Extracellular vesicles as biomarkers: flow flaws, facts and clinical - - PowerPoint PPT Presentation

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Extracellular vesicles as biomarkers: flow flaws, facts and clinical - - PowerPoint PPT Presentation

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Extracellular vesicles as biomarkers: flow flaws, facts and clinical acts Edwin van der Pol May 28th, 2019 edwinvanderpol.com 2 Outline 1. Extracellular vesicles (EVs) 3. Fluorescence 4. Flow rate 2. Light scattering image: semrock.com 3

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Edwin van der Pol May 28th, 2019

Extracellular vesicles as biomarkers: flow flaws, facts and clinical acts

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edwinvanderpol.com 2

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Outline

image: semrock.com 3

  • 1. Extracellular vesicles (EVs)
  • 2. Light scattering
  • 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 5

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EV-based “liquid biopsy”

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Extracellular vesicles are booming!

1Web of Science: “topic: exosome*” 2bioinformant.com

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Science1 Industry

Startup companies2

  • 4 large EV startups

received $ 386 million investment capital in 2018

Established companies

  • Thermo Fisher
  • Becton Dickinson
  • Beckman Coulter

Market growth factors between 6-48 %

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EV research using flow cytometry

Gardiner et al. J Extracell Vesicles 2016 8

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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 10

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 11

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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 13

  • 1. Extracellular vesicles (EVs)
  • 2. Light scattering
  • 3. Fluorescence
  • 4. Flow rate
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What is light scattering?

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Outline light scattering

Flow cytometry detection of EVs with

  • ne scatter detector

two scatter detectors

Standardization

image: Feynman lectures on physics 15

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Goal: use scatter to interpret EV flow cytometry data

van der Pol Nanomedicine 2018 16 ?

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Is a “bead size gate” a good idea?

image adopted: Robert et al. J Thromb Haemost 2008 17 Beads: 2 µm Forward scatter (a.u.) Side scatter (a.u.) EV gate 900 nm beads 500 nm beads

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Relate scatter to diameter of beads

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Relate scatter to diameter of beads

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Relate scatter to diameter of beads

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Relate scatter to diameter of EVs

10 nm

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Particles below detection limit are detected

89 nm silica beads EVs < 220 nm 22 Side scatter (a.u.) Side scatter (a.u.)

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Flow cytometry

488-nm laser electronics and computer fluorescence channels side scatter detector forward scatter detector

image: semrock.com 23

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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 29

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 30

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Goal

Obtain physical properties of particles from flow cytometry scatter signals

31 particle

  • 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 34

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

37 Flow-SR

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Validate Flow-SR with a beads mixture

38 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 41 Flow-SR No gate

lipoprotein particles? EV? 77% 23%

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Supernatant of outdated platelet concentrate

42 No gate CD61+ gate

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 43

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 44

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Standardization is boring

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Standardization is important

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van der Pol et al. J Thromb Haemost 2018 47

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 49

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EV reference sample

Platelet (CD61-PE+) EVs from cell-free platelet concentrates Trigger on most sensitive scatter channel Include EVs with CD61-PE+ fluorescence

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Exclusion of flow cytometers (FCM)

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Sensitivity of 46 flow cytometers in the field

59 = 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

61 = unable to detect EV < 1000 nm

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Results

van der Pol et al. J Thromb Haemost 2018 62

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 64

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

Yesterday you have learned about fluorescent antibody labeling, so ask Alfonso! Label EVs

Antibodies

  • Use controls: evflowcytometry.org
  • Spin down aggregates!

Membrane dyes?

De Rond et al. Clin Chem 2018 65

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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 66

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Method: Flow-SR

van der Pol et al. Nanomedicine 2018 67 Flow-SR

lipoprotein particles EVs

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Outline

image: semrock.com 68

  • 1. Extracellular vesicles (EVs)
  • 2. Light scattering
  • 3. Fluorescence
  • 4. Flow rate
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Study comprises 33 sites (64 instruments) worldwide

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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: first the flaws & facts, then the clinical acts Calibrate 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: exometry.com Reporting framework: evflowcytometry.org More info: edwinvanderpol.com 72

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