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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120104	1/1	Homo sapiens	Synovial fluid	(d)(U)C Filtration	György B	2012	29%
Study summary Full title Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases All authors György B, Szabó TG, Turiák L, Wright M, Herczeg P, Lédeczi Z, Kittel A, Polgár A, Tóth K, Dérfalvi B, Zelenák G, Böröcz I, Carr B, Nagy G, Vékey K, Gay S, Falus A, Buzás EI Journal PLoS One Abstract INTRODUCTION: Microvesicles (MVs), earlier referred to as microparticles, represent a major type of (show more...)INTRODUCTION: Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+) and CD8(+) T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction). CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures. (hide) EV-METRIC 29% (21st 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 Synovial fluid Sample origin NAY Focus vesicles microvesicles / 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 Filtration Protein markers EV: non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Synovial fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps > 0.45 µm, Characterization: Protein analysis Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV120104
species	Homo sapiens
sample type	Synovial fluid
condition	NAY
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	29