Immunosuppressive Monitoring Research Articles

Challenges for paediatric transplantation in Africa.

Transplantation is the accepted mode of treatment for patients with end-stage organ disease affecting the heart, lungs, kidney, pancreas, liver and intestine. Long-term outcomes have significantly improved and the aim of management is no longer only long-term survival, but also focuses on quality of life especially in children. Transplantation in Africa faces a number of challenges including wide socioeconomic disparity, lack of legislation around brain death and organ donation in many countries, shortage of skilled medical personnel and facilities, infectious disease burden and insecure access to and monitoring of immunosuppression. Whilst there is a need for transplantation, the establishment and sustainability of transplant programmes require careful planning with national government and institutional support. Legislation regarding brain death diagnosis and organ retrieval/donation; appropriate training of the transplant team; and transparent and equitable criteria for organ allocation are important to establish before embarking on a transplant programme. Establishing sustainable, self-sufficient transplant programmes in Africa with equal access to all citizens is an important step towards curtailing transplant tourism and organ trafficking and has a further beneficial effect in raising the level of medical and surgical care in these countries.

Monitoring of Polyomavirus BK Replication and Impact of Pre-Emptive Immunosuppression Reduction in Renal-Transplant Recipients in China: A 5-Year Single-Center Analysis.

Background:A 1-year, single-center trial demonstrated that monitoring and preemptive of immunosuppression reduction were associated with resolution of viremia.Our 5-year prospective subsequent study was conducted to determine the long-term patient/graft survival and outcome of BK viremia and BK virus-associated nephropathy (BKVAN) in renal transplant recipients under the impact of intensive monitoring and reduction of maintenance immunosuppression. Methods:Quantitative BK virus (BKV) DNA surveillance in plasma/urine and cytological test in urine were performed at 1, 3, 6, 9, and 12 months after transplantation in 229 kidney recipients.Patients with BK viremia and BKVAN treated with 30% to 50% reduction in doses of tacrolimus (Tac) or/and mycophenolate mofetil(MMF) without antiviral therapy and were monitored for BKV every 3-6 months. All the patients were followed for 5 years. Results:Overall 5-year patient survival was 95.6% and graft survival was 92.1%. There were no differences in patient/graft survival, renal function, and rejection rate by presence of decoy cells, BK-viruria, viremia, or BKVAN. Multivariate analysis showed that Tac(OR, 2.9; P=0.032)and deceased kidney donation(OR, 2.1; P=0.071) were risk factors for BK viremia. After reduction of immunosuppression, viremia (n=38) resolved in 100%, without increased acute rejection. In the duration of follow-up 2-5 posttransplant years, BK viremia was not observed again in the BK viremic patients (n=30) without BKVAN. BKVAN was diagnosed in 7 cases(3.1%). The treatment of immunosuppression reduction was effective. All the BKVAN patients cleared viremia with a mean time of 5.9 months (range 1-15 months). Three of 7 BKVAN patients cleared viruria. A repeated renal biopsy was performed in two patients and revealed resolving BKVAN. There was no decline in estimated glomerular filtration rate over time from1 month(53.1mL/min) to 5 years(58.5mL/min) after transplantation (P=0.960). Conclusions:Monitoring and preemptive of immunosuppression reduction resulted in the successful resolution of BK viremia, showed effective in BKVAN recipients at the early stage with excellent graft survival and renal function at 5-years.

Falsely elevated cyclosporin and tacrolimus concentrations over prolonged periods of time due to reversible adsorption to central venous catheters

Falsely elevated concentrations of immunosuppressants can be caused by reversible adsorption to central venous catheter (CVC) systems. If undetected, this may lead to dose reduction resulting in underdosage which may even entail graft-versus-host disease or organ rejection. We analyzed the adsorption and release for cyclosporine A (CsA) and tacrolimus (Tac) in vitro and in vivo. Four types of CVCs were examined in vitro: two made from polyurethane (PU), one from silicone and one from PU with an incorporated silver ion-based antimicrobial agent. All 26 CVCs analyzed in vitro showed significant reversible adsorption of CsA (n=13; p=0.001) and Tac (n=13; p=0.001, Wilcoxon signed rank test). Immediately after infusing the drugs, the mean concentrations of 6420ng/mL of CsA and 250ng/mL of Tac were measured. Flushing with NaCl lowered the drug release. Besides, blood samples of fifteen patients were taken simultaneously from all lumina of the CVC and via venipuncture. The samples from contaminated lumina showed the mean elevations by a factor of 11 for CsA (n=12) and 89 for Tac (n=3). Blood sampling for immunosuppressant monitoring should thus never be performed from lumina previously used for infusing the drug even after prolonged periods of time and extensive rinsing.

Pregnancy after lung and heart-lung transplantation

Advances in lung transplantation have enabled women to successfully undertake pregnancies. This study explored outcomes in this group, including changes in lung function, kidney function, and calcineurin inhibitor (CNI) levels. A retrospective review identified 19 transplant recipients who had ever become pregnant at our center, and manual reviews of their medical records were completed for 14. Results of spirometry, serum creatinine, CNI doses and trough levels, and comorbidities were collected. Eight births occurred (42% success rate). Six patients have since died, with pregnancy contributing to 1 death. Five pregnancies were unplanned, with only 1 resulting in birth. Six pregnancies ended with spontaneous termination, and 2 were terminated for medical reasons. Mean age was 31.4 years (range, 22-39 years), and mean time from transplant was 76.2 months (range, 26-139 months). Complications included preeclampsia in 2, diabetes of pregnancy in 1, and abnormal liver enzymes in 1. Within 6 months of delivery, there were 2 cases of pneumonia, 2 cases of obliterative bronchiolitis, 1 case of tuberculosis, and 1 case of mild acute rejection. Forced expiratory volume in 1 second was stable at 3 (-1.5%; p = 0.55) and 12 months (1.4%; p = 0.84) after pregnancy. Mean change in Forced expiratory volume in 1 second during full-term pregnancies was -2.4% (p = 0.29), and the mean change in forced vital capacity was -0.8% (p = 0.55). In the first trimester, 83% of patients had a fall in creatinine, and a universal fall in CNI trough levels was seen. In carefully selected patients, planned pregnancy after lung transplant can be successful. Complications are common, and close monitoring of immunosuppression and renal function is needed.

Immunosuppressive therapy in allograft transplantation: from novel insights and strategies to tolerance and challenges.

Immunosuppression therapy is the key to successful post-transplantation outcomes. The need for ideal immunosuppression became durable maintenance of long-term graft survival. In spite of current immunosuppressive therapy regimens advances, surgical procedures, and preservation methods, organ transplantation is associated with a long-term poor survival and significant mortality. This has led to an increased interest to optimize outcomes while minimizing associated toxicity by using alternative methods for maintenance immunosuppression, organ rejection treatment, and monitoring of immunosuppression. T regulatory (Treg) cells, which have immunosuppressive functions and cytokine profiles, have been studied during the last decades. Treg cells are able to inhibit the development of allergen-specific cell responses and consequently play a key role in a healthy immune response to allergens. Mature dendritic cells (DCs) play a crucial role in the differentiation of Tregs, which are known to regulate allergic inflammatory responses. Advance in long-standing allograft outcomes may depend on new drugs with novel mechanisms of action with minimal toxicity. Newer treatment techniques have been developed, including using novel stem cell-based therapies such as mesenchymal stem cells, phagosomes and exosomes. Immunoisolation techniques and salvage therapies, including photopheresis and total lymphoid irradiation have emerged as alternative therapeutic choices. The present review evaluates the recent clinical advances in immunosuppressive therapies for organ transplantation.

Comparison between ultra-performance liquid chromatography with tandem mass spectrometry and a chemiluminescence immunoassay in the determination of cyclosporin A and tacrolimus levels in whole blood

Regular immunosuppressant drug monitoring is important for maintaining the drug concentrations of organ recipients within the therapeutic range. The standardized liquid chromatography-tandem mass spectrometry (LC-TMS) technique has been used for the accurate analysis of immunosuppressive drugs. In the present study, the performance of the recently developed high-throughput, rapid ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-TMS) method was validated for the simultaneous measurement of cyclosporin A and tacrolimus in whole blood. The method of measuring cyclosporin A and tacrolimus using UPLC-TMS was established and the precision, limit of detection (LOD), limit of quantitation (LOQ) and matrix effect were validated. In addition, the performance of UPLC-TMS was compared with that of a chemiluminescence immunoassay (CLIA) in >3,400 clinical specimens. The UPLC-TMS revealed a within-run and between-run precision of <8% and showed a bias of <5%. The LOD and LOQ were 2.0 and 2.5 ng/ml for cyclosporin A, and 0.3 and 0.4 ng/ml for tacrolimus, respectively. Interference from the matrix was not observed. The CLIA measurements of cyclosporin A and tacrolimus showed correlations corresponding with the formulae: Concentration(CLIA) = 1.18 × UPLC-TMS – 5.85; [95% CI: proportional, 1.16–1.19; constant, −6.86–(−4.81)] and Concentration(CLIA) = 1.14 × UPLC-TMS – 0.38; [(95% CI: proportional, 1.13–1.14; constant, −0.35–(−0.43)], respectively. The majority of results were higher for the immunoassay than for the UPLC-TMS. The newly developed rapid UPLC-TMS method was suitable for use with a large therapeutic concentration range of the analyzed immunosuppressive drugs. Sample preparation was simple and it was possible to detect several immunosuppressants simultaneously, thus significantly lowering the cost of analysis. In conclusion, this method may contribute to improved accuracy and may be preferred to immunoassays for the routine clinical measurement of immunosuppressive drug concentrations in whole blood.

Immunosuppressant therapeutic drug monitoring by LC-MS/MS: Workflow optimization through automated processing of whole blood samples

ObjectivesAlthough, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform. MethodsSix-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler. ResultsCalibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal (n=390, r=0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6h. ConclusionsOur findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods.

ABO-Incompatible Liver Transplantation in Acute Liver Failure: A Single Portuguese Center Study

IntroductionABO-incompatible liver transplantation (ABOi LT) is considered to be a rescue option in emergency transplantation. Herein, we have reported our experience with ABOi LT including long-term survival and major complications in these situations. Patient and MethodsABOi LT was performed in cases of severe hepatic failure with imminent death. The standard immunosuppression consisted of basiliximab, corticosteroids, tacrolimus, and mycophenolate mofetil. Pretransplantation patients with anti-ABO titers above 16 underwent plasmapheresis. If the titer was above 128, intravenous immunoglobulin (IVIG) was added at the end of plasmapheresis. The therapeutic approach was based on the clinical situation, hepatic function, and titer evolution. A rapid increase in titer required five consecutive plasmapheresis sessions followed by administration of IVIG, and at the end of the fifth session, rituximab. ResultsFrom January 2009 to July 2012, 10 patients, including 4 men and 6 women of mean age 47.8 years (range, 29 to 64 years), underwent ABOi LT. At a mean follow-up of 19.6 months (range, 2 days to 39 months), 5 patients are alive including 4 with their original grafts. One patient was retransplanted at 9 months. Major complications were infections, which were responsible for 3 deaths due to multiorgan septic failure (2 during the first month); rejection episodes (4 biopsy-proven of humoral rejections in 3 patients and 1 cellular rejection) and biliary. ConclusionThe use of ABOi LT as a life-saving procedure is justifiable in emergencies when no other donor is available. With careful recipient selection close monitoring of hemagglutinins and specific immunosuppression we have obtained acceptable outcomes.

Two rapid ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods with common sample pretreatment for therapeutic drug monitoring of immunosuppressants compared to immunoassay

Therapeutic drug monitoring of immunosuppressive agents is a critical and essential part of patient therapy after organ transplantation. We have developed high-throughput, robust, and rapid liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) methods with common pretreatment procedures for simultaneous quantification of four immunosuppressive agents (everolimus, sirolimus, tacrolimus, and cyclosporin A) in whole blood and one immunosuppressant (mycophenolic acid) in plasma. The new approach used in this work is based on improved sample preparation procedures allowing the analysis of five immunosuppressive drugs. Whole blood was prepared by transferring 100μL of blood into a 1.5-mL silanized conical test tube. Zinc sulfate solution (150μL), containing deuterated internal standards, was added to perform hemolysis. The samples were vortexing for 10s, followed by the addition of 250μL acetonitrile, containing internal standard for cyclosporin A, to precipitate proteins. The mixture was vortexed for 1min and centrifuged for 2min at 14,000rpm. The whole supernatant was transferred to a vial. To prepare blood plasma, the hemolysis step involving the addition of zinc sulfate was omitted and, instead of acetonitrile, methanol was used as the solvent for the internal standard (mycophenolic acid-d3). The volumes of chemicals used in this procedure were the same as those used in the procedure for immunosuppressants in whole blood. The basic validation parameters for the analytical methods were limits of detection (0.5ng/mL for everolimus, sirolimus and tacrolimus, 25ng/mL for cyclosporin A and 100ng/mL for mycophenolic acid), precision (<15%), recovery (>84%), repeatability and reproducibility. Possible mutual ion suppression was eliminated in the presence of internal standards. The method developed for the quantitation of immunosuppressants in whole blood was used to analyze 276 patient samples containing tacrolimus and 55 samples containing cyclosporin A. The results from LC/MS/MS were compared to those obtained from immunoassays of the same samples. Immunoassays significantly overestimated the concentrations of immunosuppressants.

Clinical validation and implementation of a multiplexed immunosuppressant assay in dried blood spots by LC–MS/MS

BackgroundTherapeutic drug monitoring of immunosuppressive drugs is important in transplant patients. We developed and validated liquid chromatography–mass spectrometry (LC–MS/MS) assay for simultaneous quantitation of tacrolimus (TaC), sirolimus (SrL), and cyclosporin A (CsA) in dried blood spots (DBSs) to offer patients home sample collection, avoiding travel for blood draws. MethodsAfter extraction, samples were analyzed by LC–MS/MS in multiple reaction monitoring mode. ResultsThe assay was linear between 1.2–40ng/ml for TaC and SrL, and 30–1000ng/ml for CsA. Inter- and intra-assay CVs were ≤14.8% for all 3 drugs. This method correlated well with the existing clinical whole blood assay, with coefficients of determination >0.95 for all 3 drugs. DBS quality control samples were stable for at least 30days at −20, 4, and 25°C. Stability of patient DBS samples was at least 5days at temperatures up to 60°C, except for SrL where degradation was observed at 60°C within 24h. No effect of hematocrit level, blood spot volume or punch location was observed. ConclusionImmunosuppressant levels measured in DBS correlate with whole blood LC–MS/MS assay and may contribute to successful outcome of organ transplant and patient satisfaction.

Validation and application of a fast semi-automated whole blood extraction for LC-MS/MS simultaneous quantification of cyclosporine A, tacrolimus, sirolimus and everolimus – application to high throughput routine therapeutic drug monitoring

Numerous laboratories carried out simultaneous whole blood quantitation of everolimus, sirolimus, tacrolimus and cyclosporine-A on a liquid chromatography-tandem mass-spectrometry (LC-MS/MS) system with online solid phase extraction (SPE) after manual deproteinization and centrifugation. This method had a relatively low sample throughput due to labor intensive and time-consuming sample preparation. We develop a fast and robust method of semi-automated whole blood sample pre-treatment able to support over 120 samples daily. Whole blood samples were mixed in a V-bottom plate with 2 volumes of ZnSO4/methanol containing deuterated internal standards. After centrifugation, the supernatant was loaded on an online-SPE before being transferred and monitored on the LC-MS/MS system. Assay performance included inter- and intra-day accuracy and imprecision, relative and absolute matrix effects, extraction recovery and selectivity. Intra- and inter-day imprecision was <15% for all tested concentrations. The lower limit of quantification of cyclosporine-A, everolimus, sirolimus and tacrolimus was respectively 26.6, 1.13, 1.57 and 1.28 ng mL−1, with an accuracy between −0.63% and 11.48% and a precision <12% of the nominal concentration. Standard line slope imprecision did not exceed 20% and ion enhancement or suppression was negligible. The comparison methods were performed by Passing and Bablok regression analysis: a high correlation was found between the two pre-treatment methods for tacrolimus, everolimus and sirolimus and between LC-MS/MS vs. radio-immuno-assay for cyclosporine-A. We developed a fast, sound and less misleading analytical method for high throughput (over 120 samples daily) therapeutic drug monitoring of multiple immunosuppressive drugs in routine practice.

Executive Summary: Monitoring of Nonsteroidal Immunosuppressive Drugs in Patients With Lung Disease and Lung Transplant Recipients: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines

Objectives Immunosuppressive pharmacologic agents prescribed to patients with diffuse interstitial and inflammatory lung disease and lung transplant recipients are associated with potential risks for adverse reactions. Strategies for minimizing such risks include administering these drugs according to established, safe protocols; monitoring to detect manifestations of toxicity; and patient education. Hence, an evidence-based guideline for physicians can improve safety and optimize the likelihood of a successful outcome. To maximize the likelihood that these agents will be used safely, the American College of Chest Physicians established a committee to examine the clinical evidence for the administration and monitoring of immunosuppressive drugs (with the exception of corticosteroids) to identify associated toxicities associated with each drug and appropriate protocols for monitoring these agents. Methods Committee members developed and refined a series of questions about toxicities of immunosuppressives and current approaches to administration and monitoring. A systematic review was carried out by the American College of Chest Physicians. Committee members were supplied with this information and created this evidence-based guideline. Conclusions It is hoped that these guidelines will improve patient safety when immunosuppressive drugs are given to lung transplant recipients and to patients with diffuse interstitial lung disease.

Monitoring of Nonsteroidal Immunosuppressive Drugs in Patients With Lung Disease and Lung Transplant Recipients: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines

Immunosuppressive pharmacologic agents prescribed to patients with diffuse interstitial and inflammatory lung disease and lung transplant recipients are associated with potential risks for adverse reactions. Strategies for minimizing such risks include administering these drugs according to established, safe protocols; monitoring to detect manifestations of toxicity; and patient education. Hence, an evidence-based guideline for physicians can improve safety and optimize the likelihood of a successful outcome. To maximize the likelihood that these agents will be used safely, the American College of Chest Physicians established a committee to examine the clinical evidence for the administration and monitoring of immunosuppressive drugs (with the exception of corticosteroids) to identify associated toxicities associated with each drug and appropriate protocols for monitoring these agents. Committee members developed and refined a series of questions about toxicities of immunosuppressives and current approaches to administration and monitoring. A systematic review was carried out by the American College of Chest Physicians. Committee members were supplied with this information and created this evidence-based guideline. It is hoped that these guidelines will improve patient safety when immunosuppressive drugs are given to lung transplant recipients and to patients with diffuse interstitial lung disease.

Epstein- Barr Virus Related Malignant and Non-Malignant Transformation in Two Paediatric Liver Transplant Patients

Introduction: Epstein-Barr virus (EBV)-associated smooth muscle tumors (EBV-SMT) are a distinctive group of mesenchymal tumors that develop exclusively in patients with clinical immunosuppression, predominantly in HIV and recipients of solid organ transplantation, particularly paediatric patients. The incidence of developing neoplastic disorders after solid organ transplantation is highly dependent on the intensity of the prophylactic anti-viral regimen, the induction therapy, as well as high levels of calcineurin inhibitors. Recently 2 of our liver transplant patients have been diagnosed with both benign and malignant EBV- related smooth muscle tumours presenting as significant gastrointestinal bleeds. Since the 1970‘s, there have been a small number of cases of leiomyomas in immunosuppressed patients post transplant. Case Presentation: Patient ZH underwent a liver transplant for missed biliary atresia at one year five months of age. Her course was complicated by portal vein thrombosis and a failed Meso-Rex shunt two months post transplant. Despite this, her liver remained well perfused on hepatic arterial blood supply with no problems with rejection. A meso-caval shunt was done at six years of age when she continued having significant gastrointestinal bleeds. A mesenteric lymph node removed incidentally during surgery was consistent with leiomyosarcoma on histology. Further imaging showed lung metastatic lesions and at a subsequent laparotomy a primary ileal lesion was resected. She completed chemotherapy and six weeks later had another gastrointestinal bleed which revealed a leiomyoma on histology from a rectal biopsy. She was commenced on IVI gancyclovir and tacrolimus was changed to sirolimus. Despite the above management, a repeat colonic biopsy following another gastrointestinal bleed confirmed the presence of a leiomyoma. Patient AJG underwent a liver transplant at four years of age for biliary atresia. He had an uncomplicated course post transplant except for raised EBV levels initially treated with three months of intravenous gancyclovir and then oral valgancyclovir. He had a massive gastrointestinal bleed two years later which revealed features consistent with post transplant lymphoproliferative disorder of terminal ileum and leiomyoma in the sigmoid colon. Discussion: Smooth muscle EBV induced tumours after organ transplantation are rare, occurring more often in the paediatric population. These tumours are generally insidious and their prognosis depends on the size, site and surgical options. There is no standard treatment. Although the benign variant carries a good prognosis, leiomyosarcoma carries a poor prognosis. We are highlighting these two cases to emphasise the need for ongoing vigilance to detect EBV-associated malignancies with regular EBV PCR monitoring and adjustment of immunosuppression if EBV PCR titres rise. Management involves an interdisciplinary approach with input from transplant physicians, surgeons and oncologists. Management involves surgical resection of all lesions if possible, reduction of immunosuppression and possibly changing from a calcineurin inhibitor to a MTOR inhibitor. Once malignant transformation has occurred, the prognosis is poor.

Impact of Immunosuppression on the Development of Epstein-Barr Virus (EBV) Viremia After Pediatric Liver Transplantation

ObjectivesPediatric liver transplant (OLT) patients are at risk of posttransplant lymphoproliferative disease (PTLD) from Epstein-Barr virus (EBV). This study examined the impact of induction and immunosuppression on EBV viremia. MethodsA retrospective chart review was performed on 197 pediatric patients and induction regimen, immunosuppression levels, and EBV viremia were documented for 1 year post-OLT. Logistic regression models determined associations between induction, immunosuppression, and EBV. ResultsFifty six percent of patients developed EBV viremia. Incidence of EBV viremia was 73% with antithymocyte globulin (ATG), 63% with daclizumab, and 39% for neither, though the trend was not significant [ATG: odds ratio (OR) 0.19; 95% confidence interval (CI) 0.024–1.58; P = .125; daclizumab OR; 1.07; 95% CI 0.270–4.23; P = .925]. Tacrolimus immunosuppression levels were supratherapeutic 28.7% of the time; however, only supratherapeutic tacrolimus levels between 0 and 2 weeks increased EBV viremia at 2 to 4 weeks post-OLT (OR 1.80; 95% CI 1.10–2.94; P = .02). Three patients developed PTLD. ConclusionsThe use of ATG and daclizumab induction likely does not play a role in the development of EBV viremia. Supratherapeutic tacrolimus levels 0 to 2 weeks post-OLT impact the development of EBV viremia at 2 to 4 weeks. The incidence of PTLD was low, suggesting better EBV and immunosuppression monitoring plays an important role in reducing PTLD.

Use of immune function test in monitoring immunosuppression in liver transplant recipients

Immune function test (Immuknow™) is a measure of cell-mediated immunity based on peripheral CD4+ T cell adenosine triphosphate activity (desired range, 225-525 ng/mL). We evaluated the role of immune function test (IFT) in monitoring and adjustment of immunosuppression in orthotopic liver transplant (OLT) recipients. A total of 289 IFTs were obtained from 171 patients from March 2007 to June 2008. Graft/patient status was classified as stable, serious infection, or malignancy. IFT levels were analyzed with duration of follow-up after OLT, graft/patient status, and the presence of hepatitis C (HCV) infection. The mean age was 54±14 yr, with 62% men. The median follow-up was 65 (2-249) months. Mean IFT levels were significantly lower in patients who were <24 months than in those≥24 months post-OLT (220±19.5 vs. 257±11.3 ng/mL, p=0.03). Clinically stable patients had higher IFT levels than those with serious infection or malignancy (254±11.1 vs. 162.5±23.9, p<0.001). HCV-infected patients had lower IFT levels than uninfected patients (206.7±15.7 vs. 273±12.0 ng/mL, p<0.001). Immunosuppression was reduced in 58 patients with IFT levels<225 ng/mL, and 90% maintained stable graft function after a median follow-up of 22 (1-39) months. IFT may be a useful tool in monitoring and lowering of immunosuppression in long-term OLT recipients.

Liquid Chromatography–Tandem Mass Spectrometry or Automated Immunoassays: What Are the Future Trends in Therapeutic Drug Monitoring?

Therapeutic drug monitoring (TDM)6 of certain drugs with a narrow therapeutic index significantly improves patient outcome. For example, the quantification of immunosuppressive drugs in samples from patients after organ transplantation is an essential prerequisite for the prevention of both adverse drug reactions and rejection events. The need for accurate, precise, and standardized measurement of drugs presents a major challenge for clinical laboratories and the diagnostics industry. A plethora of different techniques have been developed in the past to meet these requirements. Currently, liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based methods and immunoassays appear to be the most prevalent approaches in clinical laboratories. Since these techniques differ in many aspects, in making the choice the laboratory must consider the technical, clinical, and economic criteria, as well as staff qualifications. In this Q&A, 4 leading experts in the field of immunosuppressive drug monitoring present their opinions on the advantages and disadvantages of the analytical methods and provide a general guide for the optimization of analytical strategies in the field of TDM. From your perspective, what are the advantages and disadvantages of mass spectrometry–based assays and immunoassays in the context of TDM? Gregory Maine: One of the main advantages of mass spectrometry is that users can develop their own assays. Mass spectrometry–based assays can be analytically sensitive, specific, and capable of measuring several compounds in a single run. The disadvantages of this technique include that the instrument is not a “turnkey” analyzer, it requires large capital investment and service costs, it is subject to matrix effects and ion suppression, and the results from different laboratories are not necessarily comparable. The advantages of drug immunoassays include their easy integration into the core laboratory and the ability to measure them along with other immunoassays, such as those for serologic factors and hormones. In addition, TDM immunoassays …

Determination of four immunosuppressive drugs in whole blood using MEPS and LC–MS/MS allowing automated sample work-up and analysis

In treatment with immunosuppressive drugs, monitoring of blood drug concentration is needed. The aim of this work was to explore micro extraction by packed sorbent (MEPS) as a possible on-line sample preparation method in combination with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for quantification of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. An automated on-line MEPS system connected with a LC–MS/MS instrument was set up. A C8 sorbent was used for the MEPS extraction. Subsequent analysis was performed with a gradient LC system. The adduct ions [M+NH4]+ of the analytes were monitored in SRM mode for quantification. Ascomycin and cyclosporine D were used as internal standards. The chromatographic run time 2.5min and the quantification ranges were 3–1500ng/mL (r2≥0.999, n=6) for cyclosporine and 0.5–50ng/mL for everolimus, sirolimus and tacrolimus (r2≥0.998, 0.994 and 0.993, respectively, n=6). Precision and accuracy were documented at three levels. Accuracy results were between 102% and 109% with precision between 2% and 13% and carry over <0.02%. Matrix effects were characterized and found to be below 20%. The quantifications obtained were in agreement with a reference LC–MS/MS method based on protein precipitation, and results obtained from external proficiency test samples compared with the mean of all other LC–mass spectrometry methods showed good agreement. This method provides an accurate, precise and automated procedure that can be applied for therapeutic drug monitoring of immunosuppressive drugs in clinical laboratories equipped with LC–MS/MS.

Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA

BackgroundTherapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated.From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards.MethodsPlasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes.ResultsLow quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 μg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards.ConclusionsThe authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid.

Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR‐inhibitors in peripheral human blood

Therapeutic drug monitoring (TDM) of immunosuppressive drugs after organ transplantation is based on measuring blood levels alone, which often results in under- or over-immunosuppression. Previous studies have shown the potential of measuring pharmacodynamic drug effects for TDM, but assessment of biomarkers for individual drugs is still not clinical routine. Therefore, we validated a specific assay to measure the pharmacodynamic effects of mammalian target of rapamycin (mTOR)-inhibitors on phosphorylated S6 ribosomal protein (p-S6RP), a downstream target of mTOR. Clinical relevant concentrations of sirolimus (SRL, 0.9-91.4 μg/L), cyclosporine A (CsA, 75.1-1202 μg/L), mycophenolate acid (MPA, 0.08-3.2 mg/L), or dexamethasone (DEX, 0.5-200 ng/mL) were added to whole-blood from healthy volunteers. Activated whole-blood was analyzed by phospho-flow cytometry to measure p-S6RP in T cells. Phospho-flow analysis revealed that SRL suppressed p-S6RP in human T cells in a dose-dependent manner with a half-maximal inhibitory concentration (IC(50)) at 19.8 nM and a maximal inhibitory effect (I(max) %) at 91.9%. Neither CsA, MPA, nor DEX inhibited mTOR-related S6RP-phosphorylation. Coefficient of variations from 0.03 to 0.05, 0.12 to 0.25, and 0.14 to 0.38 for intra-, interassay, and interindividual variability respectively, showed robustness of our assay. Furthermore, samples can be stored at RT or 4°C up to 2 h after withdrawal. We validated a robust whole-blood assay that allows the specific measurement of SRL- and everolimus-induced inhibition of T cells' function through detection of p-S6RP. Future studies in organ transplanted recipients will show if this assay has the potential to enhance a TDM for mTOR-inhibitor drugs in combination therapies.