FCM_PASS Software Aids Extracellular Vesicle Light Scatter Standardization

Abstract

The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analyzing EVs due to it being a high‐throughput, multiparametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardization exists for EV flow cytometry, which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard to interpret, compare, and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardization, utilizing flow cytometer postacquisition analysis software (FCMPASS). FCMPASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter‐diameter curve and scatter‐refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardized units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCMPASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…New, sophisticated procedures exist to derive the diameter of EVs from scatter or fluorescence signals. For example, Exometry offers a commercial kit and the National Institute of Health offers free software to determine the EV diameter from a single scatter detector. The information from two scatter detectors can be combined to determine the refractive index, which can be used for label‐free differentiation between EVs and lipoprotein particles .…”

Monolithically-integrated cytometer for measuring particle diameter in the extracellular vesicle size range using multi-angle scattering

“…Particle size was calculated from each individual particle scattering distribution by fitting a model using MATLAB® (MathWorks, US) based on Mie scattering theory. 2,12,14 This model predicts the angular scattering of light from a spherical particle, such as EVs 36 and the relative optical energy collected by each collection waveguide in the integrated cytometer. An optimal fit was achieved for each particle by minimizing the weighted sum of squared residuals…”

“…8 The technique has a relatively low throughput however at around 16 particles per second 9 compared to flow cytometry and concentration determination requires careful calibration and can be inaccurate with broad EV size distributions. 10 Conventional flow cytometry has a higher limit of detection than NTA at 200 nm but is proving to be highly practical for EV analysis as it can measure the size and concentration of polydisperse samples at kHz rates 11,12 along with detecting multiple molecular markers by antibody-based fluorescent labelling. The internal apparatus of a conventional flow cytometer has remained largely unchanged since its original inception and consists of a free space optical system of lenses, mirrors and filters aligned with a fluidic flow cell as well as photodetectors and data acquisition electronics.…”

“…Literature refractive index values for EVs can then be used with the normalised Mie scattering model to calculate EV diameter from SSC values from samples of unknown diameter. 12,14 Sizing inaccuracy and imprecision arise as the relationship between particle diameter and SSC value is non-monotonic, exhibiting plateaus and local minima at specific diameter ranges leading to ambiguity in sizing in these regions. An accurate model also requires knowledge of the system collection geometry which is often proprietary and can vary with optical alignment.…”

Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2019 Conference Workshops

“…There is as yet no consensus method for standardization of small particle analysis. The goal of this workshop was to go over some proposed methods for scatter and fluorescence standardization using scatter modeling software as well as MESF beads using FCM PASS software .…”