For this Microsamplify Podcast episode "Part 2," Neoteryx Technical Director James Rudge, PhD, interviews 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 is co-founder and Chief Scientific Officer at Sangui Bio based at the Kolling Institute at Royal North Shore Hospital in Sydney, Australia. Dr. Herbert’s recent research focus is on cytokines, and in this second part of his conversation with Dr. Rudge, he discusses the impact of cytokines on immune response in COVID-19, as well as sample preparation for microsampling studies.
This conversation continues from part one of the podcast. Neoteryx: Could you talk a little bit about the effect of the immune response that causes the ramp up towards severe COVID-19? Dr. Herbert: The cytokine that’s most discussed in this seems to be interleukin 6. One of the things about it is that there are higher levels of IL6 in the plasma [samples] of people with severe COVID. So, people with severe COVID, their levels of IL6 are high, but [what’s interesting is] the people that end up having what is called a “cytokine storm.” Their levels of IL6 are high, but not massively different to the [levels of] people who have severe COVID, but not cytokine storm events. My guess here is that there are things [happening] that are related to underlying co-morbidities that are not particularly well understood. In preparation for this [discussion], I’ve looked at some of the papers on this topic again, and they talk about things like dysregulation of the cytokine and immune response, but then they go on to say that it’s not incredibly well understood what’s going on. If you go back and look at the study that we did in 2019 in so-called healthy people, and even before then when we were looking at smaller groups of people, what we found was that you could barely even detect things like interleukin 6 in the blood of healthy people. And when you did detect it, it was in relation to either an acute or a chronic event. So, in people who were obese, what you found was that there were detectable levels of IL6. If you then went and looked at [cytokines] like the adipokines, such as leptin and adiponectin, you found those at quite different levels in those people. There have been multiple publications showing that people who are overweight and, for example, have other conditions like diabetes, are at higher risk for COVID. My assumption here, and I think the literature bears this out, is that those kinds of co-morbidities lend themselves to having a more severe kind of COVID problem. I think that’s where things like longitudinal microsampling and understanding what someone’s baseline really looks like will enable you to understand [or predict] who is more likely to have a severe COVID problem. The difficulty of doing that kind of study now is that we are still in the middle of the pandemic and finding naïve groups of people who haven’t been exposed to COVID-19 or who haven’t been vaccinated is quite difficult. I think as we come out of the pandemic, these sorts of studies where the lessons that have been learned from COVID will then be applied to how we prepare for the next pandemic. There is an awful lot of activity happening now, which has been hugely successful in terms of speed and effectiveness to address COVID, but I think there will be a lot more lessons that come out of it. [For example], what should we be doing to prepare for the next one to try and understand how to identify people who are at higher risk for the next variant of COVID or the next distinct pandemic.
Neoteryx: So, what you’re saying is that because certain individuals have their immune systems primed almost with an inflammatory response, that’s going to lead to a more severe infection. I hadn’t thought about it like that, but it makes a lot of sense. I think it makes the research that you did prior to the pandemic all the more important, because it helps us understand what those risk factors are. Dr. Herbert: I think it’s the way you see the world, and I guess the way I see it, is it’s a network. Everything inside your body is a network, even within each individual cell, there is a network of interactions going on. What that means is that there is no simple and useful way to partition things and say, “Here is a person with disease X, like diabetes, but that’s not relevant to their lung infection issues with COVID.” That’s clearly not the case. That networking and everything being connected together means that there clearly is going to be an effect of having diabetes, say, on what happens in your lungs when you get an infection like COVID.
You need more data points to understand what those data mean and, of course, that leads you to microsampling, because the way to get enough data points, from enough people, frequently enough, is to use microsampling. I guess it’s what you’ve seen, and certainly it’s what we’ve seen—there has been a huge acceleration in the number of groups interested in microsampling that’s been driven greatly by the pandemic. Groups are saying, “We really want to understand this, and this is the way to do it.”
Neoteryx: Absolutely. As you mentioned, there are groups collecting samples and bio-banking them so they can do something with them later on, once they’ve figured out what it is they want to do with those samples, which is quite remarkable! And, talking about projects…we know that you have expertise in sample preparation and sample extraction. Could you describe a research project in which you have used Mitra® devices for microsampling and discuss the pros and cons you encountered with preparing and extracting from these microsamples vs. traditional whole blood samples or serum samples…and, I suppose, what lessons can be learned for future projects?
Dr. Herbert: It’s of course everything in the world of immunoassays. That’s what we tend to use, single-plex assays like ELISAs or multi-plex immunoassays. They’re essentially set up to work in a matrix of serum or plasma, or things like cell culture, supernatant, and that kind of thing. When you go and look at the instructions on almost every immunoassay that you get, it says pretty clearly that if the blood sample you’re working with is hemolyzed, which means you’ve damaged red blood cells, then you shouldn’t analyze that sample because you’ll get the wrong answer. Of course, what we do from liquid blood samples is deliberately analyze red blood cells. So, that produces its own challenges in terms of making sure that matrix works for your immunoassay. Then, when you go to a dried blood spot, you’ve got whole blood. And what you’re extracting from Mitra is massive concentrations of hemoglobin and albumin, and all of the abundant proteins. There’s no real way of getting rid of them. And, what compounds, that is, what you’re trying to do is analyze proteins like interleukin 1b and interleukin 6, which are present in the low picogram per mil concentrations. You need to extract in quite small volumes in order to be able to even see them in your assay. So, we tend to extract [from] Mitra in quite small volumes — well under 100 microliters. We’re compounding every matrix effect that you can have. The lab team that we’ve got at Sangui Bio has done a great job and spent a lot of time making sure that the answers we get are real and they’re not just matrix-effect answers. That’s a critical aspect of this. There is no value for us in extracting a sample in 1 mil, because if we a 20-30 mL Mitra in 1 mil you are well outside the concentration to be able to detect any of these low-abundance proteins. Those things have been challenges. To then scale it up, we’ve built devices that have enabled us to do that kind of extraction in a 96-well plate format, and Mitra devices are great because they’ve been built to work in a 96-well plate format, so that has made that a whole lot easier for us as well. That whole area of extracting and making sure that you understand the matrix effect has been a big part of what we’ve done. And then comparing which analytes you see the same answer for in, say, serum or plasma from liquid blood—do you see that answer from dried blood? Looking at the markers everyone wants to understand, like C-reactive protein (CRP). We don’t really see that associated with things like red cells, so broadly, there’s the same level of CRP in a liquid blood sample as there is in a dried blood sample, but you still need to take account of things like the hematocrit of the blood to be able to calculate a ratio and get the real answer of CRP. So, things like that are important. That’s why having a liquid blood sample occasionally from people so you can actually get a handle on what their hematocrit is, is useful. Neoteryx: Like you said, what complicates matters further is the fact that the concentrations of these molecules can differ in red blood cells to plasma. So, when you’re dealing with a molecule that lives primarily in the plasma, for example, an immunoglobulin, then measuring the hemoglobin to get to a hematocrit value can allow you to approximate serum levels pretty well. But when you’re dealing with molecules that also can live in the hematocrit as well, it makes life all the more complicated, doesn’t it? Dr. Herbert: Yes. That’s why most of the projects we do now, we call a “hybrid project,” but all we really mean by that is that when we have patients that go to their doctor or come to the hospital, they have a venous blood sample taken so you get a conventional blood sample that you can do all of the “normal” things with. You can send it to a pathology lab, if you want to, to get the gold standard of things like CRP or liver enzymes, or whatever it is that you want to look at. Plus, you can do your own research panels on that liquid blood and that gives you a baseline and a model of what that blood looks like to compare it to the Mitra samples when you get them. That gives you a much better view of what you’re getting out of Mitra. Because, of course, there’s been decades of understanding what a liquid blood sample really looks like in terms of analyzing serum or plasma. Every clinician and every researcher you talk to, when they talk about a blood sample, what they really mean is a serum or plasma level of whatever marker they’re talking about. So, when you present them with data from dried blood spots, those data are quite often different. You’ve got to have a sensible explanation for why that’s the case, so that you don’t get met with, “Well, that’s not the right answer and, therefore, we’re not going to use it.” Neoteryx: You mentioned about your projects, so could you expand a little bit, if you can, about your new projects, and if you’re moving forward with microsampling? Dr. Herbert: The ones that are running at the moment for us — there are some in the planning stage — but we’ve got projects running now mostly looking at cancer. We’ve got a lung cancer study running at the hospital where people are coming in at time zero, and then at 3 months and at 6 months. They have a venous blood sample taken then. They also get a Mitra sample taken at each one of those points, so you have an exact comparison at that point of a finger-prick Mitra sample and a venous blood sample on the same day at the same time. The other advantage of that is that they are then being shown how to take a Mitra sample. The hope from that is that we get a better success rates of filling Mitra [device tips at home]. They are then sent home with Mitra devices and lancets and everything in the Mitra® Collection Kits that we got from you guys at Neoteryx. They sample themselves then at home and send those [samples] back to us through the post. That study is still recruiting and we’re tracking those people. What that’s led on to is then looking at specific sub-groups of lung cancer and specific therapeutics that are being applied. We’ve got some new studies that are about to start looking at very specific drugs in lung cancer. For us it’s been really a great journey with microsampling. The ability to demonstrate that it works, and show those data, has really widened out the group of researchers and clinicians that have started to want to work with us. That’s what you want, right? You’re doing this to try and expand and to get this reach into more projects and more diseases and more sample types. There’s another project that we’re just in the early planning of, which is looking at people with musculoskeletal trauma — whether that’s been from an accident, for example, or they have a severe joint problem, or whether they’ve got chronic back pain. There’s lots of issues with pain relief in those people. The guy we’re dealing with [on this project] has come [to us] from North America. This project is running over there, but it doesn’t have a blood sampling component, and he’s looking to get that going here in Australia. He’s looking at a whole range of things in people with chronic pain who’ve had musculoskeletal trauma. He’s excited to add in a microsampling component so that we can track those people’s blood over time, and see what kind of effect there is, depending on what kind of drugs and treatment those people might be on. So, these are the kinds of things that are coming along for us at the hospital.
Neoteryx: Thank you, Dr. Herbert, for speaking with us about how you apply microsampling in your work. We wish you great success with your ongoing cytokine studies and the other projects you’ve just mentioned! And, thanks to our audience for listening to this episode of the Microsamplify Podcast, a partner to The Microsampling Blog from Neoteryx. Thank you! Dr. Herbert: Thank you!