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.

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.

Impact of butyric acid on butanol formation by Clostridium pasteurianum.

The butanol yield of the classic fermentative acetone-butanol-ethanol (ABE) process has been enhanced in the past decades through the development of better strains and advanced process design. Nevertheless, by-product formation and the incomplete conversion of intermediates still decrease the butanol yield. This study demonstrates the potential of increasing the butanol yield from glycerol though the addition of small amounts of butyric acid. The impact of butyric acid was investigated in a 7L stirred tank reactor. The results of this study show the positive impact of butyric acid on butanol yield under pH controlled conditions and the metabolic stages were monitored via online measurement of carbon dioxide formation, pH value and redox potential. Butyric acid could significantly increase the butanol yield at low pH values if sufficient quantities of primary carbon source (glycerol) were present.

Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments.

BACKGROUND: Media containing yeast extracts and other complex raw materials are widely used for the cultivation of microorganisms. However, variations in the specific nutrient composition can occur, due to differences in the complex raw material ingredients and in the production of these components. These lot-to-lot variations can affect growth rate, product yield and product quality in laboratory investigations and biopharmaceutical production processes. In the FDA's Process Analytical Technology (PAT) initiative, the control and assessment of the quality of critical raw materials is one key aspect to maintain product quality and consistency. In this study, the Respiration Activity Monitoring System (RAMOS) was used to evaluate the impact of different yeast extracts and commercial complex auto-induction medium lots on metabolic activity and product yield of four recombinant Escherichia coli variants encoding different enzymes. RESULTS: Under non-induced conditions, the oxygen transfer rate (OTR) of E. coli was not affected by a variation of the supplemented yeast extract lot. The comparison of E. coli cultivations under induced conditions exhibited tremendous differences in OTR profiles and volumetric activity for all investigated yeast extract lots of different suppliers as well as lots of the same supplier independent of the E. coli variant. Cultivation in the commercial auto-induction medium lots revealed the same reproducible variations. In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times. Only by online monitoring of the cultures, a distinct cultivation phase (e.g. glycerol depletion) could be detected and chosen for comparable and reproducible offline analysis of the yield of functional product. CONCLUSIONS: This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked. In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity. Consequently, complex raw material lots can efficiently be assessed if the distinct effects on culture behavior and final product quality and quantity are visualized.