A Systematic Approach to Improve Scatter Sensitivity of a Flow Cytometer for Detection of Extracellular Vesicles

Extracellular vesicles (EVs) are commonly studied by flow cytometry. Due to their small size and low refractive index, the scatter intensity of most EVs is below the detection limit of common flow cytometers. Here, we aim to improve forward scatter (FSC) and side scatter (SSC) sensitivity of a common flow cytometer to detect single 100 nm EVs. The effects of the optical and fluidics configuration on scatter sensitivity of a FACSCanto (Becton Dickinson) were evaluated by the separation index (SI) and robust coefficient of variation (rCV) of polystyrene beads (BioCytex). Improvement is defined as increased SI and/or reduced rCV. Changing the obscuration bar improved the rCV 1.9‐fold for FSC. A 10‐fold increase in laser power improved the SI 19‐fold for FSC and 4.4‐fold for SSC, whereas the rCV worsened 0.8‐fold and improved 1.5‐fold, respectively. Confocalization worsened the SI 1.2‐fold for FSC, and improved the SI 5.1‐fold for SSC, while the rCV improved 1.1‐fold and worsened 1.5‐fold, respectively. Replacing the FSC photodiode with a photomultiplier tube improved the SI 66‐fold and rCV 4.2‐fold. A 2‐fold reduction in sample stream width improved both SI and rCV for FSC by 1.8‐fold, and for SSC by 1.3‐ and 2.2‐fold, respectively. Decreasing the sample flow velocity worsened rCVs. Decreasing the flow channel dimensions and the pore size of the sheath filter did not substantially change the SI or rCV. Using the optimal optical configuration and fluidics settings, the SI improved 3.8∙104‐fold on FSC and 30‐fold on SSC, resulting in estimated detection limits for EVs (assuming a refractive index of 1.40) of 246 and 91 nm on FSC and SSC, respectively. Although a 50‐fold improvement on FSC is still necessary, these adaptions have produced an operator‐friendly, high‐throughput flow cytometer with a high sensitivity on both SSC and FSC. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. To improve detection of cell‐derived extracellular vesicles and other submicrometer particles, the optics and fluidics of a common flow cytometer were systematically modified. The modifications resulted in a sensitivity increase of 3.8·104‐fold and 30‐fold for the forward and side scatter detector, respectively. The optimized flow cytometer is operator‐friendly, high throughput and can detect extracellular vesicles down to 246 nm using the forward scatter detector and down to 91 nm using the sid