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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140125	1/2	Homo sapiens	Blood plasma	(d)(U)C IAF	Shi M	2014	38%
Study summary Full title Plasma exosomal alpha-synuclein is likely CNS-derived and increased in Parkinson's disease All authors Shi M, Liu C, Cook TJ, Bullock KM, Zhao Y, Ginghina C, Li Y, Aro P, Dator R, He C, Hipp MJ, Zabetian CP, Peskind ER, Hu SC, Quinn JF, Galasko DR, Banks WA, Zhang J Journal Acta Neuropathol Abstract Extracellular ?-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a (show more...)Extracellular ?-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum ?-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived ?-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled ?-synuclein into mouse brain, that CSF ?-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of ?-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF ?-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal ?-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF ?-synuclein, a significant correlation between plasma exosomal ?-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal ?-synuclein were comparable to those determined by CSF ?-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS ?-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically. (hide) EV-METRIC 38% (72nd 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 IAF Protein markers EV: Alix/ Alpha-synuclein/ L1CAM non-EV: Proteomics yes 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 Pelleting performed No Immunoaffinity capture Selected surface protein(s) L1CAM, CD63 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ L1CAM/ Alpha-synuclein ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ L1CAM/ Alpha-synuclein Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein L1CAM Image type Close-up, Wide-field

	EV140125	2/2	Mus musculus	Blood plasma	(d)(U)C DG	Shi M	2014	14%
Study summary Full title Plasma exosomal alpha-synuclein is likely CNS-derived and increased in Parkinson's disease All authors Shi M, Liu C, Cook TJ, Bullock KM, Zhao Y, Ginghina C, Li Y, Aro P, Dator R, He C, Hipp MJ, Zabetian CP, Peskind ER, Hu SC, Quinn JF, Galasko DR, Banks WA, Zhang J Journal Acta Neuropathol Abstract Extracellular ?-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a (show more...)Extracellular ?-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum ?-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived ?-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled ?-synuclein into mouse brain, that CSF ?-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of ?-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF ?-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal ?-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF ?-synuclein, a significant correlation between plasma exosomal ?-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal ?-synuclein were comparable to those determined by CSF ?-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS ?-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically. (hide) EV-METRIC 14% (38th 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 Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Mus musculus 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) 180 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 1 Characterization: Particle analysis None

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EV-TRACK ID	EV140125
species	Homo sapiens	Mus musculus
sample type	Blood plasma	Blood plasma
condition	NAY	NAY
separation protocol	(d)(U)C IAF	(d)(U)C DG
Exp. nr.	1	2
EV-METRIC %	38	14