Innovation in detection of microparticles and exosomes

Abstract

Summary. 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.

Characterisation of particles in solution – a perspective on light scattering and comparative technologies

“…In the case of the characterisation of NPs of biological origin, the NTA technique offers the greatest flexibility and robustness [17,39,40]. The ability to analyse samples in simple and complex biological media [3,19,25,41,42] is of distinct advantage when characterising materials under physiological conditions.…”

Measurement of microvesicle levels in human blood using flow cytometry

“…Not all events detected by non-specific methods like light scatter, nano-particle tracking, scanning electron microscopy and impedance, are truly lipid microvesicles released from cells (7). Other types of "microparticles" in plasma that can be counted by nonspecific methods include immune complexes, calcium phosphate precipitates, antibody aggregates, and cell debris from freeze/thaw (1,(30)(31)(32)(33)(34).…”

“…Both the background noise threshold (lowest threshold that minimizes background noise counts) and bead resolution must be determined. Starting with polystyrene beads, decreasing size beads can be run as mixtures to determine the minimum size that can be resolved from the next larger bead and accurately counted (6,7,(44)(45)(46)(47). Figure 2 shows an example of bead resolution using forward light scatter on a dark field and a conventional flow cytometer.…”

“…A number of different standards have been used in an attempt to calibrate microvesicle size and detection limit including polystyrene and silica beads, synthetic liposomes of known size, virions of uniform size and comparisons of purified microvesicle size distributions measured using alternate technologies like dynamic light scattering or nanoparticle tracking analysis (6,7,42,48). The most straightforward calibration is for flow cytometers with resistive pulse sensing cells (impedance) where the response of the detector to a polystyrene bead and microvesicle of the same size are similar, thus bead size directly calibrates microvesicle size.…”

“…A variety of methods have been used to measure particles in blood including dynamic light scattering, nanoparticle tracking analysis, resistive pulse sensing (impedance), scanning and atomic force microscopy, but individually they are nonspecific, unable to truly distinguish lipid vesicles from other particles or evaluate microvesicle characteristics (6,7). Transmission electron microscopy is able to specifically count vesicles across a range of sizes and determine some cell characteristics, but it is labor intensive (1,6).…”