High‐fidelity detection and sorting of nanoscale vesicles in viral disease and cancer

Abstract

Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the “reference noise”). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.

Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?

“…However, only the latter technique is able to combine high-throughput and adequate speed allowing EVs evaluation in translational studies and in a routine clinical setting. Several custom-constructed flow cytometers or last generation modified cell sorter, with optimized fluidics and flow cell design, have been developed to detect extremely small particles [ 4 , 5 ]. However, these instruments are not optimally suited for other more common applications in clinical settings, mostly cell immunophenotyping.…”

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).…”

“…Non-conventional cytometer collection optics, such as the Apogee instruments, are currently not supported due to lack of testing availability. Although light scatter calibration can be applied to sorters and has been demonstrated (Morales-Kastresana et al, 2019;Welsh, Horak, et al, 2018), care must be taken in the stream alignment, and the confidence of the model fit will likely be lower. This is due to the use of laser obscuration bars in front of the collection lens that are difficult to take into account.…”

Emerging Roles of Urine-Derived Components for the Management of Bladder Cancer: One Man’s Trash is Another Man’s Treasure

“…Reliable isolation of UBC-derived EVs is dependent on recognizing markers exclusively and consistently expressed on the surface of EVs released by tumor cells. These cell-surface markers can be utilized to collect UBC-derived EVs through magnetic bead-based immunocapture or fluorescence activated particle sorting across multiple platforms 96,97 . In the absence of tumor-specific antigens, isolation of bladder-specific EVs (from both tumors and normal cells) can be acceptable as a way to enrich the diagnostic EV pool with UBC-derived EVs and to minimize confounding urinary EVs with other cellular origins.…”