Podcast: microsampling in toxicology, biomarkers of drugs and alcohol

For this episode of the Microsamplify Podcast, we spoke with toxicologist Christophe Stove, PharmD, PhD, a researcher at Ghent University in Belgium. In addition to conducting research, Professor Dr. Stove also teaches courses at the University, and collaborates in public service projects, focused primarily on forensic toxicology.

Dr. Stove and the research team at Ghent University's Laboratory of Toxicology have recently published several study papers describing their investigations of the direct alcohol biomarker PEth. A co-author of some of those papers is Dr. Katleen Van Uytfanghe, who also joins us today.

She completed her PhD in the Laboratory of Analytical Chemistry at the Faculty of Pharmaceutical Sciences at Ghent University, followed by a post-doc in the same laboratory. Since October 2014 she has been working alongside Dr. Stove as a postdoctoral fellow in the Laboratory of Toxicology.

Neoteryx: Hello Dr. Stove and Dr. Van Uytfanghe. Welcome to the Microsamplify Podcast from Neoteryx. Thanks for taking the time to speak with us about your work at Ghent University.

Drs. Stove & Van Uytfanghe: Thank you for inviting us, it's a pleasure to be here with you.

Neoteryx: Dr. Stove, I’ll direct my first question to you. The list of courses you teach at the University of Ghent — toxicology, bioanalytics in drug development, and chemical criminalistics — reveals an interesting breadth of knowledge and interests. Can you tell us a little more about yourself in terms of what drew you to this field and what aspects of your work excite you the most?

Dr. Stove: I'm a pharmacist as a background, but I did my PhD and first post-doc in cell biology and molecular biology, to then embark upon bioanalysis again in a second post-doc, before getting a position here at the Lab of Toxicology at Ghent University. So, this means I have a rather broad interest field, which is also reflected in the research that we are doing in the lab. [This work] covers what I would call Applied Pharmacology, and the set up and development of bioassays, and also the development of new assays in the context of bioanalysis, where I have a special focus on, and interest in, microsampling.

Neoteryx: You have been using microsampling for quite some time, even before you began using the Mitra^® device to apply volumetric absorptive microsampling. How and why did you first become interested in microsampling for your research studies?

Dr. Stove: It dates back to something like 2010, when I was still a post-doc. At that time, I supervised a PhD student. She worked on a compound which is called GHB, or gamma-hydroxybutyric acid, which is of interest in forensic toxicology because that is the field we are working in.

At some point, we started to work on small blood samples — 50 µL blood samples. At a given point in time we thought, "Okay, if we can work on 50 µL blood samples, can't we also work on 50 µL dried blood samples?" And, after that, [we tried] the application of blood on filter paper. Ever since, we have been working on microsampling. It wasn't something that was really planned, we sort of stumbled into this by accident.

Neoteryx: You and Dr. Van Uytfanghe recently collaborated on a couple of published papers describing your work on PEth, as a direct marker of alcohol intake. Dr. Van Uytfanghe, can you tell me why PEth is a good biomarker for determining alcohol intake and how remote specimen collection with Mitra® devices and microsampling help you to track it?

Dr. Van Uytfanghe: Yes. First of all, PEth is the abbreviation for the alcohol biomarker phosphatidylethanol. And, in the gross spectrum of alcohol biomarkers, PEth is an outstanding one for two reasons:

First, it is solely formed when ethanol is present in someone's blood — so when there is no ethanol, there will be no PEth.

Second, there are no reports yet of false-positive or false-negative results when monitoring PEth. Therefore, we promote the use of remote specimen collection among the clinical among the clinical pathologists and healthcare workers we collaborate with. [This is] because of the ease of use, the fact that sample collection can be done on the spot, and because drying of the blood immediately after sample collection protects the sample from instabilities.

Moreover, the samples can also easily be sent via regular mail. So there are several advantages of using the [remote] device.

Neoteryx: Dr. Stove, your recent PEth studies have been a collaborative effort with the laboratory research team at the National Institute of Criminalistics and Criminology (NICC). Can you explain what your two labs are trying to achieve together, from the lab perspective in terms of methodology and from a criminology perspective in terms of how your work can be applied in the world?

Dr. Stove: Both our labs are working in the field of forensic toxicology, and historically, we have had several collaborations between our labs. In the context of PEth, we have already collaborated to work on the validity of PEth as an alcohol marker using conventional dried blood spots.

In the framework of that exploratory work, we already made a comparison of the usefulness of PEth, and compared that to more established alcohol biomarkers, such as ethyl glucuronide and CDT (carbohydrate-deficient transferrin). CDT is, in Belgium and also worldwide, still one of the most used indirect biomarkers, but it has quite a few drawbacks. If it is positive — if CDT is quite high — it is reflecting, in many instances, chronic and excessive drinking.

However, a drawback is that if it is not elevated, it doesn't say anything and definitely can't be used to demonstrate abstinence. Because of that, both of our labs missions are actually the same. We aim at providing high-quality services to interested parties. In many instances, this could be the Department of Justice.

Also, combining that with what I could describe as education about these biomarkers. Meaning that we also try to make sure that the ones that need to take of the judicial aspects — for instance, driving and drinking — are also aware of the possibilities of alcohol biomarkers.

And, in addition, collaborating on getting this routine method validated really demonstrated the comparability and the transferability of the methods. It brings a lot of value to the reliability of the methods that are running in our labs, both here at Ghent University and at the NICC.

Neoteryx: Dr. Van Uytfanghe, how “portable” or transferrable is your microsampling method for PEth studies, and how easy will it be for other labs to replicate around the world?

Dr. Van Uytfanghe: For labs who have basic skills in running quantitative mass spectrometry methods, the method should be easy to implement and to copy. In essence, we combine a simple aqueous extraction of the Mitra devices combined with a liquid-liquid extraction of the PEth from the aqueous phase.

Next, the PEth from the extract is measured using mass spectrometry. So we run here in our lab, with manual sample cleanup, up to 120 samples a day. But the method could also be transferred using robotization and then in a high-throughput lab it should also be useful. In the manuscript describing the methods, we tried to be as complete as possible. So we also described the problems we ran into upon validation of the methods, and we explicitly explain the rationale why we do certain things in a certain way.

This should save other researchers a pile of work upon validation of their own methods. In addition, as already mentioned by Professor Stove, we performed an extensive method comparison with one of our peers. This proved already transferability of the methods. It also exposed the Achilles heel of the methods — Namely, standardization and calibration of the measurements. That is really a point of attention.

Neoteryx: Your work in detecting alcohol and substance use, or overuse, is so important in light of statistics that tell us that approximately three million people around the globe die every year as a result of alcohol abuse. That is roughly one in 20 alcohol-related deaths worldwide. Your research team also studies another substance of abuse that leads to nearly 600,000 deaths per year worldwide—and that is opioids. Can you tell us about your opioid studies?

Dr. Stove: Yes, we are also working on opioids. As already mentioned, we have two main research lines in our lab. One involving bioanalysis, which includes microsampling, and the other applying bioassays. These opioid studies link to the bioassay research line of the lab. In these assays we use living cells to search for the activity of a certain group of compounds.

We have, for instance, done that for cannabinoids, and also for opioids. The assay, which employs living cells and is luminescence-based, can actually be used in the lab as a screening tool to find out what biological samples contain opioids. This can be irrespective of the identity of the opioid. In a sense, it comes down to the fact that we can identify any known or any unknown, any existing or any future opioids in a biological sample.

These bioassays can be used for screening purposes, like I said, but now we also routinely use these to assess the activity of the newest opioids that are on the illicit market, or that are anticipated to enter the illicit drug market. And, like that, we can also help regulators to anticipate what compounds may soon be there.

Neoteryx: As researchers, you have been innovators in the area of remote blood collection and have shown tremendous success with Mitra^® devices based on VAMS^® technology in the field.

Your study volunteers have an impressive success rate of submitting high-quality blood samples to your lab. In fact, more than 500 volunteers recently filled out a questionnaire about their experiences with finger-prick blood collection.

Are you willing to share the secret to your success with remote blood collection, and how you have worked with your study subjects to achieve this?

Dr. Stove: Indeed, we have set up quite a large study in which people that were untrained have performed microsampling using the Mitra devices. The outcome of this study was quite successful. The success, we think, depends on a combination of factors. First, we needed something that was easily applicable for home sampling, and we did think that Mitra with volumetric absorptive microsampling was suitable for that.

Second, we paid quite a lot of attention to instructing our volunteers quite well. This was via both a video that was accessible and via a leaflet. [We also used] targeted emails. We tried not to overload them with information, but really made sure that they got the information that was important.

Also very important was that we tried to motivate the participants. We sort of made them responsible for their results. They were interested in knowing something about their alcohol markers—this was also about PEth—and in a sense it came down to the fact that we told them, "You are interested in your result, so you'd better make sure that the sample you provide us is correct." And, actually, that [approach] seemed to work!

Neoteryx: Interesting! So, you made them, sort of like a scientist, the agents of their own results.

Dr. Stove: Yes, it sort of came down to that!

Neoteryx: Excellent! I’d like to focus on the coronavirus pandemic for a moment, since at the time of this interview, we are experiencing a terrible spike in cases. Has COVID-19 sparked keener interest in remote blood collection among your colleagues and collaborators, and do you foresee Europeans adopting remote blood collection for future studies in a post COVID-19 world?

Dr. Stove: Yes. We are aware of studies, for instance in The Netherlands, where microsampling is already being applied for therapeutic drug monitoring of immunosuppressants. And, they had already a smaller project running where patients could send in their samples via regular mail.

And with the onset of COVID-19, they saw an increased interest among patients to perform home sampling and send their samples to the hospital. I think that is something that may be maintained in the future, because once someone is convinced of the advantages of home sampling, with the ease of use, that is something that can remain. This is also something that came out of the questionnaire that we performed in the framework of our large-scale study. We actually asked our participants whether they would find it acceptable to consider a monthly finger-prick sampling at home.

For instance, in the follow-up of a drug concentration. The vast majority did say that they would consider this acceptable. And, importantly, those were participants that had already done the finger-prick, so they knew what was associated with it. They knew what a finger-prick meant.

Neoteryx: Do you and your colleagues have any new projects with microsampling planned moving forward?

Dr. Stove: Microsampling remains an important part of many projects that we have running in the lab, using both conventional dried blood spots and the Mitra devices. We have several projects running. We have been working on anti-epileptics. Currently, we have a project that is assessing vitamin B1, or Thiamine, on Mitra samples.

In the future, we hope to perform a quite large-scale epidemiological study in which we want to assess the vitamin status in a larger population. This could be European, but it could also be in developing countries, like in African countries.

Neoteryx: Thank you, Dr. Stove and Dr. Van Uytfanghe, for speaking with us about how you apply microsampling in your work at the University of Ghent. We wish you great success with your many projects and collaborations! 

An overview list of publications from the research team in the Toxicology Lab at Ghent University can be found here. Read the most recent publications by this group in which Mitra^® devices were used, at the following links:

Van Uytfanghe et al, Talanta: Quantitation of phosphatidylethanol in dried blood after volumetric absorptive microsampling. https://doi.org/10.1016/j.talanta.2020.121694

Van Uytfanghe et al, Clinical Chemistry and Laboratory Medicine: Self-sampling at home using volumetric absorptive microsampling: coupling analytical evaluation to volunteers’ perception in the context of a large scale study. https://doi.org/10.1515/cclm-2020-1180

Verstraete et al, Analytical Chemistry: Patient-centric assessment of thiamine status in dried blood volumetric absorptive microsamples using LC-MS/MS analysis. In Press – preliminary link, pending publication: http://dx.doi.org/10.1021/acs.analchem.0c05018   

Publications can also be accessed at the following Google Scholar links for these authors: Christophe Stove & Katleen Van Uytfanghe

Image Credits: University of Ghent, Trajan