Detection of extracellular vesicles by flow cytometry: size does - - PowerPoint PPT Presentation

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Detection of extracellular vesicles by flow cytometry: size does - - PowerPoint PPT Presentation

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Detection of extracellular vesicles by flow cytometry: size does matter Edwin van der Pol November 6th, 2018 2 images: R. Hooke Micrographia 1665 3 image: A. van Leeuwenhoek Royal society 1675 4 image: A. van Leeuwenhoek Opera Omnia 1719 5

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Edwin van der Pol November 6th, 2018

Detection of extracellular vesicles by flow cytometry: size does matter

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images: R. Hooke Micrographia 1665 3

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image: A. van Leeuwenhoek Royal society 1675 4

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image: A. van Leeuwenhoek Opera Omnia 1719 5

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image: Österreichische Nationalbibliothek (Vienna) 6

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image: Deutsches Museum 7

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summary: Wolf Brit.J.Haemat. 1967 8

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Brazil ISAC Flow Cytometry Workshop

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Outline

image: semrock.com 15

  • 1. Extracellular vesicles (EVs)
  • 2. Light scatter
  • 3. Fluorescence
  • 4. Flow rate
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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 17

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EV‐based “liquid biopsy”

18 rare EVs all EVs

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EV research using flow cytometry

Gardiner et al. J Extracell Vesicles 2016 19

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Motivation to detect EVs by flow cytometry

EVs are heterogeneous

Flow cytometry can differentiate EV types

Study all (also rare) EVs

Flow cytometry is fast (>10,000 events s‐1)

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Problem: EV flow cytometry is difficult

Gasecka et al. Platelets 2016 21

Reported concentrations of plasma EVs differ >106‐fold Clinical data cannot be compared “Gąsecka’s law”

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Detection of EVs: size does matter

30‐fold 2‐fold power‐law relation*

*van der Pol et al. J Thromb Haemost 2014 22

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What is this and what is wrong?

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Summary extracellular vesicles (EVs)

Body fluids contain EVs with clinical information Flow cytometers can identify EV populations Size distribution and detection limit determine measured concentration: apply statistics carefully!

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Outline

image: semrock.com 25

  • 1. Extracellular vesicles (EVs)
  • 2. Light scatter
  • 3. Fluorescence
  • 4. Flow rate
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Outline light scatter

Flow cytometry detection of EVs with

  • ne scatter detector

two scatter detectors

Standardization

image: Feynman lectures on physics 26

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Goal: use scatter to interpret EV flow cytometry data

van der Pol Nanomedicine 2018 27 ?

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Is a “bead size gate” a good idea?

image adopted: Robert et al. J Thromb Haemost 2008 28

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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.)

Mie based on scripts Mätzler (Bohren and Huffman) 30

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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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detected concentration 7∙106 particles ml‐1

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

89 nm silica beads at concentration 1010 particles ml‐1 urine EVs <220 nm at concentration ≥ 1010 EVs ml‐1

Side scatter (a.u.) detected concentration 9∙105 EVs ml‐1 Side scatter (a.u.)

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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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Summary EV detection with 1 scatter detector

Single event signal attributed to scattering from multiple EVs (“Swarm detection”) Conventional flow cytometry detects <1% of all EVs

van der Pol et al. J Thromb Haemost 2012 39

lower detection limit conventional flow cytometry Side scatter (a.u.)

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Outline light scatter

Flow cytometry detection of EVs with

  • ne scatter detector

two scatter detectors

Standardization

image: Feynman lectures on physics 40

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Goal

Obtain physical properties of particles from flow cytometry scatter signals

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  • diameter
  • refractive index

laser

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Approach

Calibrate instrument (Apogee A50‐micro)

calibrate FSC and SSC derive size from Flow Scatter Ratio (Flow‐SR = SSC/FSC) derive refractive index from size and FSC

Validate Flow‐SR

beads mixture

  • il emulsion

Apply Flow‐SR

EV and lipoprotein particles from blood

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Calibrate forward scatter and side scatter

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Flow‐SR = side scatter forward scatter

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Derive size from Flow‐SR

van der Pol Nanomedicine 2018 44

Flow‐SR = side scatter forward scatter

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Derive refractive index from size and FSC

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Approach

calibrate instrument (Apogee A50‐micro)

calibrate FSC and SSC derive size from Flow Scatter Ratio (Flow‐SR = SSC/FSC) derive refractive index from size and FSC

validate Flow‐SR

beads mixture

  • il emulsion

apply Flow‐SR

EV and lipoprotein particles from blood

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Validate Flow‐SR with a beads mixture

47 Flow‐SR

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Validate Flow‐SR with a beads mixture

48 measurement error < 8% CV < 8% CV < 2%

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Validate Flow‐SR with oil emulsions

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Approach

calibrate instrument (Apogee A50‐micro)

calibrate FSC and SSC derive size from Flow Scatter Ratio (Flow‐SR = SSC/FSC) derive refractive index from size and FSC

validate Flow‐SR

beads mixture

  • il emulsion

apply Flow‐SR

EV and lipoprotein particles from blood

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Supernatant of outdated platelet concentrate

centrifuged 3‐fold, 1550 × g, 20 min 51 Flow‐SR No gate

lipoprotein particles? EV? 77% 23%

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Supernatant of outdated platelet concentrate

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97% 3% 77% 23%

Median refractive index platelet EVs >200 nm = 1.37

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Summary EV detection with 2 scatter detectors

Flow‐SR enables size and refractive index determination of nanoparticles by flow cytometry

data interpretation and comparison differentiate EVs and lipoprotein particles

van der Pol Nanomedicine 2018 53

lipoprotein particles EVs

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Outline light scatter

Flow cytometry detection of EVs with

  • ne scatter detector

two scatter detectors

Standardization

image: Feynman lectures on physics 54

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Standardization is boring (biologists, clinicians)

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Standardisation is exciting (metrologists, physicists)

0.31 nm X‐rays to size EV* (flow cytometers typically use 488 nm light) BESSYII *Varga et al. J Extracell Vesicles 2014 56

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Standardization is important (everybody)

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Goal

  • btain reproducible measurements of the EV

concentration using different flow cytometers

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Study comprises 33 sites (64 instruments) worldwide

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Approach scatter‐based standardization

Measure EV reference sample and controls Scatter (a.u.)  diameter (nm)

Measure Rosetta calibration* beads Rosetta calibration* software relates scatter to diameter and defines EV size gates

Apply EV size gate to software (e.g. FlowJo) and report concentrations

*Exometry.com 60

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EV reference sample

Platelet (CD61‐PE+) EVs from cell‐free platelet concentrates Trigger on most sensitive scatter channel Exclude EVs similar to isotype

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Exclusion of flow cytometers (FCM)

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Sensitivity of 46 flow cytometers in the field

69 = unable to detect 400 nm polystyrene beads

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400 nm polystyrene beads scatter more than 1,000 nm EV

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Sensitivity of 46 flow cytometers in the field

71 = unable to detect EV < 1000 nm

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Results

van der Pol et al. J Thromb Haemost 2018 72

Method CV* concentration (%) No scatter gate 144 Traditional bead size gate 139 1,200‐3,000 nm EV size gate 81 600‐1,200 nm EV size gate 82 300‐600 nm EV size gate 115

*CV: coefficient of variation (standard deviation / mean)

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Conclusions standardization by sizing

24% of flow cytometers in study are unable to detect EVs by scatter‐based triggering EV diameter gates by Mie theory improve reproducibility compared to no gate or bead diameter gate

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Outline

image: semrock.com 74

  • 1. Extracellular vesicles (EVs)
  • 2. Light scatter
  • 3. Fluorescence
  • 4. Flow rate
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Fluorescence

Please ask Dr. Zosia Maciorowski Label EVs

Antibodies Membrane dyes?

De Rond et al. Clin Chem 2018 75

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How specific do generic dyes label EVs?

blood contains ~1,000 lipoprotein particles (LPs) for each EV*

*Dragovic et al. Nanomedicine 2011 76

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Outline

image: semrock.com 77

  • 1. Extracellular vesicles (EVs)
  • 2. Light scatter
  • 3. Fluorescence
  • 4. Flow rate
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Determine flow rate

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concentration # of EV flow rate measurement time

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Conclusions

Detection of extracellular vesicles by flow cytometry: size does matter! Consider each flow cytometry aspect

Scatter Fluorescence Flow rate

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Acknowledgements

Vesicle Observation Center Amsterdam University Medical Centers

Ton van Leeuwen Rienk Nieuwland Frank Coumans Leonie de Rond

Software and beads by exometry.com More info: edwinvanderpol.com 80