1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Disclaimer: Given the rapid evolution of EVP technologies, this table is for general guidance only. Please check with the appropriate vendor for performance characteristics and costs of specific instruments and reagents.

 Malvern ParticleMetrix Hyperion Analytical Hitachi, Thermo-Fisher, Jeol Thermo-Fisher Wyatt IZON Spectradyne Spectradyne Luminex Luminex Beckman Coulter Cytek Apogee NanoFCM Cellarcus UW Beckman V/BI Unchained Labs ONI COH/MGH OSU HMS UND UCLA Mesoscale Mesoscale V/BI UCSF UCSF V/BI UCLA-Duke UCSD/MGH Mesoscale UCD OSU UND UND UND
Instrument NanoSight NS300 ZetaView Envision3 Transmission Electron Microscope Tecnai-f20 AF4 Exoid TRPS nCS1 ARC CellStream ImageStream Cytoflex Aurora A60 MICRO-PLUS NanoAnalyzer Digital Flow Cytometer Astrios FACSAria FAVS ExoView qSMLM qSMLM Microarray/TIRF Molecular Beacons IEM Sensor Technology SERS ECL Immunoseq FPLC SEC Gradient Density UC Ultracentrifugation AFS Immuno-Magnetic Separation Multimarker Immunomagnetic EV Selection HPLC-MALS/DLS/UV (w/SEC column TFF/iSUF NanoEX (ANM small EV isolation device) MNM immunocapturing device Continuous Isoelectric Fractionation (CIF) device
Instrument Details
Commercial or ERCC Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial ERCC Commercial ERCC Commercial Commercial ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC ERCC
Technology Type NTA NTA NTA TEM cryo-EM Filtration RPS RPS RPS/OI FC FC FC FC FC FC FC FC FC + Sorting FC + Sorting Optical Imaging OI - dStorm OI - dStorm OI - TIRFM Optical Imaging Isoelectric detection SERS IC/IF IC/seq SEC SEC DGUC UC AFS IC IC SEC + detection Filtration/Immunocapture Filtration Immunocapture IEF
Bulk or single particle Single Single Single Single Single Bulk Single Single Single Single Single Single Single Single Single Single Single Single Single Single Single Single Single Single Bulk Single Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk/Bulk Bulk Bulk Bulk
Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization Characterization
Separation Separation Separation Separation Separation Separation Separation Separation Separation Separation Separation Separation Separation Separation Separation Separation
Characterization/Separation Principle Light scatter Light scatter Light scatter Deflection of electron beam Deflection of electron beam Size Perturbation of electrical resistance Perturbation of electrical resistance Perturbation of electrical resistance Fluorescence detection Light scatter/fluorescence detection Light scatter/fluorescence detection Light scatter/fluorescence detection Light scatter/fluorescence detection Light scatter/fluorescence detection Fluorescence detection Light scatter/fluorescence detection Light scatter/fluorescence detection Light scatter/fluorescence detection Light scatter/fluorescence detection Fluorophore photoswitching Fluorophore photoswitching Fluorescence detection Nucleic acid cargo sequence Isoelectric point Raman scattering Surface marker expression Surface marker expression Size Size Buoyant Density Buoyant Density Size, other interactions with sound waves Surface marker expression Surface marker expression Size Size/Surface marker expression Size Surface marker expression Isoelectric point
Data Analysis
Fluid- phase or Solid-phase Fluid-phase Fluid-phase Fluid-phase Solid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Solid-phase Solid-phase Solid-phase Solid-phase Solid-phase Solid-phase Solid-phase Solid-phase Solid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Fluid-phase Solid-phase Solid-phase Fluid-phase Fluid-phase Fluid-phase Solid-phase Fluid-phase
Hardware (Instrument, Device) Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Requires Illumina NGS Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument Instrument
Assays (Reagents, Protocols) Protocol Protocol Protocol Protocol Protocol Protocol Protocol Protocol Reagents, Protocols Reagents, Protocols Reagents, Protocols Reagents Protocols Protocols Reagents, Protocols Reagents, Protocols Protocol Reagents, Protocols Reagents, Protocols Reagents, Protocols
Points of Contact Sven Kreutel John Nolan Duncan Griffiths Barbara Smith Rienk Nieuwland Alain Brisson Wyatt Vreeland Jared Lynch Jean-luc Fraiken, Joshua Welsh John Nolan Jean-luc Fraiken, Joshua Welsh Stephanie Brunell Brian Hall, Yoav Altman Matthew B. Goff Joshua Welsh John Tigges, John Nolan, Brian Elicieri, Robert Raffai Jennifer Jones, Joshua Welsh Desmond Pink, Min Shi, Tess Stewart, Daniel Chiu Joshua Welsh Ken Witwer John Nolan Daniel Chiu Jennifer Jones, Joshua Welsh Jim Higgenbothoam George Daaboul, Ken Witwer David Routenberg Tijana Talisman Eduardo Reategui Ionita Ghiron Hsueh-Chia Chang Ya-Hong Xie David Routenberg David Routenberg Jeff Franklin Robert Raffai Robert Raffai Robert Coffey Tony Huang Louise Laurent, Saumya Das David Routenberg Angela Zivkovic Eduardo Reategui Hsueh-Chia Chang Hsueh-Chia Chang Hsueh-Chia Chang
Input
Biofluid type
SPU 
  C 
SPU 
  C 
SPU 
  C 
    
    
    
    
    
    
SPU 
Cs C 
SPU 
Cs C 
SPU 
Cs C 
SPU 
  C 
SPU 
  C 
SPUSa
  C 
SPU 
  C 
SPU 
  C 
SPU 
  C 
SPUSa
  C 
SPU 
  C 
SPU 
  C 
 P  
  C 
SPU 
Cs C 
SPU 
  C 
SPU 
  C 
SPU 
  C 
SPU 
  C 
 P  
  C 
SPUSa
  C 
SPUSa
Cs C 
SPUSa
Cs C 
 P  
  C 
SP  
   O
SP  
   O
SP  
   O
SPUSa
  C 
SP  
  CO
 P  
Cs CO
SP  
Cs  O
SPU 
  C 
SPU 
  CO
SPU 
  CO
SP  
  C 
Is sample preparation required? Dilution or purification often needed Purification needed for scatter detection. No purification needed for fluorescence detection. Purification needed for scatter detection. No purification needed for fluorescence detection. No purification needed. Dilution may be necessary, in case of large amounts of contaminating cells. No purification needed. Dilution may be necessary, in case of large amounts of contaminating cells. No Filtration (0.22-0.45 μm filter) to remove aggregates. Dilution if sample is concentrated or viscous. Dilution if sample is concentrated or viscous. Has in-line filtration. Dilution if sample is concentrated or viscous. Has in-line filtration. Dilution often required to avoid swarm detection. Dilution often required to avoid swarm detection. Purification needed for scatter detection. No purification needed for fluorescent detection. Dilution often required to avoid swarm detection. Dilution if sample is concentrated, PQ data provides optimal sample dilution for assay to prevent coincidence Purification needed for scatter detection. Pre-analytical sample enrichment possible but not required Yes Yes Yes Yes, for complex biofluids (e.g., plasma and seum) Yes Yes Yes No No Minimal Depends on abundance of competing proteins in sample Preferably, but not always Minimal No No No Minimal Centrifugation at 2000 xg to remove cells Preferred (e.g., SEC, Captocore, or UF; depends on which markers are used). Yes, ultracentrifugation and filtration CCM, S, SA, P, U, T Minimal (e.g.. dilution or 220 nm filtration) Preferred Minimal (dilution)
Minimum input volume required (μL) 200 μL 500 μL (can recollect for additional analyses) 200 μL 1 μL 10 μL 20 μL 35 μL 3 µL 3 µL 45 μL 20 μL 5 μL 5 μL 90 μL 25 μL 5 μL 1 μL 100 μL 4 ml 25 μL (50 μL final run volume, common to dilute sample 2X or more). 5 μL 5 μL 15 μL No restrictions 200 μL 5 μL 1 μL 1 μL 1 mL (depends on column) 500 uL 1 mL 1 mL 150 μL 500 μL 25 μL 100 μL 50 µL 50 µL 100 μL 500 μL
Maximum input volume permitted (μL) 500 μL 500 μL (can recollect for additional analyses) 200 μL 20 μL 10 μL No limit 35 μL 10 µL 10 µL 500 μL 200 μL 5000 μL 300 μL 400 μL 25 μL 5 μL 10 μL 100 μL 100 ml 50 μL 150 μL 150 μL 15 μL No restrictions 200 μL 1000 μL 25μL 100μL 12 mL (depends on column) 500 uL 100 mL 40 mL 1.5 mL 1000 μL 4 mL 900 μL 1000 µL 1000 µL 2 mL 3 mL
Minimum input volume Med Med Med Low Low Low Low Low Low Low Low Low Low Low Low Low Low Med High Low Low Low Low N/A Low Low Low Low High Med Med Med Med Med Low Med Low Low Med Med
Maximum input volume Med Med Med Low Low Very high Low Low Low Med Med High Med Low Low Low Med High Low Med Med Low N/A Low Med Low Low High Med Med High Med Med High Med Med Very High High High
Speed (turnaround time/sample) 3-15 minutes 2 minutes 4-5 minutes 20 minutes hours 1 hour 15 minutes 3-5 minutes 3-5 minutes <1 minute 2 minutes 2-3 minutes 2-3 minutes 1 minute 15-30 minutes 2-3 minutes 2-4 minutes Analysis: 1-2 minutes. Sorting >1 hr hours 15-30 minutes hours hours 10-15 minutes N/A hours hours 4 hours 2 days hours 30 minutes days days 1 hour 30 minutes 3-4 hr hours hours 1 hour hours hours
Speed (turnaround time)details 5 minutes to prepare sample, 15 minutes to image 96/hour 96/3 hr 96/3-4 hr 96/3-4 hr 96/3-4 hr 96/3-4 hr
Speed (turnaround time) High High High High Med Med High High High High High High High High High High High Analysis: High; Sorting: Med Med High Med Med High N/A Med Med Med Low Med High Low Low Med High Med Med Med Med Med Med
Throughput (# samples/5 days) 150 (manual) - 500 (autoloader) 1200 800 100 20 120 100-200 250-500 250-500 3000 1000 1000 1000 480-960 100-200 1000 480-960 when fully developed Analysis: 1000. Sorting: 20 20 80-160 30 10 300 N/A 25 25 2000 96 5-10 20 2 1 50 120 960 40 30 100 15 25
Throughput details with autosampler and sample collector running 24 hr/day Each 96-well plate takes 1 hr to run Each 96 well plate take 3 hr to run at 60x Each 96 well plate take 3-4 hr to run Each 96 well plate take 3-4 hr to run Depends on number of replicate aspirations and number of washes between samples. Depends on instrument used 96 samples per half day when fully developed Analysis: 30-50 samples/ hour. Sorting: <1 sample/hr 5 hr/16 chips, 7 hr/32 chips using ididi multi area slides Depends on EV concentration, markers used,e tc. 8 samples/hr Depends on EV concentration 4 hr/96-well plate, 8 plates/day 2 days/96-well plate, 4 plates/week Depends on column can run 12 in parallel 3-4 hr/96-well plate, 2 plates/day
Throughput Med High High Med Low Med Med Med Med High High High High High Med High High when fully developed Analysis: High. Sorting: Low Low Med Low Low Med N/A Low Low High Med Low Low Low Low Med Med High Low Low Med Med Low
Separation / Purification Efficiency
Yield N/A N/A N/A N/A N/A Near quantitative N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1-5% recovery 107 EV/mg input material (assessed with fluorescent marker staining) N/A N/A N/A N/A N/A N/A N/A N/A N/A Difficult to assess (would need to quantify each subset [e.g., EVs, exomeres, supermeres] before/after) Not established Not established Scalable based on rotor size mg quantaties 0.9 To be determined Marker-dependent >90% >95% > 90% >80% >70%
Purity N/A N/A N/A N/A N/A >80% N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A >95% >95% for stained epitopes N/A N/A N/A N/A N/A N/A N/A N/A N/A Difficult to assess (would need to quantify each subset [e.g., EVs, exomeres, supermeres] in the resulting fractions) Not established Not established Variable, partially pure dependent on serial centrifugation steps 0.9 To be determined Marker-dependent, better than single-marker immunocapture TBD >95% Capable of removing >98% plasma protein and lipoprotein from plasma sample High >90%
Time/labor N/A N/A N/A N/A N/A Hands on time: minutes/sample. Time on machine: 1 hr/sample. N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 90 hr per run to isolate 2 subsets of 108 sEVs 90 hr per run to isolate 2 subsets of 108 sEVs N/A N/A N/A N/A N/A N/A N/A N/A N/A 30 min hands-on time 15-30 minutes per collection 2 days Variable, 4 hrs -3 days 1 mL/hr per device Currently a manual process that is labor-intensive 4 hr/plate, 1 hr hands-on time 1h hands-on time, 14h instrument time 20 minutes hands-on time 5 min hands-on time 20 min hands-on time 25 min hands on time, for experimental setup
Scalability (for EV production) N/A N/A N/A N/A N/A Analytical N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A suitable for viral reporter assays Practically limited to 108 EVs N/A N/A N/A N/A N/A N/A N/A N/A N/A Yes, can pool resulting fractions and concentrate by ultrafiltration. one sample at a time Up to six samples per UC run Yes with concentration steps Higher throughput with additional parallel devices 24 samples/day with full-time effort using commercially available beads, not including evaluation of performance Procedure is scalable. However, no significant economy of scale 8 samples/day, captures EV/LPP/RBP fractions in parallel Scaling to 5 L in progress Can be scaled to 1L input Can be scaled by using upstream concentration step (e.g., NanoEX) Can be scaled by running multiple CIF devices in parallel
Level of Automation. None None None None None High None None None Plate-loader option Plate-loader option Plate-loader option Plate-loader option Plate-loader option None None None None None None None None None None None None None None Moderate. automated sample collector None Partial; complicated spin perameters can be automated Automated after initial setup None Moderate None None Fully automated None Semi-automated
Carrier Characterization
Resolution (For Single EVP analysis / For Bulk analysis ) Single EVP Single EVP Single EVP Single EVP Single EVP Bulk Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Single EVP Bulk Single EVP Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk
Discriminate/separate EVs vs. other exRNA carriers No Yes (fluorescence) Yes (fluorescence) Yes Yes Yes No No Yes (fluorescence) Yes (fluorescence) Yes (fluorescence) Yes (fluorescence) Yes (fluorescence) Yes, if appropriate fluorescent markers are used; multiangle analyses permits segregation based on RI standards. Yes (fluorescence) Yes (fluorescence) Yes (fluorescence) Exploratory Yes (fluorescence) Yes (surface capture & fluorescence) Yes (surface capture & fluorescence) Yes (surface capture & fluorescence) Yes No Yes Not tested Yes Yes Yes Yes Yes Yes Yes Yes Yes (fluorescence) Yes Yes (fluorescence) Yes Yes Yes
Assess/separate EV subpopulation heterogeneity Not well Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes Yes Yes, by size No No Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used; multiangle analyses permits segregation based on RI standards. Yes, if appropriate fluorescent markers are used Yes, for CD63/CD81/CD9 Yes limited to 4-6 paramters Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes Possible Possible Yes Yes Possible
Quantification of/separation by EV size Yes Yes Yes Yes Yes Yes, with DLS and MALS detector Yes Yes Yes Exploratory with membrane dyes Exploratory with membrane dyes Yes Yes (vFC) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No Yes Yes N/A No No No No Possible No No No No
Quantification of EV concentration Yes Yes Yes Relative Relative Yes, with MALS detector Yes Yes Yes Exploratory with membrane dyes Exploratory with membrane dyes Yes Yes (vFC, light scatter) High precision, but accuracy is limited due to lack of validated standards Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Relative Relative N/A N/A N/A No No No No Yes No No No No
Cargo Type Characterization
Protein Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate gold-labeled markers are available Yes, limited to surface proteins, if appropriate gold-labeled markers are available Yes, with multi-wavelength UV detecor No No Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes, for CD63/CD81/CD9 Yes Yes, if appropriate fluorescent markers are used Yes, if appropriate fluorescent markers are used Yes (if fluorescently labeled antibody is available) Yes Yes Yes No Yes Yes Yes Yes N/A N/A N/A N/A N/A Selects for specific protein targets Selects for specific protein targets Yes, quantify total protein by UV at 280nm Selects for specific protein targets N/A N/A N/A
Nucleic Acid Yes, with non-sequence-specific nucleic acid dyes or molecular beacons Yes, with non-sequence-specific nucleic acid dyes or molecular beacons Yes, with non-sequence-specific nucleic acid dyes or molecular beacons Yes, with embedding/sectioning and in situ hybridization probes No Yes, with multi-wavelength UV detecor No No Yes, if appropriate fluorescent markers are used Yes, with non-sequence-specific nucleic acid dyes or molecular beacons Yes, with non-sequence-specific nucleic acid dyes or molecular beacons Yes, with non-sequence-specific nucleic acid dyes or molecular beacons No Yes, with non-sequence-specific nucleic acid dyes or molecular beacons Yes, with non-sequence-specific nucleic acid dyes or molecular beacons No Yes, if appropriate fluorescent markers are used Yes, with non-sequence-specific nucleic acid dyes or molecular beacons TBD In development Yes Yes Yes Yes Yes Yes No No N/A N/A N/A N/A N/A No No Yes, quantify total nucleic acid by UV at 260 nm Selects for specific RNA targets N/A N/A N/A
Lipid Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, only if specific gold-labeled markers are available. Until now limited to phosphatidylserine/ Annexin5 Yes, only if specific gold-labeled markers are available. Until now limited to phosphatidylserine/ Annexin5 No No No Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, VFred included Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains Yes, with fluorescent lipid stains No Yes Yes No No N/A N/A N/A N/A N/A No Yes, if high affinity lipid binding molecule is available. Yes, quantify relative lipid content by UV+dRI N/A N/A N/A N/A
Internal Cargo detection Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Limited information on internal EV cargo using negative staining, possible with embedding/sectioning Limited information on internal EV cargo, due to the lack of reliable and non-damaging methods for EV permeabilization Yes, UV, MALS and DLS detectors No No Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining No Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes, with permeabilization and fluorescent staining Yes Yes Yes Yes, only with membrane penetrating moiety Yes Yes Yes No N/A N/A N/A N/A N/A Yes Yes Yes Yes N/A N/A N/A
Structure No No No Yes Yes Limited No No No No No No No No No No No No No No Yes Yes No No No No No No N/A N/A N/A N/A N/A No No Yes No N/A N/A N/A
Colocalization No Yes, 2 markers at a time No Yes, if gold markers are available. Limited to 2 components Yes, if gold markers are available. Limited to 2 components No No Yes, 3 markers at a time Yes, 3 markers at a time Yes Yes, 5 or more targets depending on configuration Yes Yes, up to 8 targets Yes Yes, 2 markers at a time Yes, of CD63/CD81/CD9/lipid Yes Yes Yes Yes Yes ( laser dependent) Yes (up to 3 targets, but laser dependent) Yes Yes Yes Yes Yes Yes N/A N/A N/A N/A N/A Yes Yes No Yes N/A N/A N/A
Relative copy number quantification (case vs control) limited by photo- bleaching Yes Yes yes, with gold markers yes, with gold markers No No No No Yes, if standards used Yes, if standards used Yes, if standards used Yes, if standards used Yes, if standards used Yes Yes, if standards used Yes Yes Yes Yes, if standards used Yes Yes Yes Yes Yes Yes Yes Yes N/A N/A N/A N/A N/A Yes No No No N/A N/A N/A
"Absolute" copy number quantification No No No No No No No No No No No MESF calibration feasible, but >10 MESF limit of detection MESF calibration feasible, but >10 MESF limit of detection Yes, if standards used No MESF calibration feasible, but >10 MESF limit of detection Yes MESF calibration feasible, but >100 MESF limit of detection No No Detected fluorescent molecules/EV depends on analysis platform Detected fluorescent molecules/EV depends on analysis platform No No Yes No No No N/A N/A N/A N/A N/A No No No No N/A N/A N/A
Sensitivity
Lowest working concentration (particles/mL) 106 5x105 106 108 108 108 1-5x108 (depends on size) 107 107 No lower limit 103 No lower limit 107 LOB/LOD/LLOQ need to be defined for each assay, internal data suggest detection of 300 GFP retrovirus particles in PBS and 4000 GFP retrovirus particles when spiked into serum 107 107 108 Analysis: 2x104. Sorting: 108 Sorting: 108 needed. Analysis: 20,000 needed. 5x106 5x108 5x108 104-105 Depends on target and detection instrument used < 103/μL (in process of optimization) 106 - 107 (can be lower with surface functionalization) 105 108 N/A Not known Not known Not known N/A Not known Not known 108 Not known Not known Not known Not known
Sensitivity (particles/mL) High High High Med Med High Med Med Med High High High High Low Med Med Med Med Med Med Med Med High Depends on target and detection instrument used High High High Med N/A Not known Not known Not known N/A Not known Not known Med Not known Not known Not known Not known
Highest working concentration (particles/mL) 109 109 109 1012 for small (100 nm) EVs; 106 for large EVs (>500 nM) 1012 for small (100 nm) EVs; 106 for large EVs (>500 nM) No limit 1-5x1010 1012 1012 5x108 5x109 5x109 5x109 15,000-30,000 events/sec - depends on specific instrument - value must be qualified by empirical data. 109 5x109 1011 109 NA 109 5x1011, but depends on preparation, area, etc 5x1011, but depends on preparation, area, etc 108-1010 Depends on target and detection instrument used 1012 No Known Limit 1010 1011 N/A Not known Not known Not known N/A Depends on # target molecules/particle Not known No known limit Not known Not known Not known Not known
Sensitivity (#cargo/EV)) Low Low Low High High N/A Low Low Low High High High High LOW - again LOB/LOD/LLOQ must be defined under strict criteria, please see CLSI guideline H62 section 6.1.2.3 Detection Capability, for rare event detection please see CLSI guideline 5.3.2 Table 13. High High High Low High High High High High Depends on target and detection instrument used Med Med N/A High N/A N/A N/A N/A N/A N/A N/A Low N/A N/A N/A N/A
Limit of detection (analytical limit for size detection, nm) 30 nm 40-50 nm for scatter; <40 nm for fluorescence. 30 nm for scatter; 40 nm for fluorescence 10 nm 30 nm to several µm Configuration- specific 40 nm 50 nm 50 nm 30 nm with fluorescence 30 nm with fluorescence 70 nm by scatter 70 nm by scatter 70 nm 40 nm 40-100 nm (Depends on instrument used) ~30nm 90 nm 25 nm with fluorescence 50 nm Instrument dependent. 8 nm Instrument dependent. 8 nm 40 nm N/A 10 nm No Known Limit, single molecule sensitivity N/A N/A N/A Depends on column N/A N/A N/A N/A N/A 1-200 nm N/A N/A N/A N/A
Resolution (particle size limit of detection, nm) High High High High High High High High High High High Med Med High High Med-High (Depends on instrument used) Med High High High High N/A High N/A N/A N/A N/A Depends on column N/A N/A N/A N/A N/A High N/A N/A N/A N/A
Limit of detection (analytical sensitivity in MESF) Undefinable In progress Not known N/A N/A N/A N/A N/A FITC 50-100 MESF; PE 5-10 MESF FITC <10 MESF, PE<5 MESF FITC 4 MESF, PE 3 MESF FITC 30 MESF, PE 10 MESF, APC 25 MESF <10 MESF (fluorophore-dependent) 5-20 MESF (fluorophore-dependent) <10 MESF (fluorophore-and instrument-dependent) Depends on instrument used Dimmest <1% of Single Molecules 300 MESF PE 24 +/- 7.6 MESF Depending on specificity of the affinity reagents. Can detect a single fluorescent molecule. > 1 MESF > 1 MESF N/A Depends on instrument used N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A TBD N/A N/A N/A N/A
Specificity
Analytical Specificity Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Detects any particle Depends on specificity of morphology and markers Depends on specificity of morphology and markers Any material with refractive index different than buffer Detects any particle Detects any particle Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Depends on specificity of fluorescent affinity reagent Up to 99%, depends on specificity of affinity reagent used Based on antibody and negative control Based on antibody and negative control Based on antibody and negative control > 95 % Single-nucleotide specificity "No False Neg" 0.9 Depends on specificity of affinity reagent used Depends on specificity of affinity reagent used N/A N/A N/A N/A N/A Depends on specificity of affinity reagent used Depends on specificity of affinity reagent used Depends on sample prep, without UC and filtration before injection much lower N/A N/A N/A Depends on isoelectric point difference between targets
Output
File type csv or pdf avi (raw); txt (analysis); fcs avi (raw), csv (individual parameters), pdf (reports) Images Images .afe, .txt, .csv .idsf, .csv .h5, .xls, .pdf; (If analyzed using the open source RPSPASS software, file output options are .xlsx, .pdf, .mat, .fcs, and .json) .h5, .xls, .pdf; (If analyzed using the open source RPSPASS software, file output options are .xlsx, .pdf, .mat, .fcs, and .json) FCS Amnis image file and FCS (can be imported into FCS Express and FlowJo) fcs fcs .fcs, .pdf .fcs, .nfa Depends on instrument used Variable fcs DiVa experiments for acquisition but this is instrument specific with standard FCS output or CSV statistics files .xlsx .tiff or output from NimOS or CODI .mat, PDF, tif, or jpeg, figure can containing data or image TIFF, JPEG N/A DTA files txt file .txt NGS Can be exported as Excel file N/A N/A N/A N/A None None .cvs N/A N/A N/A N/A
Software used for data analysis Instrument software Instrument software Instrument software ImageJ/Fiji, AMT (for camera) ImageJ for size analysis ASTRA Instrument software Instrument software and/or RPSPASS Instrument software and/or RPSPASS Instrument software (CellStream Analysis) and/or FCS Express and FlowJo Instrument software (IDEAS) and/or FCS Express and FLowJo CytExpert (free acquisition and analysis from manufacturer); FCS Express/Reader (3rd party) Spectroflo (Cytek); and/or FCS Express and FlowJo Instrument software and/or third party Flow Jo, FCS express, Kaluza, etc. Yes, NF Professional FCS Express/Reader (3rd party) Yes/In House Summit (Beckman); and/or FCS Express and FlowJo DiVa experiments for acquisition but this is instrument specific with standard FCS output or CSV statistics files Yes, ExoView Yes; CODI cloud based software currently free of charge with no storage limit or lab-made python or matlab analysis code Yes; Nikon and Matlab software Yes, Matlab N/A No WIRE, PyCharm, other classification programs Methodical Mind or Discovery Workbench In-house, R No, Excel or other data processing software N/A N/A N/A N/A None None Yes, Wyatt Technologies Astra software N/A N/A N/A N/A
Additional Considerations
Accesibility for technology Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available Commercially available In House Commercially available Commercially available with custom FSC-PMT + + +++ Commercially available In development + Commercially available instruments Run on commercially available instruments Commercially available Commercially available Commercially available (requires ultracentrifuge and rotors) ++ (requires ultracentrifuge) +++ Commercially available reagents for certain targets. Uses commercially available instrument Commercially available Commercially available +++ +++ Not yet
Operating expertise required
(+, ++, +++)		 + + + ++ +++ +++ ++ ++ ++ ++ +++ ++ ++ +++ ++ ++ +++ +++ +++ + ++ - +++ +++ + ++ + + + +++ ++ + ++ ++ + ++ ++ ++ + + ++ +++
Reproducibilty and independent site validation CV 2-8% IQ/OQ In progress Ongoing N/A In progress IQ/OQ 5% precision in sizing 7% precision in sizing CV<3% CV<10% interwell CV<10%, interplate CV<20% interwell CV<10%, interplate CV<20% CV <10% (PMID: 33513846),Pink - summary for 8 different beads: repeatability MFI CV<1%, conc CV<5%, 30 day multiple operators, reproducibility MFI CV<6% , CV,<10% IQ/OQ interwell CV<10%, interplate CV<20% Not known Not known Highly reproducibly, validated with orthogonal methodology Yes Validated with orthogonal methods Yes Yes Ongoing Not known Yes Not done yet Not done yet Not done yet Not done yet Not done yet Yes Ongoing Ongoing Ongoing Completed Not done yet Not yet done Not done yet
EV size tunability No No No No No Yes Yes Yes Yes Yes, with fluorescence and Mie scatter Yes, with fluorescence and Mie scatter 80-2000 nm 80-2000 nm No Yes 30-2000 nm (Depends on instrument) N/A 90-2000 nm Separation based on size is limited. Separation is generally dependent on probes utilized No No No No No N/A N/A No No Yes, depends on selected resin Yes No No Yes No No N/A No Yes N/A N/A
Instrument cost $216K (4 lasers, syringe pump, 96-well plate "Sample Assistant") $75K (Scatter, zeta potential and 1 laser); $150K (4 lasers) $75K (Scatter); $85K (1 laser) ~$750K >$500K $250K $45K $50K $200K (Laser: 488 nm (default) or 561 nm; with user-configurable filters) $90K (10x magnification confocal with CCD camera, images not stored, 1 laser [blue]). $175K (4 lasers [blue, green, red, violet]). $300K (7 lasers) $225K (40x magnification with 1 camera, images stored, 1 laser). $350K-$500K (60x magnification with 1-2 cameras, 4 lasers). $62K (1-2 laser, autoloader) - $300K (6 lasers including UV laser, autoloader) ~$200-300K depending on number of lasers. $100-250K depending on configuration and add ons including autosampler, 3rd angle of light scatter detection, plate cooler, sorter $205K (1 excitation source), $230K (2 excitation sources) N/A N/A >$500K >$500K >$130K ~$300K (includes software, hardware, computer, etc.) >$500K $180K Standard research-grade microscope No instrument needed Raman microscope ~$200K $100K $100K for MiSeq,$350K for NextSeq $15-80K for instrument. $1000-3000/column (typically, two columns are run in tandem; columns are re-usable). Resins used include Sepharose 6 and Sephacryl S-500. $2850 for automated fraction collector (Izon) Ultracentrifuge ($60K) +rotor ($15K) Ultracentrifuge ($60K) +rotor ($15K) $50K No large equipment needed $60K $150K $7K $30k No large equipment needed No large equipment needed
Cost per sample No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). ~$35 (academic cost, includes reagents [$5] and instrument time) $5 for EM grid Minimal per sample cost $6.50-$13 per sample $13 per sample $13 per sample No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). Minimal instrument-specific consumables (tubing and sheath fluid). Depends on affinity reagents used (usually $5-10/well). No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). Minimal instrument-specific consumables (sheath fluid). Depends on reagents used (usually $5-10/well if patient sample used). No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). $5-10 per well, depending on level of cargo multiplexing N/A No instrument-specific consumables. Depends on affinity reagents used (usually $5-10/well). $150-200 >$100 Depends on imaging time, concentration of sample, and antibody price  Depends on imaging time, concentration of sample, and antibody price  $1.25-$1.50. Depends on imaging time, concentration of sample, and antibody price. $300 per beacon (50 nmol scale) 5 $5-$10 $1-5/sample/assay Assay reagents: $10/sample; Library prep and Sequencing cost: $25/sample 2 $60/column (Izon qEV columns, can be reused a limited number of times) $15/gradient for optiprep Ultracentrifuge tubes 3 50 20 $20/sample $3.50-$4.00. Dependent on imaging time, concentration of sample, and antibody price $40 per sample $15 (+ cost of antibodies) 10