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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220304	6/30	Homo sapiens	Blood plasma	DG UF SEC (non-commercial)	Dhondt B	2023	100%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 100% (99th 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD81/ Flotillin-1 non-EV: ApoA1/ Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD81/ Flotillin-1 Detected contaminants ApoA1 Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220304	3/30	Homo sapiens	Blood plasma	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	9/30	Homo sapiens	Blood plasma	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	12/30	Homo sapiens	Serum	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (99th 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 Serum 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Serum Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	15/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	18/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	21/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	24/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	30/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	83%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 83% (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 NA Sample origin NA 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-250 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220304	27/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	67%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 67% (89th 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 NA Sample origin NA 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 Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220304	5/30	Homo sapiens	Blood plasma	SEC (non-commercial)	Dhondt B	2023	43%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 43% (74th 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220304	23/30	NA	NA	SEC (non-commercial)	Dhondt B	2023	38%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 38% (72nd 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 NA Sample origin NA 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD81/ Flotillin-1/ eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD81/ Flotillin-1 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	1/30	Homo sapiens	Blood plasma	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (64th 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	4/30	Homo sapiens	Blood plasma	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (64th 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	7/30	Homo sapiens	Blood plasma	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (64th 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	10/30	Homo sapiens	Serum	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (76th 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 Serum 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Serum Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	13/30	NA	NA	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (63rd 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 NA Sample origin NA 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	16/30	NA	NA	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (63rd 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 NA Sample origin NA 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment - Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	19/30	NA	NA	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (63rd 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 NA Sample origin NA 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	22/30	NA	NA	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (63rd 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 NA Sample origin NA 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	25/30	NA	NA	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (63rd 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 NA Sample origin NA 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	28/30	NA	NA	Flow cytometric vesicle sorting	Dhondt B	2023	33%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 33% (63rd 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 NA Sample origin NA 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 Flow cytometric vesicle sorting Protein markers EV: CD61/ CD235a/ Lactadherin non-EV: IgG Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Resin type Fluorescence-activated vesicle sorting Type of flow cytometer Apogee A60-Micro Size of calibration beads (µm) 0.1-1 Fluorescent labeling Specific labelling of EV conte Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Apogee A60-Micro Calibration bead size 0.1-1 Detected EV-associated proteins CD61/ CD235a/ Lactadherin Detected contaminants IgG Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Apogee A60-Micro Hardware adjustment - Calibration bead size 0.1-1 Report type Size range/distribution Reported size (nm) 200-1000 EV concentration Yes

	EV220304	2/30	Homo sapiens	Blood plasma	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (37th 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	8/30	Homo sapiens	Blood plasma	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (37th 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	11/30	Homo sapiens	Serum	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (55th 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 Serum 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species Homo sapiens Sample Type Serum Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	14/30	NA	NA	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (45th 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 NA Sample origin NA 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	17/30	NA	NA	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (45th 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 NA Sample origin NA 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	20/30	NA	NA	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (45th 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 NA Sample origin NA 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	26/30	NA	NA	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (45th 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 NA Sample origin NA 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

	EV220304	29/30	NA	NA	SEC (non-commercial)	Dhondt B	2023	14%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 14% (45th 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 NA Sample origin NA 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 Size-exclusion chromatography (non-commercial) Protein markers EV: eGFP non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins eGFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes

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EV-TRACK ID	EV220304
species	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	NA	NA	NA	NA	NA	NA	Homo sapiens	NA	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	NA	NA	NA	NA	NA	NA	Homo sapiens	Homo sapiens	Homo sapiens	NA	NA	NA	NA	NA
sample type	Blood plasma	Blood plasma	Blood plasma	Serum	NA	NA	NA	NA	NA	NA	Blood plasma	NA	Blood plasma	Blood plasma	Blood plasma	Serum	NA	NA	NA	NA	NA	NA	Blood plasma	Blood plasma	Serum	NA	NA	NA	NA	NA
condition	Control condition	Control condition	Control condition	Control condition	NA	NA	NA	NA	NA	NA	Control condition	NA	Control condition	Control condition	Control condition	Control condition	NA	NA	NA	NA	NA	NA	Control condition	Control condition	Control condition	NA	NA	NA	NA	NA
separation protocol	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Density gradient/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Flow cytometric vesicle sorting	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)	Size-exclusion chromatography (non-commercial)
Exp. nr.	6	3	9	12	15	18	21	24	30	27	5	23	1	4	7	10	13	16	19	22	25	28	2	8	11	14	17	20	26	29
EV-METRIC %	100	83	83	83	83	83	83	83	83	67	43	38	33	33	33	33	33	33	33	33	33	33	14	14	14	14	14	14	14	14

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Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data