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

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

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

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Flow cytometric analysis of extracellular vesicle subsets in plasma : impact of swarm by particles of non-interest

BACKGROUND: Extracellular vesicles (EVs) in plasma are increasingly recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets in a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria as it allows multi-parameter analysis of individual EVs. However, analysis of plasma EVs is challenging due to their size and heterogeneity and the presence of other submicron-sized particles in plasma that could interfere in EV-analysis. OBJECTIVES: Explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labelled or differently labelled EVs and particles. METHODS: Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry using fluorescence threshold triggering. RESULTS: We found that light scatter detection of low-abundant or rare EV subsets during fluorescence threshold triggering were severely affected by particles of non-interest due to coincidence and swarm. Importantly, we show that interfering particles labelled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. CONCLUSIONS: We demonstrate how particles of non-interest in plasma can impact the light-scatter and fluorescence detection of low-abundant EVs of interest during fluorescence-based flow cytometric analysis, and provide means to prevent erroneous data interpretation. This article is protected by copyright. All rights reserved

Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry

Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs

Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest

BACKGROUND: Extracellular vesicles (EVs) in plasma are increasingly recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets in a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria as it allows multi-parameter analysis of individual EVs. However, analysis of plasma EVs is challenging due to their size and heterogeneity and the presence of other submicron-sized particles in plasma that could interfere in EV-analysis. OBJECTIVES: Explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labelled or differently labelled EVs and particles. METHODS: Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry using fluorescence threshold triggering. RESULTS: We found that light scatter detection of low-abundant or rare EV subsets during fluorescence threshold triggering were severely affected by particles of non-interest due to coincidence and swarm. Importantly, we show that interfering particles labelled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. CONCLUSIONS: We demonstrate how particles of non-interest in plasma can impact the light-scatter and fluorescence detection of low-abundant EVs of interest during fluorescence-based flow cytometric analysis, and provide means to prevent erroneous data interpretation. This article is protected by copyright. All rights reserved