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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230053	1/2	Homo sapiens	Blood plasma	(d)(U)C	Bettio V	2023	56%
Study summary Full title Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques. All authors Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D Journal PLoS One Abstract Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide) EV-METRIC 56% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ HSP70/ MHC1 non-EV: Histones/ Albumin/ APOB48/B100/ APOA1 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 (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 146 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Detected contaminants Albumin/ APOB48/B100/ APOA1 Not detected contaminants Histones Flow cytometry aspecific beads Detected EV-associated proteins CD9/ CD63/ CD81/ MHC1 Flow cytometry specific beads Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-170 EV concentration Yes Particle yield particles per milliliter of starting sample: 1000000-25000000

	EV230053	2/2	Homo sapiens	Blood plasma	(d)(U)C	Bettio V	2023	56%
Study summary Full title Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques. All authors Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D Journal PLoS One Abstract Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide) EV-METRIC 56% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ HSP70/ MHC1 non-EV: Histones/ Albumin/ APOB48/B100/ APOA1 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 (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 146 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Detected contaminants Albumin/ APOB48/B100/ APOA1 Not detected contaminants Histones Flow cytometry aspecific beads Detected EV-associated proteins CD9/ CD63/ CD81/ MHC1 Flow cytometry specific beads Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 150-210 EV concentration Yes Particle yield particles per milliliter of starting sample: 12000000-68000000

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EV-TRACK ID	EV230053
species	Homo sapiens
sample type	Blood plasma
condition	Control condition
separation protocol	dUC	dUC
Exp. nr.	1	2
EV-METRIC %	56	56