Methodological Guidelines to Study Extracellular Vesicles

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Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (ε-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8–3.0] vs 3.1 [1.7–18] A405 × 10−3/min, p = 0.02; 1.4 [1–1.6] vs 5.2 [2.2–16] A405 × 10−3/min, p = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance

Cointe

Souab

Bouriche³

et al. 2018

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“…This assay outperformed current predictive models pancreatic cancer patients ( n = 100). During the study, however, knowledge of blood handling/collection has evolved and the FGT test is amenable to great improvements [18]. Taken together, given the fact that each year about 10,000,000 new cases of VTE are described, of which 20% are associated with cancer, further exploring the coagulant properties of EVs to predict VTE in certain types of cancer, is promising.…”

Section: General Perspectives On Ev Biomarkersmentioning

confidence: 99%

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017

Clayton

Buschmann

Byrd

et al. 2018

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This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

“…UF uses a membrane filter with pressure, either directly applied or generated by a centrifuge/ultracentrifuge [65]. UF can rapidly isolate EVs from body fluids [66,67], but the yield is low because of the loss of small size EVs, which have a similar size to protein aggregates, necessary to guarantee the purity [68].…”

Section: Microfluidics-based Ev Isolationmentioning

confidence: 99%

Microfluidics‐based on‐a‐chip systems for isolating and analysing extracellular vesicles

Guo

Tao

Dawn

2018

150

111

Extracellular vesicles (EVs), which can be found in almost all body fluids, consist of a lipid bilayer enclosing proteins and nucleic acids from their cells of origin. EVs can transport their cargo to target cells and have therefore emerged as key players in intercellular communication. Their potential as either diagnostic and prognostic biomarkers or therapeutic drug delivery systems (DDSs) has generated considerable interest in recent years. However, conventional methods used to study EVs still have significant limitations including the time-consuming and low throughput techniques required, while at the same time the demand for better research tools is getting stronger and stronger. In the past few years, microfluidics-based technologies have gradually emerged and have come to play an essential role in the isolation, detection and analysis of EVs. Such technologies have several advantages, including low cost, low sample volumes, high throughput and precision. This review summarizes recent advances in microfluidics-based technologies, compares conventional and microfluidics-based technologies, and includes a brief survey of recent progress towards integrated “on-a-chip” systems. In addition, this review also discusses the potential clinical applications of “on-a-chip” systems, including both “liquid biopsies” for personalized medicine and DDS devices for precision medicine, and then anticipates the possible future participation of cloud-based portable disease diagnosis and monitoring systems, possibly with the participation of artificial intelligence (AI).