4.

EV characterization: how MISEV2014

evolves in 2018
ISEV recommends that each preparation of EVs be
1) Defined by quantitative measures of the source of Evs
(+ e.g. number of secreting cells, volume of biofluid,
mass of tissue)
2) Characterized to the extent possible to determine
abundance of EVs
(total particle number and/or protein or lipid content)
3) Tested for presence of components associated with EV
subtypes or EVs generically, depending on the specificity
one wishes to achieve
4) Tested for the presence of nonvesicular, co-isolated
components.
 Step of EV Characterization
Major recommendation of MISEV 2014 Validity and/or Update in
2018
a) No recommendation on quantification New in MISEV2018:
As a rule, both the source of
EVs and the EV preparation
must be described
quantitatively.
b) General characterization Still valid but has evolved
Show: with increasing knowledge
i. At least three positive protein markers of EVs, of the existence of different
including at least one transmembrane/lipid-bound EV types.
protein -cytosolic protein
ii. At least one negative protein marker
c) Characterization of single vesicles: use two different Still valid, but has evolved
but complementary techniques, for example: with a rapidly increasing
i. electron or atomic force microscopy (and show both number of techniques used
close-up and wide-field) to analyze
ii. single particle analyzers (not electron microscope- EVs.
based)
 Quantification of EVs
• EVs have a particulate structure and contain proteins,
lipids, nucleic acids, and other biomolecules.
Quantification of each of these components can be
used as a proxy for quantification of EVs, but none of
these values is necessarily perfectly correlated with EV
number.
 Quantification of EVs
Component of EVs Measurements
Particle number + Light scattering technologies
+ Nanoparticle tracking analysis (NTA)
+ Standard flow cytometry for larger Evs
+ High resolution flow cytometry for smaller EVs
+ By resistive pulse sensing (RPS) for a wide range of sizes,
depending on pore size
+ By cryo-EM
+ By a platform combining surface plasmon resonance (SPR)
with AFM
Total protein amount  Various colorimetric assays (BCA)
 Fluorimetric essays
 Global protein stain on SDS - PAGE
 Standard flow Cryo-transmission
cytometry for larger Evs electron microscopy

reveal the morphology, size and

Atkin-Smith GK, Tixeira R, Paone S, et al. phenotype of EVs
Nat Commun. 2015;6:7439. Arraud N, Linares R, Tan S, et al.
J Thromb Haemost. 2014;12(5):614–627.
 By resistive pulse sensing (RPS) for a wide
range of sizes, depending on pore size
• Scanning ion occlusion sensing technology,
which relies on the detection of particles upon
their movement through a nanopore, was
investigated for the ability to quantify
nanosized MVs

de Vrij J, Maas SL, van Nispen M, et al. Nanomedicine (Lond). 2013

 Typical SPR instruments
particles of around 50–150 nm, such as
small EVs, are perfectly suited to SPR
detection
Obeid S, Ceroi A, Mourey G, et al. Biosens
Bioelectron. 2017;93:250–259.
Surface plasmon resonance (SPR) is a label-
free, real-time sensor technique to detect
molecular interactions occurring in proximity
to a metal (gold/silver) surface based on
monitoring changes in refractive index
Pierce™ BCA Protein Assay Kit
 Quantification of EVs
Component of EVs Mesurement
Total lipid  Sulfophosphovanilin assay
 Measuring fluorescence of phospholipid dyes that
fluoresce only when incorporated into lipid bilayers, such
as DiR
 Total reflection Fourier-transform infrared spectroscopy
Total RNA  global RNA assays including profiles obtained by capillary
electrophoresis instruments
Specific molecules  ELISA
(tetraspanins CD9,  Bead-based flow cytometry
CD63 and/or CD81, but  Aptamer- and carbon nanotubebased colorimetric assays
sometimes tumor- SPR on surfaces such as antibody-coated nanorods
specific proteins or
other molecules such
as lipids )
Sulfophosphovanilin assay
The colorimetric reaction of sulfuric
acid and phosphovanilin with lipids
was used in a 96 well plate format
sulfophosphovanilin (SPV) assay.

Osteikoetxea X, Balogh A, Szabó-Taylor K, et al.

PLoSOne. 2015;10(3):e0121184.
 Quantification of EVs
• Quantification methods are the most informative for EVs
recovered by separation methods with the highest
expected specificity, and for these preparations, one
quantification method may suffice; in contrast, more than
one quantification should be used for EVs recovered from
low-specificity methods
 Protein:particle ratio
 Protein: lipid ratio
 RNA:particle
Absolute EV sizing and counting methods are currently
imperfect and will require further improvement, aided by
appropriate EV reference standards that are now in
development
 Protein : particle ratio

Webber, J., & Clayton, A. (2013). Journal of Extracellular Vesicles, 2(1), 19861.
 Protein: lipid ratio

Osteikoetxea X, Balogh A, Szabó-Taylor K, et al. PLoSOne. 2015;10(3):e0121184.

 Characterization of EVs by their
protein composition
• Selection of proteins for use as EV markers.
• Methods to assess presence of proteins in EV preparations.
• Non-protein components as markers of EVs.

Incorporation of any given component of the cytoplasm or

other cellular compartment into an EV is determined by
1) Proximity to the budding membrane and size of the EV
(passive loading)
2) Specific association with the membrane and any
energy-dependent processes (active loading).
 Selection of proteins for use as EV
markers.
• highlights three categories of markers that
must be analyzed in all bulk EV preparations
to demonstrate the presence of EVs
Category 1 Transmembrane or GPI-anchored proteins
localized at the external membrane of prokaryotic cells, and
plasma membrane and/or endosomes of eukaryotic cells r
Category 2 Presence of cytosolic proteins (eukaryotic cells and Gram-
positive bacteria) or periplasmic proteins (Gram-negative
bacteria) demonstrates that the analysed preparation displays
the structure of lipid bilayers enclosing intracellular material,
as expected for any EV
Category 3 Some proteins are major constituents of non-EV structures
often co-isolated with EVs. Evaluation of their presence helps
to assess the degree of purity of the EV preparation.
Category 1: Transmembrane or
GPI-anchored proteins

Junlu, Jun li and al. Oncotarget, Advance Publications 2017

 Category 3: Some proteins are major
constituents of non-EV structures
• In biofluids like blood plasma, Evs have been
reported to co-isolate with other particles:
lipoprotein, a variety of non-integral such as
albumin or soluble acetyl cholinesterase
• In urine, Tamm-Horsfall protein (uromodulin)
forms aggregates that co-precipitate with EVs
 Selection of proteins for use as EV
markers.
and two additional category
Category 4 (additional) proteins should be evaluated if authors want to claim
specificity of their study to the small EV subtype-
(s): Proteins localized in/on intracellular
compartments of eukaryotic secreting cells
other than the plasma membrane and endosomes
components of the nucleus
mitochondri , endoplasmic reticulum
Golgi apparatus autophagosomes, peroxisomes)
Category 5 secreted or luminal proteins that can associate with EVs
by binding to specific (e.g. growth factor
receptors) or to promiscuous (e.g. proteoglycan, lipid)
receptors on the EV surface
Table: Protein content – based EV characterization
 Methods to assess presence of
proteins in EV preparations.
• Western blotting is the most commonly used, and it should be
performed by loading side-by-side EV samples and source
material lysates either in specified protein amount or in cell
equivalent amounts to determine if the analyzed proteins are
enriched in EVs as compared with their producing cells
• Flow cytometry of EVs decorating beads or of bulk EV
populations (i.e. not designed to analyze single EVs) can be
used, but with care to use appropriate negative controls
• fluorescence scanning or surface plasmon resonance to
quantify EV binding to a surface coated with antibodies to
different antigens.
• Mass spectrometry has become increasingly economical and
accessible
 Western Blotting
• Purified vesicle preparations may be treated with
buffered lysis solutions which contain denaturants
and protease inhibitors. The protein lysates are then
separated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS−PAGE), before being
transferred over to a membrane for immunoblotting
of specific protein targets

Shao, H., Im, H., Castro, C. M., Breakefield, X., Weissleder, R., & Lee, H. (2018).
Chemical Reviews, 118(4), 1917–1950.
 Mass spectrometry
• Purified EV preparations undergo enzymatic digestion and
peptide separation before being ionized and analyzed by
mass spectrometer.
• Along this complex processing, multiple steps critically
affect EV proteomic profiling. Aside from effective EV
purification peptide fractionation prior to mass
spectrometry analysis is considered an important
prerequisite for identifying vesicular proteins with high
confidence.
• This is commonly achieved through three main approaches:
(1) SDS−PAGE,125,126 (2) two-dimensional liquid
chromatography,127 and (3) isoelectric-focusing-based
fractionation.

Shao, H., Im, H., Castro, C. M., Breakefield, X., Weissleder, R., & Lee, H. (2018).
Chemical Reviews, 118(4), 1917–1950.
 Non-protein components as markers
of EVs
• Phospholipids present in lipid bilayers
• Other lipids including cholesterol,
sphingomyelin, ceramide, and phosphatidyl-
choline/ethanolamine/inositol
• Dyes that are activated by intracellular
components can be used to label Evs
(Calcein, CFSE)
• Concerning nucleic acids, both DNA and RNA
Phospholipids present in lipid bilayers

Record M, Carayon K, Poirot M, et al. Biochim Biophys

Skotland T, Sandvig K, Llorente A.
Acta. 2014;1841(1):108–120.
Prog Lipid Res. 2017;66:30–41
Calcein, CFSE
Generic marker performance
on MCF7 EVs.Generic marker
fluorescence-triggered scatter
plots of EpCAM vs generic
marker fluorescence (A–E) or
side scatter (F). Data of 1
representative minute are
shown. Numbers in quadrants
indicate percentage of total
population. Venn diagrams
show mean concentrations
(×106 particles/mL) of
antibody+ (mAb+), marker+ or
scatter+, and double+particles (n
= 3–6) (G–L). Calculation of
sensitivity and PPV from Venn
diagrams (M).

de Rond L, van der Pol E, Hau CM, et al. Clin Chem. 2018;64 (4):680–689