From DBS to “Doo Dots”

When a patient presents gastrointestinal problems to their physician, a stool test is ordered before being frozen and transported to the lab for bioanalysis. This usually involves quantitative tandem mass spectrometry – a highly accurate, sensitive, and specific method for the measurement of fecal bile acids. Unfortunately, this complex, multistep process of collecting, processing, and analyzing stool samples is a significant barrier for remote clinical locations.

Enter Melinda Engevik, Donald Chace, Thomas Horvath, and Santosh Thapa – a team of researchers from across the US that sought an easier way of analyzing and diagnosing gastrointestinal ailments. Combining knowledge of metabolomics with mass spectrometry, the group developed a shelf-stable microsampling platform from repurposed credit-card sized technology that could be shipped at room temperature (1).

Here, the team discuss their exciting research discoveries and developments.

What inspired this project and the use of dried blood spot (DBS) technology in particular?
 

Thomas Horvath: The idea behind a dried fecal spot based microsampling platform evolved from a brainstorming session. We discussed the possibility of using DBS techniques to examine fecal metabolites. I brought up a recent experience where a shipment of fecal sample extracts were mailed with too little dry ice to keep the samples frozen during shipping. As you can imagine, the odor that evolved from these samples upon opening the container was quite repulsive, even with the use of a chemical fume hood. Don then proposed drying the fecal sample extracts on paper prior to shipment – I remember the smile on his face widening and the twinkle in his eye as he proclaimed, “We can call them doo dots!”

Donald Chace: I’m not so sure about the eye twinkling but the idea certainly put a smile on my face! I’ve previously performed analysis on unusual specimens collected on a cotton filter paper matrix. Examples include fruit fly homogenates applied to paper and postmortem liver tissue directly placed where the interstitial fluid would be absorbed onto the paper. Various fluid types can be analyzed on filter paper cards, from serum and urine to plasma and vitreous bile. The idea of collecting a liquid specimen, preparing it, and spotting onto the card isn’t unusual. For measuring free carnitine from dialysis patients, I have obtained plasma from whole blood in the clinic and spotted onto paper prior to shipping, which saved a great deal of money and fit nicely into our workflow. With a Capitainer™ card, the collected volume was more quantitative and protective – I’m sure this is what contributed to the excellent liquid chromatography-tandem mass spectrometry (LC-MS/MS) based quantitative data Mindy and Thomas acquired.

Melinda (Mindy) Engevik: It has been a wonderful collaborative project. Thomas and Don spearheaded the study and I was very excited to contribute with biological samples and translational applications. 

Santosh Thapa: I thoroughly agree with Mindy – I was very happy to contribute with the data analysis and method comparison graphics for our AJPGI manuscript (2). Glad to be a part of this wonderfully collaborative project.

What challenges did you face – and how did you overcome them?
 

TH: Mindy and I work closely together on all of our tissue homogenization methods, so we already had several stool-based protocols on hand that could be easily adapted to the dried fecal spot (DFS) workflow. Don is also a global expert in DBS-based bioanalysis and a pioneer in the field of newborn screening, providing us with two or three sets of conditions right off the bat. 

Thankfully, the bead-based homogenization of the DFS-laden paper worked beautifully the first time. I believe the most difficult part of developing the DFS-based bioanalytical platform thus far has to be in optimizing the LC-MS/MS based bile acid method. Optimizing the chromatographic method for retention, separation, and detection of all 16 analytes and 16 internal standards monitored in the LC-MS/MS system took several months (3). I had to pay particular attention to characterize the elution order and achieve baseline separation of the isobaric analytes.

How does your repurposed technology work?
 

TH: DFS-based sampling requires a not-so-subtle twist on DBS sampling workflows. In the DBS microsampling platform, a patient performs a finger stick, or a heel stick in the case of infants, to liberate a few drops of blood for loading onto the sampling paper stock – which are then dried to yield DBS samples. The DBS samples can then be shipped across the world for bioanalysis.

However, in the DFS sampling workflow, a stool sample has to be collected and loaded into a commercially available device or directly into sample homogenization tubes that are preloaded with homogenization beads in the lab. The stool sample is homogenized via the bead beating in the presence of an organic solvent system to limit the enzymatic biotransformation of bile acids and other metabolites. After sample homogenization, a 25 µL volume of the homogenized stool extract is loaded onto two sample channels and engineered into the Capitainer™ quantitative DBS (qDBS) devices. The samples are dried using especially designed drying pouches during shipping. Similarly to DBS sample sets, DFS-based samples can undergo ambient temperature shipping to bioanalytical labs worldwide.

Mass spectrometry (MS) is a large part of stool sample bioanalyses – do you use the same technique or an alternative method?
 

TH: For DFS-based bile acid applications, we will most likely continue to use our targeted LC-MS/MS based method to quantify bile acid targets contained in dried fecal extracts for the foreseeable future. However, I envision expanding upon our existing panel of 16 bile extracts to include a larger number of the physiologically relevant isomers, such as 7-oxolithocholic acid (7-oxoLCA), 7-oxodeoxycholic acid (7-oxoDCA), and hyodeoxycholic acid (HDCA).

Furthermore, I’m interested in building additional LC-MS/MS based methods for performing routine quantitative bioanalysis in a number of novel stool-based microbial-conjugated bile acids (MCBAs), such as leucocholic acid (Leu-CA), phenylalanocholic acid (Phe-CA), and tyrosocholic acid (Tyr-CA). These MCBAs are found in a rapidly expanding body of literature reporting on new discoveries. Additionally, we think that by slightly shifting the solvent system for stool sample homogenizations, the DFS-based microsampling platform could measure a number of stool-based protein biomarkers using colorimetric assets like ELISA in a format suitable for home-based sample collection.

What could this repurposed technology mean for patients?
 

ME: This technology opens up several exciting new avenues. In addition to bile acids, this method could be used to detect multiple different compounds, including biomarkers in GI disorders. There’s also the possibility of this technology being employed across the world. Our DFS samples loaded on qDBS devices can be stored and shipped at ambient temperatures and still yield the same bile acid profiles as traditional samples. We envision individuals in rural communities or resource-limited countries using our technology to analyze and interpret their data. 

We’re also hopeful that this technology can be transported into patients’ homes. It's exciting to think that future patients will be able to routinely send DFS samples that we can screen for gut health. This would be particularly useful for patients with inflammatory bowel disease – allowing for quicker identification of “flare ups” and treatment.

ST: Agreed. It’s well documented that understanding the relationship between gut biomarkers and human health/disease requires sampling from broad human populations. However, there’s still a disparity in which stool samples are being studied on a global level – primarily because of the lack of storage resources and struggles to maintain cold temperatures during transport in developing countries. While working in Nepal, I experienced these challenges firsthand and I believe that our repurposed technology could be the first stepping stone in addressing these challenges.