      • et al.
      Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants.
      ,
      • Andreu Z.
      • Rivas E.
      • Sanguino-Pascual A.
      • Lamana A.
      • Marazuela M.
      • González-Alvaro I.
      • et al.
      Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
      ,
      • George J.N.
      • Thoi L.L.
      • McManus L.M.
      • Reimann T.A.
      Isolation of human platelet membrane microparticles from plasma and serum.
      • Pisitkun T.
      • Shen R.-F.
      • Knepper M.A.
      Identification and proteomic profiling of exosomes in human urine.
      • Awdishu L.
      • Tsunoda S.
      • Pearlman M.
      • Kokoy-Mondragon C.
      • Ghassemian M.
      • Naviaux R.K.
      • et al.
      Identification of maltase glucoamylase as a biomarker of acute kidney injury in patients with cirrhosis.
      • Merchant M.L.
      • Rood I.M.
      • Deegens J.K.J.
      • Klein J.B.
      Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.
      • Sirica A.E.
      • Gores G.J.
      • Groopman J.D.
      • Selaru F.M.
      • Strazzabosco M.
      • Wei Wang X.
      • et al.
      Intrahepatic cholangiocarcinoma: continuing challenges and translational advances.
      • Engelmann C.
      • Splith K.
      • Krohn S.
      • Herber A.
      • Boehlig A.
      • Boehm S.
      • et al.
      Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study.
      • Li L.
      • Masica D.
      • Ishida M.
      • Tomuleasa C.
      • Umegaki S.
      • Kalloo A.N.
      • et al.
      Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis.
      • Severino V.
      • Dumonceau J.-M.
      • Delhaye M.
      • Moll S.
      • Annessi-Ramseyer I.
      • Robin X.
      • et al.
      Extracellular vesicles in bile as markers of malignant biliary Stenoses.
      • Li L.
      • Piontek K.B.
      • Kumbhari V.
      • Ishida M.
      • Selaru F.M.
      Isolation and profiling of MicroRNA-containing exosomes from human bile.
      • Ge X.
      • Wang Y.
      • Nie J.
      • Li Q.
      • Tang L.
      • Deng X.
      • et al.
      The diagnostic/prognostic potential and molecular functions of long non-coding RNAs in the exosomes derived from the bile of human cholangiocarcinoma.
      • Hogan M.C.
      • Lieske J.C.
      • Lienczewski C.C.
      • Nesbitt L.L.
      • Wickman L.T.
      • Heyer C.M.
      • et al.
      Strategy and rationale for urine collection protocols employed in the NEPTUNE study.

      Analytical techniques

      From a biomarker perspective, measurement of plasma extracellular vesicle concentrations involves a compromise between very accurate characterisation of extracellular vesicles, as proposed by MISEV 2018 guidelines,
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      and operational strategies that can be adapted to routine laboratory settings in a rapid and inexpensive manner.

      Accurate characterisation

      Several steps are proposed in the MISEV 2018 guidelines
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      in order to establish an extracellular vesicle separation method. These steps include quantification, general characterisation, single vesicle characterisation and are described in the supplementary text.
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Arraud N.
      • Linares R.
      • Tan S.
      • Gounou C.
      • Pasquet J.-M.
      • Mornet S.
      • et al.
      Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration.
      • György B.
      • Módos K.
      • Pállinger E.
      • Pálóczi K.
      • Pásztói M.
      • Misják P.
      • et al.
      Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters.
      • Takov K.
      • Yellon D.M.
      • Davidson S.M.
      Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes.
      • Sódar B.W.
      • Kittel Á.
      • Pálóczi K.
      • Vukman K.V.
      • Osteikoetxea X.
      • Szabó-Taylor K.
      • et al.
      Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.
      • Larson M.C.
      • Luthi M.R.
      • Hogg N.
      • Hillery C.A.
      Calcium-phosphate microprecipitates mimic microparticles when examined with flow cytometry.
      Available technologies that may be used to perform all these steps are listed in Table 2.
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Arraud N.
      • Linares R.
      • Tan S.
      • Gounou C.
      • Pasquet J.-M.
      • Mornet S.
      • et al.
      Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration.
      ,
      • van der Pol E.
      • Hoekstra A.G.
      • Sturk A.
      • Otto C.
      • van Leeuwen T.G.
      • Nieuwland R.
      Optical and non-optical methods for detection and characterization of microparticles and exosomes.
      • Obeid S.
      • Ceroi A.
      • Mourey G.
      • Saas P.
      • Elie-Caille C.
      • Boireau W.
      Development of a NanoBioAnalytical platform for “on-chip” qualification and quantification of platelet-derived microparticles.
      • Liang K.
      • Liu F.
      • Fan J.
      • Sun D.
      • Liu C.
      • Lyon C.J.
      • et al.
      Nanoplasmonic quantification of tumor-derived extracellular vesicles in plasma microsamples for diagnosis and treatment monitoring.
      • Koliha N.
      • Wiencek Y.
      • Heider U.
      • Jüngst C.
      • Kladt N.
      • Krauthäuser S.
      • et al.
      A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.
      • Corso G.
      • Mäger I.
      • Lee Y.
      • Görgens A.
      • Bultema J.
      • Giebel B.
      • et al.
      Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography.
      • Gool E.L.
      • Stojanovic I.
      • Schasfoort R.B.M.
      • Sturk A.
      • van Leeuwen T.G.
      • Nieuwland R.
      • et al.
      Surface plasmon resonance is an analytically sensitive method for antigen profiling of extracellular vesicles.
      • Jørgensen M.M.
      • Bæk R.
      • Varming K.
      Potentials and capabilities of the extracellular vesicle (EV) array.
      • Szatanek R.
      • Baj-Krzyworzeka M.
      • Zimoch J.
      • Lekka M.
      • Siedlar M.
      • Baran J.
      The methods of choice for extracellular vesicles (EVs) characterization.
      • Tatischeff I.
      • Larquet E.
      • Falcón-Pérez J.M.
      • Turpin P.-Y.
      • Kruglik S.G.
      Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy.
      • Wyss R.
      • Grasso L.
      • Wolf C.
      • Grosse W.
      • Demurtas D.
      • Vogel H.
      Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy.
      • Coumans F.A.W.
      • Gool E.L.
      • Nieuwland R.
      Bulk immunoassays for analysis of extracellular vesicles.
      • Ramirez M.I.
      • Amorim M.G.
      • Gadelha C.
      • Milic I.
      • Welsh J.A.
      • Freitas V.M.
      • et al.
      Technical challenges of working with extracellular vesicles.
      These steps should be considered when setting up the detection of an extracellular vesicle marker, to assess the results of separation methods and to establish the likelihood that the biomarker is associated with extracellular vesicles and not with other co-separated materials.
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      These steps do not need to be repeated, once the separation and routine measurement method have been validated.
      Table 2Summary of available technologies to perform extracellular vesicle characterisation in research settings.
      QuantificationGeneral characterisationSingle vesicle characterisation
      Aim
      • Evaluation of EV recovery
      • Evaluation of purity
      • Quantification of EV subtype
      Confirmation of EV presence (protein markers)
      • Evaluation of EV integrity
      • Confirmation of EV presence
      • Evaluation of EV subtype (size)
      TechnologyParticle number:
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      • Nanoparticle tracking analysis
        • van der Pol E.
        • Hoekstra A.G.
        • Sturk A.
        • Otto C.
        • van Leeuwen T.G.
        • Nieuwland R.
        Optical and non-optical methods for detection and characterization of microparticles and exosomes.
      • High-resolution bead-based flow cytometry
      • Resistive pulse sensing
      • Cryo-electron microscopy
        • Arraud N.
        • Linares R.
        • Tan S.
        • Gounou C.
        • Pasquet J.-M.
        • Mornet S.
        • et al.
        Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration.
      • Surface plasmon resonance coupled to atomic force microscopy
        • Obeid S.
        • Ceroi A.
        • Mourey G.
        • Saas P.
        • Elie-Caille C.
        • Boireau W.
        Development of a NanoBioAnalytical platform for “on-chip” qualification and quantification of platelet-derived microparticles.
      RNA quantification:
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      • •Bioanalyzer pico chip
      • Quant-iT RiboGreen RNA Assay
      • Quantitative reverse transcription PCR
      Total protein count :
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      • •Colorimetric assays
      • Fluoremectric assays
      • Protein stain on SDS-PAGE
      Specific protein count:
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      • •ELISA
      • Bead-based flow cytometry
      • Aptamer- carbon nanotubes colorimetric assays
      • Nanoplasmon-enhanced scattering assay
        • Liang K.
        • Liu F.
        • Fan J.
        • Sun D.
        • Liu C.
        • Lyon C.J.
        • et al.
        Nanoplasmonic quantification of tumor-derived extracellular vesicles in plasma microsamples for diagnosis and treatment monitoring.
      • Bead-based flow cytometry
        • Théry C.
        • Witwer K.W.
        • Aikawa E.
        • Alcaraz M.J.
        • Anderson J.D.
        • Andriantsitohaina R.
        • 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.
      • Western blot (mainly for cell culture media)
        • Théry C.
        • Witwer K.W.
        • Aikawa E.
        • Alcaraz M.J.
        • Anderson J.D.
        • Andriantsitohaina R.
        • 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.
      • Multiplex bead-based platforms
        • Koliha N.
        • Wiencek Y.
        • Heider U.
        • Jüngst C.
        • Kladt N.
        • Krauthäuser S.
        • et al.
        A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.
        ,
        • Corso G.
        • Mäger I.
        • Lee Y.
        • Görgens A.
        • Bultema J.
        • Giebel B.
        • et al.
        Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography.
      • Surface plasmon resonance
        • Gool E.L.
        • Stojanovic I.
        • Schasfoort R.B.M.
        • Sturk A.
        • van Leeuwen T.G.
        • Nieuwland R.
        • et al.
        Surface plasmon resonance is an analytically sensitive method for antigen profiling of extracellular vesicles.
      • Fluorescence scanning
        • Jørgensen M.M.
        • Bæk R.
        • Varming K.
        Potentials and capabilities of the extracellular vesicle (EV) array.
      • More assays are described in
        • Coumans F.A.W.
        • Gool E.L.
        • Nieuwland R.
        Bulk immunoassays for analysis of extracellular vesicles.
      High-resolution imaging technique:
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Szatanek R.
      • Baj-Krzyworzeka M.
      • Zimoch J.
      • Lekka M.
      • Siedlar M.
      • Baran J.
      The methods of choice for extracellular vesicles (EVs) characterization.
      • Transmission electron microscopy
      • Scanning electron microscopy
      • Cryo-electron microscopy
      • Atomic force microscopy
      • Super-resolution microscopy
      Estimation of biophysical features:
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Tatischeff I.
      • Larquet E.
      • Falcón-Pérez J.M.
      • Turpin P.-Y.
      • Kruglik S.G.
      Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy.
      ,
      • Wyss R.
      • Grasso L.
      • Wolf C.
      • Grosse W.
      • Demurtas D.
      • Vogel H.
      Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy.
      ,
      • Ramirez M.I.
      • Amorim M.G.
      • Gadelha C.
      • Milic I.
      • Welsh J.A.
      • Freitas V.M.
      • et al.
      Technical challenges of working with extracellular vesicles.
      • •Resistive pulse sensing
      • Nanoparticle tracking analysis
      • High-resolution flow cytometry
      • Asymmetric flow field fractionation
      • Raman spectroscopy
      ELISA, enzyme-linked immunosorbent assay; EV, extracellular vesicle.

      Routine measurement of extracellular vesicles

      For extracellular vesicles to be used as biomarkers in routine clinical settings, methods of measurement should exhibit the following characteristics: they should be able to detect small events as well as specific subpopulations; to have low inter-laboratory variability; and to use widely available devices, with quick acquisition time and limited costs. The 3 main detection methods commonly used for extracellular vesicles in this setting are summarised in Table 3.
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Rautou P.-E.
      • Bresson J.
      • Sainte-Marie Y.
      • Vion A.-C.
      • Paradis V.
      • Renard J.-M.
      • et al.
      Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.
      ,
      • Coumans F.A.W.
      • Gool E.L.
      • Nieuwland R.
      Bulk immunoassays for analysis of extracellular vesicles.
      • Ramirez M.I.
      • Amorim M.G.
      • Gadelha C.
      • Milic I.
      • Welsh J.A.
      • Freitas V.M.
      • et al.
      Technical challenges of working with extracellular vesicles.
      • van der Pol E.
      • Sturk A.
      • van Leeuwen T.
      • Nieuwland R.
      • Coumans F.
      ISTH-SSC-VB Working group
      Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation.
      • 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.
      • Groot Kormelink T.
      • Arkesteijn G.J.A.
      • Nauwelaers F.A.
      • van den Engh G.
      • Nolte-'t Hoen E.N.M.
      • Wauben M.H.M.
      Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.
      • Duijvesz D.
      • Versluis C.Y.L.
      • van der Fels C.A.M.
      • Vredenbregt-van den Berg M.S.
      • Leivo J.
      • Peltola M.T.
      • et al.
      Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.
      Table 3Extracellular vesicle detection methods with potential for clinical use.
      MethodType of detected EVAdvantagesDisadvantagesRef.
      High sensitivity-flow cytometryTotal and EV subpopulation

      Minimal diameter: 100 nm
      Detection of size and protein

      Detection of cellular origin

      Capacity of sorting different subpopulations

      Efforts in standardisation
      Only detects larger extracellular vesicles

      Inter-laboratory variability

      Dedicated cytometers

      Labour intensive

      Expensive

      Swarming: false negatives and altered linearity

      False positives: antibody aggregates, inorganic precipitates, lipoproteins
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • van der Pol E.
      • Sturk A.
      • van Leeuwen T.
      • Nieuwland R.
      • Coumans F.
      ISTH-SSC-VB Working group
      Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation.
      • 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.
      • Groot Kormelink T.
      • Arkesteijn G.J.A.
      • Nauwelaers F.A.
      • van den Engh G.
      • Nolte-'t Hoen E.N.M.
      • Wauben M.H.M.
      Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.
      Filtration/ELISAEV subpopulation

      Size: depends on filter size
      Reproducible

      High-throughput

      Absolute number and standard units
      Use of transmembrane or membrane-anchored proteins

      Filter saturation
      • Rautou P.-E.
      • Bresson J.
      • Sainte-Marie Y.
      • Vion A.-C.
      • Paradis V.
      • Renard J.-M.
      • et al.
      Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.
      ,
      • Coumans F.A.W.
      • Gool E.L.
      • Nieuwland R.
      Bulk immunoassays for analysis of extracellular vesicles.
      ,
      • Duijvesz D.
      • Versluis C.Y.L.
      • van der Fels C.A.M.
      • Vredenbregt-van den Berg M.S.
      • Leivo J.
      • Peltola M.T.
      • et al.
      Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.
      qRT-PCREV subpopulationRobust and sensitive

      Detects intact and fragmented RNA

      Low cost
      Time consuming

      Absence of endogenous controls for validation (use EV-transcriptomic data or absolute quantification)

      Average RNA copy number from total extracellular vesicles
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Ramirez M.I.
      • Amorim M.G.
      • Gadelha C.
      • Milic I.
      • Welsh J.A.
      • Freitas V.M.
      • et al.
      Technical challenges of working with extracellular vesicles.
      ELISA, enzyme-linked immunosorbent assay; EV, extracellular vesicle; qRT-PCR, quantitative reverse transcription PCR.
      High sensitivity-flow cytometry on a dedicated device can simultaneously detect light scatter (i.e. diameter) and fluorescence signal (i.e. an extracellular vesicle subpopulation) from extracellular vesicles passing one by one in front of a laser beam.
      • van der Pol E.
      • Böing A.N.
      • Gool E.L.
      • Nieuwland R.
      Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.
      It is the most commonly used technique for measuring concentrations of total extracellular vesicles, as well as subpopulations of extracellular vesicles, and can also determine cellular origin. Dedicated high-resolution flow cytometers are required, using bead-based calibrations or a scatter-diameter relationship model to detect small extracellular vesicles.
      • Groot Kormelink T.
      • Arkesteijn G.J.A.
      • Nauwelaers F.A.
      • van den Engh G.
      • Nolte-'t Hoen E.N.M.
      • Wauben M.H.M.
      Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.
      ,
      • Cointe S.
      • Judicone C.
      • Robert S.
      • Mooberry M.J.
      • Poncelet P.
      • Wauben M.
      • et al.
      Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.
      Yet, a major limitation is the variation in the smallest detectable size with different instruments, which affects the measured concentrations, and is responsible for differing sensitivities between instruments.
      • van der Pol E.
      • Böing A.N.
      • Gool E.L.
      • Nieuwland R.
      Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.
      ,
      • van der Pol E.
      • Sturk A.
      • van Leeuwen T.
      • Nieuwland R.
      • Coumans F.
      ISTH-SSC-VB Working group
      Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation.
      In response to this high inter-laboratory variability, efforts to standardise the technique are currently underway.
      • van der Pol E.
      • Sturk A.
      • van Leeuwen T.
      • Nieuwland R.
      • Coumans F.
      ISTH-SSC-VB Working group
      Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation.
      ,
      • Cointe S.
      • Judicone C.
      • Robert S.
      • Mooberry M.J.
      • Poncelet P.
      • Wauben M.
      • et al.
      Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.
      ,
      • Lacroix R.
      • Robert S.
      • Poncelet P.
      • Kasthuri R.S.
      • Key N.S.
      • Dignat-George F.
      • et al.
      Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop.
      Sandwich enzyme-linked immunosorbent assay (ELISA) quantifies specific extracellular vesicle-bound proteins.
      • Rautou P.-E.
      • Bresson J.
      • Sainte-Marie Y.
      • Vion A.-C.
      • Paradis V.
      • Renard J.-M.
      • et al.
      Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.
      ,
      • Coumans F.A.W.
      • Gool E.L.
      • Nieuwland R.
      Bulk immunoassays for analysis of extracellular vesicles.
      It is a reproducible detection method, using colorimetric or fluorescent detection, that gives results in standard units. Extracellular vesicles must be separated from soluble components. Our team has proposed an in-house filtration/ELISA method, which is highly reproducible with a variation coefficient <10%, and has proven its applicability in large cohorts.
      • Rautou P.-E.
      • Bresson J.
      • Sainte-Marie Y.
      • Vion A.-C.
      • Paradis V.
      • Renard J.-M.
      • et al.
      Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.
      • Payancé A.
      • Silva-Junior G.
      • Bissonnette J.
      • Tanguy M.
      • Pasquet B.
      • Levi C.
      • et al.
      Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis.
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • Roux O.
      • Payancé A.
      • Lebrec D.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.
      The filtration/ELISA technique is described in Fig. 2.
      Figure thumbnail gr2
      Fig. 2Extracellular vesicle subpopulation detection method by filtration/ELISA.
      (A) Graphical representation of the filtration/ELISA method. ELISA is performed on platelet-free plasma before filtration, after double filtration through 0.2 μm pores, and after filtration through 0.02 μm pores. Concentration of larger extracellular vesicles is equal to the difference between protein concentration before and after double filtration through 0.2 μm pores. Concentration of small extracellular vesicles is equal to the difference between protein concentration after double filtration through 0.2 μm pores and after filtration through 0.02 μm pores. (B) Flow cytometry graphs showing calibrated Megamix-Plus FSC beads having a 0.1, 0.3, 0.5 and 0.9 μm sizes. Filtration through 0.2 μm pores removes 0.3 μm, 0.5 and 0.9 μm beads. Filtration through 0.02 μm pores removes 0.1 μm beads. (C) Tunable Resistive Pulse Sensing (TRPS, qNano) analysis of the plasma of a patient with cirrhosis before filtration, after filtration through 0.2 μm pores and after filtration through 0.02 μm pores. After filtration through 0.2 μm pores, a decrease of 92% of events between 185 and 1,000 nm was observed (n = 2). ELISA, enzyme-linked immunosorbent assay; EV, extracellular vesicle.
      Quantitative reverse transcription PCR (qRT-PCR) is a method that quantifies a specific RNA transcript.
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      It is the most widely used method in biomarker identification as it is robust, low cost, and has high analytical sensitivity.
      • Ramirez M.I.
      • Amorim M.G.
      • Gadelha C.
      • Milic I.
      • Welsh J.A.
      • Freitas V.M.
      • et al.
      Technical challenges of working with extracellular vesicles.
      The check list of experimental details for RNA analysis has been described.
      • Hill A.F.
      • Pegtel D.M.
      • Lambertz U.
      • Leonardi T.
      • O'Driscoll L.
      • Pluchino S.
      • et al.
      ISEV position paper: extracellular vesicle RNA analysis and bioinformatics.
      Other RNA quantification methods validated for extracellular vesicles are outlined in Table 2.
      • Mateescu B.
      • Kowal E.J.K.
      • van Balkom B.W.M.
      • Bartel S.
      • Bhattacharyya S.N.
      • Buzás E.I.
      • et al.
      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.
      ,
      • Théry C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
      • Andriantsitohaina R.
      • 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.
      ,
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      ,
      • Arraud N.
      • Linares R.
      • Tan S.
      • Gounou C.
      • Pasquet J.-M.
      • Mornet S.
      • et al.
      Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration.
      ,
      • van der Pol E.
      • Hoekstra A.G.
      • Sturk A.
      • Otto C.
      • van Leeuwen T.G.
      • Nieuwland R.
      Optical and non-optical methods for detection and characterization of microparticles and exosomes.
      • Obeid S.
      • Ceroi A.
      • Mourey G.
      • Saas P.
      • Elie-Caille C.
      • Boireau W.
      Development of a NanoBioAnalytical platform for “on-chip” qualification and quantification of platelet-derived microparticles.
      • Liang K.
      • Liu F.
      • Fan J.
      • Sun D.
      • Liu C.
      • Lyon C.J.
      • et al.
      Nanoplasmonic quantification of tumor-derived extracellular vesicles in plasma microsamples for diagnosis and treatment monitoring.
      • Koliha N.
      • Wiencek Y.
      • Heider U.
      • Jüngst C.
      • Kladt N.
      • Krauthäuser S.
      • et al.
      A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.
      • Corso G.
      • Mäger I.
      • Lee Y.
      • Görgens A.
      • Bultema J.
      • Giebel B.
      • et al.
      Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography.
      • Gool E.L.
      • Stojanovic I.
      • Schasfoort R.B.M.
      • Sturk A.
      • van Leeuwen T.G.
      • Nieuwland R.
      • et al.
      Surface plasmon resonance is an analytically sensitive method for antigen profiling of extracellular vesicles.
      • Jørgensen M.M.
      • Bæk R.
      • Varming K.
      Potentials and capabilities of the extracellular vesicle (EV) array.
      • Szatanek R.
      • Baj-Krzyworzeka M.
      • Zimoch J.
      • Lekka M.
      • Siedlar M.
      • Baran J.
      The methods of choice for extracellular vesicles (EVs) characterization.
      • Tatischeff I.
      • Larquet E.
      • Falcón-Pérez J.M.
      • Turpin P.-Y.
      • Kruglik S.G.
      Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy.
      • Wyss R.
      • Grasso L.
      • Wolf C.
      • Grosse W.
      • Demurtas D.
      • Vogel H.
      Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy.
      • Coumans F.A.W.
      • Gool E.L.
      • Nieuwland R.
      Bulk immunoassays for analysis of extracellular vesicles.
      • Ramirez M.I.
      • Amorim M.G.
      • Gadelha C.
      • Milic I.
      • Welsh J.A.
      • Freitas V.M.
      • et al.
      Technical challenges of working with extracellular vesicles.
      Northern blot is a robust and sensitive method to measure specific RNA transcripts, but it is time consuming and thus not adapted to clinical laboratories.
      Other detection methods that are often used in research settings and are less applicable in clinical settings are summarised in Table 2. Nanoparticle tracking analysis, which has outperformed dynamic light scattering, is a method that determines size and concentration of extracellular vesicles by measuring their Brownian motion. However, these techniques are limited by the fact that: they are unable to detect specific extracellular vesicle subpopulations; exact concentrations can only be estimated with a risk of high inter-laboratory variability; and they require special equipment.
      • Coumans F.A.W.
      • Brisson A.R.
      • Buzas E.I.
      • Dignat-George F.
      • Drees E.E.E.
      • El-Andaloussi S.
      • et al.
      Methodological guidelines to study extracellular vesicles.
      Current development of fluorescent detection using nanoparticle tracking analysis devices may enable measurement of specific extracellular vesicle subpopulations in the future.
      • Carnell-Morris P.
      • Tannetta D.
      • Siupa A.
      • Hole P.
      • Dragovic R.
      Analysis of extracellular vesicles using fluorescence nanoparticle tracking analysis.
      ,
      • Desgeorges A.
      • Hollerweger J.
      • Lassacher T.
      • Rohde E.
      • Helmbrecht C.
      • Gimona M.
      Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.

      Extracellular vesicles in liver diseases

      Extracellular vesicles appear to be attractive biomarkers for diagnosis, as well as for estimating severity and prognosis in liver diseases, including non-alcoholic and alcoholic steatohepatitis, chronic viral hepatitis B and C infections, cirrhosis, primary liver cancers, primary sclerosing cholangitis and acute liver failure. The main clinical studies evaluating extracellular vesicle subpopulations as biomarkers in liver diseases are summarised in Table 4 (review criteria are summarised in the supplementary text). Unless otherwise mentioned, the extracellular vesicles studied were all taken from plasma.
      Table 4Summary of main clinical studies on extracellular vesicles as biomarkers in liver diseases.
      BiomarkerVarEV size & techniqueNumber (patient/control)Se/Spe (%)AUROC

      PPV/NPV
      OutcomeStudy typeRef.
      NAFLD
       T Cell (CD4+ or CD8+ or iNKT)

       Monocyte (CD14+)

       Neutrophil (CD15+)

       Platelet (CD41+)
      Larger

      FCM
      NAFLD: 65

      HC: 44
      27–59/

      90–98
      0.81–0.91

      n.a.
      Detection of NAFLDRetrospective
      • Kornek M.
      • Lynch M.
      • Mehta S.H.
      • Lai M.
      • Exley M.
      • Afdhal N.H.
      • et al.
      Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis.
      Alcoholic hepatitis
       Hepatocyte (cytokeratin-18)Larger

      Filtration/ELISA
      Confirmed AH: test: 46, validation: 48

      Ruled-out AH: test: 37, validation: 20
      76/810.82

      83/73
      Diagnosis of AHProspective
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • Roux O.
      • Payancé A.
      • Lebrec D.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.
       Haematopoietic stem cell (CD34+) & Hepatocyte (ASPGR+)Larger

      FCM
      Severe AH:

      non-responders: 71

      responders: 30

      HC: 20
      n.a.0.94

      n.a.
      Response to therapy & mortalityRetrospective
      • Sukriti S.
      • Maras J.S.
      • Bihari C.
      • Das S.
      • Vyas A.K.
      • Sharma S.
      • et al.
      Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis.
      Cirrhosis
       Platelet-derived growth factor receptor ßSmall

      ELISA
      Fibrosis 0–1: 51

      Fibrosis 2–4: 97

      HC: 14

      Validation cohort: 57
      82/410.64

      n.a.
      Detection of fibrosis F3-F4Retrospective
      • Lambrecht J.
      • Verhulst S.
      • Mannaerts I.
      • Sowa J.-P.
      • Best J.
      • Canbay A.
      • et al.
      A PDGFRβ-based score predicts significant liver fibrosis in patients with chronic alcohol abuse, NAFLD and viral liver disease.
       Leuko-endothelial (CD31+/41−)Larger

      FCM
      Cirrhosis: 91n.a.n.a.SurvivalProspective
      • Rautou P.-E.
      • Bresson J.
      • Sainte-Marie Y.
      • Vion A.-C.
      • Paradis V.
      • Renard J.-M.
      • et al.
      Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.
       Hepatocyte (cytokeratin-18)Larger

      Filtration-ELISA
      Cirrhosis:

      Test cohort: 139 Validation cohort:103
      n.a.n.a.6-month mortalityProspective Competing risk analysis
      • Payancé A.
      • Silva-Junior G.
      • Bissonnette J.
      • Tanguy M.
      • Pasquet B.
      • Levi C.
      • et al.
      Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis.
      Hepatocellular carcinoma
       Tumoural (AnnexinV+ EpCAM+ASPGR1+ +/− CD133+)Larger

      FCM
      HCC: 86

      Cirrhosis: 49

      HC: 58
      80–81/

      47–50
      0.73–0.74

      n.a.
      Detection of HCCRetrospective
      • Julich-Haertel H.
      • Urban S.K.
      • Krawczyk M.
      • Willms A.
      • Jankowski K.
      • Patkowski W.
      • et al.
      Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma.
       miRNA-519d & -595 & -939Small

      qRT-PCR
      Advanced HCC: 45

      Unifocal/small: 40

      Cirrhosis no HCC: 30
      n.a.0.82–0.84

      n.a.
      Detection of HCCRetrospective
      • Fornari F.
      • Ferracin M.
      • Trerè D.
      • Milazzo M.
      • Marinelli S.
      • Galassi M.
      • et al.
      Circulating microRNAs, miR-939, miR-595, miR-519d and miR-494, identify cirrhotic patients with HCC.
       miRNA-125Small

      qRT-PCR
      HCC: 12883/680.74

      n.a.
      Recurrence & survivalProspective
      • Liu W.
      • Hu J.
      • Zhou K.
      • Chen F.
      • Wang Z.
      • Liao B.
      • et al.
      Serum exosomal miR-125b is a novel prognostic marker for hepatocellular carcinoma.
       Signature of miRNA-122, -148a, -1246Small

      qRT-PCR
      HCC: 50

      Cirrhosis: 40
      86/880.93

      n.a.
      Detection of HCCRetrospective
      • Wang Y.
      • Zhang C.
      • Zhang P.
      • Guo G.
      • Jiang T.
      • Zhao X.
      • et al.
      Serum exosomal microRNAs combined with alpha-fetoprotein as diagnostic markers of hepatocellular carcinoma.
       miRNA-638Small

      qRT-PCR
      HCC: 126n.a.n.a.Overall survivalRetrospective
      • Shi M.
      • Jiang Y.
      • Yang L.
      • Yan S.
      • Wang Y.-G.
      • Lu X.-J.
      Decreased levels of serum exosomal miR-638 predict poor prognosis in hepatocellular carcinoma.
       miRNA-21 & miRNA-10bSmall

      qRT-PCR
      HCC: 124n.a.n.a.Prediction of recurrenceRetrospective
      • Tian X.-P.
      • Wang C.-Y.
      • Jin X.-H.
      • Li M.
      • Wang F.-W.
      • Huang W.-J.
      • et al.
      Acidic microenvironment up-regulates exosomal miR-21 and miR-10b in early-stage hepatocellular carcinoma to promote cancer cell proliferation and metastasis.
       lncRNA-RP11-513I15.6 & miRNA 1262 & RAB11ASmall

      qRT-PCR
      HCC: 54

      HCV: 42; HC: 18
      78–98/

      73–95
      n.a.

      72–95/79–97
      Detection of early HCCRetrospective
      • Abd El Gwad A.
      • Matboli M.
      • El-Tawdi A.
      • Habib E.K.
      • Shehata H.
      • Ibrahim D.
      • et al.
      Role of exosomal competing endogenous RNA in patients with hepatocellular carcinoma.
       lncRNA ENSG00000258332.1 & LINC00635Small

      qRT-PCR
      HCC: 60

      HBV: 96
      72–76

      /78–83
      0.72–0.75

      n.a.
      Detection of HCC & survivalRetrospective
      • Xu H.
      • Chen Y.
      • Dong X.
      • Wang X.
      Serum exosomal long noncoding RNAs ENSG00000258332.1 and LINC00635 for the diagnosis and prognosis of hepatocellular carcinoma.
       miRNA-92bSmall

      qRT-PCR
      After LT:

      No recurrence: 28

      Early recurrence: 43
      71/630.70

      n.a.
      Early recurrence after LTRetrospective
      • Nakano T.
      • Chen I.-H.
      • Wang C.-C.
      • Chen P.-J.
      • Tseng H.-P.
      • Huang K.-T.
      • et al.
      Circulating exosomal miR-92b: its role for cancer immunoediting and clinical value for prediction of posttransplant hepatocellular carcinoma recurrence.
       miRNA-21 & lncRNA-ATBSmall

      qRT-PCR
      HCC: 79n.a.n.a.Overall survivalProspective

      Multivariate
      • Lee Y.R.
      • Kim G.
      • Tak W.Y.
      • Jang S.Y.
      • Kweon Y.O.
      • Park J.G.
      • et al.
      Circulating exosomal noncoding RNAs as prognostic biomarkers in human hepatocellular carcinoma.
      Cholangiocarcinoma
       Total bile EVs & Total plasma EVsSmall & larger

      NTA
      Malignant stenosis: 15 (5 CCA)

      Benign stenosis: 15
      47–100 /80–1000.81–1

      70/60
      Detection of malignant stenosisProspective

      Multivariate
      • Severino V.
      • Dumonceau J.-M.
      • Delhaye M.
      • Moll S.
      • Annessi-Ramseyer I.
      • Robin X.
      • et al.
      Extracellular vesicles in bile as markers of malignant biliary Stenoses.
      Acute liver failure
       Total plasma EVLarger

      FCM
      Acute liver injury/failure: 50n.a.n.a.Prediction of mortality/LTProspective

      Multivariate
      • Stravitz R.T.
      • Bowling R.
      • Bradford R.L.
      • Key N.S.
      • Glover S.
      • Thacker L.R.
      • et al.
      Role of procoagulant microparticles in mediating complications and outcome of acute liver injury/acute liver failure.
      AH, alcoholic hepatitis; AUROC, area under the receiver-operating characteristic; CCA, cholangiocarcinoma; EV, extracellular vesicle; FCM, flow cytometry; HC, healthy control; HCC, hepatocellular carcinoma; lncRNA, long non-coding RNA; LT, liver transplantation; qRT-PCR, quantitative reverse transcription PCR; miRNA, micro-RNA; n.a., not available; ELISA, enzyme-linked immunosorbent assay; NAFLD, non-alcoholic fatty liver disease; NPV, negative predictive value; NTA, nanoparticle tracking analysis; PPV, positive predictive value; Ref, reference; Se, sensitivity; Spe, specificity; Var, variation.

      Non-alcoholic fatty liver disease

      Non-invasive tools to assess steatohepatitis and fibrosis in non-alcoholic fatty liver disease (NAFLD) do not entirely reflect the variety of liver histological changes in these patients.
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • Charlton M.
      • Cusi K.
      • Rinella M.
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      Extracellular vesicles that reflect ongoing cell damage might be able to fill this gap and better capture the disease's complexity. Table S1 summarises extracellular subpopulations as potential biomarkers in NAFLD.
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      ,
      • Kornek M.
      • Lynch M.
      • Mehta S.H.
      • Lai M.
      • Exley M.
      • Afdhal N.H.
      • et al.
      Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis.
      • Kakazu E.
      • Mauer A.S.
      • Yin M.
      • Malhi H.
      Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner.
      • Murakami Y.
      • Toyoda H.
      • Tanahashi T.
      • Tanaka J.
      • Kumada T.
      • Yoshioka Y.
      • et al.
      Comprehensive miRNA expression analysis in peripheral blood can diagnose liver disease.
      • Lee Y.-S.
      • Kim S.Y.
      • Ko E.
      • Lee J.-H.
      • Yi H.-S.
      • Yoo Y.J.
      • et al.
      Exosomes derived from palmitic acid-treated hepatocytes induce fibrotic activation of hepatic stellate cells.
      • Pirola C.J.
      • Fernández Gianotti T.
      • Castaño G.O.
      • Mallardi P.
      • San Martino J.
      • Mora Gonzalez Lopez Ledesma M.
      • et al.
      Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.
      • Akuta N.
      • Kawamura Y.
      • Watanabe C.
      • Nishimura A.
      • Okubo M.
      • Mori Y.
      • et al.
      Impact of sodium glucose cotransporter 2 inhibitor on histological features and glucose metabolism of non-alcoholic fatty liver disease complicated by diabetes mellitus.
      • Welsh J.A.
      • Scorletti E.
      • Clough G.F.
      • Englyst N.A.
      • Byrne C.D.
      Leukocyte extracellular vesicle concentration is inversely associated with liver fibrosis severity in NAFLD.
      • Guo Q.
      • Furuta K.
      • Lucien F.
      • Gutierrez Sanchez L.H.
      • Hirsova P.
      • Krishnan A.
      • et al.
      Integrin β1-enriched extracellular vesicles mediate monocyte adhesion and promote liver inflammation in murine NASH.

      Diagnosis of non-alcoholic fatty liver disease

      The largest study investigating the potential diagnostic utility of extracellular vesicles in NAFLD reported higher concentrations of T cell- and monocyte-derived extracellular vesicles in 65 patients with NAFLD compared to healthy controls.
      • Kornek M.
      • Lynch M.
      • Mehta S.H.
      • Lai M.
      • Exley M.
      • Afdhal N.H.
      • et al.
      Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis.
      A pilot study identified increased mitochondria-rich extracellular vesicles in obese patients with increased serum transaminase levels, but sample sizes were small and no histology or imaging were used to characterise NAFLD.
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      Extracellular vesicles are promising biomarkers for diagnosis of liver diseases and prediction of disease progression, complications, response to treatment and mortality. Different extracellular vesicle subpopulations have been studied, using total extracellular vesicle count, proteins, lipids, RNAs and microRNAs.
      One study showed that a pattern of 12 miRNAs carried by serum extracellular vesicles could discriminate 12 patients with non-alcoholic steatohepatitis from patients with chronic hepatitis B and C infections and from healthy controls, although limited access to microarrays in clinical routine limits the applicability of this approach.
      • Murakami Y.
      • Toyoda H.
      • Tanahashi T.
      • Tanaka J.
      • Kumada T.
      • Yoshioka Y.
      • et al.
      Comprehensive miRNA expression analysis in peripheral blood can diagnose liver disease.

      Marker of activity and fibrosis

      Regarding liver NAFLD activity, extracellular vesicles originating from natural killer T cells and monocytes (CD14+) have been shown to correlate with histological grade and with NAFLD activity score and alanine aminotransferase, respectively, unlike CD4+ and CD8+ T cell subpopulations.
      • Kornek M.
      • Lynch M.
      • Mehta S.H.
      • Lai M.
      • Exley M.
      • Afdhal N.H.
      • et al.
      Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis.
      Two lipidic extracellular vesicle markers can help to differentiate patients with non-alcoholic steatohepatitis from those with simple steatosis and from obese patients without NAFLD, but use of mass spectrometry limits their applicability in clinical settings.
      • Kakazu E.
      • Mauer A.S.
      • Yin M.
      • Malhi H.
      Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner.
      Regarding fibrosis, none of the previously cited extracellular vesicle subpopulations significantly correlated with histological fibrosis stage. On the other hand, a pilot study found that CD14+ (monocyte) and CD16+ (leukocyte) extracellular vesicles decreased with fibrosis stage.
      • Welsh J.A.
      • Scorletti E.
      • Clough G.F.
      • Englyst N.A.
      • Byrne C.D.
      Leukocyte extracellular vesicle concentration is inversely associated with liver fibrosis severity in NAFLD.
      These different results can be explained by the use of different extracellular vesicle separation methods, as well as the inclusion of patients with different non-alcoholic steatohepatitis severity.
      Two miRNAs contained in serum extracellular vesicles, miRNA-122, a major hepatic miRNA, and miRNA-192, were found to increase with activity and fibrosis stage in several studies.
      • Lee Y.-S.
      • Kim S.Y.
      • Ko E.
      • Lee J.-H.
      • Yi H.-S.
      • Yoo Y.J.
      • et al.
      Exosomes derived from palmitic acid-treated hepatocytes induce fibrotic activation of hepatic stellate cells.
      • Pirola C.J.
      • Fernández Gianotti T.
      • Castaño G.O.
      • Mallardi P.
      • San Martino J.
      • Mora Gonzalez Lopez Ledesma M.
      • et al.
      Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.
      • Akuta N.
      • Kawamura Y.
      • Watanabe C.
      • Nishimura A.
      • Okubo M.
      • Mori Y.
      • et al.
      Impact of sodium glucose cotransporter 2 inhibitor on histological features and glucose metabolism of non-alcoholic fatty liver disease complicated by diabetes mellitus.
      However, these findings should be interpreted with caution since fewer than 10 patients were included in each study, and since, in 1 study, the extracellular vesicle fraction of miRNA-122 was not specifically studied.
      To conclude, leukocyte extracellular vesicles have potential as biomarkers for NAFLD diagnosis, activity and fibrosis, providing that large prospective studies validate these findings. Further studies are required to confirm the potential utility of extracellular vesicles carrying miRNA-122 and -129.

      Alcoholic hepatitis

      The 2 main clinical challenges in alcoholic hepatitis are the non-invasive diagnosis of the disease and prediction of treatment response.

      Diagnosis of alcoholic hepatitis

      Our group tested whether plasma extracellular vesicle levels could be used to diagnose alcoholic hepatitis non-invasively. In 2 prospective cohorts of 83 and 68 patients with clinical suspicion of alcoholic hepatitis, we observed that plasma hepatocyte (cytokeratin-18+) extracellular vesicle levels were able to diagnose alcoholic hepatitis with good sensitivity (76%) and specificity (81%). Yet, total soluble cytokeratin-18 performed even better and can be measured more easily.
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • Roux O.
      • Payancé A.
      • Lebrec D.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.
      Higher levels of hepatocyte-derived (ASPGR+) extracellular vesicles were also observed in 101 patients with severe alcoholic hepatitis compared to healthy controls.
      • Sukriti S.
      • Maras J.S.
      • Bihari C.
      • Das S.
      • Vyas A.K.
      • Sharma S.
      • et al.
      Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis.
      Plasma concentrations of total extracellular vesicles, as well as of extracellular vesicles derived from T cells (plasma and serum), macrophages, neutrophils, haematopoietic stem cells and endothelial cells are found to be higher among patients with severe alcoholic hepatitis than among healthy controls.
      • Sukriti S.
      • Maras J.S.
      • Bihari C.
      • Das S.
      • Vyas A.K.
      • Sharma S.
      • et al.
      Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis.
      ,
      • Verma V.K.
      • Li H.
      • Wang R.
      • Hirsova P.
      • Mushref M.
      • Liu Y.
      • et al.
      Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles.
      However, these changes might simply reflect the consequences of alcohol intake and not alcoholic hepatitis. Indeed, our group measured plasma extracellular vesicles derived from endothelial cells (CD62e+; CD41−/31+), platelets (CD41+), and leukocytes (CD11a+) in patients with clinical suspicion of alcoholic hepatitis and did not observe any difference between patients with and without histologically proven alcoholic hepatitis.
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • Roux O.
      • Payancé A.
      • Lebrec D.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.
      Along the same lines, plasma and serum concentrations of several subpopulations of extracellular vesicles (carrying mitochondrial DNA, miRNA or hepatocyte proteins) are higher in patients after binge drinking and/or chronic excessive alcohol consumption, as summarised in Table S2.
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • Roux O.
      • Payancé A.
      • Lebrec D.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.
      ,
      • Sukriti S.
      • Maras J.S.
      • Bihari C.
      • Das S.
      • Vyas A.K.
      • Sharma S.
      • et al.
      Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis.
      • Verma V.K.
      • Li H.
      • Wang R.
      • Hirsova P.
      • Mushref M.
      • Liu Y.
      • et al.
      Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles.
      • Eguchi A.
      • Franz N.
      • Kobayashi Y.
      • Iwasa M.
      • Wagner N.
      • Hildebrand F.
      • et al.
      Circulating extracellular vesicles and their miR “barcode” differentiate alcohol drinkers with liver injury and those without liver injury in severe trauma patients.
      • Cho Y.-E.
      • Im E.-J.
      • Moon P.-G.
      • Mezey E.
      • Song B.-J.
      • Baek M.-C.
      Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury.
      • Cai Y.
      • Xu M.-J.
      • Koritzinsky E.H.
      • Zhou Z.
      • Wang W.
      • Cao H.
      • et al.
      Mitochondrial DNA-enriched microparticles promote acute-on-chronic alcoholic neutrophilia and hepatotoxicity.
      • Momen-Heravi F.
      • Bala S.
      • Kodys K.
      • Szabo G.
      Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.
      • Eguchi A.
      • Lazaro R.G.
      • Wang J.
      • Kim J.
      • Povero D.
      • Willliams B.
      • et al.
      Extracellular vesicles released by hepatocytes from gastric infusion model of alcoholic liver disease contain a MicroRNA barcode that can be detected in blood.
      • Saha B.
      • Momen-Heravi F.
      • Kodys K.
      • Szabo G.
      MicroRNA cargo of extracellular vesicles from alcohol-exposed monocytes signals naive monocytes to differentiate into M2 macrophages.
      • Wang R.
      • Ding Q.
      • Yaqoob U.
      • de Assuncao T.M.
      • Verma V.K.
      • Hirsova P.
      • et al.
      Exosome adherence and internalization by hepatic stellate cells triggers sphingosine 1-phosphate-dependent migration.
      • Sehrawat T.S.
      • Arab J.P.
      • Liu M.
      • Amrollahi P.
      • Wan M.
      • Fan J.
      • et al.
      Circulating extracellular vesicles carrying sphingolipid cargo for the diagnosis and dynamic risk profiling of alcoholic hepatitis.
      • Nielsen M.C.
      • Andersen M.N.
      • Grønbæk H.
      • Damgaard Sandahl T.
      • Møller H.J.
      Extracellular vesicle-associated soluble CD163 and CD206 in patients with acute and chronic inflammatory liver disease.
      • Babuta M.
      • Furi I.
      • Bala S.
      • Bukong T.N.
      • Lowe P.
      • Catalano D.
      • et al.
      Dysregulated autophagy and lysosome function are linked to exosome production by micro-RNA 155 in alcoholic liver disease.
      • Arab J.P.
      • Sehrawat T.S.
      • Simonetto D.A.
      • Verma V.K.
      • Feng D.
      • Tang T.
      • et al.
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      Their potential utility for diagnosis of alcoholic hepatitis should be tested in patients with a clinical suspicion of alcoholic hepatitis.

      Prediction of outcome in alcoholic hepatitis

      In a cohort of 101 patients with histologically proven alcoholic steatohepatitis, concentrations of circulating CD34+ (a haematopoietic stem cell marker) and ASGPR+ (a hepatocyte marker) extracellular vesicles were higher among non-responders to steroid therapy than among responders, and could predict 7-day and 1-month mortality.
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      Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis.
      The combination of the 2 markers could predict 7-day non-response to steroid therapy with an area under the receiver-operating characteristic (AUROC) of 0.94. Yet, no threshold that can be used in clinical practice has been established. Other extracellular vesicle subpopulations (T cell, macrophage and endothelial) evaluated in this study were higher among non-responders to steroid therapy, but were less accurate in predicting mortality.
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      To conclude, circulating hepatocyte-derived extracellular vesicles seem to be a promising biomarker for diagnosis and outcome prediction in patients with alcoholic hepatitis. Data on other subpopulations are less homogenous and need to be confirmed in future studies.

      Hepatitis B and C virus infections

      Extracellular vesicles could be useful markers to predict the risk of hepatitis B virus reactivation and detection of early fibrosis for hepatitis B and C. Results of studies on that topic are summarised in Table S3
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      Circulating ECV-associated miRNAs as potential clinical biomarkers in early stage HBV and HCV induced liver fibrosis.
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      Extracellular vesicle-associated mir-21 and mir-144 are markedly elevated in serum of patients with hepatocellular carcinoma.
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      Hepatitis B virus-encoded microRNA controls viral replication.
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      Plasma microRNA levels are associated with hepatitis B e antigen status and treatment response in chronic hepatitis B patients.
      and the supplementary text
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      Extracellular vesicles from hepatitis B patients serve as reservoir of hepatitis B virus DNA.
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      Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction.
      European Association for the Study of the Liver. Electronic address: [email protected], European Association for the Study of the Liver
      EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.
      for hepatitis B, and Table S4
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      Comprehensive miRNA expression analysis in peripheral blood can diagnose liver disease.
      ,
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      • van Grunsven L.A.
      Circulating ECV-associated miRNAs as potential clinical biomarkers in early stage HBV and HCV induced liver fibrosis.
      ,
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      Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90.
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