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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190060	1/4	Homo sapiens	Blood plasma	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (88th 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 (d)(U)C Protein markers EV: TSG101/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD41/ TSG101 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD61/ CD235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 107-145 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 EM EM-type Transmission-EM Image type Close-up

	EV190060	2/4	Homo sapiens	Blood plasma	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (88th 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 (d)(U)C Protein markers EV: TSG101/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD41/ TSG101 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD61/ CD235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 117-162 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV190060	3/4	Homo sapiens	Blood plasma	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (88th 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 (d)(U)C Protein markers EV: TSG101/ CD61/ CD41/ phosphatidylserine/ Cd235a/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ CD41 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD61/ Cd235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 106-160 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV190060	4/4	Homo sapiens	Serum	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (92nd 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 (d)(U)C Protein markers EV: TSG101/ CD235a/ CD41/ CD9/ phosphatidylserine non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD41/ CD9/ TSG101 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD41/ CD235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 88-128 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 Report type Not Reported EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV190060
species	Homo sapiens
sample type	Blood plasma	Blood plasma	Blood plasma	Serum
condition	Control condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	1	2	3	4
EV-METRIC %	56	56	56	56