Extracellular vesicles (EVs) are membrane-derived structures that include exosomes, ectosomes, microvesicles, and apoptotic bodies.1 Ranging in size from about 30 nm to 120 nm in diameter, exosomes are released through the exocytosis of multivesicular bodies, while ectosomes originate from the plasma membrane.2
Once released into the extracellular space, EVs enter body fluids. There, they interact with and transfer their molecular cargo to cells, thus influencing both physiological and pathological processes.1 EVs are released by normal, healthy cells, but recent evidence suggests that EVs may serve as mediators in the pathogenesis of neurological, oncologic,3 vascular, hematologic, and autoimmune diseases.4
Given the challenges in diagnosing, monitoring, and understanding diseases, and the potential participation of EVs in these conditions, protocols for isolating and analyzing EVs are essential for the field to progress to the translational stage.5
EV analysis
Reliably quantifying and characterizing EVs is challenging due to the particles’ small size. High-magnification microscopy, such as electron microscopy, is often used for the in-depth study of specific EVs,6 but microscopy is an inherently low-throughput technique. By contrast, flow cytometric EV analysis is a rapid, high-throughput technique suitable for characterizing discrete particles.
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Learn about the origins, functional roles, and translational significance of EVs as well as detection technology that combines the advantages of high throughput flow cytometry with high sensitivity to submicron particles to improve EV analysis.EV analysis using conventional PMT-based flow cytometers has been hampered by the dimensions of typical EVs compared with those of intact cells. A smaller size means a smaller refractive index, a property that factors into particle enumeration. Also, unlike cells that express thousands of copies of surface markers, EVs may express only tens of copies. Even when labeled with strongly fluorescent tags, label concentrations can be below the detection limits of conventional cytometers.7
Combined, these factors raise questions regarding the choice of detection method and the importance of using fluorescent markers. Another problem is validating that single vesicles are detected and not coincident events, which are known as swarms. Swarm detection is a common phenomenon that occurs when many small particles are reported as a single event, causing errors in concentration and intensity measurements.8
High sensitivity flow cytometry
To summarize, the issues with most traditional cytometry platforms for characterizing EVs are the small size of EVs and the low abundance of surface markers expressed on those EVs. To detect these small-sized particles and the low-signal markers, the Amnis® CellStream® Flow Cytometer and the ImageStream®X Mk II Imaging Flow Cytometer were used to analyze EVs.
Amnis® flow cytometers employ Time Delay Integration (TDI) combined with a charge-coupled device (CCD) camera, which offers the advantages of high-throughput flow cytometry as well high-sensitivity detection of submicron particles.
The Amnis® TDI CCD camera technology preserves sensitivity and image quality, even with fast-moving particles, and captures multiple colors of fluorescence, as well as FSC and SSC with superior photonic sensitivity. The effect is similar to physically panning a camera. TDI avoids image streaking despite signal integration times that are orders of magnitude longer than those of conventional flow cytometry.
Flow cytometers in operation
Where conventional PMT-based cytometers have high amplification noise, TDI-CCD detection offers high sensitivity with low background, provided relevant study parameters are properly controlled. Controls that must be included for reliable results are buffer only, antibody/dye only, unlabeled EVs, and labeled EVs plus a detergent. These controls are in addition to the experimentally labeled EVs. Investigators should also run dilution series of their labeled EV samples to determine if swarming has occurred.
A multi-disciplinary research group, including scientists at Luminex and academic collaborators, examined the general suitability of the Amnis® Cellstream® platform and the Amnis® ImageStream®x Mk II9 to analyze small EVs, including exosomes, and the ability of the two systems to resolve populations of smaller EVs in particular.
Using antibody-labeling approaches, investigators showed that imaging flow cytometry was capable of detecting individual small EVs and could identify distinct EV populations. They wrote that the technique “will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of small EVs as useful biomarkers in various diseases.”
Conclusion
EVs have been the subject of intense study in basic research and therapeutic and diagnostic medicine. Conventional approaches to understanding the role of EVs in disease and health, including microscopy and PMT-based flow cytometry, fall short on several fronts. Microscopy is slow, whereas flow methods, designed for particles larger than around 300 nm, miss too many details—particularly smaller EVs or EVs with rare surface markers. By utilizing a CCD-based image-acquisition detector similar to those employed in very high-end optical systems, the Amnis® platforms identify and characterize EVs with high sensitivity and specificity in a high-throughput manner.
References
1. Zaborowski MP, Balaj L, Breakefield XO, Lai CP. “Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study.” BioScience. 2015. doi:10.1093/biosci/biv084
2. Cocucci E, Meldolesi J. “Ectosomes and exosomes: shedding the confusion between extracellular vesicles.” Trends in Cell Biology. 2015. doi:10.1016/j.tcb.2015.01.004
3. Doyle LM, Wang MZ. “Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis.” Cells. 2019. doi:10.3390/cells8070727
4. Pugsley HR, Davidson BR, Morrissey P. “Immunophenotyping extracellular vesicles using the CellStream flow cytometer.” Journal of Immunology. 2019.
5. Yekula A, Muralidharan K, Kang KM, Wang L, Balaj L, Carter BS. “From laboratory to clinic: Translation of extracellular vesicle based cancer biomarkers.” Methods. 2020. doi: 10.1016/j.ymeth.2020.02.003
6. Cizmar P, Yuana Y. “Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy.” Extracellular Vesicles. doi: 10.1007/978-1-4939-7253-1_18
7. Welsh JA, Holloway JA, Wilkinson JS, Englyst NA. “Extracellular Vesicle Flow Cytometry Analysis and Standardization.” Front. Cell Dev. Biol. 2017. doi: 10.3389/fcell.2017.00078
8. Görgens A, Bremer M, Ferrer-Tur R, Murke F, Tertel T, Horn PA, Thalmann S, Welsh JA, Probst C, Guerin C, Boulanger CM, Jones JC, Hanenberg H, Erdbrügger U, Lannigan J, Ricklefs FL, El-Andaloussi S, Giebel B. “Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material.” J Extracell Vesicles. 2019. doi: 10.1080/20013078.2019.1587567
9. Nolan JP, Duggan E. “Analysis of Individual Extracellular Vesicles by Flow Cytometry.” Methods Mol Biol. 2018. doi:10.1007/978-1-4939-7346-0_5
To learn more about the importance of extracellular vesicles, including exosomes, microvesicles, and apoptotic bodies, download our free eBook Characterizing Extracellular Vesicles with Cytometry now.