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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170037	1/3	Homo sapiens	Serum	(d)(U)C	Klump, Jennifer	2018	28%
Study summary Full title Extracellular vesicles or free circulating DNA: where to search for BRAF and cKIT mutations? All authors Klump J, Phillipp U, Follo M, Eremin A, Lehmann H, Nestel S, von Bubnoff N, Nazarenko I Journal Nanomedicine Abstract Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood bi (show more...)Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. (hide) EV-METRIC 28% (70th 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 (d)(U)C Adj. k-factor 213.2 (pelleting) / 213.2 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 120000 Wash: adjusted k-factor 213.2 Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) 10-50 dependent whether healthy donors or cancer patients were analyzed Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) different populations of vesicles were detected by DLS and NTA;120-500 nm; and over 1000 nm NTA Report type Size range/distribution Reported size (nm) 90-300 EV concentration Yes Particle yield 3.00E+09 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Report size (nm) 90-120 Extra information Publication aimed to determine the content of mutated DNA oncogenes copy inside of the vesicles (post- DNase treatments) and in the free-circulating fractions. For that DNA was isolated from different EV and fc fractions and DNA was analyzed using ddPCR. Conclusion was that the mutated BRAF and c-KIT copies are preferably located in the free-circulating fractions and not in EVs.

	EV170037	2/3	Homo sapiens	Serum	(d)(U)C	Klump, Jennifer	2018	28%
Study summary Full title Extracellular vesicles or free circulating DNA: where to search for BRAF and cKIT mutations? All authors Klump J, Phillipp U, Follo M, Eremin A, Lehmann H, Nestel S, von Bubnoff N, Nazarenko I Journal Nanomedicine Abstract Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood bi (show more...)Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. (hide) EV-METRIC 28% (70th 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 Melanoma 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 (d)(U)C Adj. k-factor 213.2 (pelleting) / 213.2 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 120000 Wash: adjusted k-factor 213.2 Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) 10-50 dependent whether healthy donors or cancer patients were analyzed Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) different populations of vesicles were detected by DLS and NTA;120-500 nm; and over 1000 nm NTA Report type Size range/distribution Reported size (nm) 90-300 EV concentration Yes Particle yield 3.00E+09 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Report size (nm) 90-120 Extra information Publication aimed to determine the content of mutated DNA oncogenes copy inside of the vesicles (post- DNase treatments) and in the free-circulating fractions. For that DNA was isolated from different EV and fc fractions and DNA was analyzed using ddPCR. Conclusion was that the mutated BRAF and c-KIT copies are preferably located in the free-circulating fractions and not in EVs.

	EV170037	3/3	Homo sapiens	Serum	(d)(U)C	Klump, Jennifer	2018	28%
Study summary Full title Extracellular vesicles or free circulating DNA: where to search for BRAF and cKIT mutations? All authors Klump J, Phillipp U, Follo M, Eremin A, Lehmann H, Nestel S, von Bubnoff N, Nazarenko I Journal Nanomedicine Abstract Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood bi (show more...)Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. (hide) EV-METRIC 28% (70th 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 Mastocytosis 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 (d)(U)C Adj. k-factor 213.2 (pelleting) / 213.2 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 120000 Wash: adjusted k-factor 213.2 Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) 10-50 dependent whether healthy donors or cancer patients were analyzed Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) different populations of vesicles were detected by DLS and NTA;120-500 nm; and over 1000 nm NTA Report type Size range/distribution Reported size (nm) 90-300 EV concentration Yes Particle yield 3.00E+09 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Report size (nm) 90-120 Extra information Publication aimed to determine the content of mutated DNA oncogenes copy inside of the vesicles (post- DNase treatments) and in the free-circulating fractions. For that DNA was isolated from different EV and fc fractions and DNA was analyzed using ddPCR. Conclusion was that the mutated BRAF and c-KIT copies are preferably located in the free-circulating fractions and not in EVs.

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EV-TRACK ID	EV170037
species	Homo sapiens
sample type	Serum
condition	Control condition	Melanoma	Mastocytosis
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	1	2	3
EV-METRIC %	28	28	28