Methodological Guidelines to Study Extracellular Vesicles.

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing.

INTRODUCTION: The size of extracellular vesicles (EVs) can be determined with a tunable resistive pulse sensor (TRPS). Because the sensing pore diameter varies from pore to pore, the minimum detectable diameter also varies. The aim of this study is to determine and improve the reproducibility of TRPS measurements. METHODS: Experiments were performed with the qNano system (Izon) using beads and a standard urine vesicle sample. With a combination of voltage and stretch that yields a high blockade height, we investigate whether the minimum detected diameter is more reproducible when we configure the instrument targeting (a) fixed stretch and voltage, or (b) fixed blockade height. RESULTS: Daily measurements with a fixed stretch and voltage (n=102) on a standard urine sample show a minimum detected vesicle diameter of 128±19 nm [mean±standard deviation; coefficient of variation (CV) 14.8%]. The vesicle concentration was 2.4·109±3.8·109 vesicles/mL (range 1.4·108-1.8·1010). When we compared setting a fixed stretch and voltage to setting a fixed blockade height on 3 different pores, we found a minimum detected vesicle diameter of 118 nm (CV 15.5%, stretch), and 123 nm (CV 4.5%, blockade height). The detected vesicle concentration was 3.2-8.2·108 vesicles/mL with fixed stretch and 6.4-7.8·108 vesicles/mL with fixed blockade height. Summary/conclusion: Pore-to-pore variability is the cause of the variation in minimum detected size when setting a fixed stretch and voltage. The reproducibility of the minimum detectable diameter is much improved by setting a fixed blockade height.

Plasma markers of coagulation and endothelial activation in Fabry disease: impact of renal impairment.

BACKGROUND: In Fabry disease, storage of globotriaosylceramide (Gb3) in arterial walls is one of the main pathogenetic factors that are thought to underlie the clinical manifestations of the disease. Abnormalities of the vessel wall, haemodynamics and pro- and anticoagulant factors may play a role, though the exact pathophysiology is incompletely understood. In this study, we try to clarify inconsistencies regarding coagulation activation, fibrinolysis, platelet activation and endothelial activation in 36 patients with Fabry disease. METHODS: Cell-derived microparticles, markers for coagulation activation (F(1+2), TAT, sTF, sEPCR), fibrinolysis (D-dimer, tPA, alpha(2)-AP), platelet activation (beta-TG, PF4), endothelial activation (vWF) and acute phase response (IL-6, CRP) were studied in relation to renal function and severity of the disease and compared to data from 36 age- and sex-matched healthy controls (17 males). RESULTS: Markers for endothelial activation and fibrinolysis were normal. Male patients had elevated levels of sTF and beta-TG, with an association between sTF and renal function and severity of the disease. In female patients, levels of TAT, beta-TG, PF4, CD63-positive platelet-derived microparticles and IL-6 were somewhat increased, with no correlation with renal function or disease severity. CONCLUSIONS: Only minimal abnormalities in markers for platelet, endothelial activation and coagulation activation and fibrinolysis could be established in a large cohort of Fabry disease patients. The existing laboratory abnormalities are more likely related to renal insufficiency rather than to Fabry disease itself.