Toward Analytical Performance Specifications for Immunosuppressive Drug Quantification in Transplantation: An Opinion Article.

Background: Analytical methods require performance that meets the clinical needs. Different approaches for setting up permissible analytical imprecision goals (pCVA%) for drug analyses have been reported. The aim of this study was to calculate the pCVA% for cyclosporine, tacrolimus, everolimus, sirolimus, and mycophenolic acid using 4 alternative approaches, to compare the results and to critically discuss advantages and disadvantages of each model.

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

The Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology established the second consensus report to guide Therapeutic Drug Monitoring (TDM) of everolimus (EVR) and its optimal use in clinical practice 7 years after the first version was published in 2016. This version provides information focused on new developments that have arisen in the last 7 years. For the general aspects of the pharmacology and TDM of EVR that have retained their relevance, readers can refer to the 2016 document.

Diagnostic performance of rapid antigen testing for SARS-CoV-2: the COVid-19 AntiGen (COVAG) extension study.

Background: This study is the extension of the COVAG study. We compared two RATs, the Panbio COVID-19 Ag Rapid Test (Abbott) and the SD Biosensor Q SARS-CoV-2 Rapid Antigen Test (Roche), against RT-PCR on the foil of new variants.

Methods: We included 888 all-comers at a diagnostic center between October 20, 2021, and March 18, 2022.

Automated LC-MS/MS: Ready for the clinical routine Laboratory?

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a sensitive method with high specificity. However, its routine use in the clinical laboratory is hampered by its high complexity and lack of automation. Studies demonstrate excellent analytical performance using the first fully automated LC-MS/MS for 25-hydroxy vitamin D and immunosuppressant drugs (ISD) in hospital routine laboratories.

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.

Diagnostic Performance of Rapid Antigen Testing for SARS-CoV-2: The COVid-19 AntiGen (COVAG) study.

Background: Rapid diagnostic testing for SARS-Cov-2 antigens is used to combat the ongoing pandemic. In this study we aimed to compare two RDTs, the SD Biosensor Q SARS-CoV-2 Rapid Antigen Test (Roche) and the Panbio COVID-19 Ag Rapid Test (Abbott), against rRT-PCR.

Methods: We included 2,215 all-comers at a diagnostic center between February 1 and March 31, 2021.

Immunological Biomarkers in Blood to Monitor the Course and Therapeutic Outcomes of COVID-19.

Background: The COVID-19 pandemic has posed a great challenge to the medical community because little is known about its clinical course, therapeutic options, and laboratory monitoring tools for diagnosis, prognosis, and surveillance. This review focuses on immune biomarkers that can be measured in peripheral blood in a clinical laboratory under routine conditions to monitor the innate immune system response in the acute phase, as well as the adaptive immune response established both after infection and vaccination.

Methods: A PubMed search was performed covering January 2020 to June 2021 to extract biomarkers suitable for monitoring the immune response and outcome of COVID-19 and therapeutic interventions, including vaccination.

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

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.

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.

The Impact of on Tacrolimus Pharmacokinetics and Outcome in Clinical Practice at a Single Kidney Transplant Center.

Although there is evidence that the variant should be considered in tacrolimus dosing in renal transplantation, its impact beyond tacrolimus dose requirements remains controversial. In a cohort of 121 kidney transplant recipients, we analyzed the , , and alleles and the variants , , and for their impact on exposure and dose requirement. Relevant clinical outcome measures such as acute rejection within the first year after transplantation, delayed graft function, and renal function at discharge (estimated glomerular filtration rate) were evaluated.

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.

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.

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.

Pharmacodynamic Monitoring of mTOR Inhibitors.

Pharmacodynamic (PD) monitoring may complement routine pharmacokinetic monitoring of mTOR inhibitors (mTORis) in an attempt to better guide individualized sirolimus (SRL) or everolimus (EVR) treatment after organ transplantation. This review focuses on current knowledge about PD biomarkers for personalized mTORi therapies. Different strategies have already been used in the evaluation of the pharmacodynamics of SRL and EVR as a proxy for their effects on the immune response after transplantation.

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.

Opinion: redefining the role of the physician in laboratory medicine in the context of emerging technologies, personalised medicine and patient autonomy ('4P medicine').

The role of clinical pathologists or laboratory-based physicians is being challenged on several fronts-exponential advances in technology, increasing patient autonomy exercised in the right to directly request tests and the use of non-medical specialists as substitutes. In response, clinical pathologists have focused their energies on the pre-analytical and postanalytical phases of Laboratory Medicine thus emphasising their essential role in individualised medical interpretation of complex laboratory results. Across the European Union, the role of medical doctors is enshrined in the Medical Act.

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.

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.

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.

Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition.

Objectives: The S6 ribosomal protein (S6RP) is phosphorylated by the mammalian target of rapamycin (mTOR). The objective of this study was to assess the analytical suitability of a commercial kit-based phosphoflow cytometry protocol using whole blood (WBS) to measure the level of phosphorylated S6RP (p-S6RP) in T-cell subsets to study the pharmacodynamic effects of mTOR inhibitors (mTORi).

Design And Methods: A kit was used for fixation and permeabilization of mitogen-stimulated cells, and p-S6RP was assessed separately in CD3+CD4+ and CD3+CD8+ cells by employing an anti-phospho-Ser235/236 antibody.

Analytical evaluation of a real-time PCR-based DNA demethylation assay to assess the frequency of naturally occurring regulatory T cells in peripheral blood.

Objectives: Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR).

Design And Methods: The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR).

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.

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.

Editorial: Immune monitoring in solid organ transplantation.

Solid organ transplantation is inevitably associated with the activation of the immune system of the graft recipient. An advanced knowledge of the immunological mechanisms leading to acute and chronic rejection, the advent of powerful immunosuppressive drugs, and refined surgical techniques have made solid organ transplantation a standard therapy to replace irretrievable loss of vital functions. The immune system is a complex network involving immune cells, cytokines, chemokines, antibodies, and the complement system.