Single particle analysis: Methods for detection of platelet extracellular vesicles in suspension (excluding flow cytometry)

Abstract

Extracellular vesicles (EVs) are small membrane-bound particles released by all cell types, including abundant release by platelets. EVs are a topic of increasing interest in the academic and clinical community due to their increasingly recognized and diverse role in normal biology as well as in disease. However, typical analysis methods to characterize EVs released by cell culture or isolated from whole blood or other body fluids are restricted to bulk analysis of all EVs in a sample. In this review we discuss the motivation for analysis of individual EVs, as well as discuss three emerging methods for physical and chemical characterization of individual EVs: Nanoparticle Tracking Analysis, Tunable Resistive Pulse Sensing, and Raman Spectroscopy. We give brief descriptions of the working principles of each technique, along with a review noting the benefits and limitations of each method as applied to detection of single EVs.

Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets

“…Both ultracentrifugation and passing cell‐free saliva through a 0.2‐μ m filter reduced the number of EVs measured by flow cytometry when labeled with anti‐CD24 antibody or lactadherin (data not shown), confirming the presence of such EVs in saliva. We assume that the size distribution of salivary particles as detected by tunable resistive pulse sensing (Figure S4) reflects mostly non‐EV particles, as previously shown for human plasma .…”

Extracellular Vesicles as Biomarkers in Cardiovascular Disease; Chances and Risks

“…Knowing the viscosity and temperature of the sample medium, the EV size can be derived from their diffusion coefficients calculated using the Stokes-Einstein equation. A prerequisite is that viscosity and temperature are exactly defined and that the machine is calibrated using particles that have similar size and scatter characteristics as the EV analyzed ( 15 , 17 ). Nevertheless, the inaccuracies in EV counts obtained by NTA can be quite high, but protocols have been developed to minimize sample-to-sample variations ( 18 , 19 ).…”

“…A similar technique is dynamic light scattering, which like NTA is based on elastic Rayleigh scattering and makes use of the Stokes-Einstein equation for calculating the diffusion coefficients. A further with potential method is Raman spectroscopy, which is based on inelastic light scattering making this method attractive also for analyzing EV, since inelastic light scattering contains information about the molecular composition of the EV ( 17 , 20 ). Drawbacks are the high technical and mathematical complexity of the method and the long processing times of the analyses.…”

Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView

“…Still, most laboratories claim to investigate exosomes based on the isolation method applied, such as high‐speed differential ultracentrifugation (UC) [6] or other size‐ and density‐dependent isolating techniques yielding EVs with a characteristic diameter of 50–150 nm. As particle size plays a significant role in EV characterisation, reliable methods to determine particle size in the nanometre range are of great need [7,8]. Early in biomedical EV research, transmission electron microscopy (TEM) was used to assess EVs that are undetectable by standard means of light microscopy due to their small size and low refractive index [9,10].…”