E. van der Pol

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing

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 104 to 1012 vesicles mL–1.

Optical and non-optical methods for detection and characterization of microparticles and exosomes

Summary.  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.

Single vs. swarm detection of microparticles and exosomes by flow cytometry.

See also Harrison P, Gardiner C. Invisible vesicles swarm within the iceberg. This issue, pp 916‐8.

Innovation in detection of microparticles and exosomes

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.

Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.

The research field of extracellular vesicles (EVs), such as microparticles and exosomes, is growing exponentially. The goal of this review is to provide an overview of recent developments relevant to the readers of the Journal of Thrombosis and Haemostasis. We will discuss nomenclature, the presence of EVs in fluids, methods of isolation and detection, and emerging clinical implications. Although research on EVs has been performed within the ISTH for over a decade, most of the recent research on EVs has been brought together by the International Society on Extracellular Vesicles (ISEV). To achieve an overview of recent developments, the information provided in this review comes not only from publications, but also from latest meetings of the ISEV (April 2015, Washington, DC, USA), the International Society on Advancement of Cytometry (June 2015, Glasgow, UK), and the ISTH (June 2015, Toronto, Canada).

Dependent and multiple scattering in transmission and backscattering optical coherence tomography

We use transmission and backscattering optical coherence tomography (OCT) to distinguish and quantify dependent and multiple scattering effects in turbid media. With transmission OCT the dependent scattering coefficients for a range of monodisperse silica particle suspensions are determined. An excellent agreement is observed between the measured dependent scattering coefficients and calculations based on Mie calculations, the Percus-Yevick radial distribution function, and coherent light scattering theory. Backscattering OCT measurements are fitted using the extended Huygens-Fresnel (EHF) model with the dependent scattering coefficients obtained from the transmission OCT measurements as input parameters. Good agreement between the EHF model and the backscattering OCT measurements is observed. For large particles, the rms scattering angle θrms obtained from the EHF fit is in fair agreement with θrms calculated from the transmission OCT data.

Biological reference materials for extracellular vesicle studies

&NA; 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 200 nm), 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. Graphical abstract Figure. No caption available.

Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

Essentials Platelet extracellular vesicles (EVs) concentrations measured by flow cytometers are incomparable. A model is applied to convert ambiguous scatter units to EV diameter in nanometer. Most included flow cytometers lack the sensitivity to detect EVs of 600 nm and smaller. The model outperforms polystyrene beads for comparability of platelet EV concentrations.

Hollow organosilica beads as reference particles for optical detection of extracellular vesicles

Essentials Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement. Hollow organosilica beads were prepared, characterized, and tested as reference particles. Light scattering properties of hollow beads resemble that of platelet‐derived EVs. Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs.

Wound scabs protect regenerating tissue against harmful ultraviolet radiation

Benefits attributed to wound scabs include prevention of blood loss and protection against infection. However, when formation of a wound scab is prevented, the risk of infection is reduced. Moreover, in the absence of a wound scab, wounds heal faster and scar formation is reduced. The question arises why we develop a wound scab. Here we show that wound scabs inhibit transmission of ultraviolet radiation (UVR). We compared the UVR transmittance of human wound scabs to sunscreen by measuring the sun protection factor (SPF) with diffuse transmittance spectroscopy. Three wound scabs showed SPFs of 70, 84, and 300, which is more effective than the most protective commercially available sun block. Because our results demonstrate that a wound scab offers natural protection against UVR, and because no beneficial trait is attributed to wound scabs, we hypothesize that the main function of wound scabs is to limit DNA damage in underlying cells during regeneration of wound tissue exposed to sunlight, thereby reducing the risk of developing skin cancer.