Analytical challenges of extracellular vesicle detection – A comparison of different techniques

The interest in extracellular vesicles (EVs) has grown exponentially over the last decade. Evolving evidence is demonstrating that these EVs are playing an important role in health and disease. They are involved in intercellular communication and have been shown to transfer proteins, lipids, and nucleic acids.

This review focuses on the most commonly used techniques for detection of EVs, to include microparticles, 100-1,000 nm in size, and exosomes, 50-100 nm in size. Conventional flow cytometry is the most prevalent technique, but nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and resistive pulse sensing have also been used to detect EVs. The accurate measurement of these vesicles is challenged by size heterogeneity, low refractive index, and the lack of dynamic measurement range for most of the available technologies. Sample handling during the preanalytical phase can also affect the accuracy of measurements. Currently, there is not one single method which allows phenotyping, sizing, and enumerating the whole range of EVs and, therefore, providing all the necessary information to truly understand the biology of these particles. A combination of methods is probably needed which might also include electron and atomic force microscopy and full RNA, lipid, and protein profiling.

exosomes

Examples of techniques currently explored for detection of EVs. Panel A) demonstrates the principle of flow cytometry. A sample mixed with sheath fluid is running through a channel. The laser crosses the sample stream and generates scatter and fluorescence signals. Side scatter (SCC) is measured perpendicular to the laser beam whereas forward scatter (FSC) is collected in the direction of the laser beam. Panel B) represents a schematic of resistive pulse sensing. In this system, vesicles in fluid flow through an aperture and electrical resistance increases when a vesicle is present. This is based on the Coulter principle developed by Wallace Coulter in the 1940s and patented in 1953. As a particle suspended in an electrolyte solution enters an aperture, an equal volume of electrolytes solutes is displaced. This displaced electrolyte volume increases the impedance across the circuit, thus generating a voltage pulse that is proportional to the volume of the particle, which does not depend on shape or refractive index. Panel C) summarizes the principle of Nanoparticle tracking analysis. A laser beam is passing through a sample chamber. A camera with a microscope is capturing the particles moving under Brownian motion. Applying the Stokes Einstein equation the hydrodynamic markers of each particle can be determined and count and size of the particles be measured. Image rendition per our artist according to other published images.

Erdbrügger U, Lannigan J. (2015) Analytical challenges of extracellular vesicle detection: A comparison of different techniques. Cytometry A [Epub ahead of print]. [abstract]

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