Abstract

The characterization of clinically derived exosomes and microvesicles is becoming increasingly important. The techniques of Nanoparticle Tracking Analysis (NTA) and Fluorescence NTA (FNTA) have been demonstrated as vital to the measurement of high resolution size distributions which allows sub-populations of vesicles to be easily identified. The technique can measure the concentration of vesicles within each size class with numerous papers showing a link between concentration of vesicles and disease. The technique provides a real alternative to commonly used flow cytometric technologies which are limited by their lower limit of detection.

Introduction

Exosomes and microvesicles are small vesicles shed by cells, which play an integral role in intercellular communication. Whilst broadly accepted definitions perhaps require refinement, an exosome can be broadly defined as a 40-100 nm diameter membrane vesicle of endocytic origin released by most cells upon fusion of the multivesicular bodies with the plasma membrane – presumably as a vehicle for intercellular communication. Microvesicles are generally classed as 100nm – 1 μm vesicles which directly bud from the plasma membrane. It is their role as potential biomarkers which makes this field of research so exciting. Taylor et al. describe how the presence of tissue/cell type-specific marker proteins associated with a specific protein can be used to determine the originating cell. In lay terms, if one can understand the message being transported by these vesicles and understand where that message is originating from, one can potentially develop a diagnostic test which can detect and perhaps predict the onset of cardiac disease for example. Furthermore, it is their ubiquitous presence in a broad range of biological and physiological processes as well as their elevated levels in blood associated with many diseases such as cancer, cardiac disease and pre-eclampsia which opens avenues for the development of exosome/microvesicle based diagnostics across a broad range of conditions.

It is becoming increasingly apparent that the techniques traditionally used to both isolate and characterise biological materials are either unsuitable or have not been developed and refined for work with exosomes and microvesicles. As a result, the rate of development and discovery is hampered in what is potentially a broadly significant area of research. Van der Pol et al. provide a nice summary of the techniques currently available for the study of exosomes and microvesicles. Furthermore, Dragovic et al. complement the major conclusions of the Van der Pol review and suggest that whilst electron microscopy can demonstrate the presence of microvesicles and exosomes, it is not quantitative and requires extensive and often intrusive sample preparation. They go on discuss how ELISAs have issues with the inability to capture all of the particles present, as well as being influenced by soluble antigens. Flow cytometers would be deemed as perhaps the ‘go-to’ instruments of choice but particles smaller than 300nm cannot be detected by this technique (lower limits of detection are open to some argument and newer instruments continually push the lower limit of detection). Both of these groups discuss the potential of Nanoparticle Tracking Analysis (NTA), and perhaps more importantly, Fluorescent Nanoparticle Tracking Analysis (FNTA) as a potentially well suited technology for the measurement of these particles.

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