“The quality of the sample is the quality of the experiment” someone quipped to me recently while describing one of the key difficulties inherent in clinical translation of EV-based diagnostics. What they meant to convey was that, if the sample quality is poor, the analytical result will be compromised irrespective of the quality of the analytical device. This resonated deeply with me because it so perfectly and succinctly sums up one of the biggest problems in extracellular vesicle research today – inadequate sample quality interfering with research outcomes.
Dr. An Hendrix, a cancer researcher and professor at Ghent University, demonstrated this beautifully in a presentation she gave during Education Day at the ISEV Annual Meeting this past April. In her presentation, which was focused on how to prepare EV samples for analysis and the tradeoffs and implications inherent in choosing between methods, she shared results from her research which showed that both RNA and protein composition differ substantially depending on the isolation technique(s) used to prep the samples for analysis. She went further to show that differences in sample preparation affect functionality, as well.
“It’s really important to well reflect on how to design the preparation protocol because it will have an effect on the downstream analysis,” she summarized.
Dr. Jennifer Alexander-Brett is all too familiar with the difficulties in achieving high quality isolated EV samples. A researcher at Washington University, Dr. Alexander-Brett studies the development of chronic airway diseases, including the role played by EVs – where they come from, where they go, and the effect they have on disease development. Her team uses size exclusion chromatography columns for isolation of EVs from conditioned media.
“I feel pretty confident that we've got vesicles that come out that are what we want,” she says during a recent conversation, “but we lose so many of them – 90%. It’s very frustrating. We start with a very large amount of material, painstakingly concentrate that down, and then run a column. We’re going to lose almost all of it, but we’ll have something to work with at the end.”
So how does this affect Dr. Alexander-Brett’s research?
“It impacts how many experiments we can do. Some experiments just aren’t tractable because we can’t get enough material. We’re going to have a finite amount of sample, so we have to plan. ‘These are the three things we’re going to need to do and we can only do these three things and nothing more.’”
Exokeryx has had the pleasure and privilege of collaborating with Dr. Alexander-Brett to explore how our dielectrophoresis-based EV isolation technology compares to her size exclusion chromatography. After analyzing the replicate sample isolates from both methods, Dr. Alexander-Brett observed: “The one thing that was sort of eye opening for me was that you're purifying a really substantial population of EVs that we really can't see that well right now. I think that that's really important because it tells us that there's a whole signal that we're missing.”
And that “missing signal” will necessarily impact research outcomes, because you can’t measure what’s not there.
Dr. Alexander-Brett’s experience reflects a key point of Dr. Hendrix’s presentation – that optimizing EV isolation for purity necessarily results in a loss of subpopulations of EVs:
“If we really want to separate extracellular vesicles from these other types of particles, most commonly we would need…ideally a combination of different methods that exploit complementary biophysical or biochemical characteristics…But you also have to recognize that…the more methods you combine, the more enriched it will be in extracellular vesicles and…depleted in other types of particles…nevertheless, it also means that we will lose part of the subpopulations of extracellular vesicles and that we have a reduction in efficiency.”
The tradeoff between purity and recovery and its implications for research findings is a barrier Exokeryx is driven to tear down. Because, as Dr. Hendrix opined: “This is really…the goldmine for the EV research field – if you could find a method that is highly specific and highly efficient, that would be fantastic.”