Michael Mbughuni (Presenter)
Mayo Clinic
Bio: I was originally born in Tanzania, but I am a naturalized U.S citizen. I received my bachelor degree in biochemistry and molecular biology at the University of Wisconsin, Eau Clare and my doctorate in Biochemistry at the University of Minnesota, Twin-Cities. As a doctoral student, my research focused on mechanistic studies with non-heme oxygenase enzymes. After receiving my Ph.D, I accepted a post-doctoral research position in the department of biochemistry at the University of Wisconsin, Madison. At Madison, my project focused on studying synergistic enzyme chemistry in hydrolysis reactions before joining Mayo clinic in 2015 as a chemistry fellow.
Authorship: Mbughuni, M.M1., Penn, H.J., Langman1, L.J., Jannetto, P.J1.
Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN1.
| Short Abstract Tacrolimus and Cyclosporin A are immunosurpressants commonly prescribed for solid organ (cardiac, liver, renal) transplants. Therefore, therapeutic drug monitoring of these medications is important based on their narrow therapeutic range where suboptimal concentrations can lead to organ rejection and elevated concentrations can lead to toxicity. Since transplant patients need to take these medications for life, this study looked at the feasibility of measuring Tacrolimus and Cyclosporin A using a high-performance liquid chromatography tandem mass spectrometry assay using driedblood from a microsampling device (20 mcL) compared to a validated method using 200 mcL venous collected EDTA whole blood. |
Long Abstract
Background:
Tacrolimus (Tacro) and cyclosporin (Cyclo) are synthesized by Streptomyces tsukubaensis and Tolypocladium inflatum, respectively. Their primary clinical utility is based on immunosuppressive activities which inhibit T cell activation and proliferation. These two drugs are now commonly prescribed for the management of rejection in allogenic organ transplants, with patients being on these medications for the rest of their lives. Consequently, therapeutic drug monitoring (TDM) of Tacro/Cyclo is critical due to their narrow therapeutic windows, with increased risk of transplant rejection when drug levels are below the therapeutic range and toxic effects when levels are above the therapeutic range. These limitations make it important for clinicians to manage immunosuppressant dose adjustments using consistent and accurate TDM results. Since most patients geographically relocate from the site of their transplant, the long term management of patients is subsequently complicated with TDM results from various institutions where studies have shown significant variations between quantitative immunosuppressant results even if they use the same methodology (i.e. mass spectrometry). This study looked at the feasibility of measuring Tacro and Cyclo using a high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) using a dried whole blood microsampling device (20 mcL) that could be shipped to a laboratory for analysis. This method was compared to a validated HPLC-MS/MS method using 200 mcL EDTA whole blood.
Method/Results:
Remnant Tacro or Cyclo TDM samples spanning the quantifiable range collected for routine TDM were used for this method development and comparison. A new protocol using 20 mcL dried blood collected with the microsampling device was optimized and compared to a validated method that used 200 mcL of EDTA whole blood. The method validation focused on: accuracy, reportable range linearity, intra- and inter-assay precision, and carry-over. Our data revealed the intra- and inter-assay precision ranged from 5-10% for both Tacro and Cyclo using the new microdevice across the analytical measuring range. Furthermore, linear regression and Bland Altman analysis showed the two methods compared favorably (R2 range: 0.94 - 0.98, Slope range: 0.97 - 1.22). Carry over studies also were within acceptable limits. This new method and microsampling device opens up potential novel opportunities for patients to collect a capillary blood sample at home and mail it in for TDM analysis without having to drive to a phlebotomy draw site.
Summary:
One way to provide consistent and accurate post-transplant immunosuppressant maintenance therapy is to develop a robust method that allows patients the ability to collect samples at home and mail in their dried blood for TDM analysis to one laboratory regardless of their geographical location. This approach will help minimize the clinical variation due to lack of harmonization with laboratories using different methods for measuring immunosuppressants. As a result, physicians will be able to get consistent results as they monitor a patient serially over time and make dosage adjustments. In the end, we present a new method for quantifying Tacro/Cyclo from 20mcL of dried whole blood using HPLC-MS/MS. The new protocol is optimized from a validated protocol and the analytical performance of the new method suggests feasibility for clinical TDM on dried capillary whole blood samples which could be shipped.
References & Acknowledgements:
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