Sara Thietart

Prof Pierre-Emmanuel Rautou
email: pierre-emmanuel.rautou@inserm.fr

Extracellular vesicles in liver diseases 1 EXTRACELLULAR VESICLES AS BIOMARKERS IN LIVER DISEASES: A CLINICIAN'S POINT OF VIEW

Keywords: microvesicle, exosome, microparticle, vesicle, liver, alcohol Extracellular vesicles in liver diseases 4 ABIC: age-bilirubin-INR-creatinine, ACD: acid citrate dextrose, ALT: alanine aminotransferase, AHA: American Heart Association, AUROC: Area Under the Receiver Operating Characteristics, DNA: deoxyribose nucleic acid, DNase: deoxyribonuclease, EV: extracellular vesicle, EDTA: ethylene diamine tetraacetic acid, ELISA: sandwich enzymelinked immunosorbent assay, ISAC: International Society hepatitis C virus, HVPG: hepatic venous pressure gradient, lncRNA primary sclerosing cholangitis, MELD: Model for End-Stage Liver Disease, miRNA alcoholic fatty liver disease, NAS score: NAFLD Activity Score, NK T cell: Natural killer T cell, RNA: ribonucleic acid, RNase: ribonuclease, qRT-PCR: Quantitative reverse

Extracellular vesicles are membrane-bound vesicles, containing cell-derived biomolecules, such as proteins, lipids and RNAs.

• 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.

• Some extracellular vesicle subpopulations have already been evaluated in prospective studies in cirrhosis, alcoholic liver disease and hepatocellular carcinoma. In other liver diseases, extracellular vesicles still await validation.

• High-throughput and reproducible measurement methods of extracellular vesicles are now becoming available.

What are extracellular vesicles?

Extracellular vesicles (EVs) form a heterogeneous group of membrane-bound vesicles that contain cell-derived biomolecules, such as proteins, lipids, RNAs and microRNAs (miRNAs). [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF][START_REF] Garcia-Martinez | Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9[END_REF][START_REF] Puhm | Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells[END_REF] They sometimes also contain organelles, such as mitochondria. [START_REF] Puhm | Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells[END_REF] They can be released into the extracellular compartment by both healthy and stressed cells. [START_REF] Ridger | Microvesicles in vascular homeostasis and diseases[END_REF] All cells can form extracellular vesicles, including malignant cells. Extracellular vesicles can be found in various body fluids and tissues.

Extracellular vesicles are classified into several subtypes, each with different biogenesis mechanisms, which are thoroughly reviewed elsewhere. [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF] Briefly, exosomes (size ranging from 40 to 100-200 nm) are generated in intraluminal vesicles by inward budding of the limiting membrane of early endosomes during the maturation process of multivesicular endosomes. Exosomes are then released into the extracellular milieu by fusion of multivesicular bodies with the plasma membrane. [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF] "Microvesicles" (previously called "microparticles", with sizes ranging from 100-200 nm to 1 µm) are generated by plasma membrane budding induced by a transient phospholipid imbalance caused by externalization of phosphatidylserine (a negatively charged phospholipid), calcium dependent proteolysis of cytoskeleton and local potassium efflux responsible for cellular contraction. [START_REF] Ridger | Microvesicles in vascular homeostasis and diseases[END_REF] Apoptotic bodies (size ranging from ≈ 1 to 4 µm), which are released by dying cells, are formed by a separation of plasma membrane from the cytoskeleton. [START_REF] Doyle | Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis[END_REF] Apoptotic bodies will not be discussed in this review, as they are the same size as platelets, and are therefore eliminated during the first steps of platelet-free plasma processing. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF][START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF] Due to overlapping size, density and composition, a precise definition of these subgroups of extracellular vesicles has not yet been firmly established (Box 1). [START_REF] Théry | 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[END_REF] It is now recommended that extracellular vesicles be classified by size, density, biochemical composition or cellular origin and not by biogenesis. [START_REF] Théry | 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[END_REF] In this review, we therefore use the Thietart S & Rautou PE Extracellular vesicles in liver diseases 7

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).

Once released into the extracellular compartment, extracellular vesicles can reach target cells for intercellular communication. Communication methods of extracellular vesicles are varied and include activation of surface receptors, transfer of vesicle content into the target cell by vesicle internalization (phagocytosis, endocytosis) or membrane fusion. [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF] The role of extracellular vesicles in cell-cell communication in liver diseases has been thoroughly reviewed elsewhere. [START_REF] Szabo | Extracellular vesicles in liver disease and potential as biomarkers and therapeutic targets[END_REF][START_REF] Lemoinne | The emerging roles of microvesicles in liver diseases[END_REF] Extracellular vesicles are released following cell activation or apoptosis. Their composition depends on their cellular origin and on the stimulus responsible for their formation. [START_REF] Szabo | Extracellular vesicles in liver disease and potential as biomarkers and therapeutic targets[END_REF] Circulating extracellular vesicle concentrations and composition thus vary with disease conditions with some specificity for the ongoing detrimental process. These characteristics make extracellular vesicles useful tools for personalized medicine to diagnose disorders, evaluate prognosis, disease progression and response to treatment. Circulating extracellular vesicles have therefore emerged as attractive biomarkers in liver diseases, as

shown 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 extensively reviewed elsewhere [START_REF] Théry | 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[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF]).

Patient requirements

Thietart S & Rautou PE Extracellular vesicles in liver diseases 8

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. [START_REF] Witwer | Standardization of sample collection, isolation and analysis methods in extracellular vesicle research[END_REF] 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, standardization of plasma sample collection and processing is necessary to ensure comparability among studies and, ultimately, to permit correct interpretation of the results at an individual level. [START_REF] Clayton | Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017[END_REF] Plasma sample collection, preparation and storage Many factors can induce the release of extracellular vesicles by blood cells during or after blood draw. [START_REF] Lippi | Influence of mechanical trauma of blood and hemolysis on PFA-100 testing[END_REF][START_REF] Cheng | Plasma processing conditions substantially influence circulating microRNA biomarker levels[END_REF][START_REF] Mani | Influence of blood collection techniques on platelet function[END_REF][START_REF] Yuana | Handling and storage of human body fluids for analysis of extracellular vesicles[END_REF][START_REF] Baek | The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray[END_REF] Consequently, over the past few years, each step of sample collection and preparation has been standardized 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. [START_REF] Théry | 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[END_REF] Researchers can upload extracellular vesicle experiment protocols from a crowd-sourcing knowledge base called EV-TRACK (http://evtrack.org). [START_REF] Consortium | TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research[END_REF] Thietart S & Rautou PE Extracellular vesicles in liver diseases 9

Recommendations on blood sample collection are summarized in Box 2. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF][START_REF] Théry | 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[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Witwer | Standardization of sample collection, isolation and analysis methods in extracellular vesicle research[END_REF][START_REF] Wisgrill | Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants[END_REF] The International Society on Thrombosis and Haemostasis (ISTH) [START_REF] Lacroix | Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop[END_REF] and the International Society for Advancement of Cytometry (ISAC) [START_REF] Wisgrill | Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants[END_REF] recommend the use of citrate tubes (as opposed to ethylene diamine tetraacetic acid (EDTA) and heparin), as their use better prevents platelet activation and formation of extracellular vesicles. [START_REF] Théry | 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[END_REF][START_REF] Jayachandran | Methodology for isolation, identification and characterization of microvesicles in peripheral blood[END_REF] The American Heart Association's (AHA) 2017 guidelines [START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF] and the International Society for Extracellular Vesicles' (ISEV) 2017 position paper [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF] recommend that the choice of anticoagulants be adapted to downstream analysis. For example, acid citrate dextrose (ACD) is considered to be more suitable for RNA analysis. [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] György | Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube[END_REF] 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. [START_REF] Lacroix | Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop[END_REF] It has been shown that small extracellular vesicle miRNAs could still be assessed from samples frozen for up to 8 years. [START_REF] Andreu | Comparative analysis of EV isolation procedures for miRNAs detection in serum samples[END_REF] 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 is able to entirely separate extracellular vesicles from the remaining plasma. A balance must be found between maximizing recovery of extracellular vesicles (to avoid loss of information) and minimizing 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 labor intensive. [START_REF] Théry | 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[END_REF][START_REF] Gardiner | Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey[END_REF] Consideration should also be given to the fact that separation methods can influence important extracellular vesicle characteristics, such as size, integrity, composition

Thietart S & Rautou PE
Extracellular vesicles in liver diseases 10 and functional properties. [START_REF] Baek | The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray[END_REF][START_REF] Mol | Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation[END_REF][START_REF] Cheng | Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cellfree blood[END_REF]. Separation methods can isolate non-vesicular circulating miRNAs or RNAs together with extracellular vesicles. [START_REF] Jeppesen | PE Extracellular vesicles in liver diseases 34[END_REF][START_REF] Van Deun | The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling[END_REF] 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. [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Hill | ISEV position paper: extracellular vesicle RNA analysis and bioinformatics[END_REF] Interpretation of data should take into account isolation techniques and use of RNAse treatment. For small cohorts, it is useful to verify reliability of separation methods, by quantifying contaminants (measuring albumin, ApoB100, ApoA1 and ApoB48) and/or by characterizing the aspect of separate extracellular vesicles (for example, by using transmission electron microscopy). [START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Takov | Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential[END_REF] A ratio of at least 3 x 10 10 vesicles per µg of protein is proposed to define high purity. [START_REF] Webber | How pure are your vesicles?[END_REF] The main extracellular vesicle separation methods applicable for biomarker use are summarized in Table 1. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF][START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Witwer | Standardization of sample collection, isolation and analysis methods in extracellular vesicle research[END_REF][START_REF] Jayachandran | Methodology for isolation, identification and characterization of microvesicles in peripheral blood[END_REF][START_REF] Andreu | Comparative analysis of EV isolation procedures for miRNAs detection in serum samples[END_REF][START_REF] Van Deun | The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling[END_REF][START_REF] Momen-Heravi | Current methods for the isolation of extracellular vesicles[END_REF][START_REF] Taylor | Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes[END_REF][START_REF] Stranska | Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma[END_REF][START_REF] Momen-Heravi | Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles[END_REF][START_REF] Baranyai | Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods[END_REF][START_REF] Vergauwen | Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research[END_REF][START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF][START_REF] Bissonnette | A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis[END_REF][START_REF] Enderle | Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method[END_REF] 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 Supplementary

Text. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Witwer | Standardization of sample collection, isolation and analysis methods in extracellular vesicle research[END_REF][START_REF] Consortium | TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research[END_REF][START_REF] Gardiner | Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey[END_REF][START_REF] Momen-Heravi | Current methods for the isolation of extracellular vesicles[END_REF][START_REF] Taylor | Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes[END_REF][START_REF] Stranska | Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma[END_REF][START_REF] Enderle | Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method[END_REF] Other separation methods seem to be better adapted to clinical settings: (a) Size exclusion chromatography separates extracellular vesicles from other components depending on their size. [START_REF] Böing | Single-step isolation of extracellular vesicles by size-exclusion chromatography[END_REF] It uses a column containing porous beads: proteins are small enough to go through the pores, but not extracellular vesicles. Extracellular vesicles therefore migrate at a higher speed than soluble proteins (Fig. 1). However, if the size of a molecule (such as lipoproteins) is the same size as the size of extracellular vesicles, both will migrate at same speed and could therefore be co-isolated. [START_REF] Takov | Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential[END_REF] This technique has been shown

to give intact and functional vesicles, as well as fewer protein and lipoprotein contaminants than other methods, although its purity is not optimal. [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF][START_REF] Mol | Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation[END_REF][START_REF] Takov | Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential[END_REF][START_REF] Stranska | Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma[END_REF][START_REF] Baranyai | Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods[END_REF][START_REF] Hong | Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer[END_REF] been used, as filter type and pore size have been shown to heavily influence recovery. [START_REF] Théry | 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[END_REF][START_REF] Vergauwen | Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research[END_REF] The effect on vesicle disruption of the forces applied to push samples through the filters is unknown. [START_REF] Taylor | Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes[END_REF] (c) Precipitation kits (such as, ExoQuick™, miRCURY™ Exosome Isolation Kit or Invitrogen™ Total Exosome Isolation Kit) have a high extracellular vesicle recovery, with poor purity due to co-recovery of protein complexes and extracellular vesicle aggregates. [START_REF] Théry | 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[END_REF][START_REF] Van Deun | The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling[END_REF] Their application should be limited to samples known to be rich in small extracellular vesicles, for the study of RNA, or be used as a concentration method after using another separation method. [START_REF] Witwer | Standardization of sample collection, isolation and analysis methods in extracellular vesicle research[END_REF][START_REF] Andreu | Comparative analysis of EV isolation procedures for miRNAs detection in serum samples[END_REF][START_REF] Taylor | Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes[END_REF] Thus, as a general rule, the kits' applicability in a biomarker setting should be considered with due caution. [START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF] 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 Supplementary Text. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Witwer | Standardization of sample collection, isolation and analysis methods in extracellular vesicle research[END_REF][START_REF] Wisgrill | Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants[END_REF][START_REF] Andreu | Comparative analysis of EV isolation procedures for miRNAs detection in serum samples[END_REF][START_REF] George | Isolation of human platelet membrane microparticles from plasma and serum[END_REF][START_REF] Pisitkun | Identification and proteomic profiling of exosomes in human urine[END_REF][START_REF] Awdishu | Identification of Maltase Glucoamylase as a Biomarker of Acute Kidney Injury in Patients with Cirrhosis[END_REF][START_REF] Merchant | Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery[END_REF][START_REF] Sirica | Intrahepatic Cholangiocarcinoma: Continuing Challenges and Translational Advances[END_REF][START_REF] Engelmann | Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study[END_REF][START_REF] Li | Human bile contains microRNA-laden extracellular vesicles that can be used for Thietart S & Rautou PE Extracellular vesicles in liver diseases 37 cholangiocarcinoma diagnosis[END_REF][START_REF] Severino | Extracellular Vesicles in Bile as Markers of Malignant Biliary Stenoses[END_REF][START_REF] Li | Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile[END_REF][START_REF] Ge | The diagnostic/prognostic potential and molecular functions of long non-coding RNAs in the exosomes derived from the bile of human cholangiocarcinoma[END_REF][START_REF] Hogan | Strategy and rationale for urine collection protocols employed in the NEPTUNE study[END_REF]

Analytical techniques

From a biomarker perspective, measurement of plasma extracellular vesicle concentrations involves a compromise between very accurate characterization of extracellular vesicles, as proposed by MISEV 2018 guidelines [START_REF] Théry | 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[END_REF], and operational strategies that can be adapted to routine laboratory settings in a rapid and inexpensive manner.

Accurate characterization

Thietart S & Rautou PE Extracellular vesicles in liver diseases 12

Several steps are proposed in the MISEV 2018 guidelines [START_REF] Théry | 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[END_REF] in order to establish an extracellular vesicle separation method. These steps include quantification, general characterization, single vesicle characterization and are described in Supplementary

Text. [START_REF] Théry | 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[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Arraud | Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration[END_REF][START_REF] György | Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters[END_REF][START_REF] Takov | Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes[END_REF][START_REF] Sódar | Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection[END_REF][START_REF] Larson | Calcium-phosphate microprecipitates mimic microparticles when examined with flow cytometry[END_REF] Available technologies that may be used to perform all these steps are listed in Table 2. [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF][START_REF] Théry | 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[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Arraud | Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration[END_REF][START_REF] Van Der Pol | Optical and non-optical methods for detection and characterization of microparticles and exosomes[END_REF][START_REF] Obeid | Development of a NanoBioAnalytical platform for "on-chip" qualification and quantification of plateletderived microparticles[END_REF][START_REF] Liang | Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring[END_REF][START_REF] Koliha | A novel multiplex bead-based platform highlights the diversity of extracellular vesicles[END_REF][START_REF] Corso | Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size Thietart S & Rautou PE Extracellular vesicles in liver diseases 39 exclusion chromatography[END_REF][START_REF] Gool | Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles[END_REF][START_REF] Jørgensen | Potentials and capabilities of the Extracellular Vesicle (EV) Array[END_REF][START_REF] Szatanek | The Methods of Choice for Extracellular Vesicles (EVs) Characterization[END_REF][START_REF] Tatischeff | Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy[END_REF][START_REF] Wyss | Molecular and Dimensional Profiling of Highly Purified Extracellular Vesicles by Fluorescence Fluctuation Spectroscopy[END_REF][START_REF] Coumans | Bulk immunoassays for analysis of extracellular vesicles[END_REF][START_REF] Ramirez | Technical challenges of working with extracellular vesicles[END_REF] 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. [START_REF] Théry | 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[END_REF] These steps do not need to be repeated, once the separation and routine measurement method has been validated.

Routine measurement of extracellular vesicles

Measurement methods for extracellular vesicles as biomarkers in routine clinical settings should exhibit the following characteristics: they should be able to detect small events as well as specific sub-populations; 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 as biomarkers are summarized in Table 3. [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Coumans | Bulk immunoassays for analysis of extracellular vesicles[END_REF][START_REF] Ramirez | Technical challenges of working with extracellular vesicles[END_REF][START_REF] Van Der Pol | Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation[END_REF][START_REF] Van Der Pol | Single vs. swarm detection of microparticles and exosomes by flow cytometry[END_REF][START_REF] Kormelink | Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry[END_REF][START_REF] Duijvesz | Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer[END_REF] High sensitivity-flow cytometry on a dedicated device can simultaneously detect light scatter (i.e. diameter) and fluorescence signal (i.e. an extracellular vesicle sub-population)

from extracellular vesicles passing one by one in front of a laser beam. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF] It is the most commonly used technique for measuring concentrations of total extracellular vesicles as well as sub-populations of extracellular vesicles, and can also determine cellular origin. Dedicated high-resolution flow cytometers are required, using bead-based calibrations or a scatterdiameter relationship model in order to detect small extracellular vesicles. [START_REF] Kormelink | Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry[END_REF][START_REF] Cointe | Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop[END_REF] Yet, a major limitation resides in the variability among instruments of the smallest detectable size, which

Thietart S & Rautou PE
Extracellular vesicles in liver diseases 13 affects the measured concentrations, and is responsible for differing sensitivities between instruments. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF][START_REF] Van Der Pol | Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation[END_REF] In response to this high interlaboratory variability, efforts to standardize the technique are currently underway. [START_REF] Van Der Pol | Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation[END_REF][START_REF] Cointe | Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop[END_REF][START_REF] Lacroix | Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis Thietart S & Rautou PE Extracellular vesicles in liver diseases 41 SSC Collaborative workshop[END_REF] Sandwich enzyme-linked immunosorbent assay (ELISA) quantifies specific extracellular vesicle-bound proteins. [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Coumans | Bulk immunoassays for analysis of extracellular vesicles[END_REF] It is a reproducible detection method, using colorimetric or fluorescent detection, giving 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. [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF][START_REF] Bissonnette | A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis[END_REF] Filtration/ELISA technique is described in Fig. 2.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a method that quantifies a specific RNA transcript. [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF] It is the most widely used method in biomarker identification as it is robust, low-cost, and has high analytical sensitivity. [START_REF] Ramirez | Technical challenges of working with extracellular vesicles[END_REF] The check list of experimental details for RNA analysis has been previously described. [START_REF] Hill | ISEV position paper: extracellular vesicle RNA analysis and bioinformatics[END_REF] Other RNA quantification methods validated for extracellular vesicles are enumerated in Table 2. [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNAan ISEV position paper[END_REF][START_REF] Théry | 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[END_REF][START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF][START_REF] Arraud | Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration[END_REF][START_REF] Van Der Pol | Optical and non-optical methods for detection and characterization of microparticles and exosomes[END_REF][START_REF] Obeid | Development of a NanoBioAnalytical platform for "on-chip" qualification and quantification of plateletderived microparticles[END_REF][START_REF] Liang | Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring[END_REF][START_REF] Koliha | A novel multiplex bead-based platform highlights the diversity of extracellular vesicles[END_REF][START_REF] Corso | Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size Thietart S & Rautou PE Extracellular vesicles in liver diseases 39 exclusion chromatography[END_REF][START_REF] Gool | Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles[END_REF][START_REF] Jørgensen | Potentials and capabilities of the Extracellular Vesicle (EV) Array[END_REF][START_REF] Szatanek | The Methods of Choice for Extracellular Vesicles (EVs) Characterization[END_REF][START_REF] Tatischeff | Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy[END_REF][START_REF] Wyss | Molecular and Dimensional Profiling of Highly Purified Extracellular Vesicles by Fluorescence Fluctuation Spectroscopy[END_REF][START_REF] Coumans | Bulk immunoassays for analysis of extracellular vesicles[END_REF][START_REF] Ramirez | Technical challenges of working with extracellular vesicles[END_REF] Northern blot is a robust and sensitive method to measure specific RNA transcripts, but 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 summarized 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. [START_REF] Coumans | Methodological Guidelines to Study Extracellular Vesicles[END_REF] Current development of fluorescent

Extracellular vesicles in liver diseases

Extracellular vesicles appear to be attractive biomarkers for diagnosis, 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 sub-populations as biomarkers in liver diseases are summarized in Table 4 (review criteria are summarized in Supplementary Text). Unless otherwise mentioned, the studied extracellular vesicles were all derived from plasma.

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. [START_REF] Chalasani | The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases[END_REF] Extracellular vesicles that reflect ongoing cell damage might be able to fill this gap and better capture the disease's complexity. Supplementary Table 1

Alcoholic hepatitis

The two main clinical challenges in alcoholic hepatitis are the elaboration of tests for non-invasive diagnosis of the disease and prediction of response to treatment.

Diagnosis of alcoholic hepatitis

Our group tested whether plasma extracellular vesicle levels could diagnose alcoholic hepatitis non-invasively. In two 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. [START_REF] Bissonnette | A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis[END_REF] Higher levels of hepatocyte-derived (ASPGR+) extracellular vesicles have also been found in 101 patients with severe alcoholic hepatitis, in comparison with healthy controls. [START_REF] Sukriti | Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis[END_REF] Plasma concentrations of total extracellular vesicles, as well as of extracellular vesicles derived from T cells (plasma and serum), macrophages, neutrophils, hematopoietic stem cells and endothelial cells are found to be higher among patients with severe alcoholic hepatitis than among healthy controls. [START_REF] Sukriti | Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis[END_REF][START_REF] Verma | Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of Thietart S & Rautou PE Extracellular vesicles in liver diseases 43 CD40L containing extracellular vesicles[END_REF] 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. [START_REF] Bissonnette | A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis[END_REF] Along the same lines, plasma and serum concentrations of several sub-populations of extracellular vesicles (carrying mitochondrial DNA, miRNA or hepatocyte proteins) are higher in patients with binge drinking and/or chronic excessive alcohol consumption, as summarized in Supplementary Table 2. [START_REF] Bissonnette | A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis[END_REF][START_REF] Sukriti | Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis[END_REF][START_REF] Verma | Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of Thietart S & Rautou PE Extracellular vesicles in liver diseases 43 CD40L containing extracellular vesicles[END_REF][START_REF] Eguchi | Circulating Extracellular Vesicles and Their miR "Barcode" Differentiate Alcohol Drinkers With Liver Injury and Those Without Liver Injury in Severe Trauma Patients[END_REF][START_REF] Cho | Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug-and alcohol-induced liver injury[END_REF][START_REF] Cai | Mitochondrial DNAenriched microparticles promote acute-on-chronic alcoholic neutrophilia and hepatotoxicity[END_REF][START_REF] Momen-Heravi | Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS[END_REF][START_REF] Eguchi | Extracellular vesicles released by hepatocytes from gastric infusion model of alcoholic liver disease contain a MicroRNA barcode that can be detected in blood[END_REF][START_REF] Saha | MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages[END_REF][START_REF] Wang | Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration[END_REF][START_REF] Sehrawat | Circulating extracellular vesicles carrying sphingolipid cargo for the diagnosis and dynamic risk profiling of alcoholic hepatitis[END_REF][START_REF] Nielsen | Extracellular vesicle-associated soluble CD163 and CD206 in patients with acute and chronic inflammatory liver disease[END_REF][START_REF] Babuta | Dysregulated Autophagy and Lysosome Function Are Linked to Exosome Production by Micro-RNA 155 in Alcoholic Liver Disease[END_REF][START_REF] Arab | An Open-Label, Dose-Escalation Study to Assess the Safety and Efficacy of IL-22 Agonist F-652 in Patients With Alcohol-associated Hepatitis[END_REF][START_REF] Cho | Increased ethanolinducible cytochrome P450-2E1 and cytochrome P450 isoforms in exosomes of alcohol-exposed rodents and patients with alcoholism through oxidative and endoplasmic reticulum stress[END_REF] 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 hematopoietic 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 one-month mortality. [START_REF] Sukriti | Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis[END_REF] The combination of the two 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 sub-populations (T cell, macrophage and endothelial) evaluated in this study were higher among nonresponders to steroid therapy, but were less accurate in predicting mortality. [START_REF] Sukriti | Microvesicles in hepatic and peripheral vein can predict nonresponse to corticosteroid therapy in severe alcoholic hepatitis[END_REF] To conclude, circulating hepatocyte-derived extracellular vesicles seem to be a promising biomarker for diagnosis and outcome of patients with alcoholic hepatitis. Data on other sub-populations are less homogenous and need to be confirmed in future studies.

Hepatitis B and C virus infections

Cirrhosis

Different extracellular vesicle sub-populations increase in patients with cirrhosis, and are predictive of disease progression, complications and mortality (Supplementary Table 5). [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF][START_REF] Awdishu | Identification of Maltase Glucoamylase as a Biomarker of Acute Kidney Injury in Patients with Cirrhosis[END_REF][START_REF] Engelmann | Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study[END_REF][START_REF] Nielsen | Extracellular vesicle-associated soluble CD163 and CD206 in patients with acute and chronic inflammatory liver disease[END_REF][START_REF] Wang | Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma[END_REF][START_REF] Brodsky | Dynamics of circulating microparticles in liver transplant patients[END_REF][125][START_REF] Campello | Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis[END_REF][START_REF] Ogasawara | Platelet activation in patients with alcoholic liver disease[END_REF][START_REF] Fusegawa | Platelet activation in patients with chronic hepatitis C[END_REF][START_REF] Sayed | Monocyte-platelet aggregates and platelet microparticles in patients with post-hepatitic liver cirrhosis[END_REF][START_REF] Eyraud | Evolution of platelet functions in cirrhotic patients undergoing liver transplantation: A prospective exploration over a month[END_REF][START_REF] Taleb | Quantitative proteome analysis of plasma microparticles for the characterization of HCV-induced hepatic cirrhosis and hepatocellular carcinoma[END_REF][START_REF] Kostallari | Hepatic stellate cell-derived platelet-derived growth factor receptor-alpha-enriched Thietart S & Rautou PE Extracellular vesicles in liver diseases 49 extracellular vesicles promote liver fibrosis in mice through SHP2[END_REF][START_REF] Brandon-Warner | Processing of miR17-92 Cluster in Hepatic Stellate Cells Promotes Hepatic Fibrogenesis During Alcohol-Induced Injury[END_REF][START_REF] Lambrecht | A PDGFRβbased score predicts significant liver fibrosis in patients with chronic alcohol abuse, NAFLD and viral liver disease[END_REF][START_REF] Rautou | Circulating microparticle tissue factor activity is increased in patients with cirrhosis[END_REF] Doubt remains, however, on whether this increase is due to excess production, decreased clearance, or, most likely, both.

[10]

Estimation of cirrhosis severity

Hepatocyte extracellular vesicle concentrations, characterized by cytokeratin-18 expression, significantly increased with Child-Pugh score in 3 independent prospective cohorts of 40, 139 and 103 patients. [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF] Their concentrations also correlated with Model for End-Stage Liver Disease (MELD) and its components. [START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF] In the same study, hepatocyte larger extracellular vesicle concentrations increased with histological liver necroinflammatory activity, suggesting that hepatocyte injury is the driver for extracellular vesicle release. [START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF] Cirrhosis is known to be responsible for vascular changes including systemic with plasma leuko-endothelial (CD31+/41-) larger extracellular vesicles in an initial cohort of 91 patients [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF], but was subsequently not confirmed using the same markers (CD31+/41-) [START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF] or other markers (CD31+/42b-).

[125]

With systemic inflammation being a key feature of cirrhosis, several sub-populations of leukocyte-derived extracellular vesicles (CD45+, CD11a+ and CD4+) have been analyzed

in different studies and found to be higher than in healthy individuals. [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF][START_REF] Campello | Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis[END_REF] However, no link with cirrhosis severity was observed.

Data on platelet extracellular vesicles are controversial. Three studies have found a significant increase in platelet extracellular vesicles in patients with cirrhosis, compared with healthy controls, using CD62P+ as a marker. [START_REF] Campello | Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis[END_REF][START_REF] Ogasawara | Platelet activation in patients with alcoholic liver disease[END_REF][START_REF] Fusegawa | Platelet activation in patients with chronic hepatitis C[END_REF] Four other studies have found no significant difference using the CD41+ marker. [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF][START_REF] Sayed | Monocyte-platelet aggregates and platelet microparticles in patients with post-hepatitic liver cirrhosis[END_REF][START_REF] Eyraud | Evolution of platelet functions in cirrhotic patients undergoing liver transplantation: A prospective exploration over a month[END_REF] CD41+ extracellular vesicle levels did not change with cirrhosis severity. [START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF][START_REF] Sayed | Monocyte-platelet aggregates and platelet microparticles in patients with post-hepatitic liver cirrhosis[END_REF] Other platelet extracellular vesicles were studied and their levels were found to overlap with healthy controls. [START_REF] Chandler | Patients with cirrhosis show a relative increase in thrombin generation that is correlated with lower antithrombin levels[END_REF] These contradictory results could be due to the use of different platelet markers, as well as numerous factors influencing platelet extracellular vesicle release during plasma sample preparation, therefore making it an unreliable marker. [START_REF] Berckmans | Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation[END_REF] Pilot studies, described in Supplementary

Prediction of mortality

In a prospective collaborative study, including a validation cohort, we demonstrated that plasma hepatocyte-derived extracellular vesicle levels can predict 6-month mortality, independently of Child-Pugh and MELD scores, using a threshold of 65 UI/L. [START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF] In this study, other sub-populations of plasma extracellular vesicles, including pan-leukocyte (CD11+) and endothelial (CD144+, CD62E+), were not useful for predicting mortality. Only small leuko-endothelial (CD31+/41-) extracellular vesicles also predicted 6-month mortality independently of Child-Pugh score, in two studies. [START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF] The potential interest of ascites extracellular vesicles has been investigated in 163 patients with cirrhosis. [START_REF] Engelmann | Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study[END_REF] The study found that low total ascites extracellular vesicle levels predicted 30-day mortality independently of MELD and antibiotic treatment. High percentages of ascites extracellular vesicles derived from neutrophil (CD66b+) or lymphocyte (CD3) were more commonly

Thietart S & Rautou PE
Extracellular vesicles in liver diseases 21 observed in patients with poor outcomes, but survival analyses were not performed with these sub-populations. [START_REF] Engelmann | Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study[END_REF] In conclusion, several sub-populations of extracellular vesicles seem to be promising candidate biomarkers to evaluate cirrhosis severity or predict mortality. Data are missing on the prediction of more specific complications and this data gap will need to be filled if personalized monitoring strategies are to be achieved.

Hepatocellular carcinoma

An unmet need is the development of tools to predict the risk of occurrence of hepatocellular carcinoma (HCC) among patients with chronic liver diseases, in order to identify those requiring closer disease monitoring. However, no study has evaluated the potential interest of extracellular vesicles in meeting this need. Conversely, abundant data suggest that extracellular vesicles could help detect HCC, predict HCC recurrence, survival and might also be associated with tumor aggressiveness.

Detection of hepatocellular carcinoma

A major challenge is to find markers facilitating early detection of HCC, among patients with chronic liver diseases. Ideally, studies should include patients with early HCC and use as controls patients with chronic liver diseases similar to those with HCC, but this study design has rarely been applied. [START_REF] Pu | Extracellular Vesicle-Associated mir-21 and mir-144 Are Markedly Elevated in Serum of Patients With Hepatocellular Carcinoma[END_REF][START_REF] Wang | Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma[END_REF][START_REF] Brodsky | Dynamics of circulating microparticles in liver transplant patients[END_REF][START_REF] Campello | Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis[END_REF][START_REF] Julich-Haertel | Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma[END_REF][START_REF] Abbate | HepPar1-Positive Circulating Microparticles Are Increased in Subjects with Hepatocellular Carcinoma and Predict Early Recurrence after Liver Resection[END_REF][START_REF] Sohn | Serum exosomal microRNAs as novel biomarkers for hepatocellular carcinoma[END_REF][START_REF] Wang | Expression of serum exosomal microRNA-21 in human hepatocellular carcinoma[END_REF][START_REF] Xu | Serum Exosomal Long Noncoding RNAs ENSG00000258332.1 and LINC00635 for the Diagnosis and Prognosis of Thietart S & Rautou PE Extracellular vesicles in liver diseases 51 Hepatocellular Carcinoma[END_REF][START_REF] Xu | Serum exosomal hnRNPH1 mRNA as a novel marker for hepatocellular carcinoma[END_REF][START_REF] Wang | Serum exosomal microRNAs combined with alpha-fetoprotein as diagnostic markers of hepatocellular carcinoma[END_REF][START_REF] Liu | Serum exosomal miR-125b is a novel prognostic marker for hepatocellular carcinoma[END_REF][START_REF] Fornari | Circulating microRNAs, miR-939, miR-595, miR-519d and miR-494, Identify Cirrhotic Patients with HCC[END_REF][START_REF] El Gwad | Role of exosomal competing endogenous RNA in patients with hepatocellular carcinoma[END_REF][START_REF] Ma | X-inactive-specific transcript of peripheral blood cells is regulated by exosomal Jpx and acts as a biomarker for female patients with hepatocellular carcinoma[END_REF][START_REF] Li | Microvesicle microRNA profiles and functional roles between chronic hepatitis B and hepatocellular carcinoma[END_REF] Total extracellular vesicles, as well as some sub-populations, increase in the plasma [START_REF] Brodsky | Dynamics of circulating microparticles in liver transplant patients[END_REF] and sera [START_REF] Wang | Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma[END_REF] Other circulating extracellular vesicle sub-populations have been tested, but use of low-throughput measurement techniques, absence of precise description of measurement methods or patient characteristics, small sample sizes, use of healthy controls or advanced HCC, or absence of confirmation on whether the marker truly circulates in the extracellular vesicle fraction limits interpretation of the results (Supplementary Table 6). [START_REF] Wang | Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma[END_REF][START_REF] Brodsky | Dynamics of circulating microparticles in liver transplant patients[END_REF][START_REF] Campello | Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis[END_REF][START_REF] Taleb | Quantitative proteome analysis of plasma microparticles for the characterization of HCV-induced hepatic cirrhosis and hepatocellular carcinoma[END_REF][START_REF] Brandon-Warner | Processing of miR17-92 Cluster in Hepatic Stellate Cells Promotes Hepatic Fibrogenesis During Alcohol-Induced Injury[END_REF][START_REF] Julich-Haertel | Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma[END_REF][START_REF] Abbate | HepPar1-Positive Circulating Microparticles Are Increased in Subjects with Hepatocellular Carcinoma and Predict Early Recurrence after Liver Resection[END_REF][START_REF] Sohn | Serum exosomal microRNAs as novel biomarkers for hepatocellular carcinoma[END_REF][START_REF] Wang | Expression of serum exosomal microRNA-21 in human hepatocellular carcinoma[END_REF][START_REF] Xu | Serum Exosomal Long Noncoding RNAs ENSG00000258332.1 and LINC00635 for the Diagnosis and Prognosis of Thietart S & Rautou PE Extracellular vesicles in liver diseases 51 Hepatocellular Carcinoma[END_REF][START_REF] Xu | Serum exosomal hnRNPH1 mRNA as a novel marker for hepatocellular carcinoma[END_REF][START_REF] Wang | Serum exosomal microRNAs combined with alpha-fetoprotein as diagnostic markers of hepatocellular carcinoma[END_REF][START_REF] Liu | Serum exosomal miR-125b is a novel prognostic marker for hepatocellular carcinoma[END_REF][START_REF] Fornari | Circulating microRNAs, miR-939, miR-595, miR-519d and miR-494, Identify Cirrhotic Patients with HCC[END_REF][START_REF] El Gwad | Role of exosomal competing endogenous RNA in patients with hepatocellular carcinoma[END_REF][START_REF] Ma | X-inactive-specific transcript of peripheral blood cells is regulated by exosomal Jpx and acts as a biomarker for female patients with hepatocellular carcinoma[END_REF][START_REF] Li | Microvesicle microRNA profiles and functional roles between chronic hepatitis B and hepatocellular carcinoma[END_REF] Predictor of recurrence and survival Extracellular vesicles could help predict HCC recurrence after liver resection. High levels of pre-operative plasma small extracellular vesicle miRNA-155 were predictive of lower relapse-free survival in a retrospective study of 40 patients with mainly unifocal HCC treated with 1 st line curative hepatectomy, although no difference in 3-and 5-year overall survival was found. [START_REF] Matsuura | Exosomal miR-155 Derived from Hepatocellular Carcinoma Cells Under Hypoxia Promotes Angiogenesis in Endothelial Cells[END_REF] In another study, recurrence was higher when serum extracellular vesicle miRNA-125 expression was low among 128 patients with HCC treated by surgical resection, in multivariate analysis. [START_REF] Liu | Serum exosomal miR-125b is a novel prognostic marker for hepatocellular carcinoma[END_REF] Circulating extracellular vesicle RNA expression can also predict overall survival. For example, higher levels of serum small extracellular vesicle RNA RAB11A was associated with lower relapse-free survival in multivariate analysis among 60 patients with mainly early HCC. [START_REF] El Gwad | Role of exosomal competing endogenous RNA in patients with hepatocellular carcinoma[END_REF] Several studies have found serum small extracellular vesicle miRNA-21 to be a potentially interesting marker of outcome. [START_REF] Tian | Acidic Microenvironment Up-Regulates Exosomal miR-21 and miR-10b in Early-Stage Hepatocellular Carcinoma to Promote Cancer Cell Proliferation and Metastasis[END_REF]191,[START_REF] Zhou | Hepatocellular carcinoma-derived exosomal miRNA-21 contributes to tumor progression by converting hepatocyte stellate cells to cancer-associated fibroblasts[END_REF] Notably, a prospective study of 79 patients with HCC observed that serum small extracellular vesicle miRNA-21 expression was independently associated with overall survival and disease progression. [191] Thietart S & Rautou PE

Extracellular vesicles in liver diseases 24

To conclude, many proteins and RNAs contained in extracellular vesicles have been identified as predictors of outcome among patients with HCC (Supplementary Tables 7 and8). [START_REF] Itami-Matsumoto | Circulating Exosomal miRNA Profiles Predict the Occurrence and Recurrence of Hepatocellular Carcinoma in Patients with Direct-Acting Antiviral-Induced Sustained Viral Response[END_REF][START_REF] Matsuura | Exosomal miR-155 Derived from Hepatocellular Carcinoma Cells Under Hypoxia Promotes Angiogenesis in Endothelial Cells[END_REF][START_REF] Abbate | HepPar1-Positive Circulating Microparticles Are Increased in Subjects with Hepatocellular Carcinoma and Predict Early Recurrence after Liver Resection[END_REF][START_REF] Xu | Serum Exosomal Long Noncoding RNAs ENSG00000258332.1 and LINC00635 for the Diagnosis and Prognosis of Thietart S & Rautou PE Extracellular vesicles in liver diseases 51 Hepatocellular Carcinoma[END_REF][START_REF] Xu | Serum exosomal hnRNPH1 mRNA as a novel marker for hepatocellular carcinoma[END_REF][START_REF] Liu | Serum exosomal miR-125b is a novel prognostic marker for hepatocellular carcinoma[END_REF][START_REF] El Gwad | Role of exosomal competing endogenous RNA in patients with hepatocellular carcinoma[END_REF][START_REF] Tian | Acidic Microenvironment Up-Regulates Exosomal miR-21 and miR-10b in Early-Stage Hepatocellular Carcinoma to Promote Cancer Cell Proliferation and Metastasis[END_REF][START_REF] Xue | Development and validation of serum exosomal microRNAs as diagnostic and prognostic biomarkers for hepatocellular carcinoma[END_REF][START_REF] Suehiro | Serum exosomal microRNA-122 and microRNA-21 as predictive biomarkers in transarterial chemoembolization-treated hepatocellular carcinoma patients[END_REF][START_REF] Fu | Primary tumor-derived exosomes facilitate metastasis by regulating adhesion of circulating tumor cells via SMAD3 in liver cancer[END_REF][START_REF] Cui | Mechanism of exosomal microRNA-224 in development of hepatocellular carcinoma and its diagnostic and prognostic value[END_REF][START_REF] Xue | Exosomal miR-93 promotes proliferation and invasion in hepatocellular carcinoma by directly inhibiting Thietart S & Rautou PE Extracellular vesicles in liver diseases 54 TIMP2/TP53INP1/CDKN1A[END_REF][START_REF] Shi | Decreased levels of serum exosomal miR-638 predict poor prognosis in hepatocellular carcinoma[END_REF][START_REF] Wang | Three isoforms of exosomal circPTGR1 promote hepatocellular carcinoma metastasis via the miR449a-MET pathway[END_REF]183,[START_REF] Cho | Serum Exosomal MicroRNA, miR-10b-5p, as a Potential Diagnostic Biomarker for Early-Stage Hepatocellular Carcinoma[END_REF][START_REF] Li | Downregulation of serum exosomal miR-320d predicts poor prognosis in hepatocellular carcinoma[END_REF][191][START_REF] Zhou | Hepatocellular carcinoma-derived exosomal miRNA-21 contributes to tumor progression by converting hepatocyte stellate cells to cancer-associated fibroblasts[END_REF][START_REF] Gramantieri | LncRNAs as novel players in hepatocellular carcinoma recurrence[END_REF][START_REF] Nakano | Circulating exosomal miR-92b: Its role for cancer immunoediting and clinical value for prediction of posttransplant hepatocellular carcinoma recurrence[END_REF][START_REF] Fang | Hepatoma cellsecreted exosomal microRNA-103 increases vascular permeability and promotes metastasis by targeting junction proteins[END_REF][START_REF] Fang | Tumor-derived exosomal miR-1247-3p induces cancer-associated fibroblast activation to foster lung metastasis of liver cancer[END_REF][START_REF] Sugimachi | Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation[END_REF][198][START_REF] Huang | Exosomal circRNA-100338 promotes hepatocellular carcinoma metastasis via enhancing invasiveness and angiogenesis[END_REF] These findings should nevertheless be confirmed in patients with all stages of HCC progression, taking into account confounding factors such as comorbidities and underlying liver disease.

Primary sclerosing cholangitis & cholangiocarcinoma

Major challenges in clinical management of patients with primary sclerosing cholangitis include the unpredictable outcome of this liver disease in terms of progression and evolution towards cholangiocarcinoma. [START_REF] Lazaridis | Primary Sclerosing Cholangitis[END_REF] Data on serum extracellular vesicles as markers in this setting are limited to a proteomic analysis of serum extracellular vesicles: 10 proteins were found to be differentially expressed in patients with cholangiocarcinoma (n = 43), as compared with patients with primary sclerosing cholangitis (n = 30), as well as with HCC (n = 29). [START_REF] Arbelaiz | Serum extracellular vesicles contain protein biomarkers for primary sclerosing Thietart S & Rautou PE Extracellular vesicles in liver diseases 55 cholangitis and cholangiocarcinoma[END_REF] Western blot analyses confirmed these results for 6 proteins, but clinical validation is needed. One team has also analyzed both serum and urine extracellular vesicles, and found different RNA transcriptomic profiles between patients with cholangiocarcinoma (n = 12) and those with primary sclerosing cholangitis (n = 6), although, here again, clinical confirmation is needed. [START_REF] Lapitz | Patients with Cholangiocarcinoma Present Specific RNA Profiles in Serum and Urine Extracellular Vesicles Mirroring the Tumor Expression: Novel Liquid Biopsy Biomarkers for Disease Diagnosis[END_REF] One of the main challenges in biliary diseases is the difficult differentiation of benign strictures from malignant ones.

Two independent groups demonstrated that bile or serum extracellular vesicles could efficiently differentiate cholangiocarcinoma from benign biliary stenosis. One group reported that total bile and serum extracellular vesicles, measured using nanoparticle tracking analysis, could distinguish malignant biliary stenosis (pancreatic cancer and cholangiocarcinoma) from

Thietart S & Rautou PE
Extracellular vesicles in liver diseases 25 benign stenosis (chronic pancreatitis), bile extracellular vesicles having a significantly better discriminating ability than serum CA19-9. [START_REF] Severino | Extracellular Vesicles in Bile as Markers of Malignant Biliary Stenoses[END_REF] Others have observed an overexpression of an small extracellular vesicle miRNA panel in bile extracellular vesicles from 26 patients with cholangiocarcinoma, as compared with 50 controls with benign stenosis (including 13 primary sclerosing cholangitis). [START_REF] Li | Human bile contains microRNA-laden extracellular vesicles that can be used for Thietart S & Rautou PE Extracellular vesicles in liver diseases 37 cholangiocarcinoma diagnosis[END_REF] Beyond the setting of biliary strictures, other studies have analyzed extracellular vesicles in patients with cholangiocarcinoma. One study observed that circulating tumor cellderived (AnnexinV+EpCAM+ASGPR1+) extracellular vesicles were 3-fold higher in the serum of 38 patients with cholangiocarcinoma than in 49 patients with cirrhosis. [START_REF] Julich-Haertel | Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma[END_REF] These tumor cell-derived extracellular vesicles were, however, unable to differentiate HCC from cholangiocarcinoma. [START_REF] Julich-Haertel | Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma[END_REF] A second study identified 2 small extracellular vesicle lncRNAs that were significantly overexpressed in the bile of 35 patients with cholangiocarcinoma, as compared with 56 healthy controls. [START_REF] Ge | The diagnostic/prognostic potential and molecular functions of long non-coding RNAs in the exosomes derived from the bile of human cholangiocarcinoma[END_REF] Their combination yielded higher sensitivity and specificity than CA19-9, and was associated with lower overall survival. A third study compared the expression of small extracellular vesicle miRNA-200c-3p between 36 patients with cholangiocarcinoma and 12 healthy controls. [START_REF] Shen | Exosomal miR-200 family as serum biomarkers for early detection and prognostic prediction of cholangiocarcinoma[END_REF] Expression of small extracellular vesicle miRNA-200c-3p was higher among patients with cholangiocarcinoma, and increased with tumor stage and the presence of lymph node metastasis. Patients with higher levels of miRNA-200c-3p had lower disease-free and overall-free survivals. [START_REF] Shen | Exosomal miR-200 family as serum biomarkers for early detection and prognostic prediction of cholangiocarcinoma[END_REF] Two other preclinical studies found differentially expressed extracellular vesicle RNAs between patients with cholangiocarcinoma and healthy controls. [START_REF] Wang | Circ-0000284 arouses malignant phenotype of cholangiocarcinoma cells and regulates the biological functions of peripheral cells through cellular communication[END_REF][START_REF] Gu | Exosomal piRNA profiling revealed unique circulating piRNA signatures of cholangiocarcinoma and gallbladder carcinoma[END_REF] To conclude, several sub-populations of extracellular vesicles have the potential to become very useful tools to differentiate cholangiocarcinoma from PSC and other tumors.

The use of demanding techniques to measure extracellular vesicles would likely not be a

Acute liver failure

Detection of acute liver failure

Diagnosis of acute liver injury/failure is usually easy, as it is based on readily available clinical and laboratory data.

[205] In contrast, prediction or early identification of drug-induced liver injury remains an unmet need. Since extracellular vesicles are sensitive markers of cell injury, we can hypothesize that their circulating levels could be an early marker of drug-induced liver injury, even before liver blood test abnormalities appear. So far, data on this hypothesis are very limited. Most studies compared patients with acute liver injury with healthy controls, thereby limiting their clinical relevance (Supplementary Table 9). [START_REF] Stravitz | Role of procoagulant microparticles in mediating complications and outcome of acute liver injury/acute liver failure[END_REF][START_REF] Duan | Role of extracellular vesicles in release of protein adducts after acetaminophen-induced liver injury in mice and humans[END_REF][START_REF] Schmelzle | Increased plasma levels of microparticles expressing CD39 and CD133 in acute liver injury[END_REF][START_REF] Zhang | Identification of exosomal miRNAs associated with the anthracycline-induced liver injury in postoperative breast cancer patients by small RNA sequencing[END_REF]

Predictor of outcome

The interest of extracellular vesicles in the acute liver injury/acute liver failure setting has been analyzed by the "Acute Liver Failure Study Group" in a group of 50 patients.

Plasma concentrations of total larger-size extracellular vesicles significantly increased with hepatic encephalopathy grade, as well as with systemic inflammatory response syndrome. [START_REF] Stravitz | Role of procoagulant microparticles in mediating complications and outcome of acute liver injury/acute liver failure[END_REF] Moreover, concentrations of these extracellular vesicles were associated with death or liver transplantation at day-21 after admission, independently of other predictors (age, gender, and etiology). [START_REF] Stravitz | Role of procoagulant microparticles in mediating complications and outcome of acute liver injury/acute liver failure[END_REF] Extracellular vesicles were mainly of platelet (CD41+)

origin, but also of hepatocyte (ASPGR+), monocyte (CD18+) and endothelial cell (CD144+) origin and were tissue-factor positive, although each specific sub-population was not significantly associated with severity. [START_REF] Stravitz | Role of procoagulant microparticles in mediating complications and outcome of acute liver injury/acute liver failure[END_REF] Thietart S & Rautou PE Extracellular vesicles in liver diseases 27

Conclusion

Extracellular vesicles hold promise in liver diseases as biomarkers for diagnosis and prediction of disease progression, complications, response to treatment and mortality.

Although data so far are derived from pilot studies, some sub-populations of extracellular vesicles have been robustly evaluated in independent large multicentric prospective cohorts, as summarized in Fig. 3.

Guidelines on extracellular vesicle research stress the importance of parameters such as purity, extracellular vesicle structure, physical characteristics and marker localization, while their importance in clinical research is less explicitly covered. [START_REF] Théry | 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[END_REF] Other parameters which are less often considered in basic research, such as cost, assay time, laborintensiveness, accessibility of measuring devices, inter-laboratory variability, characteristics of study population and control group, are more relevant for application in clinical settings.

Several challenges must be overcome prior to considering the use of extracellular vesicles in clinical routines. [START_REF] Ayers | Clinical requirements for extracellular vesicle assays[END_REF] First, the reliability and reproducibility of the quantification methods have to be strengthened. In the current state of affairs, results are likely to reflect the settings of several preanalytical variables. For example, measured vesicle concentrations can be influenced by the minimum vesicle size that the technique can detect. [START_REF] Van Der Pol | Particle size distribution of exosomes and microvesicles determined by transmission[END_REF] The variation coefficient is often not mentioned in studies or is high and variable. There is no international reference preparation (gold standard) to assess trueness of assay. Second, elaboration of highthroughput detection methods, not affecting sample stability and diversity, would facilitate the development of extracellular vesicle measurement. Currently, many characterization techniques are prohibitively expensive and/or labor-intensive. Third, determination of cut-off values is needed to help physicians make decisions. Forth, validation of results is needed using studies involving large cohorts of patients with comorbidities. Graphical representation of the filtration/ELSA 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 1000 nm was observed (n=2). Abbreviations: ELISA: sandwich enzyme-linked immunosorbent assay; EV: extracellular vesicle. Over the last 20 years, terms used for naming different types of extracellular vesicles varied.

Extracellular vesicle subtypes were previously defined by their biogenesis pathway, "exosomes" having an endosomal origin, "microvesicles" deriving from plasma membrane and "apoptotic bodies" being released by dying cells. However, no specific marker of biogenesis pathway has been found, and extracellular vesicle size and protein composition overlap between "exosomes" and "microvesicles". Accordingly, both "Minimal information for studies of extracellular vesicles" (MISEV) guidelines 2018 and American Heart Association guidelines 2017 recommend using the term "extracellular vesicles" to avoid confusion. [START_REF] Van Der Pol | Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles[END_REF][START_REF] Théry | 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[END_REF] Remove cuff rapidly after venipuncture and discard first 2-3 mL.

Properly fill tubes to obtain correct blood/anticoagulant ratio. Invert tubes 8-10 times without shaking.

Stabilize tubes and store vertically. Minimize bench-top storage time < 30-60 minutes.

Avoid analyzing hemolyzed samples. Double centrifugate at 2500 g for 15 minutes at room temperature, set the lowest deceleration of centrifuge.

Use a clean plastic tube for second centrifugation. Do not collect the last 0.5 cm of plasma above the buffy coat.

Freeze and store at -80 °C.

Fig. 1 :

 1 Fig. 1: Size 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, separating progressively 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.

Fig. 2 :

 2 Fig. 2: Extracellular vesicle subpopulation detection method by filtration/ELISA. A.

Fig. 1

 1 Fig. 1

  Extracellular vesicles could be potential markers for prediction risk of hepatitis B virus reactivation and detection of early fibrosis for hepatitis B and C. Results of studies on that topic are summarized in Supplementary and Supplementary Text[104][START_REF] Lambrecht | Circulating ECV-Associated miRNAs as Potential Clinical Biomarkers in Early Stage HBV and HCV Induced Liver Fibrosis[END_REF][START_REF] Pu | Extracellular Vesicle-Associated mir-21 and mir-144 Are Markedly Elevated in Serum of Patients With Hepatocellular Carcinoma[END_REF][START_REF] Yang | Hepatitis B Virus-Encoded MicroRNA Controls Viral Replication[END_REF][START_REF] Van Der Ree | Plasma MicroRNA Levels Are Associated With Hepatitis B e Antigen Status and Treatment Response in Chronic Hepatitis B Patients[END_REF][START_REF] Yang | Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction[END_REF][110] for hepatitis B, and Supplementary Table 4[START_REF] Kornek | Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis[END_REF][START_REF] Murakami | Comprehensive miRNA expression analysis in peripheral blood can diagnose liver disease[END_REF][START_REF] Lambrecht | Circulating ECV-Associated miRNAs as Potential Clinical Biomarkers in Early Stage HBV and HCV Induced Liver Fibrosis[END_REF][111][START_REF] Liu | Exosome-associated hepatitis C virus in cell cultures and patient plasma[END_REF][START_REF] Bukong | Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90[END_REF][START_REF] Jiao | Serum and exosomal miR-122 and miR-199a as a biomarker to predict therapeutic efficacy of hepatitis C patients[END_REF][START_REF] Fan | Circulating microRNAs as a biomarker to predict therapy efficacy in hepatitis C patients with different genotypes[END_REF][START_REF] Devhare | Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells[END_REF][START_REF] Welker | Soluble serum CD81 is elevated in patients with chronic hepatitis C and correlates with alanine aminotransferase serum activity[END_REF][118][START_REF] Itami-Matsumoto | Circulating Exosomal miRNA Profiles Predict the Occurrence and Recurrence of Hepatocellular Carcinoma in Patients with Direct-Acting Antiviral-Induced Sustained Viral Response[END_REF][START_REF] Matsuura | Exosomal miR-155 Derived from Hepatocellular Carcinoma Cells Under Hypoxia Promotes Angiogenesis in Endothelial Cells[END_REF][START_REF] Matsuura | Circulating let-7 levels in plasma and extracellular vesicles correlate with hepatic fibrosis progression in chronic hepatitis C[END_REF] and Supplementary Text[START_REF] Kornek | Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis[END_REF][START_REF] Murakami | Comprehensive miRNA expression analysis in peripheral blood can diagnose liver disease[END_REF][START_REF] Lambrecht | Circulating ECV-Associated miRNAs as Potential Clinical Biomarkers in Early Stage HBV and HCV Induced Liver Fibrosis[END_REF][111][START_REF] Liu | Exosome-associated hepatitis C virus in cell cultures and patient plasma[END_REF][START_REF] Bukong | Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90[END_REF][START_REF] Jiao | Serum and exosomal miR-122 and miR-199a as a biomarker to predict therapeutic efficacy of hepatitis C patients[END_REF][START_REF] Fan | Circulating microRNAs as a biomarker to predict therapy efficacy in hepatitis C patients with different genotypes[END_REF][START_REF] Devhare | Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells[END_REF][START_REF] Welker | Soluble serum CD81 is elevated in patients with chronic hepatitis C and correlates with alanine aminotransferase serum activity[END_REF][118][START_REF] Itami-Matsumoto | Circulating Exosomal miRNA Profiles Predict the Occurrence and Recurrence of Hepatocellular Carcinoma in Patients with Direct-Acting Antiviral-Induced Sustained Viral Response[END_REF][START_REF] Matsuura | Exosomal miR-155 Derived from Hepatocellular Carcinoma Cells Under Hypoxia Promotes Angiogenesis in Endothelial Cells[END_REF][START_REF] Matsuura | Circulating let-7 levels in plasma and extracellular vesicles correlate with hepatic fibrosis progression in chronic hepatitis C[END_REF][START_REF] Panel | Hepatitis C Guidance 2018 Update: AASLD-IDSA Recommendations for Testing, Managing, and Treating Hepatitis C Virus Infection[END_REF] for hepatitis C.

	Thietart S & Rautou PE	Extracellular vesicles in liver diseases	18

  The link with cirrhosis severity (Child-Pugh score or MELD) has been observed

	Thietart S & Rautou PE	Extracellular vesicles in liver diseases	19
	endothelial-derived extracellular vesicles (CD31+/41-, CD31+/42b-or CD62e) than healthy
	individuals.
	vasodilation, increased cardiac output and increased intra-hepatic vascular resistance. For this
	reason,	several	studies	have	analyzed	endothelial-derived	extracellular
	vesicles.[39,40,125,126] They all concluded that patients with cirrhosis have higher

Table 5 ,

 5 have identified several plasma and serum miRNAs that are up-or down-regulated during advanced cirrhosis, but the association with cirrhosis severity has not been investigated.[START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF][START_REF] Awdishu | Identification of Maltase Glucoamylase as a Biomarker of Acute Kidney Injury in Patients with Cirrhosis[END_REF][START_REF] Engelmann | Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study[END_REF][START_REF] Nielsen | Extracellular vesicle-associated soluble CD163 and CD206 in patients with acute and chronic inflammatory liver disease[END_REF][START_REF] Wang | Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma[END_REF][START_REF] Brodsky | Dynamics of circulating microparticles in liver transplant patients[END_REF][125][START_REF] Campello | Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis[END_REF][START_REF] Ogasawara | Platelet activation in patients with alcoholic liver disease[END_REF][START_REF] Fusegawa | Platelet activation in patients with chronic hepatitis C[END_REF][START_REF] Sayed | Monocyte-platelet aggregates and platelet microparticles in patients with post-hepatitic liver cirrhosis[END_REF][START_REF] Eyraud | Evolution of platelet functions in cirrhotic patients undergoing liver transplantation: A prospective exploration over a month[END_REF][START_REF] Taleb | Quantitative proteome analysis of plasma microparticles for the characterization of HCV-induced hepatic cirrhosis and hepatocellular carcinoma[END_REF][START_REF] Kostallari | Hepatic stellate cell-derived platelet-derived growth factor receptor-alpha-enriched Thietart S & Rautou PE Extracellular vesicles in liver diseases 49 extracellular vesicles promote liver fibrosis in mice through SHP2[END_REF][START_REF] Brandon-Warner | Processing of miR17-92 Cluster in Hepatic Stellate Cells Promotes Hepatic Fibrogenesis During Alcohol-Induced Injury[END_REF][START_REF] Lambrecht | A PDGFRβbased score predicts significant liver fibrosis in patients with chronic alcohol abuse, NAFLD and viral liver disease[END_REF][START_REF] Rautou | Circulating microparticle tissue factor activity is increased in patients with cirrhosis[END_REF] Furthermore, validation is needed, given the small sample sizes and other methodological limitations.For the time being, no extracellular vesicle sub-population has been found to reliably estimate portal hypertension. Hepatocyte cytokeratin-18 positive extracellular vesicles weakly correlated with hepatic venous pressure gradients (HVPG), but could not discriminate patients having a HVPG ≥10 mmHg.[START_REF] Payancé | Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis[END_REF] Prediction of complications of cirrhosisThe procoagulant activity of some extracellular vesicles could play a role in portal vein thrombosis. Few studies have evaluated the capacity of extracellular vesicles for predicting portal vein thrombosis as well as other complications of cirrhosis, including ascites, hepatic encephalopathy, hepatopulmonary and hepatorenal syndromes. These studies are discussed in the Supplementary Text.[START_REF] Rautou | Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis[END_REF][START_REF] Awdishu | Identification of Maltase Glucoamylase as a Biomarker of Acute Kidney Injury in Patients with Cirrhosis[END_REF]125,[START_REF] Campello | Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis[END_REF][START_REF] Rautou | Circulating microparticle tissue factor activity is increased in patients with cirrhosis[END_REF][START_REF] Rautou | Microvesicles as risk markers for venous thrombosis[END_REF][START_REF] Zanetto | Cancer-Associated Thrombosis in Cirrhotic Patients with Hepatocellular Carcinoma[END_REF][140][START_REF] Mooberry | Microparticle analysis in disorders of hemostasis and thrombosis[END_REF][START_REF] Campello | Changes in plasma circulating microvesicles in patients with HCV-related cirrhosis after treatment with direct-acting antivirals[END_REF]

	Estimation of portal hypertension:

Table 1 :

 1 Main separation methods of extracellular vesicles. 1364

	Thietart S & Rautou PE	Extracellular vesicles in liver diseases	28
	These limitations are currently being progressively overcome, thanks to
	standardization guidelines and recommendations. Moreover, the number of studies on
	extracellular vesicles as biomarkers of liver diseases is growing exponentially. In the near
	future, these studies will make convincing levels of evidence available to both physicians and
	scientists.

Table 4 : Summary of main clinical studies on extracellular vesicles as biomarkers in 1375 liver diseases. 1376

 4

	Biomarker	Var	EV size &	Number (patient/	Se/Spe	AUROC	Outcome	Study type	Ref.
			Technique	control)	(%)	PPV/NPV
	Nonalcoholic fatty liver disease
	T Cell (CD4+ or CD8+ or iNKT)	↑	Larger FCM	NAFLD: 65 HC: 44	27-59 / 90-98	0.81-0.91 NA	Detection of NAFLD	Retrospective	[82]
	Monocyte (CD14+)
	Neutrophil (CD15+)
	Platelet (CD41+)
	Alcoholic hepatitis
	Hepatocyte (cytokeratin-18)	↑	Larger Filtration/ELISA	Confirmed AH: test: 46,	76/81	0.82 83/73	Diagnosis of AH	Prospective	[41]
				validation: 48
				Ruled-out AH:
				test: 37,
				validation: 20
	Hematopoietic stem cell (CD34+) &	↑	Larger FCM	Severe AH: non-responders:	NA	0.94 NA	Response to therapy &	Retrospective	[90]
	Hepatocyte (ASPGR+)			71			mortality
				responders: 30
				HC: 20
	Cirrhosis
	Platelet-derived growth factor receptor ß	↑	Small ELISA	Fibrosis 0-1: 51 Fibrosis 2-4: 97	82/41	0.64 NA	Detection of fibrosis F3-	Retrospective [134]
				HC: 14			F4
				Validation cohort:
				57
	Leuko-endothelial (CD31+/41-)	↑	Larger FCM	Cirrhosis: 91	NA	NA	Survival	Prospective	[39]
	Hepatocyte (cytokeratin-18)	↑	Larger Filtration-	Cirrhosis: Test cohort: 139	NA	NA	6-month mortality	Prospective Competing	[40]
			ELISA	Validation				risk analysis
				cohort:103
	Hepatocellular
	carcinoma
	Tumoral (AnnexinV+ EpCAM+ASPGR1+ +/-	↑	Larger FCM	HCC: 86 Cirrhosis: 49	80-81 / 47-50	0.73-0.74 NA	Detection of HCC	Retrospective [143]
	CD133+)			HC: 58
	miRNA-519d & -595 & -939	↑	Small qRT-PCR	Advanced HCC: 45	NA	0.82-0.84 NA	Detection of HCC	Retrospective [151]
				Unifocal/small: 40
				Cirrhosis no HCC:
				30
	miRNA-125	↓	Small qRT-PCR	HCC: 128	83/68	0.74 NA	Recurrence & survival	Prospective	[150]
	Signature of miRNA-122, -148a, -1246	↑	Small qRT-PCR	HCC: 50 Cirrhosis: 40	86/88	0.93 NA	Detection of HCC	Retrospective [149]
	miRNA-638	↓	Small qRT-PCR	HCC: 126	NA	NA	Overall survival	Retrospective [171]
	miRNA-21 & miRNA-10b	↑	Small qRT-PCR	HCC: 124	NA	NA	Prediction of recurrence	Retrospective [156]
	lncRNA-RP11-513I15.6 & miRNA	↑	Small qRT-PCR	HCC: 54 HCV: 42; HC: 18	78-98 / 73-95	NA 72-95/79-	Detection of early HCC	Retrospective [152]
	1262 & RAB11A					97
	lncRNA ENSG00000258332.1	↑	Small qRT-PCR	HCC: 60 HBV: 96	72-76 / 78-83	0.72-0.75 NA	Detection of HCC	Retrospective [147]

Phases of development of extracellular vesicles as biomarkers for diagnosis, 1409 severity and prognosis in liver diseases. Abbreviations

		Thietart S & Rautou PE Thietart S & Rautou PE	Extracellular vesicles in liver diseases Extracellular vesicles in liver diseases	67 68
	1410	Fig. 3: : extracellular vesicle. Box 1.

How to name extracellular vesicles?

Blood sample collection, platelet free plasma preparation and storage, adapted from ISEV 2013, AHA 2017, ISAC 2016 and ISTH 2015 guidelines.[6,11,12,20] ISEV

  Sub-classification of these extracellular vesicles can be based on operational aspects including size (small, i.e. inferior to 100 or 200 nm vs. medium/large, i.e. superior to 100 or 200 nm), density, biochemical composition, and cell condition or origin (e.g. hepatocyte extracellular vesicle, apoptotic body, hypoxic extracellular vesicle). : International Society of Extracellular Vesicles; AHA: American Heart Society; ISAC: International Society for Advancement of Cytometry; ISTH: International Society of Thrombosis and Hemostasis; EDTA: ethylene diamine tetraacetic acid Prefer fasting blood samples. If possible, collect samples during same time period. Adapt anticoagulant to downstream analysis: citrate, EDTA or sodium fluoride/potassium. Avoid heparin-based anticoagulants.For venipuncture, use 21-gauge needles or larger. Avoid butterfly systems.

	Thietart S & Rautou PE	Extracellular vesicles in liver diseases	69
	Box 2.

Acknowledgments.

We thank Marion Tanguy for providing images and for her helpful advice and Camille Florimond for superb technical assistance. We thank Kathryn Gordon for her thorough English re-reading of the paper.

Thietart S & Rautou PE

Extracellular vesicles in liver diseases

This work was supported by the "Institut National de la Santé et de la Recherche Médicale" (ATIP AVENIR), the "Agence Nationale pour la Recherche" (ANR-18-CE14-0006-01, RHU QUID-NASH) and by « Émergence, Ville de Paris ».

Authors' contributions: Sara Thietart and Pierre-Emmanuel Rautou wrote the manuscript.

Bead-based flow cytometry [START_REF] Théry | 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[END_REF] Western blot (mainly for cell culture media) [START_REF] Théry | 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[END_REF] Multiplex bead-based platforms [START_REF] Koliha | A novel multiplex bead-based platform highlights the diversity of extracellular vesicles[END_REF][START_REF] Corso | Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size Thietart S & Rautou PE Extracellular vesicles in liver diseases 39 exclusion chromatography[END_REF] Surface plasmon resonance [START_REF] Gool | Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles[END_REF] Fluorescence scanning [START_REF] Jørgensen | Potentials and capabilities of the Extracellular Vesicle (EV) Array[END_REF] More assays are described in [START_REF] Coumans | Bulk immunoassays for analysis of extracellular vesicles[END_REF] High