Dried blood microsampling-assisted therapeutic drug monitoring of immunosuppressants: An overview.

In the field of solid organ transplantation, chemotherapy and autoimmune disorders, treatment with immunosuppressant drugs requires intensive follow-up of the blood concentrations via therapeutic drug monitoring (TDM) because of their narrow therapeutic window and high intra- and inter-subject variability. This requires frequent hospital visits and venepunctures to allow the determination of these analytes, putting a high burden on the patients. In the context of patient-centric thinking, it is becoming increasingly established that at least part of these conventional blood draws could be replaced by microsampling, allowing home-sampling and increasing the quality of life for these patients. In this review we discuss the published methods - mostly using liquid chromatography coupled to tandem mass spectrometry - that have utilized (volumetric) dried blood samples as an alternative for conventional liquid whole blood for the TDM of immunosuppressant drugs. Furthermore, some pre-analytical considerations using DBS or volumetric alternatives are considered, as well as the applicability on clinical samples. The implementation status in clinical practice is also discussed, including (1) the cost-effectiveness of this approach compared to venepuncture, (2) the availability of multiplexed methods, (3) the status of harmonization and (4) patient perception. A brief perspective on potential future developments for the dried blood-based TDM of immunosuppressant drugs is provided, by considering how obstacles for the implementation of these strategies into clinical practice might be overcome.

Liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of immunosuppressants and creatinine from a single dried blood spot using the Capitainer® qDBS device.

In recent years, a lot of attention has been given to a more patient-centric therapeutic drug monitoring (TDM) of immunosuppressant drugs (tacrolimus, sirolimus, everolimus and cyclosporin A) by the use of microsampling techniques. By adopting Dried Blood Spots (DBS) after a finger prick, instead of conventional venous blood draws, follow-up can (partially) be established from patients' homes. Despite the many advantages of DBS, one of the major disadvantages associated with this technique is the well described hematocrit (hct) effect. In order to overcome the hct area bias, different strategies have been proposed, amongst which the use of dried blood sampling techniques based on the volumetric collection of blood. The aim of this study was to evaluate the use of the Capitainer® qDBS (quantitative Dried Blood Spot) device for the combined TDM of four immunosuppressants and creatinine from a single qDBS. The set-up of an adequate sample preparation allowing both immunosuppressants and creatinine quantification was one of the key challenges in the method development due to device-specific interferences. Liquid chromatography tandem-mass spectrometry methods for the quantification of tacrolimus, sirolimus, everolimus, cyclosporin A and creatinine from qDBS (10 µL) were developed and validated based on international guidelines, also taking into account DBS-specific parameters. The methods proved to be accurate and reproducible, with absolute biases below 10% and within-run CVs (%) below 8% over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus, 20-1500 ng/mL for cyclosporin A, and 15-700 µmol/L for creatinine. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained, showing no hematocrit-dependence (compared to a hct of 0.37), except for cyclosporin A at higher hct values. Application on venous blood left-over patient samples showed good agreement between the results of Capitainer® qDBS and whole blood with 98% (47/48), 93% (41/44), 89% (41/46), 88% (38/43) and 89% (116/131) of the samples lying within 20% of the whole blood result for tacrolimus, sirolimus, everolimus, cyclosporin A and plasma/serum for creatinine, respectively. For creatinine a blood/plasma ratio of 0.85 was found and used to convert qDBS results to plasma/serum results. As a next step, capillary finger prick samples will need to demonstrate the clinical applicability of the method in a real life setting.

Application of non-contact hematocrit prediction technologies to overcome hematocrit effects on immunosuppressant quantification from dried blood spots.

Fully automated dried blood spot (DBS) analysis for therapeutic drug monitoring (TDM) of the immunosuppressants tacrolimus, sirolimus, everolimus and cyclosporin A suffers from a so-called hematocrit (hct) effect. This effect is related to the analysis of a partial DBS punch and extractability differences imposed by blood with different hcts. As this is intrinsic to automated DBS analysis, this poses a serious drawback for accurate immunosuppressant quantification. Knowledge of a sample's hct allows to correct the derived immunosuppressant concentrations for this effect. Unfortunately, when using the DBS approach for sampling at patients' homes, this hct will typically not be available. The aim of this study was to investigate the validity of a correction algorithm during fully automated DBS analysis of immunosuppressants, based on knowledge of the DBS' hct, obtained via two distinct non-contact hematocrit prediction strategies, using either near-infrared (NIR) or ultra-violet/visible (UV/VIS) spectroscopy. For tacrolimus, sirolimus, everolimus, and cyclosporin A, 48, 47, 58 and 48 paired venous whole blood and venous DBS patient samples were collected, respectively, and analyzed using an automated DBS-MS 500 HCT extraction unit coupled to a liquid chromatography tandem mass spectrometry system. Additionally, for all 201 samples the hct of the DBS was predicted based on NIR and UV/VIS spectroscopy. For tacrolimus and cyclosporin A, both hct prediction strategies allowed for adequate correction of the hct effect. Also for sirolimus and everolimus the results greatly improved after hct correction, although a hct bias remained for sirolimus and for everolimus a slightly significant hct effect was observed after NIR- and UV/VIS-based correction. Application of both hct prediction strategies ensured that clinical acceptance limits (i.e. ≥ 80% of the samples within 20% difference compared to whole blood) were met for all analytes. In conclusion, we demonstrated that non-contact hct prediction strategies, applied in tandem with fully automated DBS analysis, can be used to adequately correct immunosuppressant concentrations, yielding a good agreement with whole blood.

Application of a Fully Automated Dried Blood Spot Method for Therapeutic Drug Monitoring of Immunosuppressants: Another Step Toward Implementation of Dried Blood Spot Analysis.

CONTEXT.­: The follow-up of patients under lifelong immunosuppressant therapy is pivotal to prevent allograft rejection after transplant. Part of the difficulties associated with routine monitoring of immunosuppressant concentrations can be alleviated by home sampling using dried blood spots (DBSs). OBJECTIVE.­: To evaluate the applicability of a DBS method for the determination of immunosuppressants in venous blood samples, making use of an automated extraction platform. DESIGN.­: Paired venous DBSs and whole blood samples were analyzed for tacrolimus (n = 162), sirolimus (n = 47), everolimus (n = 45), and cyclosporin A (n = 61) with liquid chromatography coupled to tandem mass spectrometry, using fully automated extraction for DBSs. Agreement between the automated DBS and whole blood method was assessed by using Bland-Altman comparison. Both an analytical and a clinical acceptance limit were predefined at more than 67% of all paired samples within 20% of the mean of both samples and more than 80% of all paired samples within 20% of the whole blood concentration, respectively. RESULTS.­: An impact of the hematocrit (hct) on DBS quantitation was observed for all analytes, which could be alleviated for all analytes by using a hct conversion formula based on a tacrolimus data subset: [DBScorrected] = [DBSmeasured]/(1.6305 - 1.559*hct). After correction, both analytical and clinical acceptance criteria were met for all analytes. CONCLUSIONS.­: Automated DBS analysis shows great potential for routine therapeutic drug monitoring of immunosuppressants, avoiding any manual sample handling.

Fully Automated Dried Blood Spot Extraction coupled to Liquid Chromatography-tandem Mass Spectrometry for Therapeutic Drug Monitoring of Immunosuppressants.

Patients receiving immunosuppressant therapy, require intensive follow-up via therapeutic drug monitoring (TDM). This puts quite a burden on the patient involving frequent hospital visits and venipunctures and could (partially) be resolved by the use of dried blood microsamples (e.g. dried blood spots, DBS). One of the drawbacks of the use of DBS is the requirement for a dedicated, manual sample preparation. Fully automated DBS extraction systems, online coupled to standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) configurations, could provide a solution for that. The aim of this study was to evaluate the use of the DBS-MS 500, online coupled to an LC-MS/MS system, for the TDM of immunosuppressants using DBS. Two methods for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A, in both DBS and whole blood, were developed and validated based on international guidelines. For the DBS method also DBS-specific parameters were taken into account. Both methods proved to be accurate and reproducible with biases below 11% (20% for the LLOQ) and CVs (%) below 14% (with a single exception) (20% for the LLOQ) over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus and 20 to 1500 ng/mL for cyclosporin A. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained. However, a hematocrit-dependent relative recovery was observed for DBS, with lower hematocrit values yielding higher relative recoveries (and vice versa). Relative to the reference hematocrit of 0.37, this difference exceeded 15% at hematocrit extremes (0.18 and 0.60). Application on venous left-over patient samples showed reasonable agreement between the results of both methodologies (8,6,9 and 9/10 mean DBS results within 20% of the mean whole blood result for tacrolimus, sirolimus, everolimus and cyclosporin A, respectively), although also here an impact of the hematocrit could be discerned. As a next step, larger patient sets are needed to allow a better insight on how (correction for) the hct effect affects the quantification of immunosuppressants via fully automated DBS analysis.

Evaluation of the Performance and Hematocrit Independence of the HemaPEN as a Volumetric Dried Blood Spot Collection Device.

Dried blood spots (DBS) are often used as a less invasive alternative to venous blood sampling. Despite its numerous advantages, the use of conventional DBS suffers from the hematocrit (hct) effect when analyzing a subpunch. This effect could be avoided by using hct-independent sampling devices, of which the hemaPEN is a recent example. This device collects the blood via four integrated 2.74 µL microcapillaries, each depositing the blood on a prepunched paper disc. In this study, we evaluated the technical performance of the hemaPEN devices, using an extensive bioanalytical validation and application on authentic patient samples. An LC-MS/MS method quantifying caffeine and its metabolite paraxanthine in dried whole blood (using the hemaPEN device) was fully validated, meeting all preset acceptance criteria. A comparative analysis of 91 authentic patient samples (hct range: 0.17-0.53) of hemaPEN, 3 mm DBS subpunches, and whole blood revealed a limited hct dependence (≤7% concentration difference over a 0.20-0.50 hct range) for the hemaPEN devices, which we could not attribute to the analytical procedure. Using conventional partial-punch DBS (3 mm punches), concentration differences of ≥25% over this hct range were found. The hemaPEN showed to be robust to the effects of blood sample volume, device lot, analytical operator, and storage stability. The technical performance of the hemaPEN when dealing with patients having a high hct and in cases where a large blood drop is present should be further investigated. Based on the successful validation and application on patient samples, we conclude that the hemaPEN device shows good potential for the volumetric collection of DBS.

Fully automated therapeutic drug monitoring of anti-epileptic drugs making use of dried blood spots.

Fully automated dried blood spot (DBS) extraction systems, online coupled to standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) configurations, decrease the hands-on time associated with conventional DBS analysis, resulting in a higher sample throughput, making the technique more compatible with a high-capacity bioanalytical workflow. The aim of this study was to develop and validate an LC-MS/MS method, using a DBS-MS 500 autosampler, for the determination and quantification of four anti-epileptic drugs (carbamazepine, valproic acid, phenobarbital and phenytoin) and one active metabolite (carbamazepine-10,11-epoxide) in DBS samples. Method development included thorough optimization of the fully automated extraction procedure (i.e. extraction solvent, extraction (loop) volume, internal standard application, internal standard drying time, etc.). The method was fully validated based on international guidelines. Accuracy (%bias), as well as precision (%RSD) (with a single exception) were below 13%. Neither carry-over nor unacceptable interferences were observed. All compounds were stable in DBS for at least 1 month when stored at room temperature, 4 °C and -20 °C and for at least 4 days when stored at 60 °C. Internal standard-corrected matrix effects were below 8%, with %RSDs below 9.1%. Reproducible relative recovery values (around 60% for all analytes) were obtained and the effect of the hematocrit on the relative recovery was overall limited. Successful application on capillary patient samples originating from developing countries demonstrated the applicability of the developed procedure in a remote setting.