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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210443	2/2	Homo sapiens	CSF	(d)(U)C Filtration	Akers JC	2016	29%
Study summary Full title Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF). All authors Akers JC, Ramakrishnan V, Nolan JP, Duggan E, Fu CC, Hochberg FH, Chen CC, Carter BS Journal PLoS One Abstract Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patient (show more...)Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations. (hide) EV-METRIC 29% (60th 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 CSF Sample origin Glioblastoma Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type CSF Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps > 0.45 µm, Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes TRPS Report type Not Reported EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Custom build flow cytometer Hardware adjustment Stoner SA, Duggan E, Condello D, Guerrero A, Turk JR, Narayanan PK, et al. High sensitivity flow cytometry of membrane vesicles. Cytometry A. 2015. doi: 10.1002/cyto.a.22787 PMID: 26484737 Calibration bead size 0.12 Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Wide-field EV concentration Yes

	EV210443	1/2	Homo sapiens	CSF	(d)(U)C Filtration	Akers JC	2016	14%
Study summary Full title Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF). All authors Akers JC, Ramakrishnan V, Nolan JP, Duggan E, Fu CC, Hochberg FH, Chen CC, Carter BS Journal PLoS One Abstract Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patient (show more...)Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations. (hide) EV-METRIC 14% (53rd 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 CSF Sample origin Glioblastoma Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type CSF Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 10000 Filtration steps > 0.45 µm, Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes TRPS Report type Not Reported EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Custom build flow cytometer Hardware adjustment Stoner SA, Duggan E, Condello D, Guerrero A, Turk JR, Narayanan PK, et al. High sensitivity flow cytometry of membrane vesicles. Cytometry A. 2015. doi: 10.1002/cyto.a.22787 PMID: 26484737 Calibration bead size 0.12 Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Wide-field EV concentration Yes

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EV-TRACK ID	EV210443
species	Homo sapiens
sample type	CSF
condition	Glioblastoma
separation protocol	dUC/ Filtration	dUC/ Filtration
vesicle related term	exosome	(shedding) microvesicle
Exp. nr.	2	1
EV-METRIC %	29	14