Flow cytometer optimisation and standardisation for the study of extracellular vesicles as translational biomarkers

• Main Author: Welsh, Joshua
• Format: Dissertation
• Language: English
• Published: University of Southampton 2016

Background: The term ‘extracellular vesicles’ (EVs) encompasses a range of vesicles. These include apoptotic vesicles (1000-300nm), microvesicles (30-1000nm), exosomes (~30-120nm) and retrovirus like vesicles (90-100nm). EVs have been linked to promising diagnostic, and therapeutic potentials. Their characterisation is poorly understood due to the lack of resolution and standardisation in detection equipment currently used. Aims & Methods: In this thesis, I have developed methods for flow cytometer (FCM) resolution quantification, improvement, and standardisation. This involved building, testing and validating FCM optical models for EV analysis standardisation, and optimising FCM settings and protocols to increase resolution and decreasing variation in results. I then tested the benefits of these optimisations on EV analysis, which involved comparing optimised to non-optimised EV analysis protocols utilising clinical samples. Finally, EVs potential as translational biomarkers in non-alcoholic fatty liver disease (NAFLD) was investigated, employing the previously developed protocols in this thesis. Results: FCM optimisations combined with a novel fluorescent assay resulted in a validated modelling technique, that allows diameter of EVs in plasma samples to be approximated using their scatter power, and separation of microvesicles, apoptotic vesicles, and residual platelets. Comparison of EV optimised to non-optimised protocols showed the FCM optimisation protocol to have increased EV absolute count reliability, and lower variation between results, when compared to a non-optimised FCM analysis protocol. Upon applying these methods to a biobank of clinical samples from individuals with NAFLD, novel insights were gained between the association of platelet-, endothelial-, and leukocyte-derived EVs in the progression of the disease. A clinically relevant finding being leukocyte EVs showing potential as a diagnostic marker of liver fibrosis severity.