Jul 07, 2021
Kenneth D. R. Setchell, PhD, joined the tenured faculty of the Department of Pediatrics, University of Cincinnati College of Medicine, in 1984, having moved from the Clinical Research Centre of the Medical Research Council (UK), where he previously held tenure. In 1973, he obtained a PhD in steroid biochemistry from the University of London and was then awarded a fellowship from The Royal Society for postdoctoral studies at the Karolinska Institute in Stockholm, Sweden (1974-1975), in the application of mass spectrometry to clinical and biomedical problems specifically related to the steroid hormone field. In 1984, Dr. Setchell moved to Cincinnati to become director of a new clinical mass spectrometry facility at Children’s Hospital Medical Center (CCHMC), where he is now a tenured professor in the Division of Pathology and Laboratory Medicine of the Department of Pediatrics and University of Cincinnati College of Medicine. In 2016, he received the Distinguished Contributor Award from MSACL for lifetime contributions to the application of mass spectrometry to the clinical laboratory presented at the International Congress on Mass Spectrometry Applications to the Clinical Laboratory. He received an Entrepreneurial Achievement Award at the 5th Annual Faculty Awards Ceremony of the Department of Pediatrics and Cincinnati Children’s Hospital Medical Center. In 2016, he received the NAMS/ Pfrizer Inc. Wulf H. Utian Translational Science Endowed Lecture award at the North American Menopause Society annual meeting in Orlando. In 2014, he was listed in the Thomson Reuters publication “The World’s Most Influential Scientific Minds,” which lists researchers in all fields of science and medicine to have published the greatest number of highly cited papers over the previous decade.
Thomas D. Ryan, MD, PhD, is the director of clinical operations in the Cardiomyopathy and Heart Failure Programs and the codirector of the Cardio-Oncology Program at Cincinnati Children’s Hospital Medical Center. He is an associate professor of Pediatrics-Clinical at the University of Cincinnati College of Medicine. Dr. Ryan graduated magna cum laude from Wichita State University with a bachelor’s degree in biological sciences and earned his medical and philosophy doctorate degrees at the University of Alabama at Birmingham. He completed his general pediatrics residency and pediatric cardiology fellowship at Cincinnati Children’s Hospital followed by his advanced fellowship in the Heart Institute’s Heart Failure and Transplant Program. Dr. Ryan’s clinical and research interests focus on heart failure, cardiomyopathy, and cardiac transplantation. He also focuses on patients who have developed heart dysfunction as a result of cancer-related therapies. He has published over 50 manuscripts in peer-reviewed journals and is author of 10 textbook chapters. He is also the chair of the Pediatric Working Group of the Cardio- Oncology Member Section in the American College of Cardiology.
Q: Why did the mass spectrometry lab at Cincinnati Children’s Hospital Medical Center develop a mail-in blood test for organ transplant patients last year?
KS: I’ve been working with establishing a number of therapeutic drug monitoring tests over the past few years. Several years ago, we used volumetric absorptive microsampling (VAMS) devices to measure concentrations of hydroxyurea administered as a treatment in sickle cell anemia patients in Africa. The idea was to try to develop tests that we could do remotely, so we needed to find a way of having the patients collect samples and have them shipped. The mail-in blood test for organ transplant patients was an extension of that work. Spurred on by the COVID-19 pandemic, Dr. Ryan contacted me about his particular patient population and the problem of what to do given that, being immunosuppressed, they’re at much higher risk of picking up infections, and also because many of the outpatient clinics were closed.
TR: Dr. Setchell is right that COVID really spurred this on. But even before COVID, I had gone to a couple of lectures by a pharmacology colleague of ours, and she talked about using blood spots to help set doses. I asked her about it, and she referred me to Dr. Setchell, who explained that he and his colleagues had already done all of the work in the sickle cell anemia study he mentioned. He said, “I can do you one better than blood spots—there’s some interesting new technologies that we can use.” So, that was really the beginning of the conversation about setting up a home monitoring program for our patients, so they don’t have to come in every time they need a blood draw.
Q: Can you tell me about the patient population that you developed these mail-in blood tests for, and what types of testing they normally undergo?
TR: We take care of patients who have had transplants and who need their immunosuppression levels checked pretty regularly. Some of them live two, three, or four hours away, so if the patient has to drive to a lab that’s part of our hospital system, it can be a significant drive for them. We often have them go to a local lab and get the level drawn there and either sent to a reference lab or sometimes a hospital, and that can sometimes take a week for us to get results. We’re making clinical decisions based on these results, so the sooner we can get them, the better.
The testing is pretty similar across organs. For heart transplant, we normally have patients on some combination of two different types of immunosuppressants—but one type, the calcineurin inhibitors, we really have to follow the levels. If they’re too high it will have side effects like kidney injury, infections, increased risk for cancer, tremors, seizures, and all sorts of bad things, and if they’re too low then the patient is at risk for organ rejection, which we’re obviously trying to avoid. So, the main tests that we have to do early on as frequently as weekly or more than once weekly measure levels of these medications in their blood.
KS: We are routinely measuring these immunosuppressants. We started with sirolimus for kidney transplant and liver transplant patients. The methodology we developed was based on venipunctures and whole blood draws done in the clinic or in the hospital immediately after these patients were transplanted. We’ve since worked on developing those assays for all of the suppressants, including sirolimus, tacrolimus, and cyclosporine. We routinely run those assays.
What we do that’s been very helpful to the physicians is offer very fast turnaround time on reporting of the assays. For example, if the patients were here in the hospital and the sample was received by 10 in the morning, we’d have the results by the end of the day. That’s a big help because prior to us establishing this testing with mass spectrometry, the transplant physicians were waiting in many cases five to 10 days to get answers because the samples would be shipped out to other labs. We made it a routine service, and that was based on whole blood.
Q: Why did you decide to use VAMS devices for your mail-in test rather than dried blood spots?
A: Dried blood spot technology has been around a very long time. It’s used in DNA screening; the Guthrie cards were used way back for diagnosis of genetic diseases in the first few days of life. That’s old technology and we initially investigated that.
Then along came the opportunity to work with these VAMS devices. I first saw them at a conference many years ago where the small startup company that developed them gave me samples and we started to play with them. We first validated for hydroxyurea and showed that we got it almost essentially identical data as we did from a dried blood spot.
From a lab point of view, it’s a lot easier to handle the sample on the VAMS device. The issue with the dried blood spot is that you have to punch out a circle of the paper; typically we would do a 3-millimeter punch, and we would do three of these and that would generally generate about 10–11 microliters of blood, which we would then have to extract. The nice thing about the VAMS device is that we don’t have that tedious punching out of paper—we just snap the tip off the device and put it into a test tube, and we extract directly from the tip.
The other nice thing is that the VAMS devices are made to absorb a fixed volume. When we started this work, we used 10-microliter tips, and subsequently the company has produced devices that are 20 microliters, and I think even up to 50 microliters now.
Another advantage of VAMS is that the concentrations that you determine are independent of the hematocrit, whereas you often have to correct for hematocrit when it’s off a dried blood spot.
I think from a lab perspective, we find the VAMS devices a little more convenient in terms of sample handling and sample extraction than dealing with dried blood spots. But having said that, we also do work with both.
TR: What I’ve learned through working with Dr. Setchell on this program is that it’s a lot easier to get a poor sample with the blood spot than with the VAMS. The VAMS can certainly be under or oversampled, but the blood spot is really tough to get just right.
It takes more blood to get a good sample on the paper than in the VAMS devices, and even though 10–30 millimeters doesn’t sound like a big difference, when you have a screaming kid who you just stuck and you’re trying to milk the heel or finger for blood, the less you can get away with needing to collect the better. I know that the coordinators from my team spend a significant amount of time on the phone trying to explain to the families how to do it, and still the paper blood spots were challenging. We would regularly get updates that the samples were sent in but they just weren’t enough or they were in the wrong spot, and even though the VAMS has challenges, it’s more user friendly for the families because there are more specific instructions and it’s just designed better.
Q: Can you tell me more about how the mail-in blood test you developed works?
KS: Dr. Ryan’s team developed a kit that gets mailed out to patients that are remote from the hospital, and we’ve done the same from our end with other studies. The kit contains a lancet to do a finger prick, the VAMS device in a shell, the requisition form that has to be filled out, and a return envelope.
TR: We have also been working with kits that the company produces, though in some cases, it’s easier for us to put together basically the same kit ourselves. That kit is mailed to the family and then our team calls with instructions on the phone that go over how you use it. The company also has online tutorials on how you collect a sample. Then we instruct the patients on when we would like them to take it based on when they would normally get a blood draw, because these are timed blood draws that have to happen before their dose so that it’s a trough level that gets measured. That’s how we make our clinical decisions—we aim for certain goal troughs based on how far out from transplantation the patient is. We’re using these when patients are at home where a turnaround of a couple of days is reasonable, especially since if they go to an outside lab, it’s sometimes a week before we get the labs back.
Q: What challenges did you face in developing and rolling out these tests?
KS: From an analytical point of view, the challenge is typical of any assay that we’re confronted with in pediatrics—small sample volumes. With newborns and infants, it’s not so easy to stick them so we have a limitation on sample volume and that pushes sensitivity. Attaining sensitivity to get to low levels of any compound becomes one of our major challenges in any assay development.
The matrix effect was a challenge because blood is quite dirty when you extract. The background or matrix is a critical factor in determining sensitivity in any assay. One of the ways we got around that was to develop a high-resolution mass spectrometry approach, so we would measure accurate mass of the compound, which really enhances specificity and cuts through the noise and reduces the matrix effect. We got comparable data with a lower resolution using a triple quadrupole or tandem mass spectrometer, but I think we just get cleaner mass spectra when we go to the higher resolution.
Then the validation of the methodology is always challenging. We wanted to be sure that the levels we were reporting were comparable to the levels we typically would be reporting when receiving venipuncture samples. They turned out to be very nice comparisons.
The other big challenge, as in any clinical study, is always sampling. Where we have failures, they’re usually failures of the sampling of the blood. You have to really educate the parents on how to handle these devices because sometimes a non-optimized sample arrives where the tip of the VAMS device is not completely stained with the blood. I always used to say, the biggest challenge is not always developing an assay; the biggest challenge is collecting an accurate sample.