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Abstract
ABSTRACT Accurate characterization of extracellular vesicles (EVs) is critical to explore their diagnostic and therapeutic applications. As the EV research field has developed, so too have the techniques used to characterize them. The development of reference materials are required for the standardization of these techniques. This work, initiated from the ISEV 2017 Biomarker Workshop in Birmingham, UK, and with further discussion during the ISEV 2019 Standardization Workshop in Ghent, Belgium, sets out to elucidate which reference materials are required and which are currently available to standardize commonly used analysis platforms for characterizing EV refractive index, epitope abundance, size and concentration. Due to their predominant use among EV researchers, a particular focus is placed on the optical methods nanoparticle tracking analysis and flow cytometry.
Highlights
Background to reference materialsAt present, many investigators in the extracellular vesicles (EVs) field use the term “reference material” to refer to any material that assists with evaluation of reproducibility of a measurement
The development of standard EV reference materials that can be used across optical techniques for validation of size distributions using light scatter requires knowledge of refrac tive index, which is not fixed across EV diameter and is not currently a traceable measurement using available methods
The characterization of reliable EV reference materials requires the calibration of measurements obtained from any EV analysis technique
Summary
“Extracellular vesicles” is an umbrella term for lipid bilayerdelimited particles derived from cells through a number of pathways; these include exosomes and ectosomes [1]. In the above example with NTA, the limit of detection could be reported in terms of the number of photons needed to resolve a signal from background, or as the diameter of a particle with a given refractive index It is one of the few single-particle measurement techniques that does not have a definable sensitiv ity limit with regard to light scatter intensity or fluor escence intensity in standard units. Only single-EV flow cytometry combines abilities for sizing, concentration measurement, affinity-based phenotyp ing and high throughput with calibration into standard units to provide a limit of sensitivity for each para meter [26–29].
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