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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140123	2/2	Homo sapiens	Blood plasma	(d)(U)C DG Filtration SEC	Hong CS	2014	44%
Study summary Full title Plasma exosomes as markers of therapeutic response in patients with acute myeloid leukemia All authors Hong CS, Muller L, Whiteside TL, Boyiadzis M Journal Front Immunol Abstract PURPOSE: Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients (show more...)PURPOSE: Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients have elevated protein and transforming growth factor-beta 1 (TGF-?1) contents and inhibit natural killer (NK) cell cytotoxicity (Haematologica 96, p. 1302, 2011). A potential role of exosomes in predicting responses to chemotherapy (CT) was evaluated in AML patients undergoing treatment. EXPERIMENTAL DESIGN: Plasma was obtained from AML patients at diagnosis (n = 16); post-induction CT (n = 9); during consolidation CT (n = 10); in long-term remission (Lt-CR, n = 5); and from healthy volunteers (n = 7). Exosomes were isolated by size-exclusion chromatography and ultracentrifugation. The exosomal protein, soluble TGF?-1 levels (ELISA), and the TGF-?1 profiles (western blots) were compared among patients' cohorts. The results were correlated with the patients' cytogenetic profile, percentage of leukemic blast, and outcome. RESULTS: At diagnosis, protein and TGF-?1 levels were higher (p < 0.009 and p < 0.004) in AML than control exosomes. These values decreased after induction CT (p < 0.05 and p < 0.004), increased during consolidation CT (p < 0.02 and p < 0.005), and normalized in Lt-CR. While TGF-?1 and protein levels tracked one another, TGF-?1 pro-peptide, latency-associated peptide (LAP), or mature TGF-?1 differentially decorated exosomes isolated before, during, and after CT. Only TGF-?1 pro-peptide was seen in exosomes of controls or Lt-CR patients. During consolidation CT, exosomes carried TGF-?1 pro-peptide, LAP, and low levels of mature TGF-?1. NK cell co-incubation with AML exosomes carrying all three TGF-?1 forms induced down-regulation of NKG2D expression. CONCLUSION: Changes in exosomal protein and/or TGF-?1 content may reflect responses to CT. The exosomal profile may suggest the presence of residual disease in patients considered to have achieved complete remission. (hide) EV-METRIC 44% (77th 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 NAY Focus vesicles exosomes 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 DG Filtration SEC Protein markers EV: CD81 non-EV: Proteomics no EV density (g/ml) 1.16-1.19 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma 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: time(min) 120 Density gradient Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD81 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 Close-up

	EV140123	1/2	Homo sapiens	Blood plasma	(d)(U)C Filtration SEC	Hong CS	2014	0%
Study summary Full title Plasma exosomes as markers of therapeutic response in patients with acute myeloid leukemia All authors Hong CS, Muller L, Whiteside TL, Boyiadzis M Journal Front Immunol Abstract PURPOSE: Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients (show more...)PURPOSE: Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients have elevated protein and transforming growth factor-beta 1 (TGF-?1) contents and inhibit natural killer (NK) cell cytotoxicity (Haematologica 96, p. 1302, 2011). A potential role of exosomes in predicting responses to chemotherapy (CT) was evaluated in AML patients undergoing treatment. EXPERIMENTAL DESIGN: Plasma was obtained from AML patients at diagnosis (n = 16); post-induction CT (n = 9); during consolidation CT (n = 10); in long-term remission (Lt-CR, n = 5); and from healthy volunteers (n = 7). Exosomes were isolated by size-exclusion chromatography and ultracentrifugation. The exosomal protein, soluble TGF?-1 levels (ELISA), and the TGF-?1 profiles (western blots) were compared among patients' cohorts. The results were correlated with the patients' cytogenetic profile, percentage of leukemic blast, and outcome. RESULTS: At diagnosis, protein and TGF-?1 levels were higher (p < 0.009 and p < 0.004) in AML than control exosomes. These values decreased after induction CT (p < 0.05 and p < 0.004), increased during consolidation CT (p < 0.02 and p < 0.005), and normalized in Lt-CR. While TGF-?1 and protein levels tracked one another, TGF-?1 pro-peptide, latency-associated peptide (LAP), or mature TGF-?1 differentially decorated exosomes isolated before, during, and after CT. Only TGF-?1 pro-peptide was seen in exosomes of controls or Lt-CR patients. During consolidation CT, exosomes carried TGF-?1 pro-peptide, LAP, and low levels of mature TGF-?1. NK cell co-incubation with AML exosomes carrying all three TGF-?1 forms induced down-regulation of NKG2D expression. CONCLUSION: Changes in exosomal protein and/or TGF-?1 content may reflect responses to CT. The exosomal profile may suggest the presence of residual disease in patients considered to have achieved complete remission. (hide) EV-METRIC 0% (median: 22% 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 NAY Focus vesicles exosomes 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 SEC Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma 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: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis None

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EV-TRACK ID	EV140123
species	Homo sapiens
sample type	Blood plasma
condition	NAY
separation protocol	(d)(U)C DG Filtration SEC	(d)(U)C Filtration SEC
Exp. nr.	2	1
EV-METRIC %	44	0