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microsampling to investigate cytokines: Dr. Ben Herbert (pt.1 )

Dr. Ben Herbert discusses cytokines

For this episode of the Microsamplify Podcast, Neoteryx Technical Director James Rudge, PhD, spoke with Ben Herbert, PhD, a research scientist with nearly 40 years of experience in protein chemistry, sample preparation, fractionation, and the use of proteomics.

Dr. Herbert also has many years of experience in technology development and commercialization of biotechnology products and has co-founded companies in the biotech & healthcare industries. Currently, he is co-founder and Chief Scientific Officer at Sangui Bio in Sydney, Australia. They are based at the Kolling Institute at Royal North Shore Hospital. Dr. Herbert’s recent research focus is on cytokines.

This is part one of a two-part podcast.

Neoteryx: Hello Dr. Herbert, and welcome to the Microsamplify Podcast from Neoteryx. Thanks for taking the time to speak with us about your work at Sangui Bio.

Dr. Herbert: Thanks James, nice to be with you.

Neoteryx: Chronic inflammation, often studied via cytokines, is a predictor of many conditions, such as diabetes, cardiovascular disease, cancer, and neurological disorders. Your company, Sangui Bio, is a pioneer in the field of inflammatory biomarkers, and your studies have shown that red blood cells have a role to play as carriers of cytokines. Can you discuss the role of cytokines in this regard, and how microsampling has helped you identify biomarkers or risk factors for developing chronic inflammation and certain health conditions?

Dr. Herbert: Sure. Like you say, cytokines are incredibly important and, if you look at the literature, there are hundreds of thousands of papers that talk about cytokines and quantify them in almost every disease that you can think of. The work that led us to the studies we’re doing now was to do with arthritis. [We were] looking at the role of cytokines in people with osteoarthritis—they were being treated with stem cells for osteoarthritis.

My group was interested in blood levels of inflammatory and anti-inflammatory cytokines. Ultimately, we were looking at things that were irregular, depending on how we prepared blood samples, either plasma or serum. And the experiment that we did, way back in 2014, was to freeze whole blood and then centrifuge out the cell debris, and then analyze the cytokines in that. And there were a range of cytokines that turned out to be at much higher levels than we’d expected. It turned out that red blood cells were the driver of that. The student working on that, Dr. Elisabeth Karsten, is still working for us, and she ended up being one of the co-founders of Sangui Bio.

So, what she found when she went to the flow cytometer and purified different cell populations was that red blood cells had much higher levels of cytokines and chemokines that we typically look at, particularly in the blood of the people that we were looking at. At that stage, we were looking at our own blood because it was the easiest thing to get. We looked at a pretty wide range of cytokines, and we’ve published a few papers now that show that in many people — not every single person — there can be between 3 or 4 times and up to 1,000 times higher levels of some cytokines and chemokines associated with the red blood cell component. That, then, becomes really important if you want to think about those in relation to disease.

For a number of years after that, the work we did was really focused on fractionating blood simply and quickly, and looking at the red blood cell component and, sometimes, the platelets and white cells and the plasma. That’s interesting, and you get quite deep and detailed data doing that. However, it’s very difficult to scale that looking into hundreds or thousands of people. And that is what really led us to look at microsampling, because our idea was that microsampling, of course, is whole blood, particularly if you use a dried blood spot. Ultimately, that led us to Neoteryx and using Mitra devices. We’ve been working with Mitra, exclusively really, for a few years.

Our theory was, and still is, that if you can get liquid blood from people occasionally when they come into a clinic, you can then get dried blood spot from them when they’re at home. The combination of the liquid blood and the dried blood spot gives you the best of all worlds. You’ve got longitudinal sampling [with dried microsamples] where you’re seeing everything, and you’ve got periodic liquid samples where you can investigate the blood more deeply to get a better understanding of what happens and why are you seeing the answers that you’re getting from the dried blood spot, because often, they’re different.

Neoteryx: And it allows you to do that fractionation of the cells against the plasma, but equally illustrates from your previous work how important measuring the whole blood sample is, because of these concentrations of cytokines and other molecules that would normally be centrifuged away in favor of measuring just plasma levels.

Dr. Herbert: That’s right. And, of course, none of these things are static. So, there’s not really a situation that we can see where the level that is in the plasma, the white cells, and platelets and the red cells is static and will just happily sit there while you process the sample. So, time is important in sample processing to make sure that you’re getting reproducible, sensible, and biologically relevant answers.

But then, inside the body there is this dynamic exchange, and that’s been well documented in studies looking at other cell types. That’s what cytokines are, they’re proteins involved in cell to cell signaling, so of course that’s dynamic and we should expect that will change over time. That’s why microsampling and longitudinal sampling is so relevant and important because it enables us to do that in a way that you just simply can’t do by bringing people into a clinic.

Neoteryx: You’re making the individual become their own control in a sense, aren’t you, by doing that? So, you’ve got that longitudinal measurement and then, obviously, the fractionation as well. This kind of brings us to our next question, actually, which is…

I believe you provided microsamples and developed protein profiles that included cytokines as a way to identify biomarkers for obesity and hereditary risk factors of disease. Could you expand a little bit more on this work?

Dr. Herbert: We had a partner we were working with in 2018 and 2019, and their interest was really the role of chronic inflammation in the development of the kinds of lifestyle diseases that you see now—notwithstanding that there’s a global pandemic, but in normal times what you’ve seen — in the developed world over the last 50 years. [There has been] a real acceleration towards people dying of lifestyle diseases and the chronic effect that has on their life, the health systems, productivity, and all those things. Chronic inflammation, or elevated inflammation over a long period plays a role in the development of those diseases, like cardiovascular disease, kidney failure, and diabetes.

The partner we were working with wanted to look at: how would you measure these things in people using microsampling, and would the answers that you got be relevant and similar to what had been seen in other studies where people had clearly shown that chronic levels of inflammation were associated with these sorts of diseases?

This study was completely done remotely. The people were all healthy, so the initial stages of this [study] were done in healthy people, but we accepted any weight range from healthy up to obese. As long as they said that they were not heavily reliant on medical care, they could join the study. The recruitment was all done over social media. We never saw these people [in person]. They were sent the instructions and the kits through the post, and they posted them back.

The success rates that we had in terms of [correctly] filling the device tips was great. We were at about 85% success with people filling at least one Mitra device [tip] completely, and we were using 30 µL Mitra devices. At that start, we sampled people more frequently—perhaps half a dozen times in the first 2 ½ weeks—to try and get a view of how they were varying and how we could get a baseline. Then we went to monthly sampling after that. We genotyped everybody from dried blood spots from Mitra as well, and we had 100% success rate with extracting enough DNA from a Mitra tip to get genotyping done. That was done using a Thermo snip array, which was one of the ones based on the UK biobank studies. That was done at the Ramashanti Center, which is here at the University of New South Wales in Sydney.

We didn’t have enough people in the study to do a genome-wide association. What we really wanted to prove was that “Yes, in our hands this would work—you could get sufficient DNA of good quality to do genotyping.” That was clearly the case. And since then, there have been other groups that we’ve interacted with who are very interested in being able to genotype from Mitra [devices], so that was useful to prove that it really worked. And that was all done with just off-the-shelf kits.

The reason we were interested in genotyping was because an Italian group had published some genome-wide association studies looking at inflammation in quite large populations of people from Sardinia (5,000-6,000 people at a time). They’d shown that there are a range of genes associated with chronic inflammation. We were of the view that if you were doing large studies, you should be looking at that to enable you to stratify people in that way to understand the genetic predispositions and then look at lifestyle factors and longitudinal blood sampling.

The results we found were broadly what we had expected to see. There were correlations between obesity and chronic inflammatory protein levels. Within that, of course, there are always subgroups of people. Broadly speaking, this study was quite successful. We found what we expected to see, that there were correlations. We showed that Mitra was a really effective way of getting samples from people. [Mitra] was broadly stable, so you could collect and receive samples, store them in the lab until you’re ready to go, and it worked.

What this led us to is a range of other studies that are still ongoing, looking at: stability of particular analytes, [whether] there are good or bad ways of storing or shipping samples, and which individual analytes vary the most—so that we have got a good understanding of all that.

That was probably our first large-scale study that really confirmed to us that this worked and that Mitra was a good way to go.

Neoteryx: Yeah, it’s incredible, when you think of it, given current technology that you were able to do proteo-genomic measurements from tiny little volumetrically collected blood samples. You know, when you think back to what things were like just 10 or 20 years ago, it’s amazing how technology has allowed us to move forward. You mentioned also that we’re in a global pandemic, and … we understand that cytokines are small proteins that are important in cell signaling and that one thing they do is mediate communication among immune and non-immune cells to help the body’s immune and inflammatory responses. What is the significance of this in COVID-19?

Dr. Herbert: It’s interesting [with] COVID-19 — you mentioned the non-immune cells — COVID-19 is a respiratory problem, and it’s really affecting people’s respiratory systems and their lungs. When you look in the literature, the two cell types that come up most often are the endothelial and epithelial cells of the respiratory system and the lungs. Those are the cells that are non-immune, but they are releasing cytokines. It seems to be that what’s occurring in COVID-19 is a dysregulation of cytokine signaling, and macrophages appear to be pretty central to this. You’re getting a shift in the polarization of macrophages from inflammatory to anti-inflammatory, or the other way round. So, when you think of non-immune cells, that’s what’s in the literature.

Of course, our interest here is what role might red blood cells play in this. With red blood cells, their primary job is [to be] carriers of carbon dioxide and oxygen. Where does that happen? In the lungs. What are they interacting with? They’re interacting with the endothelial and epithelial cells in your lungs. We think there is likely to be a role for red blood cells and their activities in terms of being carriers of cytokines.

We’ve been involved in a project getting COVID-19 blood samples, and we’ve processed a bunch of samples, which are [stored] in the freezers here at the hospital. One of the great things about what has happened in Australia is that there aren’t too many people who have got COVID-19 and had very severe [cases] and not many people have died of COVID-19. And so that’s meant that getting access to large numbers of people with severe disease hasn’t been that easy.

But what’s turned out to happen is that this business of “long-COVID” that people are suffering from seems to be less related to how acute and severe their disease was in the initial phases. People with relatively mild initial COVID-19 are still having long-term effects, and I don’t know that that’s particularly well understood. We’re waiting now for the clinicians we’re working with to sort out which samples we need to go ahead and look at. I think, ultimately, whilst Mitra hasn’t played a huge role in this so far, I suspect that it will once we start to understand what kinds of questions need to be asked. There will be an expanded role for microsampling in trying to understand all this.

There is a new study here that is looking at the effects of COVID-19 vaccines on people, particularly ones who already have a co-morbidity, like cancer. What happens when they get vaccinated? And we’ve got some involvement in that, as well.

This discussion is continued in Part 2 of the Microsamplify Podcast, linked here.

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