Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

Abstract

Improvements in identification and assessment of extracellular vesicles (EVs) have fuelled a recent surge in EV publications investigating their roles as biomarkers and mediators of disease. Meaningful scientific comparisons are, however, hampered by difficulties in accurate, reproducible enumeration and characterization of EVs in biological fluids. High-sensitivity flow cytometry (FCM) is presently the most commonly applied strategy to assess EVs, yet its utility is limited by variant ability to resolve smaller EVs. Here, we propose the use of 405 nm (violet) wavelength lasers in place of 488 nm (blue) for side scatter (SSC) detection to obtain greater resolution of EVs using high-sensitivity FCM. To test this hypothesis, we modelled EV resolution by violet versus blue SSC in silico and compared resolution of reference beads and biological EVs from plasma and bronchoalveolar lavage (BAL) fluid using either violet or blue wavelength SSC EV detection. Mie scatter modelling predicted that violet as compared to blue SSC increases resolution of small (100–500 nm) spherical particles with refractive indices (1.34–1.46) similar to EVs by approximately twofold in terms of light intensity and by nearly 20% in SSC signal quantum efficiency. Resolution of reference beads was improved by violet instead of blue SSC with two- and fivefold decreases in coefficients of variation for particles of 300–500 nm and 180–240 nm size, respectively. Resolution was similarly improved for detection of EVs from plasma or BAL fluid. Violet SSC detection for high-sensitivity FCM allows for significantly greater resolution of EVs in plasma and BAL compared to conventional blue SSC and particularly improves resolution of smaller EVs. Notably, the proposed strategy is readily implementable and inexpensive for machines already equipped with 405 nm SSC or the ability to accommodate 405/10 nm bandpass filters in their violet detector arrays.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

“…Particle number can be measured by light scattering technologies, such as nanoparticle tracking analysis (NTA); by standard flow cytometry for larger EVs [164][165][166][167] or high resolution flow cytometry for smaller EVs [127,[168][169][170][171][172][173][174][175][176]; by resistive pulse sensing (RPS) for a wide range of sizes, depending on pore size [177]; by cryo-EM [174]; by a platform combining surface plasmon resonance (SPR) with AFM [178]; or by other techniques with similar capabilities. Accurate quantitation may be possible only within a certain concentration and size range that varies by platform; where possible, this range (or the minimum and maximum diameter measured) should be reported along with concentration.…”

The potential diagnostic power of CD138+ microparticles from the plasma analysis for multiple myeloma clinical monitoring

“…Because the diameter of the MPs is too smaller to detect with conventional method (488‐nm SSC laser light). So in order to improve the sensitivity for MP detection, we adjusted the side scatter (SSC) with 405‐nm (instead of 488 nm) laser light to avoid background noise during acquisition . The predefined MP gate was applied to all samples during analysis.…”

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

“…Lower EV detection limits of 45 nm with NTA and 100 nm by flow virometry were reported in this work. The sample composition and also the implementation of the V-SSC instead of the 488 nm SSC in flow virometry could contribute to explain these differences [34]. Still, a higher level of accuracy is possible by using National Institute of Standards and Technology (NIST)-traceable beads in the Mie correlation with flow virometry.…”

“…Still, a significant loss of scattered light that fall in the range of the background noise of the instrument and different sensitivities between equipments are a general concern [33]. To address this issue, the implementation of the violet (405 nm) side scatter (V-SSC) has been reported to improve the sensitivity but also the resolution of the technique [34]. Owing to the specific features of each analytical method, characterization results such as particle concentration obtained by different techniques are often difficult to compare.…”