Detection of extracellular vesicles: size does matter Edwin van der - - PowerPoint PPT Presentation

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Detection of extracellular vesicles: size does matter Edwin van der - - PowerPoint PPT Presentation

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Detection of extracellular vesicles: size does matter Edwin van der Pol November 16 th , 2018 Outline PhD research Extracellular vesicles Size does matter Flow cytometry Outlook Standardisation Clinical studies Small and fast 2 200 nm

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Edwin van der Pol November 16th, 2018

Detection of extracellular vesicles: size does matter

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Outline

PhD research

Extracellular vesicles Size does matter Flow cytometry

Outlook

Standardisation Clinical studies Small and fast

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200 nm

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Extracellular vesicles

Cells release EVs: biological nanoparticles with receptors, DNA, RNA Specialized functions Clinically relevant

van der Pol et al. Pharmacol Rev 2012 4

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Vesicle‐based “liquid biopsy”

5 rare EVs all EVs

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Problem: counting vesicles is difficult

Gasecka et al. Platelets 2016 6

Reported concentrations of blood vesicles differ >106‐fold Clinical data cannot be compared

“Gąsecka’s law”

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Outline

PhD research

Extracellular vesicles Size does matter Flow cytometry

Outlook

Standardisation Clinical studies Small and fast

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Size distribution of extracellular vesicles?

8 Extracellular vesicles Reference particles

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Size distribution of extracellular vesicles

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Size distribution of extracellular vesicles

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Size distribution of extracellular vesicles

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Size distribution of extracellular vesicles

van der Pol et al. J Thromb Haemost (2014) 12

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Size distribution of extracellular vesicles

van der Pol et al. J Thromb Haemost (2014) 13 smaller more vesicles 3‐fold 12‐fold

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Outline

PhD research

Extracellular vesicles Size does matter Flow cytometry

Outlook

Standardisation Clinical studies Small and fast

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Problem: count the black grains

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Solution: flow cytometry

image: semrock.com 16

laser electronics and computer fluorescence channels side scatter detector forward scatter detector

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Problem: which size of vesicles do I measure?

17 ? Flow cytometry:

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Flow cytometry calibration before 2012

18 ? Flow cytometry: 500 nm polystyrene particles 900 nm polystyrene particles

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Relate scatter to diameter of beads

Side scatter (a.u.)

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Relate scatter to diameter of beads

Side scatter (a.u.)

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Relate scatter to diameter of beads

Side scatter (a.u.)

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Relate scatter to diameter of vesicles

10 nm Side scatter (a.u.)

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Particles that are too small to be detected generate a signal!

89 nm silica beads urine EVs <220 nm

Side scatter (a.u.) Side scatter (a.u.)

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Flow cytometry

image: semrock.com 24

laser electronics and computer fluorescence channels side scatter detector forward scatter detector

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beam volume ≈ 54 pl At a concentration of 1010 vesicles ml‐1, >800 vesicles are simultaneously present in the beam.

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Invisible vesicles swarm within the iceberg Harrison & Gardiner J Thromb Haemost (2012)

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Outline

PhD research

Extracellular vesicles Size does matter Flow cytometry

Outlook

Standardisation Clinical studies Small and fast

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How to calibrate flow cytometers?

image: semrock.com 31

laser fluorescence (MESF) ? side scatter (nm2) ? forward scatter (nm2) ? flow rate (µL/min) ?

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Solution

One reference material to calibrate them all Properties similar to extracellular vesicles

scatter (size + refractive index) fluorescence concentration

Tolkien (1954) 32

500 nm

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Outline

PhD research

Extracellular vesicles Size does matter Flow cytometry

Outlook

Standardisation Clinical studies Small and fast

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Clinical studies on extracellular vesicles

Myocardial infarction

Therapy monitoring

Stroke

Differentiate between ischemic stroke and hemorrhage stroke in acute phase

Prostate cancer

Diagnoses

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Outline

PhD research

Extracellular vesicles Size does matter Flow cytometry

Outlook

Standardisation Clinical studies Small and fast

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Problem: small or fast

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vesicles diameter (nm) Measurement time >150 15 minutes >30 24 hours

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vesicles diameter (nm) Measurement time >150 15 minutes >30 24 hours

Solution: EV‐Radar, small and fast

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Conclusion

Body fluids contain extracellular vesicles with clinical information The size of extracellular vesicle matters!

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Acknowledgements

My parents My wife Vesicle Observation Center Amsterdam University Medical Centers

Ton van Leeuwen Rienk Nieuwland Guus Sturk Frank Coumans

More info about extracellular vesicles: edwinvanderpol.com 40

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