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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120092	1/1	Homo sapiens	Urine	(d)(U)C	van der Pol E	2012	14%
Study summary Full title Single vs swarm detection of microparticles and exosomes by flow cytometry All authors van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG Journal J Thromb Haemost Abstract BACKGROUND: Microparticles and exosomes are cell-derived vesicles and potential biomarkers for disea (show more...)BACKGROUND: Microparticles and exosomes are cell-derived vesicles and potential biomarkers for disease. Recently, the Scientific Standardization Committee collaborative workshop of the ISTH initiated standardization of vesicle detection by flow cytometry with polystyrene beads. Because polystyrene beads have different optical properties from biological vesicles, and because the mechanisms causing the detection signal are incompletely understood, there are contradictions between expected and observed results. OBJECTIVES: To develop a model with which to relate the detection signal of a flow cytometer to the diameter of vesicles and clarify observed discrepancies. METHODS: We combined measurements of polystyrene and silica beads with an estimated refractive index of vesicles and performed Mie calculations of light scattering. RESULTS: We established the relationship between measured light scattering and the diameter of vesicles. The Megamix gating strategy proposed by the Scientific Standardization Committee selects single vesicles and cells with diameters between 800 and 2400 nm when applied on the forward-scattering detector of regular flow cytometers. Nevertheless, we demonstrated that, irrespective of the applied gating, multiple vesicles smaller than 220 nm or multiple 89-nm silica beads were counted as a single event signal at sufficiently high concentrations. CONCLUSIONS: Vesicle detection by flow cytometry is attributed to large single vesicles and swarm detection of smaller vesicles; that is, multiple vesicles are simultaneously illuminated by the laser beam and counted as a single event signal. Swarm detection allows the detection of smaller vesicles than previously thought possible, and explains the finding that flow cytometry underestimates the concentration of vesicles. (hide) EV-METRIC 14% (40th 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 Urine Sample origin NAY Focus vesicles exosomes / microparticles 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: non-EV: Proteomics no TEM measurements 55(mode) Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field Report size (nm) 55(mode)

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EV-TRACK ID	EV120092
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	14