MISEV Extracellular Vesicle Analysis Guidelines

MISEV: Minimum Information for the Study of Extracellular Vesicles

Studying vesicles is challenging. As of yet, there is no consensus on the most appropriate methods for characterizing or isolating (also described as purifying, separating, or concentrating) extracellular vesicles (EVs). The typical vesicle researcher (studying exosomes, microvesicles, or similar particles) characterizes vesicles in terms of functional, physical, and biochemical makeup, but suffers from data reliability due to shortcomings in current vesicle analysis assays. To cope with ambiguity in appropriate methods, the International Society of Extracellular Vesicles (ISEV) has developed a set of criteria for studying EVs to drive publication of accurate and reproducible data. First published in 2014 as the Minimal Information  for the Study of EVs (MISEV) in the Journal of Extracellular Vesicles, these guidelines are now updated and published regularly to keep pace with the rapidly evolving EV research landscape. The original  guidelines were developed solely by leadership from ISEV, but as interest expanded, consensus guidelines were sought and subsequent updates have been drafted starting with an initial survey of hundreds of researchers in the field.  These results are distilled by a panel of experts and incorporated into existing guidelines. The latest MISEV guidelines were published by Witwer et. al, 2017. For our work, which is primarily focused on assays for characterizing vesicles, we’ve distilled these recommendations down into three categories with the most focus on appropriate methods for EV characterization.

Cryo EM Image of microvesicle.

EV Characterization

Characterization of vesicles also presents with its own set of challenges. MISEV reporting guidelines remain approach agnostic and focus on pushing requirements for multiple orthogonal measurements. Triangulating multiple measurements (different by application or target being measured) is the best way to address shortcomings of current approaches.

Existing assays can be divided up into two categories, total or aggregate EV measurements and individual EV measurements.  In general, MISEV recommendations defer to the researcher to select appropriate measurements based on their research requirements. Measurements looking at all vesicles in aggregate are generally reserved for where sensitivity of existing assays is not currently suitable. Individual vesicle measurements are important in understanding heterogeneity.

Assay Type Measurements
Vesicle Flow Cytometry Individual and Total Vesicles Concentration, Size, Cargo
NTA Individual Vesicles Concentration*, Size
RPS Individual Vesicles Concentration*, Size
Electron Microscopy Individual Vesicles Visual Confirmation of Characterization
Traditional Flow Cytometry Individual and Total Vesicles Has many issues with coincidence and sensitivity
Western Blot Total Vesicles Cargo
ELISA Total Vesicles Cargo
*Particles. Not Vesicle Specific.

EV Separation

There are many methods currently available to researchers separating (formerly referred to as purifying or isolating) vesicles. Each method has its own strengths and limitations. Unfortunately, there is no accepted practice for EV isolation, therefore, MISEV defers to the investigator. According to the most recently published guidelines, isolation methods are to be selected based on their suitability for a set of experimental aims. Commonly employed methods currently include:

  • Ultracentrifugation
  • Gradient Ultracentrifugation
  • Immunocapture (DynaBeads™ or Similar)
  • PEG (or similar) precipitation such as ExoQuick
  • Chromatography (SEC)
Ultracentrifuge commonly used for vesicle isolation

EV Functional Studies

In general, MISEV guidelines around functional studies focus around demonstrating that vesicle isolates are of sufficient purity to attribute observations to vesicle biology. In the absence of suitable measurements of purity, control requirements are outlaid to include control vesicles, vesicle depleted vehicles, and other similar samples.

Mouse representing functional study.