The Answer- Extracellular Vesicles (EVs)
The study of EVs pushes the limits of biological detection and for much of the history of their study it was difficult to compare EV research. In 2014 the Board of Directors of the International Society of Extracellular Vesicles published MISEV2014 (the Minimal Information for Studies of Extracellular Vesicles) to give EV researchers a set of standards to follow. Previously in 2008 the International Society for the Advancement of Cytometry adopted MIFlowCyt (Minimum Information about a Flow Cytometry Experiment) as its minimum data standards. Specifically for EV Researchers using flow cytometry, those frameworks have been merged to create MIFlowCyt-EV. It lays out the framework for Flow EV Researchers (aka Microflowers, aka Nanoflowers, aka Signal to Noise Champions, aka Threshold Jockies or TJ’s) to create thoroughly controlled and reproducible data. The guidelines are comprehensive and address every step of research from preanalytical variables (example, what gauge needle was used if using blood as a sample source) to flow cytometry data reporting and sharing (example, converting fluorescence to molecules of equivalent soluble fluorochrome). But what exactly are EVs?
What are EVs?
Extracellular vesicles (EVs) have been called many things. Ectosomes, exosomes, microvesicles, microparticles, oncosomes, apoptotic bodies, shedding vesicles, membrane fragments, plasma membrane vesicles, inclusion vesicles, and protasomes are among them. Although they have gone by many names, they share a few commonalities. They are generally small – they are thought to have a size range of 30nm to 10 microns with the vast majority being smaller than 200nm. They are bound by a bilayer lipid membrane. They are released from cells, cannot replicate, and are anucleoid.
What do EVs Do?
The diversity of nomenclature stems from a diversity of function and genesis. EVs are thought to play roles in many cellular activities, both in natural and disease states. They’re often thought of as cargo carriers, transporting proteins, nucleic acids, lipids, metabolites, and organelles from their origination cell. Functionally they can play roles in management of cellular waste, transfer of functional proteins, molecular recycling or nutrition, cell signaling, creation of the metastatic niche for cancer, pathfinding, quorum sensing, and mediating host commensal or parasite pathogen interaction. It is believed almost all cells release EVs and EVs as a cellular function have been shown across taxa, including bacteria, plants, fungi, and animals.
How are EVs isolated?
There are a number of ways of enriching EVs (sometimes known as isolating, sometimes known as purifying). Popular methodologies include differential ultra-centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, and immunoaffinity capture. Each methodology carries with it its own considerations, such as ratio of true EVs to co-isolates, percentage of EVs obtained out of the original source population, contaminant factors, and pre-analytic variables that could potentially influence EV isolate. Tradeoffs must be considered when designing a study of EVs. Ultracentrifugation will result in a high percentage of EVs retained from the source material but will contain co-isolates. Size exclusion chromatography will contain fewer co-isolates but may limit the size range of the original EV population. Immunoaffinity beads provide a convenient and easy way to do bulk EV analysis without the use of high-speed centrifugation or specialized flow cytometers however this sort of analysis may obscure dynamics of heterogenous EV populations.
How do we characterize EVs?
Typically, we seek to measure EVs by size, cargo, and concentration. Conventional bulk analyses (ELISAs, Western Blot, qPCR, Mass Spec) are important tools in the EV researcher tool kit but report average values. These averages may obscure important information that only single particle analysis can elucidate. Electron microscopy provides the highest resolution and can phenotype individual vesicles however it is low throughput, time consuming, labor intensive and is not readily available for many EV researchers. Resistive Pulse Spectroscopy and Nanoparticle Tracking Analysis estimate particle size and are capable of estimating EV sized particles but are not specific, cannot measure EV cargo, and are relatively low throughput.
So… How does FlowMetric characterize them?
The answer is always flow cytometry but, in this case, it is flow cytometry with careful experimental design, more controls, and more beads! Remember, EV researchers typically seek to measure EVs by size, cargo, and concentration. Flow cytometry is capable of single particle phenotyping (cargo – check), quantitative with the right protocols (concentration and size – check and check), and it is high throughput and relatively ubiquitous in academic, clinical, and pharma labs.
Good Vesiculometry = More Controls, More Beads and More Experimental Design
Good flow vesiculometry means appropriate experimental design which means volumetric measurement and appropriate threshold triggering. There are three components to making your cytometry volumetric: 1) document concentration / dilution of your sample from start to finish, 2) run your samples at a consistent flow rate and 3) characterize that flow rate in µL/s. Taken all together this will allow you to report EVs as number of EVs per starting volume of sample. For thresholding, traditional forward scatter will not do the job. With a high-powered blue laser vesicles can be triggered well on side scatter although the events that trigger will not be specific to EVs. The gold standard is to use a lipophilic dye and trigger on fluorescence.
Assay controls for flow EVs are different than your typical flow experiment. I’m going to warp a few cytometrist brains right now – in the context of EVs, we don’t wash. Non-specific staining of EVs is negligible. With that, buffer only and buffer with reagents controls are necessary to make sure the buffer and reagents are free of EVs or anything that could look like an EV (aka antibody aggregates). Detergent treatment is necessary to determine vesicle lability or the question of – did my vesicle pop? The idea being that EVs should be susceptible to detergent lysing and background material should not. So, in the question of “Did it pop?” if it did, congratulations you had a vesicle and sincere regards to the happy parents! If it did not, then you have something else and you should subtract that something else from that which popped to determine an accurate accounting of EVs in your sample. Furthermore, you must control for coincidence. Doublets of cells are easy to gate out in normal flow cytometry, however doublets or swarms of EVs not so much. Swarm is the occurrence of multiple EVs passing through the point of interrogation simultaneously. Because EVs reside at the lower limit of scatter detection a typical doublet discrimination gate is not suitable and the only way to control for swarm (aka coincidence) is dilution. A serial dilution of stained EVs should show no decrease in fluorescence intensity, if it does then you have swarm or coincident EV events. Procedural controls are a way of controlling against artifacts that may have arisen during processing. As an example, say you want to use ExoFuge brand EV isolation kit. An appropriate procedural control would be to run EV free buffer through your ExoFuge kit to determine if any EV-sized artifacts were generated by the kit itself. In addition to these controls, are many of the standard controls that flow cytometry users will be familiar with, including but not limited to: unstained, isotype, and single stained controls.
Qualifying a flow cytometer for use with EVs takes more than your standard CS&T. To calibrate fluorescence intensity, a combination of Rainbow and MESF beads can be used to provide NIST standardized measurements of fluorescence of every detector and calibrate across cytometers. Size bead calibrators or lipoparticles can be used for estimating EV size but not without understanding the nuance of light scatter, importance of refractive indices, and mie theory. Utilizing mie theory to estimate EV size is the only valid way of estimating EV size via flow cytometry.
Extracellular vesicles are a thrilling field of study filled with opportunities to push the limits of science and advance medicine. It provides an opportunity for new diagnostics and new avenues of treatment. The difficulty in studying these particles is also the driver for innovation that crosses disciplines, continuing a tradition in flow cytometry as a place where engineers, biologists, chemists, mathematicians, statisticians, physicists, and clinicians intersect. The potential of EV research at present has been likened to that of T cell and B cell research in the 1960s and 1970s. In recent years there has been a significant increase of EV publications. In one study, nearly 5500 English language EV articles were published between 2000 and 2016, with over 20% of those articles publishing in 2016 alone. Comparing scientific literature as a whole, EV literature has expanded by 81% each year, whereas general scientific literature expands by about 8-9% year over year. In other words, year over year EVs are carving out a larger portion of all scientific publications. In 2016 the global market for EV diagnostics and therapeutics was estimated to be about $16 million and projected to be about $112 million by 2021. EV research and the steps outlined in the MIFlowCyt-EV may seem daunting, but EV research is necessary to the progress of science and MIFlowCyt-EV is necessary to create robust and reproducible data for this burgeoning field and as a wise president of ISAC once said to me (and many others), “It’s not easy… but nothing in science worth doing is!”
Authored by: Michael McGrane
Michael McGrane's career in science started in the foggy redwoods of Northern California's Humboldt State University where he graduated with a Bachelor's in Zoology in 2010. He began studying EVs and Flow Cytometry working as a Research Technician at University at Buffalo in 2014. In 2017 he became an ASCP certified Specialist in Cytometry while working with CytoVas in Philadelphia. He joined FlowMetric in 2018 where he continues his career in Flow Cytometry research today. In his free time, he enjoys watching television, eating food, watching television while eating food, and watching television about food while eating food. Turn offs include yardwork and poor experimental design.