An article published by Nils T. Vethe et al in the October 2019 issue of Therapeutic Drug Monitoring, reported on the successful validation of 10 µL Mitra® devices from 26-27 renal transplant recipients to measure therapeutic drug levels of the anti-rejection drug, tacrolimus. The paper, entitled “Tacrolimus can be reliably measured with volumetric absorptive capillary microsampling throughout the dose interval in renal transplant recipients,” describes an analytical and clinical validation comparing wet blood to dried Mitra microsamples. During the study, over 600 blood microsamples collected by study participants at home were compared to a similar number of samples taken in the clinic and sent directly to the Oslo University Hospital laboratory.
At-home TDM Using Dried Capillary Blood for Transplant Recipients
This therapeutic drug monitoring (TDM) study showed that both trough level measurements and PK measurements provided equivalent results from dried capillary blood samples compared to venous samples collected via venipuncture blood draws. The results demonstrated both accuracy and precision within accepted criteria. Further, the Mitra dried blood microsamples showed stability over 1-2 months when stored at ambient temperatures. Additionally, the researchers found that processing of Mitra samples was more efficient than processing of traditional wet samples, with increased options for automation.
A Brief History of Organ Transplantation
The very first solid organ transplant was conducted in 1954 at at the Peter Bent Brigham Hospital in Boston by Dr. Joseph E. Murray at Harvard Medical School in Massachusetts, where he conducted a kidney transplant between identical twins, Richard and Ronald Herrick. Receiving a new kidney from his brother via the successful operation added years to Richard Herrick’s life and ushered in a new dawn of transplant science. Over the ensuing years, this area of medical science has advanced even further. Indeed, in 2019 it was estimated that as many as 39,500 kidney transplantations were conducted in the US alone. Similar numbers were reported for Europe.
Preventing Organ Rejection with Therapeutic Drugs
The success of the world’s first successful organ transplant was attributed to the genetic similarity and histocompatibility of the identical Herrick twins. However, organ rejection was a real concern even when tissue typing between the donor and recipient was so close. Indeed, back in the 1950s, very little was known about organ rejection and how to overcome it. In the past 50 years, much research on preventing organ rejection in transplant patients has been conducted. In 1971, the calcineurin inhibitor cyclosporine was discovered and isolated from the fungus Tolypocladium inflatum. In 1983, after additional research and development, the drug was approved for clinical use as a preventative against organ rejection. Cyclosporine is still used for this purpose to this day.
Calcineurin inhibitors continue to be a key class of drugs used in the prevention of graft rejection. These inhibitors work by actively suppressing specific T-cell immune interactions. The most popular calcineurin inhibitor used in renal transplants is the macrolide, tacrolimus. Tacrolimus was first isolated from a soil sample in the Tsukuba region of Japan in 1984. The drug was commercialized by Astellas Pharmaceuticals. In 1994, tacrolimus was approved by the US Food and Drug Administration (FDA) as a therapeutic drug for patients who had undergone liver transplantation. This application was later approved for use in patients with other transplanted organs.
TDM of Tacrolimus and Other Calcineurin Inhibitors
Graft recipients in present times are often given a cocktail of drugs which include steroids, mycophenolate and a calcineurin inhibitor such as tacrolimus. Due to the narrow therapeutic index of calcineurin inhibitors, tacrolimus and similar drugs need to be carefully monitored. In fact, as highlighted in the paper by Vethe et al, there can be a 10-fold difference in dosing between patients. For this reason, post-transplant dose changes are often required throughout a patient’s lifetime, thus requiring regular therapeutic drug monitoring (TDM).
In the initial days of a successful graft, blood testing is carried out several times per week to assess drug levels. As the graft stabilizes over time, drug monitoring is gradually reduced to occur about every 2-3 months. This transition typically happens around 6-12 months post-transplant. This regular TDM schedule (and related dose adjustments), helps to prevent drug-induced chemotoxicity which would otherwise result in the following complications: hypertension, post-transplant diabetes mellitus, neurotoxicity, and nephrotoxicity. Alternatively, too low a dose of the administered tacrolimus can lead to acute organ rejection due to over-activity of donor-specific antibodies.
The Oslo University Hospital Researchers Explored the Benefits of Remote TDM
There are two primary reasons for taking TDM and testing out of the clinic. The first reason is logistics. The Oslo team highlighted the following challenges with the current in-clinic system of blood draws for therapeutic drug monitoring.
Patients have to travel to the clinic (sometimes making overnight stays) for morning phlebotomy procedures, and then must wait hours for results. They also have in-clinic meetings with their specialist. If tacrolimus levels are not returned in time, then a follow up call is needed.
If dose adjustments are required, then follow-up blood samples are needed. This requires the patient to travel back to the clinic and repeat the process.
The second reason for taking TDM out of the clinic is to minimize exposure of immunosuppressed patients to contagions that could lead to dangerous infections, such as COVID-19 caused by the SARS-CoV-2 virus. Enabling these vulnerable patients to complete a majority of their maintenance care at home keeps them out of high-risk environments. The Oslo study illustrates that the current “wet sampling” situation is time-consuming and carries a higher risk for transplant recipients, so Vethe et al proposed that home sampling would be more patient-friendly and would save time and money. Indeed, they highlighted a previous study that had conducted a cost evaluation of home monitoring in pediatric TDM. In the conclusions of their 2016 paper the authors stated, “From a societal perspective, total costs per blood draw decrease drastically with home sampling. Patient costs are reduced to zero and costs related to loss of productivity are decreased by >95%.”
The team from Oslo also highlighted that home sampling would allow for limited sampling strategies by measuring AUC. The team also noted that dried blood spot (DBS, which was used in previous studies for tacrolimus measurements), was hampered by a type of “hematocrit effect,” or HCT bias. This was particularly an issue for renal grafts, due to observed large shifts in HCT levels post transplantation. However, because Mitra devices enable a volumetric approach to microsampling with their VAMS® tips, it was proposed that these devices would overcome the observed HCT hurdle and indeed they did.
Object of the Tacrolimus Study
To 1) develop a VAMS method for measuring trough levels with the same performance as methods for liquid blood, and 2) to develop a future model requiring quantification throughout the dose model for renal transplant patients.
Tacrolimus Study Methods & Findings
The study team developed a method from VAMS extracts involving the same reagents for protein precipitation dried blood sample preparation compared to standard liquid blood samples. This made the method very compatible when compared to the standard LC-MS/MS method.
The analytical method was fully validated according to EMA guidelines, including the inclusion of external QC samples from LGC in the UK.
For the clinical study, adult transplant patients (n=27) were recruited and put on the following regime: tacrolimus, mycophenolate and prednisolone. One patient was dropped from the study due to a change in medication.
The aim of the study was to compare at-home vs. clinical collection of capillary samples with Mitra vs. venous samples for 2 x 12h PK investigations separated by at least 1 week with no dose adjustments between PK1 and PK2.
For each PK curve, 12 blood samples were collected at specific times after tacrolimus administration. At each PK sample time, two Mitra samples (10uL) and two venous samples were taken.
To assess the journey of the sample, one set of clinic samples was sent directly to the lab, while the other was mailed from the hospital to the lab. Finally, in between clinic collections, the participants mailed in trough samples from home to assess home self-collection vs. in-clinic sampling.
Home sampling was found to be acceptable and similar in precision to in-clinic sampling.
The agreement between wet and dry samples was consistent throughout the dose intervals and was thought to be underestimated, due to differences in time of sampling between wet and dry sampling times.
Because the dried assay shared the same standards, reagents, and LCMS conditions as the liquid assay, it was shown to be quicker than the wet method due to less individual mixing of the wet samples. This would also allow for future automation of Mitra samples.
During this study, a 96 well plate of Mitra samplers was processed from extraction to data reporting within around 3 hours.
No HCT dependence was observed across the HCT range tested (0.25-0.44).
Mean accuracy for the VAMS samples was within acceptable criteria (85-115%).
Measurement and CVs were ≤11%
The team observed a bias of -3.1% (95% CI ranging from -3.9% to -2.3) from the shipped Mitra samples that were analyzed 3-7 days post-shipping, compared to liquid samples sent directly to the lab and stored overnight before analysis.
Of the dried samples, they observed 6.9% individual deviations outside of ±20%.
The team highlighted in the manuscript that this was seen as an acceptable bias considering a mean deviation of -7% had been previously observed with another device that used dried paper (HemaXis) for renal/pancreas patients.
They reported a -8% bias for external QCs dried on Mitra devices. In comparison, no bias was observed from liquid QC samples. The authors recommended that external QCs should be shipped as dried Mitra devices to overcome this observed bias when compared to wet QC samples.
They observed a -ve bias (10-20%) when calibrators and QCs were dried for just 3 hours compared to drying for ~ 1 day. The group thus recommended drying these standards for at least 1 day, where the reported bias was observed to be minimal. They commented that this would be compatible with measuring home collected samples that were mailed and would be naturally dried for a number of days prior to analyzing.
The Mitra samples were stable for at least 1 month with mean deviation of -9%.
The mean difference between the 2-parallel VAMS tips in the Mitra cartridge was generally less than 10% for hospital samples and also for home samples.
The upper CI limited for the difference was ≤11% and all differences were <20%, so the interpretation of this was that analysis from a single microsampling tip would be sufficient. However, they advised collecting repeat samples for reanalysis.
They recommended, as a pre-analytical precaution, that VAMS tips on the Mitra devices are visually inspected before extraction to observe for any under- or over-sampling.
Study Authors' Discussion and Conclusions
10 µL Mitra sampling for LCMS demonstrated a method performance that was acceptable compared to standard wet sampling.
A plate of 96 samples could be extracted and analyzed within 3h. This was found to be more efficient than processing the same number of wet samples.
Tacrolimus concentrations were reliably quantified throughout the dose intervals of the PK studies.
Methodology was considered suitable for trough and rich strategies for sampling transplant patients.
The work reported by Nils T. Vethe et al in Oslo demonstrated that a rich dataset could be obtained from Mitra devices which was comparable to data from wet samples. This, coupled with the benefits of home sampling, could usher in an AUC approach to TDM of anti-rejection drugs possibly interspersed with trough measurements over the course of each 12 months. Sampling from home will allow the patient to spend less time travelling to clinic and waiting for blood results.
Given the success that other centers have had in developing similar methodologies, such as a program at Wythenshawe Hospital in the UK, home sampling for tacrolimus measurements may become the gold standard for combining convenience with accuracy in TDM. Furthermore, patients in the Oslo study reported no difficulties in using Mitra devices when sampling at home, as reported in a follow-up paper by the same group in Oslo focusing on the PK data. This finding was echoed in a paper from the Mayo Clinic in 2020, where 100 transplant patients were surveyed and 82% preferred capillary sampling with Mitra devices compared to 3% who preferred venous sampling and only 13% who had no preference. Furthermore, this patient preference also was reported in a recent paper published in Australia. The findings from Oslo and other transplant groups globally certainly paint a very bright future for at-home TDM sampling for research of transplant recipients. This study paper was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this review, visit the original article published in Therapeutic Drug Monitoring.
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