Quantification of midostaurin in plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry: Application to a cohort of patients with acute myeloid leukemia.

OBJECTIVES: Midostaurin is an oral multitargeted tyrosine kinase inhibitor for the treatment of acute myeloid leukemia (AML). Therapeutic drug monitoring of midostaurin may support its safe use when suspecting toxicity or combined with strong CYP3A4 inhibitors. METHODS: A stable isotope dilution liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and quantification of midostaurin in human plasma and serum. Midostaurin serum concentrations were analyzed in 12 patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML during induction chemotherapy with cytarabine, daunorubicin, and midostaurin. Posaconazole was used as prophylaxis of invasive fungal infections. RESULTS: Linear quantification of midostaurin was demonstrated across a concentration range of 0.01-8.00 mg/L. Inter- and intraday imprecisions of the proposed method were well within ±10%. Venous blood samples were taken in nine and three patients in the first and second cycle of induction chemotherapy. Median (range) midostaurin serum concentration was 7.9 mg/L (1.5-26.1 mg/L) as determined in 37 independent serum specimens. CONCLUSION: In a real-life cohort of AML patients, interindividual variability in midostaurin serum concentrations was high, highlighting issues concerning optimal drug dosing in AML patients. A personalized dosage approach may maximize the safety of midostaurin. Prospective studies and standardization of analytical methods to support such an approach are needed.

Comparison of the Performance of GPT-3.5 and GPT-4 With That of Medical Students on the Written German Medical Licensing Examination: Observational Study.

BACKGROUND: The potential of artificial intelligence (AI)-based large language models, such as ChatGPT, has gained significant attention in the medical field. This enthusiasm is driven not only by recent breakthroughs and improved accessibility, but also by the prospect of democratizing medical knowledge and promoting equitable health care. However, the performance of ChatGPT is substantially influenced by the input language, and given the growing public trust in this AI tool compared to that in traditional sources of information, investigating its medical accuracy across different languages is of particular importance. OBJECTIVE: This study aimed to compare the performance of GPT-3.5 and GPT-4 with that of medical students on the written German medical licensing examination. METHODS: To assess GPT-3.5's and GPT-4's medical proficiency, we used 937 original multiple-choice questions from 3 written German medical licensing examinations in October 2021, April 2022, and October 2022. RESULTS: GPT-4 achieved an average score of 85% and ranked in the 92.8th, 99.5th, and 92.6th percentiles among medical students who took the same examinations in October 2021, April 2022, and October 2022, respectively. This represents a substantial improvement of 27% compared to GPT-3.5, which only passed 1 out of the 3 examinations. While GPT-3.5 performed well in psychiatry questions, GPT-4 exhibited strengths in internal medicine and surgery but showed weakness in academic research. CONCLUSIONS: The study results highlight ChatGPT's remarkable improvement from moderate (GPT-3.5) to high competency (GPT-4) in answering medical licensing examination questions in German. While GPT-4's predecessor (GPT-3.5) was imprecise and inconsistent, it demonstrates considerable potential to improve medical education and patient care, provided that medically trained users critically evaluate its results. As the replacement of search engines by AI tools seems possible in the future, further studies with nonprofessional questions are needed to assess the safety and accuracy of ChatGPT for the general population.

Diagnostic sample transport via pneumatic tube systems: data logger and their algorithms are sensitive to transport effects.

OBJECTIVES: Many hospitals use pneumatic tube systems (PTS) for transport of diagnostic samples. Continuous monitoring of PTS and evaluation prior to clinical use is recommended. Data loggers with specifically developed algorithms have been suggested as an additional tool in PTS evaluation. We compared two different data loggers. METHODS: Transport types - courier, conventional (cPTS) and innovative PTS (iPTS) - were monitored using two data loggers (MSR145® logger, CiK Solutions GmbH, Karlsruhe, Germany, and a prototype developed at the University Medicine Greifswald). Data loggers differ in algorithm, recording frequencies and limit of acceleration detection. Samples from apparently healthy volunteers were split among the transport types and results for 37 laboratory measurands were compared. RESULTS: For each logger specific arbitrary units were calculated. Area-under-the-curve (AUC)-values (MSR145®) were lowest for courier and highest for iPTS and increased with increasing recording frequencies. Stress (St)-values (prototype logger) were obtained in kmsu (1,000*mechanical stress unit) and were highest for iPTS as well. Statistical differences between laboratory measurement results of transport types were observed for three measurands sensitive for hemolysis. CONCLUSIONS: The statistical, but not clinical, differences in the results for hemolysis sensitive measurands may be regarded as an early sign of preanalytical impairment. Both data loggers record this important interval of beginning mechanical stress with a high resolution indicating their potential to facilitate early detection of preanalytical impairment. Further studies should identify suitable recording frequencies. Currently, evaluation and monitoring of diagnostic sample transport should not only rely on data loggers but also include diagnostic samples.

Modified CLEC3A-Derived Antimicrobial Peptides Lead to Enhanced Antimicrobial Activity against Drug-Resistant Bacteria.

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics. Sequence changes can significantly improve the therapeutic properties of antimicrobial peptides. In our study, we apply different sequence modifications to enhance the performance of the CLEC3A-derived AMPs HT-16 and HT-47. We truncated their sequences, inserting a triple-glycine linker, adding an N-terminal tryptophan residue, and generating a D-amino acid variant, resulting in the generation of seven new peptides. We investigated their antimicrobial activity against gram-positive and gram-negative bacteria, their cytotoxicity to murine cells, and the biostability of the modified peptides in serum. We identified a novel antimicrobial peptide, WRK-30, with enhanced antimicrobial potency against S. aureus and MRSA. Additionally, WRK-30 was less cytotoxic to eukaryotic cells, allowing its application in higher concentrations in an in vivo setting. In conclusion, we identified a novel CLEC3A-derived antimicrobial peptide WRK-30 with significantly improved therapeutic properties and the potential to widen the repertoire of conventional antibiotics.

Measuring capillary blood glucose concentration: Is the first blood drop really the right blood drop?

BACKGROUND: Capillary blood glucose (CBG) is fundamental for diabetes mellitus management. However, it is still unclear whether the first or the subsequent blood drops most accurately reflect patients' blood glucose levels. METHODS: 128 healthy volunteers were included in this prospective cohort study from November 2021 to December 2021. Capillary blood glucose concentrations of the first four blood drops, measured using glucose meters were compared with venous blood concentrations of the respective donors, measured using an in-lab hexokinase method. ANOVA, the Spearman correlation coefficient and Bland-Altman plots were used to analyze the data. RESULTS: The mean plasma glucose concentration was 90.46 mg/dl with an SD of ± 14.416 (5.02 ± 0.8 mmol/l). There were statistically strong correlations among the glucose concentrations of all capillary blood drops (correlation coefficients of r > 0.8). The first capillary blood drops showed the lowest mean difference to plasma blood glucose concentrations (+4.92 mg/dl; +0.27 mmol/l), followed by the third drop (+7.28 mg/dl; +0.4 mmol/l), second drop (+7.30 mg/dl; +0.4 mmol/l) and fourth drop (+8.87 mg/dl; +0.49 mmol/l). CONCLUSION: There is a strong correlation and good agreement between the different capillary blood drops, making all blood drops equally suitable for blood glucose monitoring. In practice, the given setting (clinical or patient self-monitoring) should be considered upon choosing a specific blood drop.

SHIP1 Is Present but Strongly Downregulated in T-ALL, and after Restoration Suppresses Leukemia Growth in a T-ALL Xenotransplantation Mouse Model.

Acute lymphoblastic leukemia (ALL) is the most common cause of cancer-related death in children. Despite significantly increased chances of cure, especially for high-risk ALL patients, it still represents a poor prognosis for a substantial fraction of patients. Misregulated proteins in central switching points of the cellular signaling pathways represent potentially important therapeutic targets. Recently, the inositol phosphatase SHIP1 (SH2-containing inositol 5-phosphatase) has been considered as a tumor suppressor in leukemia. SHIP1 serves as an important negative regulator of the PI3K/AKT signaling pathway, which is frequently constitutively activated in primary T-ALL. In contrast to other reports, we show for the first time that SHIP1 has not been lost in T-ALL cells, but is strongly downregulated. Reduced expression of SHIP1 leads to an increased activation of the PI3K/AKT signaling pathway. SHIP1-mRNA expression is frequently reduced in primary T-ALL samples, which is recapitulated by the decrease in SHIP1 expression at the protein level in seven out of eight available T-ALL patient samples. In addition, we investigated the change in the activity profile of tyrosine and serine/threonine kinases after the restoration of SHIP1 expression in Jurkat T-ALL cells. The tyrosine kinase receptor subfamilies of NTRK and PDGFR, which are upregulated in T-ALL subgroups with low SHIP1 expression, are significantly disabled after SHIP1 reconstitution. Lentiviral-mediated reconstitution of SHIP1 expression in Jurkat cells points to a decreased cellular proliferation upon transplantation into NSG mice in comparison to the control cohort. Together, our findings will help to elucidate the complex network of cell signaling proteins, further support a functional role for SHIP1 as tumor suppressor in T-ALL and, much more importantly, show that full-length SHIP1 is expressed in T-ALL samples.

ISO 15189 is a sufficient instrument to guarantee high-quality manufacture of laboratory developed tests for in-house-use conform requirements of the European In-Vitro-Diagnostics Regulation.

The EU In-Vitro Diagnostic Device Regulation (IVDR) aims for transparent risk-and purpose-based validation of diagnostic devices, traceability of results to uniquely identified devices, and post-market surveillance. The IVDR regulates design, manufacture and putting into use of devices, but not medical services using these devices. In the absence of suitable commercial devices, the laboratory can resort to laboratory-developed tests (LDT) for in-house use. Documentary obligations (IVDR Art 5.5), the performance and safety specifications of ANNEX I, and development and manufacture under an ISO 15189-equivalent quality system apply. LDTs serve specific clinical needs, often for low volume niche applications, or correspond to the translational phase of new tests and treatments, often extremely relevant for patient care. As some commercial tests may disappear with the IVDR roll-out, many will require urgent LDT replacement. The workload will also depend on which modifications to commercial tests turns them into an LDT, and on how national legislators and competent authorities (CA) will handle new competences and responsibilities. We discuss appropriate interpretation of ISO 15189 to cover IVDR requirements. Selected cases illustrate LDT implementation covering medical needs with commensurate management of risk emanating from intended use and/or design of devices. Unintended collateral damage of the IVDR comprises loss of non-profitable niche applications, increases of costs and wasted resources, and migration of innovative research to more cost-efficient environments. Taking into account local specifics, the legislative framework should reduce the burden on and associated opportunity costs for the health care system, by making diligent use of existing frameworks.

The impact of COVID-19 on medical students' practical skills and hygiene behavior regarding venipuncture: a case control study.

BACKGROUND: Despite their importance to current and future patient care, medical students' hygiene behaviors and acquisition of practical skills have rarely been studied in previous observational study. Thus, the aim of this study was to investigate the potential impact of the COVID-19 pandemic on medical student's hygiene and practical skills. METHODS: This case-control study assessed the effect of the COVID-19 pandemic on hygiene behavior by contrasting the practical skills and hygiene adherence of 371 medical students post the pandemic associated lockdown in March 2020 with that of 355 medical students prior to the SARS-CoV-2 outbreak. Students' skills were assessed using an objective structured clinical examination (OSCE). Their skills were then compared based on their results in hygienic venipuncture and the total OSCE score. RESULTS: During the SARS-CoV-2 pandemic, medical students demonstrated an increased level of compliance regarding hand hygiene before (prior COVID-19: 83.7%; during COVID-19: 94.9%; p < 0.001) and after patient contact (prior COVID-19: 19.4%; during COVID-19: 57.2%; p = 0.000) as well as disinfecting the puncture site correctly (prior COVID-19: 83.4%; during COVID-19: 92.7%; p < 0.001). Prior to the pandemic, students were more proficient in practical skills, such as initial venipuncture (prior COVID-19: 47.6%; during COVID-19: 38%; p < 0.041), patient communication (prior COVID-19: 85.9%; during COVID-19: 74.1%; p < 0.001) and structuring their work process (prior COVID-19: 74.4%; during COVID-19: 67.4%; p < 0.024). CONCLUSION: Overall, the COVID-19 pandemic sensitized medical students' attention and adherence to hygiene requirements, while simultaneously reducing the amount of practice opportunities, thus negatively affecting their practical skills. The latter development may have to be addressed by providing additional practice opportunities for students as soon as the pandemic situation allows.

The ratio of serum LL-37 levels to blood leucocyte count correlates with COVID-19 severity.

Beneficial effects of vitamin D on COVID-19 progression have been discussed in several studies. Vitamin D stimulates the expression of the antimicrobial peptide LL-37, and evidence shows that LL-37 can antagonize SARS-CoV-2. Therefore, we investigated the association between LL-37 and vitamin D serum levels and the severity of COVID-19. To this end, 78 COVID-19 patients were divided into 5 groups according to disease severity. We determined serum levels of LL-37, vitamin D, and routine laboratory parameters. We demonstrated a correlation of CRP, IL-6, PCT, leukocyte count, and LDH with the severity of COVID-19. Our study did not demonstrate a direct relationship between serum levels of LL-37 and vitamin D and the severity of COVID-19. LL-37 is produced by granulocytes and released at the site of inflammation. Therefore, the analysis of LL-37 in broncho-alvelolar lavage rather than in patient serum seems critical. However, since LL-37 is produced by granulocytes, we determined serum LL-37 levels as a function of leukocyte count. The LL-37/leukocyte count ratio correlates highly significantly inversely proportional with COVID-19 severity. Our results indicate that the LL-37/leukocyte count ratio could be used to assess the risk of COVID-19 progression as early as hospital admission.

Implementation of the new EU IVD regulation - urgent initiatives are needed to avert impending crisis.

Laboratory medicine in the European Union is at the dawn of a regulatory revolution as it reaches the end of the transition from IVDD 98/79/EC (https://eur-lex.eur-opa.eu/legal-content/EN/TXT/?uri=CELEX%3A31998L0079&qid=1628781352814) to IVDR 2017/746 https://eur-lex.europa.eu/eli/reg/2017/746. Without amendments and contingency plans, implementation of the IVDR in May 2022 will lead the healthcare sector into uncharted waters due to unpreparedness of the EU regulatory infrastructure. Prospective risk analyses were not made by the European Commission, and if nothing happens it can be anticipated that the consequences will impact all stakeholders of the medical test pipeline, may seriously harm patients and may prevent caregivers from making appropriate clinical decisions due to non-availability of medical tests. Finally, it also may discourage manufacturers and academia from developing specialty tests, thereby hampering innovation in medical diagnostic care. We hereby inform laboratory professionals about the imminent diagnostic collapse using testimonies from representative stakeholders of the diagnostic supply chain and from academia developing innovative in-house tests in domains of unmet clinical needs. Steps taken by the EFLM Task Force on European Regulatory Affairs, under the umbrella of the Biomedical Alliance in Europe, will be highlighted, as well as the search for solutions through dialogue with the European Commission. Although we recognize that the IVDR promotes positive goals such as increased clinical evidence, surveillance, and transparency, we need to ensure that the capabilities of the diagnostic sector are not damaged by infrastructural unpreparedness, while at the same time being forced to submit to a growing bureaucratic and unsupportive structure that will not support its "droit d'exister".

Preparing Laboratories for Interconnected Health Care.

In an increasingly interconnected health care system, laboratory medicine can facilitate diagnosis and treatment of patients effectively. This article describes necessary changes and points to potential challenges on a technical, content, and organizational level. As a technical precondition, electronic laboratory reports have to become machine-readable and interpretable. Terminologies such as Logical Observation Identifiers Names and Codes (LOINC), Nomenclature for Properties and Units (NPU), Unified Code for Units of Measure (UCUM), and SNOMED-CT can lead to the necessary semantic interoperability. Even if only single "atomized" results of the whole report are extracted, the necessary information for correct interpretation must be available. Therefore, interpretive comments, e.g., concerns about an increased measurement uncertainty must be electronically attached to every affected measurement result. Standardization of laboratory analyses with traceable standards and reference materials will enable knowledge transfer and safe interpretation of laboratory analyses from multiple laboratories. In an interconnected health care system, laboratories should strive to transform themselves into a data hub that not only receives samples but also extensive information about the patient. On that basis, they can return measurement results enriched with high-quality interpretive comments tailored to the individual patient and unlock the full potential of laboratory medicine.

Deterioration in hygiene behavior among fifth-year medical students during the placement of intravenous catheters: a prospective cohort comparison of practical skills.

BACKGROUND: The American Association of Medical Colleges has defined peripheral intravenous cannulation as one of the eight practical skills that a medical student should possess upon graduation. Since following a standard hygiene protocol can reduce the rate of complications such as bloodstream infections, the medical student's compliance to hygienic standards is highly relevant. METHODS: This unicentric longitudinal cohort study included 177 medical students undergoing OSCE 1 in the winter semesters 2016/2017 and 2017/2018 as well as OSCE 2 during the winter semesters 2018/2019 and 2019/2020 at the University of Cologne. Their performance in peripheral intravenous cannulation was rated by trained student supervisors using a scaled 13-item questionnaire and compared between OSCE 1 and OSCE 2. RESULTS: Overall, a decline in the correct placement of peripheral intravenous catheters was observed among advanced medical students during OSCE 2 (mean total score: 6.27 ± 1.84) in comparison to their results in OSCE 1 (mean total score: 7.67 ± 1.7). During OSCE 2, the students were more negligent in regard to hygienic behavior, such as disinfection of the puncture site as well as hand disinfection before and after venipuncture. Their patients were also less likely to be informed about the procedure as compared to OSCE 1. CONCLUSIONS: An unsatisfying performance in regard to peripheral intravenous cannulation was observed in medical students with hygiene compliance deteriorating between the third and fifth year of their study. Thus, we promote an extension of practical hygiene and stress management training in medical school to reduce complications associated with intravenous catheters, such as bloodstream infections.

Advisory opinion of the AWMF Ad hoc Commission In-vitro Diagnostic Medical Devices regarding in-vitro diagnostic medical devices manufactured and used only within health institutions established in the Union according to Regulation (EU) 2017/746 (IVDR).

In view of the approaching application date of Regulation (EU) 2017/746 ("IVDR") and the resulting EU-wide, harmonized requirements for in-vitro diagnostic medical devices (IVD) manufactured and used within European health institutions, the Ad hoc Commission IVD of the German Association of the Scientific Medical Societies (AWMF) takes a national position on the details of the requirements and conditions related to the use of these IVD products. The Ad hoc Commission IVD emphasizes the relevance of examination procedures developed in medical laboratories, especially in the field of orphan diseases and new diagnostic markers. The IVDR sets an adequate regulatory framework for IVD manufactured and used within health institutions as long as these requirements are fulfilled with reliability and in accordance with the current state of the art in medical laboratory sciences. At the same time, the IVDR requirements have to be regarded under a pragmatic view and in accordance with the quality management systems approved within the different EU Member States. On the one hand, the mandatory requirements of the RiLiBÄK play an essential role in Germany. On the other hand, elements of voluntarily applicable international standards may support the fulfilment of product requirements for safety and performance according to Annex I of the IVDR. Both the complexity and possible solutions for the implementation of the IVDR requirements are discussed on the basis of examples such as the required documentation, performance evaluation and software validation. The Ad hoc Commission IVD recommends that, while aiming at a preferably EU-wide harmonized interpretation of the IVDR requirements, the flexibility in medical laboratory diagnostics necessary for patient care, including the use of IVD from in-house production, should be emphasized.

Comparison of reference intervals derived by direct and indirect methods based on compatible datasets obtained in Turkey.

BACKGROUND: Indirect derivation of reference intervals (RIs) from the laboratory information system (LIS) has been recently pursued. We aimed at evaluating the accuracy of indirectly predicted RIs compared to the RIs established directly from healthy subjects in the nationwide RI study in Turkey, targeting 25 major chemistry analytes. METHODS: LIS data were retrieved from the laboratory that performed measurements for the direct study. They were cleaned by limiting to outpatients with age 18-65 years, and by allowing only one record per year per patient. Evaluated were four indirect methods of univariate approach: Hoffmann, Bhattacharya, Arzideh, and Wosniok methods. Power transformation of the LIS dataset was performed either using the power (λ) reported by the IFCC global RI study (the first two methods) or using a λ predicted (the last two). RESULTS: Compared to the direct study dataset, the LIS dataset showed a variable degree of alterations in peak location and shape. Consequently, lower-side peak-shifts observed in sodium, albumin, etc. led to lowered RI limits, whereas higher-side peak-shift observed in triglyceride, low-density lipoprotein cholesterol, etc. led to raised RI limits. Overall, 72% (62-81) of the RI limits predicted by indirect methods showed significant biases from direct RIs. However, the biases observed in total cholesterol, lactic dehydrogenase, etc. were attributed to a higher-side age-bias in LIS dataset. After excluding them, the overall proportion of biased RIs was reduced to 47% (38-54). CONCLUSION: To reduce prediction biases that remained after age adjustment, it is necessary to apply more rigorous data-cleaning before applying indirect methods.

Analysis of IgM, IgA, and IgG isotype antibodies Directed against SARS-CoV-2 spike glycoprotein and ORF8 in the course of COVID-19.

Immunoassays are a standard diagnostic tool that assesses immunity in severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection. However, immunoassays do not provide information about contaminating antigens or cross-reactions and might exhibit inaccurately high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS-CoV-2 patients by immunoblot analysis. We analyzed serum immunoglobulins IgM, -A, and -G directed against SARS-CoV-2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA and assessed the clinical parameters of inflammation status and kidney and liver injury. Unexpectedly, we found no correlation between the presence of antibodies and the future course of the disease. However, attention should be paid to the parameters CRP, IL-6, and LDH. We found evidence of antibody cross-reactivity, which questions the reliability of results for serum samples that tested negative for anti-SARS-CoV-2 antibodies when assessed by immunoassays. Nevertheless, for the detection of IgG anti-SARS-CoV-2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best way of detecting anti-SARS-CoV-2 IgM antibodies.

High-resolution pediatric reference intervals for 15 biochemical analytes described using fractional polynomials.

OBJECTIVES: Assessment of children's laboratory test results requires consideration of the extensive changes that occur during physiological development and result in pronounced sex- and age-specific dynamics in many biochemical analytes. Pediatric reference intervals have to account for these dynamics, but ethical and practical challenges limit the availability of appropriate pediatric reference intervals that cover children from birth to adulthood. We have therefore initiated the multi-center data-driven PEDREF project (Next-Generation Pediatric Reference Intervals) to create pediatric reference intervals using data from laboratory information systems. METHODS: We analyzed laboratory test results from 638,683 patients (217,883-982,548 samples per analyte, a median of 603,745 test results per analyte, and 10,298,067 test results in total) performed during patient care in 13 German centers. Test results from children with repeat measurements were discarded, and we estimated the distribution of physiological test results using a validated statistical approach (kosmic). RESULTS: We report continuous pediatric reference intervals and percentile charts for alanine transaminase, aspartate transaminase, lactate dehydrogenase, alkaline phosphatase, γ-glutamyl-transferase, total protein, albumin, creatinine, urea, sodium, potassium, calcium, chloride, anorganic phosphate, and magnesium. Reference intervals are provided as tables and fractional polynomial functions (i.e., mathematical equations) that can be integrated into laboratory information systems. Additionally, Z-scores and percentiles enable the normalization of test results by age and sex to facilitate their interpretation across age groups. CONCLUSIONS: The provided reference intervals and percentile charts enable precise assessment of laboratory test results in children from birth to adulthood. Our findings highlight the pronounced dynamics in many biochemical analytes in neonates, which require particular consideration in reference intervals to support clinical decision making most effectively.

Quantification of direct-acting oral anticoagulants: Application of a clinically validated liquid chromatography-tandem mass spectrometry method to forensic cases.

In certain forensic cases, a quantification of direct-acting oral anticoagulants (DOACs) can be necessary. We evaluate the applicability of a previously described liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the determination of DOACs in plasma to postmortem specimen. Postmortem internal quality control (PIQC) samples were prepared in pooled blank postmortem heart blood, femoral blood, cerebrospinal fluid (CSF), and urine as well in plasma. To examine the application of the clinical method to forensic cases, the main validation parameters were reinvestigated using PIQC samples. Postmortem samples of 12 forensic cases with evidence of previous rivaroxaban intake and unknown bleeding disorders were analyzed. Interday variability remained within the acceptance criterion of ±15%. Matrix effects were comparable in blank plasma and postmortem matrix extracts. After 4 weeks of storage in the refrigerator, no relevant decrease of DOACs was evident. After 96 h of storage at room temperature, a slight decrease in edoxaban concentration was observed in CSF and urine, while plasma edoxaban decreased by about 50%. Median (range) rivaroxaban concentrations determined in specimen of forensic cases were as follows: heart blood (n = 6), 17.2 ng/ml (

Osteoidosis leads to altered differentiation and function of osteoclasts.

In patients with osteomalacia, a defect in bone mineralization leads to changed characteristics of the bone surface. Considering that the properties of the surrounding matrix influence function and differentiation of cells, we aimed to investigate the effect of osteoidosis on differentiation and function of osteoclasts. Based on osteomalacic bone biopsies, a model for osteoidosis in vitro (OIV) was established. Peripheral blood mononuclear cells were differentiated to osteoclasts on mineralized surfaces (MS) as internal control and on OIV. We observed a significantly reduced number of osteoclasts and surface resorption on OIV. Atomic force microscopy revealed a significant effect of the altered degree of mineralization on surface mechanics and an unmasking of collagen fibres on the surface. Indeed, coating of MS with RGD peptides mimicked the resorption phenotype observed in OIV, suggesting that the altered differentiation of osteoclasts on OIV might be associated with an interaction of the cells with amino acid sequences of unmasked extracellular matrix proteins containing RGD sequences. Transcriptome analysis uncovered a strong significant up-regulation of transmembrane glycoprotein TROP2 in osteoclastic cultures on OIV. TROP2 expression on OIV was also confirmed on the protein level and found on the bone surface of patients with osteomalacia. Taken together, our results show a direct influence of the mineralization state of the extracellular matrix surface on differentiation and function of osteoclasts on this surface which may be important for the pathophysiology of osteomalacia and other bone disorders with changed ratio of osteoid to bone.

Pharmacodynamic Monitoring of Mycophenolic Acid Therapy: Improved Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Inosin-5'-Monophosphate Dehydrogenase Activity.

BACKGROUND: Mycophenolic acid (MPA), a powerful inhibitor of lymphocyte proliferation, is widely used in transplantation medicine and as a glucocorticoid-sparing agent in rheumatic and inflammatory diseases. As inosine-5'-monophosphate dehydrogenase (IMPDH), the target enzyme of MPA, shows high interindividual variability in its basal activity, the assessment of IMPDH activity in addition to pharmacokinetic monitoring has emerged as a strategy to individualize MPA pharmacotherapy. METHODS: A liquid chromatography-tandem mass spectrometry method was developed to measure IMPDH activity in peripheral blood mononuclear cells from lithium-heparinized blood. Stable isotope-labeled analogs of analytes were used as internal standards for the quantitative analyses of xanthosine-5'-monophosphate (XMP) and adenosine-5'-monophosphate (AMP). IMPDH activity was expressed as enzymatic production of XMP per time normalized to the AMP concentration. Validation and evaluation of the new method were performed by using blood samples from healthy volunteers (n = 10). RESULTS: Linearity was demonstrated over the concentration ranges of 0.25-80 µM for XMP and 4-80 µM for AMP (R > 0.99). Between-day and within-day assay precisions and accuracies were within the acceptance criterion of ±15%. Matrix effects were fully compensated by the coelution of internal standards. Specific and linear XMP production (R > 0.99) and the inhibition of IMPDH activity by MPA at clinically relevant doses were demonstrated. CONCLUSIONS: In this study, a liquid chromatography-tandem mass spectrometry method to measure IMPDH activity was established and fully evaluated for matrix and ion suppression effects. The method enabled precise quantification of IMPDH activity for the improvement of pharmacokinetic/pharmacodynamic therapeutic drug monitoring approaches to optimize immunosuppressive treatment with MPA.