Single Vesicle Flow Cytometry (vFC TM ) JOHN NOLAN, CEO CELLARCUS BIOSCIENCES SELECTBIO EV-BASED DIAGNOSTICS, DELIVERY & THERAPEUTICS FEBRUARY 18, 2020
Workshop Overview MIFlowCyt EV – Minimum Information Guidelines for Reporting EV FC Methods and Results Vesicle Flow Cytometry (vFC TM ) overview Instrument considerations and setup Assay protocol Data analysis protocol Beckman Coulter CytoFlex Live demo
EV Isolation Methods
MISEV 2018: EV Characterization EV Quantification Single vesicle analysis ◦ Particle number ◦ Imaging ◦ Total protein (lipid, RNA) ◦ Impedance ◦ Flow cytometry Protein composition ◦ EV markers Protein topology ◦ Non-EV markers ◦ Exofacial ◦ “Small EV” markers ◦ Cytoplasmic Non-protein components
Conventional FC of Individual EVs: Pitfalls Lack of sensitivity, specificity Lack of appropriate calibration Difficulty of standardization Irreproducibility Artifacts!!!
Limitations of Conventional FC for EV Analysis SSC Calibur 1e+6 1. Light scatter as a trigger channel PS Beads 1e+5 ➢ Depends on size, shape, λ , collection angle, refractive 1e+4 index Vesicle Intensity 1e+3 ➢ Well described by Mie theory 1e+2 ➢ Vesicles scatter 10-100x <beads 1e+1 1e+0 1e-1 0 1 2 3 4 2. Coincidence (aka “swarm”) Diameter (um) ➢ Depends on [EV], probe volume ➢ Frequency is readily calculated ➢ Can be identified/eliminated by dilution 3. Fluorescence sensitivity and calibration ➢ Required for data/methods sharing 250 29740 MESF FITC ➢ 200 Well-established protocols, reagents ST 9210 MESF FITC Counts 150 3789 MESF FITC ➢ Not widely practiced 1252 MESF FITC 245 MESF FITC 100 (Blank) 50 0 1 10 100 1000 488, 530/30 MFI (log)
ISEV-ISAC-ISTH EV FC Working Group • Need for high throughput EV analysis • Technology development • Knowledge on biological samples • Education and EV isolation • Strong connections with Industry • Development of EV analysis • Interface with users technology • Many end-users SSC on Vascular Biology • Development of guidelines for plasma EV isolation • Standardization of plasma EV analysis and functional assays (e.g. coagulation) • Many end-users Marca Wauben, Ger Arkesteijn, Sten Libregts, Romaric Lacroix, Stpahne Robert, Fracoise Dignat- EstefaniaLozano Andres (Utrecht) George (Marseilles) Rienk Nieuwland, Edwin van der Pol, Frank Coumans, John Tigges, Ionita Ghiron, Vasilis Toxivaidis (Boston) Leonie de Rond (Amsterdam) Bernd Giebel, Andre Goergens, Tobias Tertel (Essen) John Nolan, Erika Duggan (San Diego) James Higgenbotham, Bob Coffey (Vanderbilt) Jennifer Jones, Aizea Morales-Kastresana, Joshua Welsh An Hendrix, Oliver de Wever (Ghent) (Bethesda) Xiaomei Yan (Xiamen) Joanne Lannigan, Uta Erdbrugger (Charlottesville) Alain Brisson (Bordeaux)
ISEV-ISAC-ISTH EV FC Working Group • Need for high throughput EV analysis • Technology development • Knowledge on biological samples • Education and EV isolation • Strong connections with Industry • Development of EV analysis • Interface with users technology • Many end-users SSC on Vascular Biology • Development of guidelines for plasma EV isolation • Standardization of plasma EV analysis and functional assays (e.g. coagulation) • Many end-users
Standardized Methods and Results Reporting Reproducibility Reproducibility Confirm single EV detection Standardization Advanced standardization Reproducibility Reproducibility
3. Assay controls 1. Buffer only background 2. Buffer with reagents background 3. Unstained controls autofluorescence 4. Isotype controls Fc receptor binding 5. Single stained controls positivity, compensation 6. Procedural controls unexpected artifacts 7. Serial dilution coincidence 8. Detergent-treatment vesicle lability
Workshop Overview MIFlowCyt EV – Minimum Information Guidelines for Reporting EV FC Methods and Results Vesicle Flow Cytometry (vFC TM ) overview Instrument considerations and setup Assay protocol Data analysis protocol Beckman Coulter CytoFlex Live demo
Vesicle Flow Cytometry (vFC ™) 1. Dilute 3. Dilute and measure 2. Stain: 4. Calibrate and report Membrane Fluor Diameter (nm) Diameter (nm) Membrane Fluor Fluorescence- vFC measures EVs vFluorRed selectively triggered flow stains membrane- directly in diluted cytometry biofluid, or after bound particles CD81 PE (MESF) purification mAb Fluorescence • • Membrane probe provides specificity Sensitive and specific detection: vesicle • Uses commercially-available flow • Homogeneous assay: no wash steps size to ~70 nm, cargo to >25 molecules cytometers • • Measures EVs directly in biofluid: Calibrated measurements for inter-lab, • Lab automation-compatible no isolation/purification required longitudinal, cross-platform comparisons CELLARCUS BIOSCIENCES INC
vFC ™: Standards for EV Analysis Measurement Standard Uses Data 3e+10 Nanoparticle Concentration (nps/mL) Lipo100 TM : synthetic vesicle, Lipo100 TM Vesicle size Calibrate VFC measurements, NTA 3e+10 Vesicle Size 2e+10 extruded through nanopore Immunofluorescence negative Standard 2e+10 filters, extensively control 1e+10 5e+9 characterized 0 0 100 200 300 400 500 600 Diameter (nm) Diameter (nm) vCal TM MESF beads: Fluorescence Calibrate fluorescence (MESF vCal TM MESF intensity Polymer beads (800 nm) with units) calibration calibrated levels of Enable cross-platform beads fluorescence fluorescence measurements CD41 [HIP8] PE vCal TM mAb binding beads: 151 Antibody binding Qualify antibody conjugates, vCal TM mAb capture 113 Polymer beads (800 nm) with Calibrate antibody binding, Count beads stained with PE- 76 calibrated mAb capture Enable cross-platform anti-CD41 38 capacity measurements 0 1 2 3 4 10 10 10 10 PE Fluorescence (MESF) PE-H Molecules PE 600.0 PE MFI: 1607 Cell-derived EVs EVs prepared from specific Cargo expression positive vCal TM RBC EVs PE+ : 42063 ( 97.84 %) Diameter (nm) 500.0 Pos MFI: 1624 Diameter (nm) 400.0 staining with PE- cells types expressing control, size and concentration 300.0 anti-CD235ab characteristic cargo standard, enable cross- 200.0 100.0 platform measurements 0 0 1 2 3 10 10 10 10 PE Fluorescence (MESF) PE-A
vFC TM : Getting Started Instrument evaluation, configuration and set up ◦ Protocol 0 – Essential standards to qualify and calibrate an instrument Sample preparation and staining ◦ Protocol 1 – Sample serial dilutions to establish assay dynamic range, EV concentration, lack of coincidence/swarm ◦ Protocol 2 – EV counting, sizing and cargo analysis Data processing and analysis ◦ Gating ◦ Diameter estimation ◦ Immunofluorescence calibration ◦ Batch export of data to spreadsheet, summary and plots to PPT/PDF
Instrument performance vCal TM nanoRainbow beads ◦ 0.5 nm multifluorophore, multipeak beads ◦ Cross calibrated vs MESF, vesicle standards ◦ Fixed concentration: counting standard Protocol and template evaluates: ◦ Laser alignment ◦ Vesicle detection performance ◦ Immunofluorescence calibration ◦ Volumetric measurement Beckman Coulter CytoFlex CONFIDENTIAL CELLARCUS BIOSCIENCES INC
vCal TM nanoRainbow beads MESF Cross Calibration Bangs Quantum FITC BD QuantiBrite PE FITC MESF PE MESF Bead 1: 98 Bead 1: 10 Bead 2: 258 Bead 2: 62 Bead 3: 987 Bead 3: 169 Bead 4: 3730 Bead 4: 923
vFC TM : Getting Started Instrument evaluation, configuration and set up ◦ Protocol 0 – Essential standards to qualify and calibrate an instrument Sample preparation and staining ◦ Protocol 1 – Sample serial dilutions to establish assay dynamic range, EV concentration, lack of coincidence/swarm ◦ Protocol 2 – EV counting, sizing and cargo analysis Data processing and analysis ◦ Gating ◦ Diameter estimation ◦ Immunofluorescence calibration ◦ Batch export of data to spreadsheet, summary and plots to PPT/PDF
CONFIDENTIAL CELLARCUS BIOSCIENCES INC
vFC TM : Getting Started Instrument evaluation, configuration and set up ◦ Protocol 0 – Essential standards to qualify and calibrate an instrument Sample preparation and staining ◦ Protocol 1 – Sample serial dilutions to establish assay dynamic range, EV concentration, lack of coincidence/swarm ◦ Protocol 2 – EV counting, sizing and cargo analysis Data processing and analysis ◦ Gating ◦ Diameter estimation ◦ Immunofluorescence calibration ◦ Batch export of data to spreadsheet, summary and plots to PPT/PDF
CONFIDENTIAL CELLARCUS BIOSCIENCES INC
vFC ™: Controls HEKB1020 Dilution Series 497 408 1:10 Establishes dynamic range and 1:20 1:40 373 allows assessment of coincidence 306 1:80 1:160 (“swarm”) artifact (multiple EVs). Count Count 1:320 249 204 102 124 0 0 0 100.0 200.0 300.0 400.0 500.0 600.0 1 2 3 4 5 6 7 10 10 10 10 10 10 10 Diameter (nm) Violet SSC-A 1x Lipo100 Detergent treatment 1200 4000 Detergent solubilizes EV Detergent 900 and other vesicles, 3000 confirming their vesicular Count Count 2000 600 nature 1000 300 0 0 0 100.0 200.0 300.0 400.0 500.0 600.0 0 1 2 3 4 5 6 7 10 10 10 10 10 10 10 10 Diameter (nm) Violet SSC-H
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