Factors influencing voriconazole plasma level in intensive care patients.

Background: In clinical routine, voriconazole plasma trough levels (Cmin) out of target range are often observed with little knowledge about predisposing influences. Objectives: To determine the distribution and influencing factors on voriconazole blood levels of patients treated on intensive- or intermediate care units (ICU/IMC). Patients and methods: Data were collected retrospectively from patients with at least one voriconazole trough plasma level on ICU/IMC (n = 153) to determine the proportion of sub-, supra- or therapeutic plasma levels. Ordinal logistic regression analysis was used to assess factors hindering patients to reach voriconazole target range. Results: Of 153 patients, only 71 (46%) reached the target range at the first therapeutic drug monitoring, whereas 66 (43%) patients experienced too-low and 16 (10%) too-high plasma levels. Ordinal logistic regression analysis identified the use of extra corporeal membrane oxygenation (ECMO), low international normalized ratio (INR) and aspartate-aminotransferase (AST) serum levels as predictors for too-low plasma levels. Conclusion: Our data highlight an association of ECMO, INR and AST levels with voriconazole plasma levels, which should be considered in the care of critically ill patients to optimize antifungal therapy with voriconazole.

Pharmacokinetics of a single orally administered therapeutic dosage of cyclosporine A in healthy cats.

This study aimed to determine a pharmacokinetic profile for a single dosage of cyclosporine A (CsA) clinically used for immunosuppression in cats. Blood-CsA-concentrations were measured before and 1, 2, 4, 6, 8, 12 and 24 h after oral administration of 7 mg/kg body weight (BW) CsA (Atopica® oral solution) to 8 healthy adult cats using high-performance liquid chromatography coupled to mass spectrometry. Pharmacokinetic parameters were calculated using WinNonLin software based on a 1-compartment-model. The median maximum plasma-concentration of 1466 ng/ml (530-2235 ng/ml; minimum-maximum) was reached after 2.0 h (1.0-4.7 h). The area under the curve was 12,568 h x ng/ml (5732-20,820 h x ng/ml) and the apparent total clearance of the drug from plasma was 557 ml/h/kg (336-1221 ml/h/kg). Half-life of absorption into the central compartment was 0.6 h (0.4-2.6 h), half-life of elimination from the central compartment was 4.6 h (1.4-7.5 h).

Immunosuppressant Monitoring-Performance of the First Mass Spectrometry-Based Automated Clinical Analyzer Cascadion.

BACKGROUND: Automatic analyzers simplify processes and may help improve standardization. The first automated analyzer based on mass spectrometry is available and offers a panel for monitoring cyclosporin A, tacrolimus, sirolimus, and everolimus. Method comparisons and evaluation tests are presented to verify the capability of the Cascadion system for use in a clinical laboratory. METHODS: Sample preparation and measurements were performed using the Cascadion clinical analyzer. More than 1000 measurement values of patient samples were compared with an in vitro diagnostic-certified assay run on a liquid chromatography tandem mass spectrometry instrument. Precision and accuracy were determined using commercial quality control and external quality assessment (EQA) samples. RESULTS: A good correlation between the 2 instruments was observed (Pearson correlation r = 0.956-0.996). Deming regression revealed 95% confidence intervals of slopes and intercepts covering the values 1 and 0, for sirolimus and everolimus, respectively, indicating equivalence of both measuring systems. However, for cyclosporin A, a bias was observed and confirmed using a Bland-Altman plot (-9.1%). Measurement repeatability and intermediate measurement precision were appropriate showing coefficients of variation of 0.9%-6.1% and 2.0%-5.3%, respectively. Accuracy according to internal quality controls was 85%-111% and 81%-100% in the EQA samples of Reference Institute of Bioanalytics and Laboratory of the Government Chemist, respectively. High robustness was found with regard to the linearity of the calibration lines (linear regression coefficient r2 > 0.99). Carryover was negligible (0.1%). CONCLUSIONS: The Cascadion automatic analyzer produced convincing results in the measurement of patient, control, and EQA samples. The throughput was sufficient for routine use. Overall, it can be used as an alternative to open liquid chromatography tandem mass spectrometry instruments for immunosuppressant monitoring, simplifying processes without the need for specially trained personnel.

Pharmacokinetics of Meropenem in People with Cystic Fibrosis-A Proof of Concept Clinical Trial.

Anti-infective treatment of pulmonary exacerbations is a major issue in people with cystic fibrosis (CF). Individualized dosing strategies and adaptation of infusion times are important concepts to optimize anti-infective therapy. In this prospective non-randomized controlled open-label trial, we compared pharmacokinetics of meropenem in 12 people with CF experiencing a pulmonary exacerbation, of whom six received parenteral meropenem 2 g tid as short infusion over 30 min and six extended infusion over 120 min. We measured blood concentrations of meropenem at five predetermined time points over 240 min and calculated differences in the percentages of the time above the minimal inhibitory concentration (fT > MIC) for meropenem concentrations >16 and >32 mg/L, respectively. Mean percentages of fT > 16 and fT > 32 mg/L were higher in the extended compared to the short infusion group (83 and 56% vs. 59% and 34%), with a statistically significant prolongation of the fT > 32 mg/L (mean 134 vs. 82 min; p = 0.037). Our results demonstrate that, in people with CF, longer fT > MIC can be achieved with a simple modification of meropenem dosing. Further studies are needed to clarify if this may translate into improved microbiological and clinical outcomes, in particular in adults with difficult-to-treat chronic infection by carbapenem-resistant Pseudomonas aeruginosa.

Using ISO/TS 20914:2019 to calculate the measurement uncertainty of immunosuppressive drugs in a clinical laboratory.

According to the standard ISO 15189 clinical routine laboratories shall estimate measurement uncertainty (MU) of patient results of their provided measurands. Up to now there was no accepted description on how to perform. Recently, the ISO technical standard ISO/TS 20914 was published giving a practical guide for uncertainty estimation. The immunosuppressive drugs Everolimus, Ciclosporin, Sirolimus and Tacrolimus have narrow therapeutic windows. Hence, their MU should be considered for deducing clinical decisions. Here, a pathway is presented in detail on how to estimate MU measuring immunosuppressants using a widespread CE certified assay via LC-MS/MS technology. Namely, the expanded measurement uncertainties are from 13% to 27% depending on analyte and concentration. The calculation based on n > 2000 measurements each of four control levels within one year. Lower uncertainties were observed if the material was native pooled blood (13% to 17%, n > 300 measurements, one year).

Total haemoglobin - a reference measuring system for improvement of standardisation.

Background Total haemoglobin (Hb) concentration in blood belongs to the most requested measurands, and the HiCN method (hemiglobincyanide) is accepted as a reference. Although the reaction principle is clearly characterised, measurement conditions and settings are not consistently defined, some of them influencing the results. An improvement of standardisation is the object. Methods After method optimization, measurement results between different calibration laboratories (CL) were compared with each other and also with results of the National Metrology Institute of Germany (PTB), with target values of certified reference material, within the RELA scheme, and to >1500 results from routine laboratories. Results Overall deviations between three CLs were ≤0.5% (n = 24 samples) in a measurement range of 20 g/L to 300 g/L. A CV of 0.4% was determined in pooled blood (1 year long-term imprecision, 99.0%-101.1% recovery of the mean). For selected measurements (n = 4 samples) the PTB participated without significant differences to three CLs, and no significant differences were observed comparing CLs to certified values of reference materials. The expanded measurement uncertainty (probability 95%) was estimated as 1.1%. Conclusions A reference measuring system, comprising measuring instruments and other devices, including reagents and supply, to generate reference measurement values for total Hb concentration of high accuracy and low measurement uncertainty is presented. Measurement parameters are investigated and defined. The reference measuring system is ready to offer service to EQA providers and to the IVD industry for certifying control materials or calibrators.

Comprehensive characterization and resolution of discrepant spectrophotometric bilirubin results in patients on eltrombopag therapy.

Background Eltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at ~450 nm (bilirubin), (ii) absorbance at ~550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels. Methods We collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites. Results Forty-nine of 52 samples from patients on ≥50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 µmol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 µmol/L (0.4-3.8 mg/dL) by different routine analyzers, with direct bilirubin ≤4 µmol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r = 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites. Conclusions Eltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

In vivo efficacy of mutant IDH1 inhibitor HMS-101 and structural resolution of distinct binding site.

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone- and DNA-hypermethylation through the inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. Here we report the biochemical characterization, in vivo antileukemic effects, structural binding, and molecular mechanism of the inhibitor HMS-101, which inhibits the enzymatic activity of mutant IDH1 (IDH1mut). Treatment of IDH1mut primary AML cells reduced 2-hydroxyglutarate levels (2HG) and induced myeloid differentiation in vitro. Co-crystallization of HMS-101 and mutant IDH1 revealed that HMS-101 binds to the active site of IDH1mut in close proximity to the regulatory segment of the enzyme in contrast to other IDH1 inhibitors. HMS-101 also suppressed 2HG production, induced cellular differentiation and prolonged survival in a syngeneic mutant IDH1 mouse model and a patient-derived human AML xenograft model in vivo. Cells treated with HMS-101 showed a marked upregulation of the differentiation-associated transcription factors CEBPA and PU.1, and a decrease in cell cycle regulator cyclin A2. In addition, the compound attenuated histone hypermethylation. Together, HMS-101 is a unique inhibitor that binds to the active site of IDH1mut directly and is active in IDH1mut preclinical models.

Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

Higher HbA1c Measurement Quality Standards are Needed for Follow-Up and Diagnosis: Experience and Analyses from Germany.

Measurement of HbA1c is an essential laboratory measure for the follow-up and therapy decision-making in patients with diabetes. HbA1c is one of the measurands in laboratory medicine that have to be successfully checked according to the criteria of the guidelines of the German Medical Association (Rili-BAEK) in external quality assurance using the reference method value concept, when applied in patient care. The allowed deviation of ±18% in external quality assessment (EQA) and ± 10% in internal quality control has been ultimately met by virtually all the different manufacturers and methods. However, such broad limits for permissible deviations are not suitable in view of medical requirements in patient care. The low-level acceptance criteria also depends on the previously used EQA materials used in Germany. In fact, HbA1c measurement results that are imprecisely measured or come from incorrectly calibrated devices are difficult to identify. With implementation of unprocessed fresh EDTA blood, the situation has changed. Until now systems with unit use reagents for point-of-care testing (POCT) of HbA1c are not mandatory to participate in EQA schemes in Germany. This paper outlines why there was a need to narrow the acceptance limits listed within the Rili-BAEK for HbA1c's internal (to ± 3%) and external (to ± 8%) quality controls in EQA schemes for Germany, which will take place after a transition period in the next years. Higher quality in HbA1c measurements will help to avoid misdiagnosis of diabetes as well as potential over- or undertreatment of patients at risk for diabetes.

Commutability Assessment of Candidate Reference Materials for Pancreatic α-Amylase.

BACKGROUND: Measurement standardization of the catalytic concentration of α-amylase in serum is based on 3 pillars: the primary reference measurement procedure (PRMP), reference laboratories, and suitable certified reference materials (CRMs). Commutability is a prerequisite when using a CRM for calibration and trueness control of routine methods or for value transfer from the PRMP to end-user calibrators of routine methods through a calibration hierarchy. METHODS: We performed a commutability study with 30 serum pools and 5 candidate reference materials (RMs) for pancreatic α-amylase using an automated version of the PRMP and 5 different routine methods. Four candidate RMs had an artificial matrix, each with a different composition, and 1 candidate RM was based on human serum. Data were analyzed according to a linear regression analysis with prediction interval as described in the Clinical and Laboratory Standards Institute guideline EP30-A and a difference in bias analysis as described in the recommendations of the IFCC Working Group on Commutability. RESULTS: The commutability profile of the 4 candidate RMs with an artificial matrix was variable. Only 1 candidate RM, with human serum albumin in the matrix, showed a good profile like that of the candidate RM based on serum. The comparison of both commutability assessment approaches indicated some differences because of inconclusive results for the difference in bias approach, suggesting a large uncertainty on the commutability assessment. CONCLUSIONS: A CRM for pancreatic amylase in an artificial matrix can be commutable for routine methods using the same substrate as the PRMP, but the matrix composition is crucial.

Reference measurement procedure for total bilirubin in serum re-evaluated and measurement uncertainty determined.

BACKGROUND: For the determination of total bilirubin in serum the candidate reference method developed by Doumas et al. has international recognition. The primary standard SRM 916a (NIST) was recommended for use as the primary reference material for calibration. Nowadays, no primary standard is anymore commercially available. Further, a description of uncertainty components was missing. METHODS: Two reference laboratories have re-investigated the candidate reference measurement procedure. Beside minor modifications, mainly the use of a molar absorption coefficient instead of calibration by use of bilirubin standard solutions has facilitated the operating, and improved the analytical performance. All relevant sources of measurement uncertainty were investigated. RESULTS: A measurement range of 5-525 µmol/L and a CV of 0.5% to 1.4% (long term imprecision) were determined. Excellent agreement was obtained comparing to Doumas procedure (r = 0.9999) and during a two laboratory comparison participating at IFCC RELA ring trials (mean deviation: 0.6%). The combined expanded measurement uncertainty (probability 95%) for bilirubin concentrations >30 µmol/L was estimated as 2.2%. CONCLUSION: A reference system for total bilirubin based on the described reference procedure shall enable metrological traceability and optimized standardization of the values obtained in clinical routine laboratories.

Performance of the New RapidFire System for Therapeutic Monitoring of Immunosuppressants.

BACKGROUND: Typical runtimes for quantitation of immunosuppressants in whole blood based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) are in the order of a few minutes per sample. The Agilent RapidFire-MS/MS system uses solid phase extraction cartridges instead of chromatography columns and benefits from runtimes below 15 seconds. The purpose of this study was to figure out if this high-throughput instrument could be capable for application in the clinical laboratory to quantify cyclosporine A, everolimus, sirolimus, and tacrolimus. METHODS: 1172 measurement results from patient samples were compared between our routinely used LC-MS/MS system and the RapidFire 360 coupled to a tandem mass spectrometer. Sample preparation and routine measurement were performed using a certified kit. Imprecision and accuracy were investigated analyzing 3 commercial quality controls of different concentrations. RESULTS: Both measurement procedures showed excellent agreement (Pearson correlation coefficients r = 0.964-0.995). Deming regression revealed confidence intervals (95%) for slopes and intercepts of the linear equation, which covered the values 1 and 0, respectively, in almost all cases. The relative differences between both methods were marginal (1.7%-2.9%). Good intraday precision (coefficients of variation, 0.7%-9.0%) and interday precision (coefficients of variation, 2.8%-8.4%) for all immunosuppressants were achieved. The recovery of target concentrations was 81%-116%. High robustness was found with regard to linearity of the calibration lines (linear regression coefficients r ≥ 0.99). There was only negligible carry-over (cyclosporine A, 0.07%) and high endurance of the solid phase extraction cartridge (>3000 injections). CONCLUSIONS: The RapidFire-MS/MS system provided convincing results measuring patient samples and quality control materials. Together with a high throughput and a high robustness, it could represent an alternative to LC-MS/MS instruments in therapeutic drug monitoring.