Technical Note
A standardised protocol for the evaluation of small extracellular vesicles in plasma by imaging flow cytometry

https://doi.org/10.1016/j.jim.2019.03.006Get rights and content

Highlights

  • Imaging flow cytometry provides a sensitivity platform for quantification of small extracellular vesicles (sEVs)

  • Challenges surrounding detection of single sEV particles hinder downstream analysis

  • Utilisation of advanced imaging flow cytometry features permits accurate single particle characterisation.

Abstract

Flow cytometry provides robust, multi-parametric and quantitative information on single cells which also exhibits enormous potential as a tool for small particle characterisation. Small extracellular vesicle (sEV) detection by flow cytometry remains compromised due to the high prevalence of swarm detection, which is defined by the simultaneous illumination of more than one sEV, recorded as a single event. Detection of sEVs by imaging flow cytometry presents a major advantage by having the ability to resolve single particles from swarm detection based on the image features recorded for each event. In this study, we provide a simplified protocol that facilitates the removal of both swarm events and aggregated particles to improve the accuracy of sEV analysis. Our results indicate that imaging flow cytometry should be at the forefront as a robust and sensitive technique for sEV characterisation.

Introduction

Small extracellular vesicles (sEVs) vary from 30 to 200 nm in size, and are enriched in a variety of bioactive molecules and surface markers including CD9, CD63, CD81 and tumour suppressor gene (TSG)-101 (Raposo and Stoorvogel, 2013). SEVs arise from several sources during health and disease and may serve as biomarkers of pathological processes as well as providing options for delivery of therapeutic cargoes to sites of injury (Inamdar et al., 2017). However, despite significant research there are major challenges in standardising methods to quantify and characterise sEVs.

Single sEV flow cytometry has the potential to provide rapid quantitative data on sEVs based on their surface expression, however there are still unresolved limitations surrounding the sensitivity of the technique due to the low refractive index of sEVs (Gardiner et al., 2014). Furthermore, the accuracy of flow-based characterisation becomes compromised due to the prevalence of coincident sEV (“swarm”) detection, which describes the phenomenon where more than one particle is detected at the same time, processing it as a single event (Van Der Pol et al., 2012).

The Amnis ImageStream®X Mark II imaging flow cytometer (ISXmkII) combines the use of conventional flow cytometry with fluorescent microscopy. The ISXmkII possesses a host of components which improves small particle detection, including stronger excitation lasers (100–200 mW) charged couple device detectors, time delay integration, and ultra-precise fluidics. For these reasons, the use of the ISXmkII has become a well described method for flow-based sEV quantification (Erdbrügger et al., 2014; Headland et al., 2014; Lannigan and Erdbruegger, 2017; Mastoridis et al., 2018). Despite the advances of sEV flow cytometry, challenges still remain. At present, gating strategies cannot discriminate single particles versus. Multiple particles based on conventional fluorescent or scatter parameters. The most unique feature of the ISXmkII, is its ability to capture high resolution images of every recorded event which provides positional information of the fluorescence within the image. Such images possess the potential to discriminate swarm from single particles (i.e. multiple fluorescent positions versus. a single fluorescent position within the image). The use of this advantage remains underutilised for EV work.

One of the biggest limitations surrounding the use of flow cytometry for EV analysis is a lack of standardised protocols that can be easily replicated. This study provides a simplified protocol that can be utilised to rapidly quantitate single sEV particles from either clarified (filtered, platelet-poor) plasma or sEVs isolated and enriched by size-exclusion chromatography while accurately quantifying EVS through the exclusion of swarm detection. This would be an important methodological advancement in understanding sEV physiology and pathology.

Section snippets

Isolation of plasma-derived sEVs

Lithium heparin plasma was first clarified by serial low speed centrifugation (2000 xg; 10 mins and 10,000 xg; 30 mins) followed by 0.22 μm centrifugal filtration (14,000 xg; 2 mins) at room temperature (RT). Clarified plasma (500 μL) was carefully applied to a qEV column (Izon Sciences, Christchurch, NZ) which is a size-exclusion chromatography column. Elution buffer (sterile, 0.22 μm-filtered phosphate-buffered saline; PBS; Sigma-Aldrich, Castle Hill, NSW, Australia) is sequentially added

SEV validation and characterisation by imaging flow cytometry

Plasma-derived sEVs were purified by qEV and quantitated by NTA (modal particle diameter of ~80 nm). A representative image displays the visualisation of particles during sample acquisition (Fig. 2a). Visual confirmation was perform by TEM showing the presence of nanoparticles around 100 nm in qEV preparations (Fig. 2b). Western blots also confirm the presence of a CD9+CD81+Calnexin- sEV fraction (Fig. 2c). To remove Speedbeads from the analysis, which are used to calibrate the instrument, sEV

Discussion

The ISXmkII has been proposed as the preferred platform for performing sensitive measurements by flow cytometry due to its specialised features. Thus, it has been implicated as a useful diagnostic and prognostic tool, particularly in a cancer context (Doan et al., 2018). The ISXmkII has been used in a number of applications related to EVs, which includes the study of interactions between EVs and target cells (Ofir-Birin et al., 2018; Ohno et al., 2013), the subsetting of EVs in plasma based on

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

All authors have no conflict of interest to declare.

Acknowledgements

The authors thank all healthy donors used in this study. The authors also acknowledge the facilities, and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, University of Western Australia, a facility funded by the University, State and Commonwealth Governments. This study was supported by funding a National Health and Medical Research Council grant. The authors also thank Dr. Katie

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