Fluorescence and Light Scatter Calibration Allow Comparisons of Small Particle Data in Standard Units across Different Flow Cytometry Platforms and Detector Settings

Abstract

Flow cytometers have been utilized for the analysis of submicron‐sized particles since the late 1970s. Initially, virus analyses preceded extracellular vesicle (EV), which began in the 1990s. Despite decades of documented use, the lack of standardization in data reporting has resulted in a growing body of literature that cannot be easily interpreted, validated, or reproduced. This has made it difficult for objective assessments of both assays and instruments, in‐turn leading to significant hindrances in scientific progress, specifically in the study of EVs, where the phenotypic analysis of these submicron‐sized vesicles is becoming common‐place in every biomedical field. Methods for fluorescence and light scatter standardization are well established and the reagents to perform these analyses are commercially available. However, fluorescence and light scatter calibration are not widely adopted by the small particle community as methods to standardize flow cytometry (FCM) data. In this proof‐of‐concept study carried out as a resource for use at the CYTO2019 workshop, we demonstrate for the first‐time simultaneous fluorescence and light scatter calibration of small particle data to show the ease and feasibility of this method for standardized FCM data reporting. This data was acquired using standard configuration commercial flow cytometers, with commercially available materials, published methods, and freely available software tools. We show that application of light scatter, fluorescence, and concentration calibration can result in highly concordant data between FCM platforms independent of instrument collection angle, gain/voltage settings, and flow rate; thus, providing a means of cross comparison in standard units. © 2020 International Society for Advancement of Cytometry

Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration

“…This is also the case for other techniques where affinity-based phenotyping is not or cannot be done. Currently, only single-EV flow cytometry combines abilities for sizing, concentration measurement, affinity-based phenotyping and high throughput with calibration into standard units to provide a limit of sensitivity for each parameter [26][27][28][29]. While not all techniques have a discernable limit of sensitivity that can be derived from a calibration and expressed in standard units, it is possible to perform indirect assessments of sensitivity using reference materials.…”

“…Ideally, the calibration reference materials would also be certified, so as to give a traceable measurement back to SI units and limiting the potential for bias in calibration accuracy. The development of certified calibration reference particles is crucial for instrument measurement calibration, instrument sensitivity quantification and in turn instrumentation standardization and comparisons [29]. Calibration reference materials allow standardized reporting and consequently, validation of published EV studies between assays, instruments and laboratories.…”

“…Calibration reference materials do not necessarily need to mimic all characteristics of the downstream analysis particles, such as EVs. Materials such as polystyrene beads, hollow organosilica beads, liposomes, etc., could all be conceivably used as size standards, so long as the measurement technique sizing ability is based on physical size and not on other properties such as light scatter intensity (which would also depend on refractive index) [29]. Calibration reference materials for some parameters, such as fluorescence and light scatter, typically require a set of standards for multiple populations [19,32,33].…”

“…Reporting data in quantitative units (e.g. molecules of equivalent soluble fluorophore, MESF) instead of as relative expression or in arbitrary units (fluorescence intensity) allows for comparison of data across scientific institutions and platforms [29,39]. Where this is impossible, many assays are still able to indicate the relative amount of an epitope, comparing single EVs or EV samples.…”

Small Particle Fluorescence and Light Scatter Calibration Using FCMPASSSoftware

“…The lack of clarity within the nomenclature is in part due to the lack of an ability to separate and characterize all EVs. Today, as flow cytometers become ever more sensitive due to technological advancements, the ability to characterize particles of 100 nm in diameter or smaller is feasible using some commercially available instruments (de Rond et al, 2019;Gasecka et al, 2020;Morales-Kastresana et al, 2019;Stoner et al, 2016;Tian et al, 2020;Tian et al, 2018;Welsh, Jones, & Tang, 2020;Zhu et al, 2014). As the use of small particle flow cytometry increases, it is critical for researchers to understand the particular controls and considerations that are distinctively necessary in small particle flow cytometry, as compared to what is necessary in cellular flow cytometry (see Current Protocols article; Nolan, 2015).…”