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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210044	1/1	Homo sapiens	Serum	qEV	Newman, Lauren	2021	71%
Study summary Full title Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses All authors Lauren A. Newman, Alia Fahmy, Michael J. Sorich, Oliver G. Best, Andrew Rowland, Zivile Useckaite Journal Cells Abstract Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human bl (show more...)Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease. (hide) EV-METRIC 71% (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 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 qEV Protein markers EV: CD81/ ASGR1/ CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Flow cytometry Type of Flow cytometry CytoFlex S Hardware adaptation to ~100nm EV's 1. Filter configuration as per Beckman Coulter website 2. Violet filter used for small particle detection 3. Calibration beads were used to determine particle size gating strategy 4. Callibration beads used Megamix Plus SSC and Megaamix Plus FSC Calibration bead size 0.1-0.9 Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ASGR1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 85.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.25E+11 Particle analysis: flow cytometry Flow cytometer type CytoFlex S Hardware adjustment 1. Laser configuration to detect small particles, violet laser use, hardware set up according to Beckman Coulter website 2. Use of MegaMix beads to determine particle size for gating purposes 3. MegaMix plus beads were used: MegaMix Plus SSC and MegaMix Plus Forward FSC Calibration bead size 0.1-0.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV210044
species	Homo sapiens
sample type	Serum
condition	Control condition
separation protocol	qEV
Exp. nr.	1
EV-METRIC %	71