Engineered Retroviruses as Fluorescent Biological Reference Particles for Small Particle Flow Cytometry

Abstract

There has been renewed interest in the use of flow cytometry for single particle phenotypic analysis of particles in the nanometer size-range such as viruses, organelles, bacteria and extracellular vesicles (EVs). However, many of these particles are smaller than 200 nm in diameter, which places them at the limit of detection for many commercial flow cytometers. The use of reference particles of diameter, fluorescence, and light-scattering properties akin to those of the small biological particles being studied is therefore imperative for accurate and reproducible data acquisition and reporting across different instruments and analytical technologies. We show here that an engineered murine leukemia virus (MLV) can act as a fluorescence reference particle for other small particles such as retroviruses and EVs. More specifically, we show that engineered MLV is a highly monodisperse enveloped particle that can act as a surrogate to demonstrate the various effects of antibody labeling on the physical properties of small biological particles in a similar diameter range.

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

“…Inversely, the assay specificity for negative results could be validated using CD41a certified negative particles for a negative result. Validation reference materials appear to be the most sought-after within the EV field in order to test detection methods and assays [34][35][36]. These types of reference materials can be used as positive and negative controls within assays by having previously characterized properties, such as protein expression, concentration, diameter distribution, composition, etc.…”

“…Several efforts are being undertaken to develop reference materials as EV mimetics for use as validation of assays and quality controls [34][35][36][37]. Some come in the form of synthetic materials meant to mimic biological materials, such as hollow organosilica beads and liposomes, while others are derived from biological sources [34,35,37,38]. Synthetic materials such as hollow beads have the benefit of utilizing characteristics of EVs with fine control, such as low refractive indices and a coreshell structure, whilst being in the format of a tightly defined population which will likely be unambiguous in its detectability using optical methods.…”

“…While refractometers exist, they tend to be designed for liquids or films and are therefore not optimized for single particles or polydisperse mixtures of spheres. The application of Mie theory modelling requires calibration with spherical particles of known diameter and refractive index in order to fit predicted scattering models to detection equipment [22,32,35]. Many certified polystyrene reference particles exist.…”

“…The approximation of particle diameter from light scatter or fluorescence intensity in flow cytometry requires the use of light scatter calibration and fluorescence calibration, respectively [26,32,35]. Certified size standards are available for various sizes and compositions.…”

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

“…In order to engage the audience, a link to the FCM PASS software (https://nanopass.ccr.cancer.gov) was included with the workshop abstract, so that attendees would have an opportunity to familiarize themselves with the software, and also get a chance to test it with their own data. A sample data set including MESF beads (PE QuantiBrite, Becton Dickinson, San Jose, CA), polystyrene and silica NIST‐traceable scatter calibration beads (ThermoFisher Scientific, Waltham, MA), and a fluorescent virus test sample (ViroFlow Technologies, Inc, Canada) was uploaded onto FlowRepository (https://flowrepository.org/id/FR-FCM-Z24E) prior to the workshop for attendees who currently do not conduct small particle research . The presentation demonstrated using the FCM PASS software how to perform fluorescence and light scatter calibration on the sample data set provided on FlowRepository (Figure ).…”