Therapeutic Drug Monitoring of Antibiotic Drugs: The Role of the Clinical Laboratory.

BACKGROUND: Therapeutic drug monitoring (TDM) of anti-infective drugs is an increasingly complex field, given that in addition to the patient and drug as 2 usual determinants, its success is driven by the pathogen. Pharmacodynamics is related both to the patient (toxicity) and bacterium (efficacy or antibiotic susceptibility). The specifics of TDM of antimicrobial drugs stress the need for multidisciplinary knowledge and expertise, as in any other field. The role and the responsibility of the laboratory in this interplay are both central and multifaceted. This narrative review highlights the role of the clinical laboratory in the TDM process. METHODS: A literature search was conducted in PubMed and Google Scholar, focusing on the past 5 years (studies published since 2016) to limit redundancy with previously published review articles. Furthermore, the references cited in identified publications of interest were screened for additional relevant studies and articles. RESULTS: The authors addressed microbiological methods to determine antibiotic susceptibility, immunochemical and chromatographic methods to measure drug concentrations (primarily in blood samples), and endogenous clinical laboratory biomarkers to monitor treatment efficacy and toxicity. The advantages and disadvantages of these methods are critically discussed, along with existing gaps and future perspectives on strategies to provide clinicians with as reliable and useful results as possible. CONCLUSIONS: Although interest in the field has been the driver for certain progress in analytical technology and quality in recent years, laboratory professionals and commercial providers persistently encounter numerous unresolved challenges. The main tasks that need tackling include broadly and continuously available, easily operated, and cost-effective tests that offer short turnaround times, combined with reliable and easy-to-interpret results. Various fields of research are currently addressing these features.

Assessment of urine sample quality by the simultaneous measurement of urinary γ-glutamyltransferase and lactate dehydrogenase enzyme activities: possible application to unravel cheating in drugs of abuse testing.

OBJECTIVES: Evaluation of the simultaneous measurement of urinary γ-glutamyltransferase (γGT) and lactate dehydrogenase (LDH) to discriminate fresh from previously frozen specimens in urine drug monitoring. METHODS: Two widely available photometric tests (Siemens Healthineers Atellica) were used to determine the range of urinary γGT and LDH excretion and to study the decay in urinary enzyme activity under various storage conditions (room temperature, 4-8 °C, -18 °C, -80 °C). From these data, cut-off values were established and evaluated in split (fresh/frozen) specimens. RESULTS: Both assays allow robust, reliable, and simultaneous determination of urinary γGT and LDH. In healthy subjects, the 95% reference intervals for enzyme activity in native urine were γGT: 24.4-100.4 U/g Crea (creatinine) and LDH: 2.5-45.8 U/g Crea. Frozen storage for at least 7 days at -18 °C resulted in a loss of activity to less than 50% in both enzymes. Cut-offs for frozen samples were γGT≤33.2 U/g Crea and LDH≤ 8.4 U/g Crea. When applied to 100 sample pairs (fresh/frozen), 86.5% (173/200) of the measurements were conclusive and the combination of concordant enzyme measurements (low γGT/low LDH or high γGT/high LDH) was able to predict the mode of storage with a sensitivity of 96.3% and a specificity of 96.7%. CONCLUSIONS: The additional measurements of urinary γGT and LDH can be used to detect previously frozen urine specimens. A simple protocol is proposed to provide additional information on sample quality when deceit is suspected. The procedure can be easily integrated into the standard workflow of urinary drug monitoring.

Personalized Therapy for Mycophenolate: Consensus Report by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.

ABSTRACT: When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.

Time-Dependent Apparent Increase in dd-cfDNA Percentage in Clinically Stable Patients Between One and Five Years Following Kidney Transplantation.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is reportedly a valuable tool for graft surveillance following kidney transplantation (KTx). Possible changes in dd-cfDNA(%) reference values over time have not been evaluated. For long-term monitoring after KTx, changes in host cfDNA might represent a biasing factor in dd-cfDNA(%) determinations. METHODS: Plasma samples were obtained (n = 929) 12-60 months after engraftment in a cross-sectional cohort of 303 clinically stable KTx recipients. Total cfDNA(copies/mL), dd-cfDNA(%), and dd-cfDNA(copies/mL) were determined using droplet-digital PCR. Stability of threshold values in these stable KTx recipients over time was assessed by 80th, 85th, and 90th quantile regression. RESULTS: Upper percentiles of total cfDNA showed a significant decline of -1902, -3589, and -4753 cp/mL/log(month) (P = 0.014, <0.001, and 0.017, respectively), resulting in increasing dd-cfDNA(%) percentiles by 0.25, 0.46, and 0.72%/log(month) (P = 0.04, 0.001, and 0.002, respectively), with doubling of the 85th percentile value by 5 years. In contrast, dd-cfDNA(cp/mL) was stable during the observation period (P = 0.52, 0.29, and 0.39). In parallel increasing white blood cell counts and decreasing tacrolimus concentrations over time were observed. After 5 years, the median total cfDNA was still 1.6-fold (P < 0.001) higher in KTx recipients than in healthy controls (n = 135) and 1.4-fold (P < 0.001) higher than patients with other medical conditions (n = 364). CONCLUSIONS: The time-dependent decrease of host cfDNA resulted in an apparent increase of dd-cfDNA fraction in stable KTx patients. For long-term surveillance, measurement of absolute dd-cfDNA concentrations appears to be superior to percentages to minimize false positive results.

Absolute quantification of donor-derived cell-free DNA as a marker of rejection and graft injury in kidney transplantation: Results from a prospective observational study.

Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for comprehensive monitoring of allograft injury and rejection in kidney transplantation (KTx). dd-cfDNA quantification of copies/mL plasma (dd-cfDNA[cp/mL]) was compared to dd-cfDNA fraction (dd-cfDNA[%]) at prespecified visits in 189 patients over 1 year post KTx. In patients (N = 15, n = 22 samples) with biopsy-proven rejection (BPR), median dd-cfDNA(cp/mL) was 3.3-fold and median dd-cfDNA(%) 2.0-fold higher (82 cp/mL; 0.57%, respectively) than medians in Stable Phase patients (N = 83, n = 408) without rejection (25 cp/mL; 0.29%). Results for acute tubular necrosis (ATN) were not significantly different from those with biopsy-proven rejection (BPR). dd-cfDNA identified unnecessary biopsies triggered by a rise in plasma creatinine. Receiver operating characteristic (ROC) analysis showed superior performance (P = .02) of measuring dd-cfDNA(cp/mL) (AUC = 0.83) compared to dd-cfDNA(%) (area under the curve [AUC] = 0.73). Diagnostic odds ratios were 7.31 for dd-cfDNA(cp/mL), and 6.02 for dd-cfDNA(%) at thresholds of 52 cp/mL and 0.43%, respectively. Plasma creatinine showed a low correlation (r = 0.37) with dd-cfDNA(cp/mL). In a patient subset (N = 24) there was a significantly higher rate of patients with elevated dd-cfDNA(cp/mL) with lower tacrolimus levels (<8 µg/L) compared to the group with higher tacrolimus concentrations (P = .0036) suggesting that dd-cfDNA may detect inadequate immunosuppression resulting in subclinical graft damage. Absolute dd-cfDNA(cp/mL) allowed for better discrimination than dd-cfDNA(%) of KTx patients with BPR and is useful to avoid unnecessary biopsies.

Improving Therapeutic Decisions: Pharmacodynamic Monitoring as an Integral Part of Therapeutic Drug Monitoring.

Although the monitoring of drug therapies based on the determination of drug concentrations in biological materials is certainly an important instrument for individualized dosing and dose adjustment with a broad variety of pharmaceuticals, its role is limited by the fact that it does not reflect pharmacodynamic (PD) and toxicodynamic interactions such as those caused by individual and environment-related factors. However, these interactions are important for both the efficacy and the safety of the drug therapy. Therefore, during recent years, there is an increased interest in personalized drug therapy as reflected by the development and clinical implementation of molecular "biomarkers" that are direct or surrogate markers of pharmacological effects [PD therapeutic drug monitoring (TDM)]. Moreover, this process is driven by new developments in instrumentation, such as mass spectrometry and array technologies, and in computational biology/pharmacology, databases, and bioinformatics. This Focus Issue of the journal focuses on current achievements in and status of PD TDM with different classes of drugs. The contributions to the present issue of Therapeutic Drug Monitoring provide a critical analysis of current practices of TDM with their limitations, introduce newer promising biomarkers in the field of PD TDM, discuss the challenges faced to date in translating preclinical tools into clinical settings, and point out recent advances in the establishment of modeling approaches that apply to pharmacokinetics (PK)/PD as well as pharmacogenetic information.

Pharmacokinetic and Pharmacodynamic Drug Monitoring of Direct-Acting Oral Anticoagulants: Where Do We Stand?

For decades, oral anticoagulation has been based on vitamin K antagonist such as warfarin, which requires pharmacodynamic (PD) drug monitoring to guide the therapy. The drug effect is measured by the clotting test prothrombin time and expressed as international normalized ratio. New direct oral anticoagulants are increasingly used in fixed-dose regimens but are licensed without any therapy monitoring. However, extensive clinical experiences have demonstrated that interindividual variations in the response to the therapy with direct oral anticoagulants do exist. In situations such as bleeding or thrombosis, therapeutic drug monitoring could be useful. Unfortunately, global coagulation assays such as the prothrombin time or the activated partial thrombin time are not suitable for this purpose. To measure drug concentrations, more specific coagulation test can be used if they are externally calibrated with the respective drugs. For the direct thrombin inhibitor dabigatran etexilate, a calibrated diluted thrombin time or ecarin clotting time can be used, whereas for anti-factor Xa drugs such as rivaroxaban, apixaban, edoxaban, and betrixaban, calibrated anti-factor Xa assays are appropriate. However, the gold standard to measure drug concentrations is LC-MS/MS. The variation in bleeding and thrombotic events noted with both drug classes under fixed-dose conditions suggests additional interindividual PD differences. Therefore, PD monitoring to individualize the therapy may be an option. For dabigatran, this is the inhibition of thrombin formation and for anti-factor Xa drugs, the inhibition of factor Xa activity, which can be followed using the functional assays mentioned above but without calibration. Alternatively, thrombin generation assays have been proposed for both drug classes. So far, not many clinical data have been published about the potentially beneficial effects of PD monitoring for dose individualization. The assay platforms for PD monitoring are present in many clinical laboratories, but efforts are needed to validate and standardize available assays to perform appropriate clinical trials.

Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report.

Ten years ago, a consensus report on the optimization of tacrolimus was published in this journal. In 2017, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicity (IATDMCT) decided to issue an updated consensus report considering the most relevant advances in tacrolimus pharmacokinetics (PK), pharmacogenetics (PG), pharmacodynamics, and immunologic biomarkers, with the aim to provide analytical and drug-exposure recommendations to assist TDM professionals and clinicians to individualize tacrolimus TDM and treatment. The consensus is based on in-depth literature searches regarding each topic that is addressed in this document. Thirty-seven international experts in the field of TDM of tacrolimus as well as its PG and biomarkers contributed to the drafting of sections most relevant for their expertise. Whenever applicable, the quality of evidence and the strength of recommendations were graded according to a published grading guide. After iterated editing, the final version of the complete document was approved by all authors. For each category of solid organ and stem cell transplantation, the current state of PK monitoring is discussed and the specific targets of tacrolimus trough concentrations (predose sample C0) are presented for subgroups of patients along with the grading of these recommendations. In addition, tacrolimus area under the concentration-time curve determination is proposed as the best TDM option early after transplantation, at the time of immunosuppression minimization, for special populations, and specific clinical situations. For indications other than transplantation, the potentially effective tacrolimus concentrations in systemic treatment are discussed without formal grading. The importance of consistency, calibration, proficiency testing, and the requirement for standardization and need for traceability and reference materials is highlighted. The status for alternative approaches for tacrolimus TDM is presented including dried blood spots, volumetric absorptive microsampling, and the development of intracellular measurements of tacrolimus. The association between CYP3A5 genotype and tacrolimus dose requirement is consistent (Grading A I). So far, pharmacodynamic and immunologic biomarkers have not entered routine monitoring, but determination of residual nuclear factor of activated T cells-regulated gene expression supports the identification of renal transplant recipients at risk of rejection, infections, and malignancy (B II). In addition, monitoring intracellular T-cell IFN-g production can help to identify kidney and liver transplant recipients at high risk of acute rejection (B II) and select good candidates for immunosuppression minimization (B II). Although cell-free DNA seems a promising biomarker of acute donor injury and to assess the minimally effective C0 of tacrolimus, multicenter prospective interventional studies are required to better evaluate its clinical utility in solid organ transplantation. Population PK models including CYP3A5 and CYP3A4 genotypes will be considered to guide initial tacrolimus dosing. Future studies should investigate the clinical benefit of time-to-event models to better evaluate biomarkers as predictive of personal response, the risk of rejection, and graft outcome. The Expert Committee concludes that considerable advances in the different fields of tacrolimus monitoring have been achieved during this last decade. Continued efforts should focus on the opportunities to implement in clinical routine the combination of new standardized PK approaches with PG, and valid biomarkers to further personalize tacrolimus therapy and to improve long-term outcomes for treated patients.

Evaluation of an Ion Trap Toxtyper Liquid Chromatography With An Ion Trap Mass Spectrometric Instrument (Toxtyper) for Drug of Abuse Screening in Oral Fluid.

BACKGROUND: Oral fluid (OF) is increasingly used as an alternative sample matrix in drug of abuse screening. Screening is commonly performed by immunoassays and results confirmed using laborious gas chromatography-mass spectrometry (GC-MS)-based methods. Therefore, an easy to operate ion trap mass spectrometric (IT-MS) commercial screening method (Toxtyper; Bruker Daltronik, Bremen, Germany) combined with a laboratory-developed sample preparation procedure has been evaluated for their application to OF. METHODS: OF samples were subjected to protein precipitation followed by HybridSPE-Phospholipid extraction. Chromatographic separation was achieved by ultra-high-performance liquid chromatography; MS2/MS3 spectra were recorded by IT-MS and analyzed using a library provided by the manufacturer (Bruker Daltronik). The lower limit of detection, linearity, imprecision, inaccuracy, and specificity (interferences and matrix effects) were investigated for methadone, buprenorphine, pregabalin, fentanyl, amphetamine, 3,4-methylendioxy-N-methylamphetamine, cocaine, acetylcodeine, and nordiazepam, after spiking drug-free OF with these test substances. In addition, concordance between IT-MS results and gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry, or immunoassay (buprenorphine) results was investigated. RESULTS: No interferences or matrix effects were observed. The lower limit of detection for acetylcodeine, amphetamine, benzoylecgonine, methadone, and nordiazepam was below the common cutoffs for immunological screening assays and comparable to that of GC-MS. Imprecision and inaccuracy, both in- and between-series, were consistently <25%, except for buprenorphine. Toxtyper screening for pregabalin and fentanyl was less sensitive than a targeted liquid chromatography-tandem mass spectrometry assay. A very good concordance was found between the previous analytical approach and the new IT-MS method. CONCLUSIONS: The Toxtyper IT-MS is easy to use and can be applied for the screening of drug of abuse and the qualitative confirmation analysis in OF in a clinical toxicology service. Although intended for qualitative analysis, performance data suggest that the methods investigated may also be applicable for semiquantitative longitudinal follow-up.

Multicenter Evaluation of a New Electrochemiluminescence Immunoassay for Everolimus Concentrations in Whole Blood.

BACKGROUND: The precise monitoring of everolimus, an immunosuppressant drug, is vital for transplant recipients due to its narrow therapeutic range. This study evaluated the analytical performance of a new electrochemiluminescence immunoassay (ECLIA) for everolimus concentrations in whole blood. METHODS: Accuracy, imprecision, and sensitivity studies for the Roche Elecsys everolimus ECLIA were performed at 5 European laboratories. The ECLIA was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, as well as the Quantitative Microsphere System everolimus assay. RESULTS: Everolimus ECLIA accuracies were within the range 100% ± 9%. Coefficients of variation (CVs) across the target range were ≤4.8% for repeatability and ≤8.4% for intermediate imprecision, whereas multisite reproducibility at lower (2.71 mcg/L) and higher everolimus concentrations (3.0-30.0 mcg/L) resulted in CVs of ≤13.7% and ≤12.4%, respectively. The CV at the assay's lower limit of quantification without considering bias was excellent, estimated as ≤9.3% at 0.5 mcg/L. The weighted Deming regression analysis, used for comparison of the results obtained by everolimus ECLIA and by LC-MS/MS methods, yielded a slope of 1.21 [95% confidence interval (CI): 1.15-1.26], intercept of 0.478 mcg/L (95% CI: 0.241-0.716), and a Pearson correlation coefficient (r) of 0.91. A single-site comparison between the ECLIA and the Quantitative Microsphere System assay revealed a slope of 1.05 (95% CI: 0.917-1.17), intercept of 1.03 mcg/L (95% CI: 0.351-1.70), and r of 0.91. CONCLUSIONS: Based on these results, the Roche Elecsys everolimus ECLIA can be considered suitable for routine therapeutic drug monitoring. A positive bias was observed with respect to LC-MS/MS methods, suggesting that it may be necessary to rebaseline individual patients when switching from LC-MS/MS to the ECLIA; however, this must also be considered for any change of method for everolimus measurement.

Measurement of sirolimus concentrations in human blood using an automated electrochemiluminescence immunoassay (ECLIA): a multicenter evaluation.

BACKGROUND: Therapeutic drug monitoring (TDM) of sirolimus is essential in transplant recipients. We evaluated the performance of a new electrochemiluminescence immunoassay (ECLIA) for measuring sirolimus concentrations in whole blood at five European laboratories. METHODS: Study assessments included repeatability, intermediate precision and functional sensitivity (concentration at coefficient of variation [CV] of 20%) experiments. Method comparisons with liquid chromatography-tandem mass spectrometry (LC-MS/MS; reference method) and two immunoassays (chemiluminescent microparticle immunoassay [CMIA] and antibody-conjugated magnetic immunoassay [ACMIA]) were performed using native samples from patients with kidney transplants. RESULTS: Imprecision testing CVs were ≤6.4% and ≤10.7% across the sirolimus concentration range for both repeatability and intermediate precision, respectively. The ECLIA showed excellent functional sensitivity: the CV did not reach 20%; the CV at the assay's limit of quantitation (1.5 µg/L) was 7.0%. Agreement between the ECLIA and LC-MS/MS using native kidney samples was close, with weighted Deming regression analysis yielding a slope of 1.05, an intercept of 0.154 µg/L and a Pearson's correlation coefficient (r) of 0.94, while Bland-Altman analysis showed a combined mean bias of 0.41 µg/L (±2 standard deviation [SD], -1.96 to 2.68). The ECLIA also showed good correlation with the two other immunoassays: the CMIA (slope=0.91, intercept=0.112 µg/L and r=0.89) and the ACMIA (slope=0.99, intercept=0.319 µg/L and r=0.97). CONCLUSIONS: The ECLIA showed good precision, functional sensitivity and agreement with other methods of sirolimus measurement used in clinical practice, suggesting that the assay is suitable for TDM in transplant recipients and provides an alternative to LC-MS/MS.

Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

BACKGROUND: Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy. METHODS: We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor. RESULTS: The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h). CONCLUSIONS: This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Therapeutic Drug Monitoring of Everolimus: Comparability of Concentrations Determined by 2 Immunoassays and a Liquid Chromatography Tandem Mass Spectrometry Method.

BACKGROUND: Therapeutic drug monitoring is recommended to guide therapy with the immunosuppressant everolimus (EVL) in solid organ transplantation to prevent rejections and to limit toxicity. For therapeutic drug monitoring, predose EVL concentrations are measured in whole blood mainly by liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, 2 immunoassays [Quantitative Microsphere System (QMS) EVL and Elecsys EVL] are commercially available. The aim of this study was to evaluate the comparability of EVL results determined with the 2 immunoassays and a validated LC-MS/MS test using samples from kidney, liver, and heart transplant (KT, LT, and HT, respectively) recipients. METHODS: Analysis of predose samples from KT (n = 56), LT (n = 60), and HT (n = 59) recipients, obtained at variable time points after transplantation, was performed by LC-MS/MS and with the 2 immunoassays. The QMS EVL assay was applied on Dimension Xpand Plus and the Elecsys EVL assay on cobas e 411 analyzer. Results were compared by the Spearman's rank correlation coefficient, unbiased Passing and Bablok linear regression test, and Bland-Altman plot. RESULTS: Results generated with both immunoassays correlated well with those of LC-MS/MS. An overestimation of EVL concentrations by the Elecsys EVL compared with LC-MS/MS was observed (mean bias: 34.2%). Using the QMS EVL, a small but significant negative deviation (mean bias: -8.0%) was found. Looking at KT, HT, and LT samples separately, the bias to LC-MS/MS seen with the Elecsys EVL was similar. With the QMS EVL, the best agreement was observed with the KT samples followed by LT and HT. CONCLUSIONS: Results generated by the 3 methods are not consistent regarding their diagnostic value. Both laboratories and manufacturers should take care to inform their costumers about the between-method differences to avoid misinterpretation of the results in clinical practice.

T-Cell Surface Antigens and sCD30 as Biomarkers of the Risk of Rejection in Solid Organ Transplantation.

T-cell activation is a characteristic of organ rejection. T cells, located in the draining lymph nodes of the transplant recipient, are faced with non-self-molecules presented by antigen presenting cells and become activated. Activated T cells are characterized by up-regulated surface antigens, such as costimulatory molecules, adhesion molecules, chemokine receptors, and major histocompatibility complex class II molecules. Surface antigen expression can be followed by flow cytometry using monoclonal antibodies in either cell function assays using donor-specific or nonspecific stimulation of isolated cells or whole blood and without stimulation on circulating lymphocytes. Molecules such as CD30 can be proteolytically cleaved off the surface of activated cells in vivo, and the determination of the soluble protein (sCD30) in serum or plasma is performed by immunoassays. As promising biomarkers for rejection and long-term transplant outcome, CD28 (costimulatory receptor for CD80 and CD86), CD154 (CD40 ligand), and sCD30 (tumor necrosis factor receptor superfamily, member 8) have been identified. Whereas cell function assays are time-consuming laboratory-developed tests which are difficult to standardize, commercial assays are frequently available for soluble proteins. Therefore, more data from clinical trials have been published for sCD30 compared with the surface antigens on activated T cells. This short review summarizes the association between selected surface antigens and immunosuppression, and rejection in solid organ transplantation.

Analytical Validation and Cross-Validation of an NFAT-Regulated Gene Expression Assay for Pharmacodynamic Monitoring of Therapy With Calcineurin Inhibitors.

BACKGROUND: Analysis of residual gene expression of the nuclear factor of activated T cell (NFAT)-regulated genes has been developed as a pharmacodynamic biomarker to monitor therapy with calcineurin inhibitors. The availability of commercial primer sets (Search-LC) and the well-established assay protocol makes this biomarker a promising candidate to be used clinically in different laboratories. However, implementation of the method in routine practice requires analytical robustness and comparable results across laboratories. Therefore, a protocol originally established at the Institute of Immunology, Heidelberg was verified at the Institute of Laboratory Medicine, Klinikum Stuttgart, and a comparison study was conducted between the 2 laboratories. METHODS: For the analytical verification, whole blood samples of healthy individuals were incubated with tacrolimus in vitro. Linearity, imprecision, and limit of quantification, as well as sample stability, were investigated. For interlaboratory comparison, samples of patients under cyclosporine A therapy were analyzed in Heidelberg and then reanalyzed in Stuttgart within 24 hours. RESULTS: Tacrolimus (6.25-50 mcg/L) decreased the expression of NFAT-regulated genes in vitro dose dependently (15%-89%). Within- and between-assay coefficient of variations (n = 6 each) were <17%. The limit of quantification was <200 cDNA copies for each of the interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor genes. Samples were stable for 24 hours. Interlaboratory comparison using patient samples correlated well (r = 0.951) but showed an inconsistent bias depending on the magnitude of residual gene expression. CONCLUSIONS: The assay can be set up with a satisfactory analytical performance in a routine molecular biological laboratory and shows comparable results between laboratories. The reproducibility of the NFAT-regulated gene expression assay across laboratories can facilitate the implementation of this assay for pharmacodynamic routine monitoring of calcineurin inhibitors in different centers.

Analytical Aspects of the Implementation of Biomarkers in Clinical Transplantation.

In response to the urgent need for new reliable biomarkers to complement the guidance of the immunosuppressive therapy, a huge number of biomarker candidates to be implemented in clinical practice have been introduced to the transplant community. This includes a diverse range of molecules with very different molecular weights, chemical and physical properties, ex vivo stabilities, in vivo kinetic behaviors, and levels of similarity to other molecules, etc. In addition, a large body of different analytical techniques and assay protocols can be used to measure biomarkers. Sometimes, a complex software-based data evaluation is a prerequisite for appropriate interpretation of the results and for their reporting. Although some analytical procedures are of great value for research purposes, they may be too complex for implementation in a clinical setting. Whereas the proof of "fitness for purpose" is appropriate for validation of biomarker assays used in exploratory drug development studies, a higher level of analytical validation must be achieved and eventually advanced analytical performance might be necessary before diagnostic application in transplantation medicine. A high level of consistency of results between laboratories and between methods (if applicable) should be obtained and maintained to make biomarkers effective instruments in support of therapeutic decisions. This overview focuses on preanalytical and analytical aspects to be considered for the implementation of new biomarkers for adjusting immunosuppression in a clinical setting and highlights critical points to be addressed on the way to make them suitable as diagnostic tools. These include but are not limited to appropriate method validation, standardization, education, automation, and commercialization.

Assuring the Proper Analytical Performance of Measurement Procedures for Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee.

Monitoring immunosuppressive drugs (ISDs) in blood or plasma is still a key therapeutic drug monitoring (TDM) application in clinical settings. Narrow target ranges and severe side effects at drug underexposure or overexposure make accurate and precise measurements a must. This overview prepared by the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology is intended to serve as a summary and guidance document describing the current state-of-the-art in the TDM of ISDs.

Therapeutic Drug Monitoring of Everolimus: A Consensus Report.

In 2014, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology called a meeting of international experts to provide recommendations to guide therapeutic drug monitoring (TDM) of everolimus (EVR) and its optimal use in clinical practice. EVR is a potent inhibitor of the mammalian target of rapamycin, approved for the prevention of organ transplant rejection and for the treatment of various types of cancer and tuberous sclerosis complex. EVR fulfills the prerequisites for TDM, having a narrow therapeutic range, high interindividual pharmacokinetic variability, and established drug exposure-response relationships. EVR trough concentrations (C0) demonstrate a good relationship with overall exposure, providing a simple and reliable index for TDM. Whole-blood samples should be used for measurement of EVR C0, and sampling times should be standardized to occur within 1 hour before the next dose, which should be taken at the same time everyday and preferably without food. In transplantation settings, EVR should be generally targeted to a C0 of 3-8 ng/mL when used in combination with other immunosuppressive drugs (calcineurin inhibitors and glucocorticoids); in calcineurin inhibitor-free regimens, the EVR target C0 range should be 6-10 ng/mL. Further studies are required to determine the clinical utility of TDM in nontransplantation settings. The choice of analytical method and differences between methods should be carefully considered when determining EVR concentrations, and when comparing and interpreting clinical trial outcomes. At present, a fully validated liquid chromatography tandem mass spectrometry assay is the preferred method for determination of EVR C0, with a lower limit of quantification close to 1 ng/mL. Use of certified commercially available whole-blood calibrators to avoid calibration bias and participation in external proficiency-testing programs to allow continuous cross-validation and proof of analytical quality are highly recommended. Development of alternative assays to facilitate on-site measurement of EVR C0 is encouraged.

Barcelona Consensus on Biomarker-Based Immunosuppressive Drugs Management in Solid Organ Transplantation.

With current treatment regimens, a relatively high proportion of transplant recipients experience underimmunosuppression or overimmunosuppression. Recently, several promising biomarkers have been identified for determining patient alloreactivity, which help in assessing the risk of rejection and personal response to the drug; others correlate with graft dysfunction and clinical outcome, offering a realistic opportunity for personalized immunosuppression. This consensus document aims to help tailor immunosuppression to the needs of the individual patient. It examines current knowledge on biomarkers associated with patient risk stratification and immunosuppression requirements that have been generally accepted as promising. It is based on a comprehensive review of the literature and the expert opinion of the Biomarker Working Group of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology. The quality of evidence was systematically weighted, and the strength of recommendations was rated according to the GRADE system. Three types of biomarkers are discussed: (1) those associated with the risk of rejection (alloreactivity/tolerance), (2) those reflecting individual response to immunosuppressants, and (3) those associated with graft dysfunction. Analytical aspects of biomarker measurement and novel pharmacokinetic-pharmacodynamic models accessible to the transplant community are also addressed. Conventional pharmacokinetic biomarkers may be used in combination with those discussed in this article to achieve better outcomes and improve long-term graft survival. Our group of experts has made recommendations for the most appropriate analysis of a proposed panel of preliminary biomarkers, most of which are currently under clinical evaluation in ongoing multicentre clinical trials. A section of Next Steps was also included, in which the Expert Committee is committed to sharing this knowledge with the Transplant Community in the form of triennial updates.