ISEV2014 - Introduction to flow cytometry analysis of EV's (M. Wauben)
1. Flow cytometric analysis of
individual extracellular vesicles
Marca Wauben
Utrecht University
Dept. Biochemistry & Cell Biology
Fac. Veterinary Medicine
The Netherlands
2. Extracellular vesicles have changed the way we
look at communication in and between
biological systems
EV offer tremendous opportunities for clinical applications
ranging from biomarkers for diagnosis or prognosis
to therapeutic application of EV or mimics for drug delivery
3. Therapeutic application of
extracellular vesicles or mimics
Next hurdle to take: Large scale preparation and isolation of
well-defined vesicles
Quality control: Quantitative & qualitative analysis
Multiparameter analysis of individual vesicles
4. Vesicle-based biomarkers: A novel class
between small molecule and cellular
biomarkers
High potential biomarkers
BUT………
Major technical problem is the analysis of specific subsets (rare
events) of vesicles in complex body fluids
5. EVs are heterogeneous in size &
composition
Vast majority of EVs released by living cells <200nm in size
NO unique markers for different EV-subsets available
Cellular and Molecular Life Sciences 2011; 68(16):2667-88
6. Great challenge in the EV-field
• Cargo incorporation into EVs is dynamic
Individual EV analysis
can discriminate
• Mixed population of EVs
Bulk-based analysis methods e.g. Western-blotting,
proteomics, (bead)capture assays, ELISA
To monitor quantitative and qualitative changes in
EV-subsets
7. Great challenge in the EV-field
High throughput analysis at the particle level
Flow cytometer Designed for high throughput analysis
of cells applied for EV analysis
8. EV analysis by flow cytometry
Size:>300 nm
Conventional flow
cytometry-based analysis
Size: <300 nm
High resolution flow
cytometry-based analysis
Optimized BD Influx:
Nolte-’t Hoen et al.
Nanomedicine, 2012
8:712
Van der Vlist et al. Nat.
Protocols, 2012 7:1311
9. Trigger signal
• Uniform parameter to detect all EVs of
interest
• Light scatter is useful as a trigger parameter
for cells and large EVs
• Small EVs (<300nm) background problems
14. Any flow cytometer can measure something
when concentrations are high enough…….
BUT what does the signal mean?
15. Van der Pol et al. : Theoretical model for vesicle detection by flow cytometry (Single vs. Swarm
detection of microparticles and exosomes by flow cytometry) J. Thromb. Haemost. 2012 10:919
Swarm vs. single detection of nano-sized
extracellular vesicles by flow cytometry
Regular flow cytometers
•Large single EV detection (>300 nm)
•Nano-sized EVs detected as ‘swarm’ =
multiple vesicles counted as single event
High resolution flow cytometry
•Large and nano-sized (~100 nm)
single EV detection
16. Swarm detection influences quantitative and
qualitative flow cytometric analysis of nano-
sized EVs
• Regular flow cytometers can be used for swarm detection of
nano-sized EVs ‘Bulk-based’ analysis (no information on EV-
subsets, no quantitative analysis)
• For high resolution flow cytometry proper concentrations
should be used for genuine single nano-sized vesicle-based
analysis
17. -Reproducibility and comparison of results
-Development and evaluation of (novel)techniques
No gold standard technique available
Need for EV-like standards to calibrate and compare
Need for sample preparation guidelines
Need for sample analysis by several techniques
Need for standardization of high
throughput EV-analysis
Need for comprehensive reporting
of well-controlled experiments