Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in ...concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) .
To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques.
The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS).
Each technique gave a different size distribution and a different concentration for the same vesicle sample.
Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.
Cell-derived or extracellular vesicles, including microparticles and exosomes, are abundantly present in body fluids such as blood. Although such vesicles have gained strong clinical and scientific ...interest, their detection is difficult because many vesicles are extremely small with a diameter of less than 100 nm, and, moreover, these vesicles have a low refractive index and are heterogeneous in both size and composition. In this review, we focus on the relatively high throughput detection of vesicles in suspension by flow cytometry, resistive pulse sensing, and nanoparticle tracking analysis, and we will discuss their applicability and limitations. Finally, we discuss four methods that are not commercially available: Raman microspectroscopy, micro nuclear magnetic resonance, small-angle X-ray scattering (SAXS), and anomalous SAXS. These methods are currently being explored to study vesicles and are likely to offer novel information for future developments.
Microparticles and exosomes are cell-derived vesicles and potential biomarkers for disease. Recently, the Scientific Standardization Committee collaborative workshop of the ISTH initiated ...standardization of vesicle detection by flow cytometry with polystyrene beads. Because polystyrene beads have different optical properties from biological vesicles, and because the mechanisms causing the detection signal are incompletely understood, there are contradictions between expected and observed results.
To develop a model with which to relate the detection signal of a flow cytometer to the diameter of vesicles and clarify observed discrepancies.
We combined measurements of polystyrene and silica beads with an estimated refractive index of vesicles and performed Mie calculations of light scattering.
We established the relationship between measured light scattering and the diameter of vesicles. The Megamix gating strategy proposed by the Scientific Standardization Committee selects single vesicles and cells with diameters between 800 and 2400 nm when applied on the forward-scattering detector of regular flow cytometers. Nevertheless, we demonstrated that, irrespective of the applied gating, multiple vesicles smaller than 220 nm or multiple 89-nm silica beads were counted as a single event signal at sufficiently high concentrations.
Vesicle detection by flow cytometry is attributed to large single vesicles and swarm detection of smaller vesicles; that is, multiple vesicles are simultaneously illuminated by the laser beam and counted as a single event signal. Swarm detection allows the detection of smaller vesicles than previously thought possible, and explains the finding that flow cytometry underestimates the concentration of vesicles.
Microparticles and exosomes are cell-derived microvesicles present in body fluids that play a role in coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and ...transport. Despite increasing scientific and clinical interest, no standard procedures are available for the isolation, detection and characterization of microparticles and exosomes, because their size is below the reach of conventional detection methods. Our objective is to give an overview of currently available and potentially applicable methods for optical and non-optical determination of the size, concentration, morphology, biochemical composition and cellular origin of microparticles and exosomes. The working principle of all methods is briefly discussed, as well as their applications and limitations based on the underlying physical parameters of the technique. For most methods, the expected size distribution for a given microvesicle population is determined. The explanations of the physical background and the outcomes of our calculations provide insights into the capabilities of each method and make a comparison possible between the discussed methods. In conclusion, several (combinations of) methods can detect clinically relevant properties of microparticles and exosomes. These methods should be further explored and validated by comparing measurement results so that accurate, reliable and fast solutions come within reach.
Abstract Background Membrane abnormalities in polyunsaturated fatty acids (PUFAs) have been reported in schizophrenia and have been associated with brain tissue loss in normal ageing. Therefore PUFA ...may be involved in the excessive brain tissue loss reported in schizophrenia. Methods A systematic MEDLINE database search was conducted to identify studies that compared PUFAs in erythrocyte membranes in patients and controls. Patients were categorized by medication regime in medication naive first-episode patients, and patients receiving typical or atypical antipsychotics. Sample Fourteen studies were included, comprising a total of 429 patients with schizophrenia and 444 healthy control subjects. Cohen's d effect sizes were calculated for PUFAs in erythrocyte membranes using the random-effects model. Combined Cohen's d was calculated separately for patients on different medication regime. Results Medication-naive patients and patients taking typical antipsychotics showed significantly (p < 0.01) decreased concentrations of arachidonic (AA), docosahexaenoic (DHA), and docosapentaenoic (DPA) acid. In addition, patients taking typical antipsychotics showed decreased linoleic (LA), dihomo-γ-linolenic acid (DGLA), eicosapentaenoic (EPA) and docosatetraenoic (DTA) acid (p < 0.01). Patients taking atypical antipsychotics showed decreased DHA (p < 0.01) only. Conclusions PUFA concentrations in erythrocyte membranes are decreased in schizophrenia. Of particular importance in patients are lower concentrations of DHA and AA, two fatty acids that are abundant in the brain and important precursors in the cell-signalling cascade.
The purpose of this study was to assess whether management of heart failure (HF) guided by an individualized N-terminal pro-B-type natriuretic peptide (NT-proBNP) target would lead to improved ...outcome compared with HF management guided by clinical assessment alone.
Natriuretic peptides may be attractive biomarkers to guide management of heart failure (HF) and help select patients in need of more aggressive therapy. The PRIMA (Can PRo-brain-natriuretic peptide guided therapy of chronic heart failure IMprove heart fAilure morbidity and mortality?) study is, to our knowledge, the first large, prospective randomized study to address whether management of HF guided by an individualized target NT-proBNP level improves outcome.
A total of 345 patients hospitalized for decompensated, symptomatic HF with elevated NT-proBNP levels at admission were included. After discharge, patients were randomized to either clinically-guided outpatient management (n = 171), or management guided by an individually set NT-proBNP (n = 174) defined by the lowest level at discharge or 2 weeks thereafter. The primary end point was defined as number of days alive outside the hospital after index admission.
HF management guided by this individualized NT-proBNP target increased the use of HF medication (p = 0.006), and 64% of HF-related events were preceded by an increase in NT-proBNP. Nevertheless, HF management guided by this individualized NT-proBNP target did not significantly improve the primary end point (685 vs. 664 days, p = 0.49), nor did it significantly improve any of the secondary end points. In the NT-proBNP-guided group mortality was lower, as 46 patients died (26.5%) versus 57 (33.3%) in the clinically-guided group, but this was not statistically significant (p = 0.206).
Serial NT-proBNP measurement and targeting to an individual NT-proBNP value did result in advanced detection of HF-related events and importantly influenced HF-therapy, but failed to provide significant clinical improvement in terms of mortality and morbidity. (Effect of NT-proBNP Guided Treatment of Chronic Heart Failure PRIMA; NCT00149422).
Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we ...showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) < 1.42 that included the majority of EVs, and a second population with an RI > 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI < 1.42, and the lipoprotein-enriched fractions (1.04 to 1.03 g/mL) contained the highest concentration of particles with an RI > 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs.
•This article describes the rationale, design, and procedures of the YOUth cohort.•YOUth is set up to investigate what drives the development of social competence and self-regulation in ...children.•YOUth specifically investigates the role of neurocognitive development in child development.•YOUth has a flexible longitudinal design with repeated measurements throughout childhood, starting prenatally.
Behavioral development in children shows large inter-individual variation, and is driven by the interplay between biological, psychological, and environmental processes. However, there is still little insight into how these processes interact. The YOUth cohort specifically focuses on two core characteristics of behavioral development: social competence and self-regulation. Social competence refers to the ability to engage in meaningful interactions with others, whereas self-regulation is the ability to control one’s emotions, behavior, and impulses, to balance between reactivity and control of the reaction, and to adjust to the prevailing environment. YOUth is an accelerated population-based longitudinal cohort study with repeated measurements, centering on two groups: YOUth Baby & Child and YOUth Child & Adolescent. YOUth Baby & Child aims to include 3,000 pregnant women, their partners and children, wheras YOUth Child & Adolescent aims to include 2,000 children aged between 8 and 10 years old and their parents. All participants will be followed for at least 6 years, and potentially longer.
In this paper we describe in detail the design of this study, the population included, the determinants, intermediate neurocognitive measures and outcomes included in the study. Furthermore, we describe in detail the procedures of inclusion, informed consent, and study participation.
Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings ...requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated.
The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively).
Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.
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