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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140210	1/1	Equus caballus	Serum	(d)(U)C DG	Rout ED	2014	33%
Study summary Full title Transferrin receptor expression in serum exosomes as a marker of regenerative anaemia in the horse All authors Rout ED, Webb TL, Laurence HM, Long L, Olver CS Journal Equine Vet J Abstract REASONS FOR PERFORMING STUDY: Evaluation of erythrocyte regeneration in horses is challenging, as th (show more...)REASONS FOR PERFORMING STUDY: Evaluation of erythrocyte regeneration in horses is challenging, as they do not release reticulocytes into the peripheral blood. This study investigated transferrin receptor 1 (TfR1) expression in exosomes as a noninvasive method of characterising the regenerative response in anaemic horses. OBJECTIVES: To quantify TfR1 in ultraprecipitate of serum in horses before and after phlebotomy-induced anaemia, and to identify exosomes as the source of TfR1. The hypothesis was that serum exosomal TfR1 expression would increase during a regenerative response. STUDY DESIGN: Experimental model of anaemia. METHODS: Six horses were phlebotomised to achieve a 25% decrease in packed cell volume. Transferrin receptor 1 quantity in exosomes was determined by western blot and relative densitometry before and after phlebotomy. The size and density of the TfR1-associated particles were confirmed by transmission electron microscopy and density gradient centrifugation, respectively. RESULTS: Regenerative anaemia was confirmed by decreased packed cell volumes and decreased myeloid:erythroid ratios in the bone marrow. In all 6 horses, TfR1 expression increased between Days 7 and 10. Mean TfR1 levels peaked on Day 10 and at 3-fold higher than levels on Day 0. Appropriately sized particles were evident on transmission electron microscopy and sucrose density gradient fractions expected to contain exosomes also contained TfR1. CONCLUSIONS: These data indicate that TfR1 expression in serum exosomes may provide a marker for regeneration in anaemic horses. (hide) EV-METRIC 33% (76th 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 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 Adj. k-factor 255.8 (pelleting) Protein markers EV: Tf-receptor non-EV: Proteomics no EV density (g/ml) 1.125-1.16 Show all info Study aim Biomarker Sample Species Equus caballus Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Wash: volume per pellet (ml) 1 Density gradient Only used for validation of main results Yes Highest density fraction 60 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Tf-receptor ELISA Antibody details provided? No Detected EV-associated proteins Tf-receptor Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140210
species	Equus caballus
sample type	Serum
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	33