The document discusses the advancements and challenges in the analysis of extracellular vesicles (EVs) through flow cytometry, highlighting their potential as biomarkers and therapeutic agents. It addresses the technical difficulties in analyzing specific subsets of EVs in complex fluids, emphasizing the need for high throughput and high resolution techniques. Furthermore, it underscores the importance of standardization and comprehensive reporting in EV analysis to ensure reproducibility and comparison of results.

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

10. Scatter-based thresholding
Detection of fluorescent nano-sized beads
FSC-based
thresholding
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threshold
SSC
Reduced wide-angle FSC
Nolte-’t Hoen et al. Nanomedicine, 2012 8:712
Van der Vlist et al. Nat. Protocols, 2012 7:1311
100 nm
200 nm

11. Fluorescence-based thresholding
Detection of fluorescent nano-sized beads
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threshold
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threshold
100 nm
beads
200 nm
beads
Reduced wide-angle FSC
Fluorescence
noise
Nolte-’t Hoen et al. Nanomedicine, 2012 8:712
Van der Vlist et al. Nat. Protocols, 2012 7:1311

12. Vesicle isolation:
Cell culture supernatant

2x 200g

2x 500g

1x 10,000g

1x 100,000g
Pelleted vesicles
Generic label PKH-67
Collection of
density
gradient
fractions
Flow cytometry
(Generic) fluorescent labeling of cell-derived vesicles
Specific protein
labeling
(FL-Ab)
Nolte-’t Hoen et al. Nanomedicine, 2012 8:712
Van der Vlist et al. Nat. Protocols, 2012 7:1311

13. High-resolution flow cytometric
analysis of nano-sized EVs
Reduced wide-angle FSC
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PKH67Fluorescence
threshold 0
10000
20000
30000
40000
50000
60000
1,26
1,24
1,22
1,20
1,18
1,15
1,11
1,08
1,07
1,06
DC
Numberofevents
Density (g/ml)
QuantificationDetection
Nolte-’t Hoen et al., Nanomedicine 2012; Van der Vlist/Nolte-’t Hoen et al., Nature Protocols 2012; Van der Vlist et al., J Extracellular Vesicles 2012
MFG-E8 (B-PE)
MHCII(APC)
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vesicles
act-DC
vesicles
non-act-DC
Reduced wide-angle FSC
PHK67fluorescence
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Characterization
Proteins
T cell extracellular vesicles
Light scattering

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