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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV231013	1/5	Homo sapiens	Blood plasma	(d)(U)C	Robinson, Stephen	2024	78%
Study summary Full title Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods All authors Stephen David Robinson, Mark Samuels, William Jones, Nicolas Stewart, Murat Eravci, Nektarios K Mazarakis, Duncan Gilbert, Giles Critchley, Georgios Giamas Journal Abstract Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing bl (show more...)Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation. (hide) EV-METRIC 78% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ HSP70/ TSG101/ Syntenin non-EV: Albumin/ GM130/ ApoA1/ ApoB/ ApoE Proteomics yes Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 10 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70/ TSG101/ Syntenin Detected contaminants ApoB/ ApoE Not detected contaminants Albumin/ GM130/ ApoA1 Detected contaminants Albumin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(3) Single particle interferometric reflectance imaging sensing (Leprechaun) Report type Mean

	EV231013	4/5	Homo sapiens	Blood plasma	(d)(U)C Filtration	Robinson, Stephen	2024	78%
Study summary Full title Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods All authors Stephen David Robinson, Mark Samuels, William Jones, Nicolas Stewart, Murat Eravci, Nektarios K Mazarakis, Duncan Gilbert, Giles Critchley, Georgios Giamas Journal Abstract Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing bl (show more...)Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation. (hide) EV-METRIC 78% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD9/ CD63/ CD81/ HSP70/ TSG101/ Syntenin non-EV: GM130/ ApoA1/ Albumin/ ApoB/ ApoE Proteomics yes Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 10 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100,000 Filtration steps 0.2 or 0.22 ðm Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70/ TSG101/ Syntenin Detected contaminants Albumin/ ApoB/ ApoE Not detected contaminants GM130/ ApoA1 Detected contaminants Albumin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 128 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.15E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(3) Single particle interferometric reflectance imaging sensing (Leprechaun) Report type Mean

	EV231013	2/5	Homo sapiens	Blood plasma	Total Exosome Isolation	Robinson, Stephen	2024	75%
Study summary Full title Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods All authors Stephen David Robinson, Mark Samuels, William Jones, Nicolas Stewart, Murat Eravci, Nektarios K Mazarakis, Duncan Gilbert, Giles Critchley, Georgios Giamas Journal Abstract Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing bl (show more...)Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Total Exosome Isolation Protein markers EV: TSG101/ CD9/ CD63/ CD81/ HSP70/ Syntenin non-EV: Albumin/ GM130/ ApoB/ ApoE/ ApoA1 Proteomics yes Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins TSG101 Not detected EV-associated proteins CD9/ CD63/ CD81/ HSP70/ Syntenin Detected contaminants ApoA1 Not detected contaminants Albumin/ GM130/ ApoB/ ApoE Detected contaminants Albumin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 92 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.18E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(3) Single particle interferometric reflectance imaging sensing (Leprechaun) Report type Mean

	EV231013	3/5	Homo sapiens	Blood plasma	qEV	Robinson, Stephen	2024	75%
Study summary Full title Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods All authors Stephen David Robinson, Mark Samuels, William Jones, Nicolas Stewart, Murat Eravci, Nektarios K Mazarakis, Duncan Gilbert, Giles Critchley, Georgios Giamas Journal Abstract Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing bl (show more...)Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: CD9/ CD63/ CD81/ HSP70/ TSG101/ Syntenin non-EV: Albumin/ GM130/ ApoA1/ ApoE/ ApoB Proteomics yes Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70/ TSG101/ Syntenin Detected contaminants ApoB Not detected contaminants Albumin/ GM130/ ApoA1/ ApoE Detected contaminants Albumin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 103 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.95E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(3) Single particle interferometric reflectance imaging sensing (Leprechaun) Report type Mean

	EV231013	5/5	Homo sapiens	Blood plasma	MagCapture Exosome Isolation Kit PS	Robinson, Stephen	2024	75%
Study summary Full title Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods All authors Stephen David Robinson, Mark Samuels, William Jones, Nicolas Stewart, Murat Eravci, Nektarios K Mazarakis, Duncan Gilbert, Giles Critchley, Georgios Giamas Journal Abstract Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing bl (show more...)Background Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: CD9/ HSP70/ CD63/ CD81/ TSG101/ Syntenin non-EV: GM130/ ApoA1/ ApoB/ ApoE/ Albumin Proteomics yes Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit MagCapture Exosome Isolation Kit PS Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected EV-associated proteins CD63/ CD81/ TSG101/ Syntenin Detected contaminants Albumin Not detected contaminants GM130/ ApoA1/ ApoB/ ApoE Detected contaminants Albumin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.92E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(3) Single particle interferometric reflectance imaging sensing (Leprechaun) Report type Mean

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EV-TRACK ID	EV231013
species	Homo sapiens
sample type	Blood plasma
condition	Control condition
separation protocol	dUC	dUC/ Filtration	Total Exosome Isolation	qEV	MagCapture Exosome Isolation Kit PS
Exp. nr.	1	4	2	3	5
EV-METRIC %	78	78	75	75	75