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Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the \"protein corona\" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.

Blood is the most commonly used body fluid for obtaining and studying extracellular vesicles (EVs). While blood is a standard choice for clinical analysis, using blood as a source of EVs introduces multiple layers of complexity. At the Blood Extracellular Vesicle Workshop organized by the International Society for Extracellular Vesicles in Helsinki (2022), it became evident that beginner researchers lack trustworthy information on how to initiate their research and avoid common pitfalls. This educational guide explains the composition and frequently used terminology of blood, provides guidelines for blood collection, and the preparation of plasma and serum. It also introduces the basic principles of isolating and detecting blood EVs while considering blood‐related factors. The goal of this guide is to assist beginners by offering a concise and evidence‐based introduction to the current knowledge and available resources to study blood EVs.

Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca 2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. The main EV populations were 100-250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca 2+ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100-250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.

Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor‐dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre‐analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood‐EV), a tool to record and report information about pre‐analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre‐analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence‐based optimization of pre‐analytical protocols through meta‐analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter‐laboratory comparative EV studies, and (iv) improve the peer review process.

Previously thought to be nothing more than cellular debris, extracellular vesicles (EVs) are now known to mediate physiological and pathological functions throughout the body. We now understand more about their capacity to transfer nucleic acids and proteins between distant organs, the interaction of their surface proteins with target cells, and the role of vesicle‐bound lipids in health and disease. To date, most observations have been made in reductionist cell culture systems, or as snapshots from patient cohorts. The heterogenous population of vesicles produced in vivo likely act in concert to mediate both beneficial and detrimental effects. EVs play crucial roles in both the pathogenesis of diseases, from cancer to neurodegenerative disease, as well as in the maintenance of system and organ homeostasis. This two‐part review draws on the expertise of researchers working in the field of EV biology and aims to cover the functional role of EVs in physiology and pathology. Part I will outline the role of EVs in normal physiology.

It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change—size, number, and physicochemical composition—in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention.

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities—particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies.

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor‐mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C‐type lectin‐like receptor 2 and combining all thrombin‐collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor‐induced PEVs. Our findings underscore that PEVs are tunable through receptor‐mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative. This study investigated the immunomodulatory profiles of platelet‐derived extracellular vesicles (PEVs) generated through activation by different platelet receptors: glycoprotein VI (GPVI), C‐type lectin‐like receptor 2 (CLEC‐2), and thrombin‐collagen receptors (TC). In vivo (zebrafish) and in vitro (human macrophage) assays revealed distinct homing patterns and secretory responses for each PEV type, respectively. While particle characterization and analysis of protein and miRNA cargoes identified subtle differences between the PEV types, these variations were insufficient to predict their immunomodulatory effects. Notably, PEVs from unstimulated platelets (US PEVs) exhibited a distinct profile compared to receptor‐induced PEVs. Overall, the findings suggest that PEV immunomodulatory functions are finely tuned, emphasizing the need for functional assays to fully elucidate their roles in platelet‐mediated immune responses.

Extracellular vesicles (EVs), including microvesicles and exosomes, mediate intercellular signalling which has a profound role in cancer progression and in the development of metastasis. Internalisation of EVs can prompt functional changes in the recipient cells, the nature of which depends on the molecular composition and the cargo of the EVs. We hypothesised that the metastatic stage of cancerous parent cells would determine the uptake efficacy and the subsequent functional effects of the respective cancer cell-derived EVs. To address this question, we compared the internalisation of EVs derived from two metastatic site-derived prostate cancer cell lines (PC-3 and LNCaP), human telomerase reverse transcriptase immortalised primary malignant prostate epithelial cells (RC92a/hTERT), and a benign epithelial prostate cell line (PNT2). EVs isolated from the metastatic site-derived PC-3 and LNCaP cells were more efficiently internalised by the PC-3 and PNT2 cells compared to the EVs from the primary malignant RC92a/hTERT cells or the benign PNT2 cells, as determined by high content microscopy, confocal microscopy, and flow cytometry. EV uptake was also influenced by the phase of the cell cycle, so that an increased EV-derived fluorescence signal was observed in the cells at the G 2 /M phase compared to the G 0 /G 1 or S phases. Finally, differences were also observed in the functions of the recipient cells based on the EV source. Proliferation of PNT2 cells and to a lesser extent also PC-3 cells was enhanced particularly by the EVs from the metastatic-site-derived prostate cancer cells in comparison to the EVs from the benign cells or primary cancer cells, whereas migration of PC-3 cells was enhanced by all cancerous EVs.

The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell‐derived EVs using flow cytometry. To address the orders‐of‐magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %–31 %. The calibration methodology was then used to determine reference ranges of erythrocyte‐, leukocyte‐, and platelet‐derived EVs in human blood plasma in a cohort of healthy individuals (n = 224). This study demonstrates that calibration enables comparable concentration measurements of blood cell‐derived EVs, thereby bringing EVs one step closer to clinical applications.