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 ChatGPT, Gemini, and Le Chat with physician interpretations of medical laboratory questions from an online health forum.

OBJECTIVES: Laboratory medical reports are often not intuitively comprehensible to non-medical professionals. Given their recent advancements, easier accessibility and remarkable performance on medical licensing exams, patients are therefore likely to turn to artificial intelligence-based chatbots to understand their laboratory results. However, empirical studies assessing the efficacy of these chatbots in responding to real-life patient queries regarding laboratory medicine are scarce. METHODS: Thus, this investigation included 100 patient inquiries from an online health forum, specifically addressing Complete Blood Count interpretation. The aim was to evaluate the proficiency of three artificial intelligence-based chatbots (ChatGPT, Gemini and Le Chat) against the online responses from certified physicians. RESULTS: The findings revealed that the chatbots' interpretations of laboratory results were inferior to those from online medical professionals. While the chatbots exhibited a higher degree of empathetic communication, they frequently produced erroneous or overly generalized responses to complex patient questions. The appropriateness of chatbot responses ranged from 51 to 64 %, with 22 to 33 % of responses overestimating patient conditions. A notable positive aspect was the chatbots' consistent inclusion of disclaimers regarding its non-medical nature and recommendations to seek professional medical advice. CONCLUSIONS: The chatbots' interpretations of laboratory results from real patient queries highlight a dangerous dichotomy - a perceived trustworthiness potentially obscuring factual inaccuracies. Given the growing inclination towards self-diagnosis using AI platforms, further research and improvement of these chatbots is imperative to increase patients' awareness and avoid future burdens on the healthcare system.

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.

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.

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".

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.

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.

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.

The cartilage-specific lectin C-type lectin domain family 3 member A (CLEC3A) enhances tissue plasminogen activator-mediated plasminogen activation.

C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation.

Precision and reliability of liver iodine quantification from spectral detector CT: evidence from phantom and patient data.

OBJECTIVE: To comprehensively assess precision, reproducibility, and repeatability of iodine maps from spectral detector CT (SDCT) in a phantom and in patients with repetitive examination of the abdomen. METHODS: Seventy-seven patients who underwent examination two (n = 52) or three (n = 25) times according to clinical indications were included in this IRB-approved, retrospective study. The anthropomorphic liver phantom and all patients were scanned with a standardized protocol (SSDE in patients 15.8 mGy). In patients, i.v. contrast was administered and portal venous images were acquired using bolus-tracking technique. The phantom was scanned three times at three time points; in one acquisition, image reconstruction was repeated three times. Region of interest (ROI) were placed automatically (phantom) or manually (patients) in the liver parenchyma (mimic) and the portal vein; attenuation in conventional images (CI [HU]) and iodine map concentrations (IM [mg/ml]) were recorded. The coefficient of variation (CV [%]) was used to compare between repetitive acquisitions. If present, additional ROI were placed in cysts (n = 29) and hemangioma (n = 29). RESULTS: Differences throughout all phantom examinations were < 2%. In patients, differences between two examinations were higher (CV for CI/IM: portal vein, 2.5%/3.2%; liver parenchyma, -0.5%/-3.0% for CI/IM). In 80% of patients, these differences were within a ± 20% limit. Differences in benign liver lesions were even higher (68% and 38%, for CI and IM, respectively). CONCLUSIONS: Iodine maps from SDCT allow for reliable quantification of iodine content in phantoms; while in patients, rather large differences between repetitive examinations are likely due to differences in biological distribution. This underlines the need for careful clinical interpretation and further protocol optimization. KEY POINTS: • Spectral detector computed tomography allows for reliable quantification of iodine in phantoms. • In patients, the offset between repetitive examinations varies by 20%, likely due to differences in biological distribution. • Clinically, iodine maps should be interpreted with caution and should take the intra-individual variability of iodine distribution over time into account.

Next-generation reference intervals for pediatric hematology.

Background Interpreting hematology analytes in children is challenging due to the extensive changes in hematopoiesis that accompany physiological development and lead to pronounced sex- and age-specific dynamics. Continuous percentile charts from birth to adulthood allow accurate consideration of these dynamics. However, the ethical and practical challenges unique to pediatric reference intervals have restricted the creation of such percentile charts, and limitations in current approaches to laboratory test result displays restrict their use when guiding clinical decisions. Methods We employed an improved data-driven approach to create percentile charts from laboratory data collected during patient care in 10 German centers (9,576,910 samples from 358,292 patients, 412,905-1,278,987 samples per analyte). We demonstrate visualization of hematology test results using percentile charts and z-scores (www.pedref.org/hematology) and assess the potential of percentiles and z-scores to support diagnosis of different hematological diseases. Results We created percentile charts for hemoglobin, hematocrit, red cell indices, red cell count, red cell distribution width, white cell count and platelet count in girls and boys from birth to 18 years of age. Comparison of pediatricians evaluating complex clinical scenarios using percentile charts versus conventional/tabular representations shows that percentile charts can enhance physician assessment in selected example cases. Age-specific percentiles and z-scores, compared with absolute test results, improve the identification of children with blood count abnormalities and the discrimination between different hematological diseases. Conclusions The provided reference intervals enable precise assessment of pediatric hematology test results. Representation of test results using percentiles and z-scores facilitates their interpretation and demonstrates the potential of digital approaches to improve clinical decision-making.

Distinguishing reference intervals and clinical decision limits - A review by the IFCC Committee on Reference Intervals and Decision Limits.

Reference Intervals (RIs) and clinical decision limits (CDLs) are a vital part of the information supplied by laboratories to support the interpretation of numerical clinical pathology results. RIs describe the typical distribution of results seen in a healthy reference population while CDLs are associated with a significantly higher risk of adverse clinical outcomes or are diagnostic for the presence of a specific disease. However, as the two concepts are sometimes confused, there is a need to clarify the differences between these terms and to ensure they are easily distinguished, especially because CDLs have a clinical association with specific diseases and risks, thereby implying that effective clinical interventions are available. It is important to note that, because population-based RIs are derived from the range of values expected in a typical community population, laboratory results that fall outside a RI do not necessarily indicate a disease but rather that additional medical follow-up and/or treatment may be warranted. In contrast, CDLs are associated with a risk of specific adverse outcomes, and are commonly used to interpret laboratory test results, including lipid parameters, glucose, hemoglobin A1c (HbA1c), and tumor markers, to determine risk of disease, to diagnose or to treat. In recent years, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Reference Intervals and Decision Limits (C-RIDL) has focused primarily on RIs and has performed multicenter studies to obtain common RIs. However, the broader responsibility of the Committee, from its name, includes "decision limits". C-RIDL now aims to emphasize the importance of the correct use of both RIs and CDLs and to encourage laboratories to specify the appropriate information to clinicians as needed. This review discusses RIs and CDLs in detail, describes the similarities and the differences between these two important tools in laboratory medicine, and clearly explains the processes used to define them. C-RIDL encourages the involvement of laboratory professionals in the establishment of both RIs and CDLs.

Reliable and Easy-To-Use Liquid Chromatography-Tandem Mass Spectrometry Assay for Quantification of Olorofim (F901318), a Novel Antifungal Drug, in Human Plasma and Serum.

A fast and easy-to-use liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of a novel antifungal drug, olorofim (F901318), a member of the novel class of orotomides, in human plasma and serum was developed and validated. Sample preparation was based on protein precipitation with acetonitrile and subsequent centrifugation. An isotope-labeled analogue of F901318 was employed as an internal standard. Chromatographic separation was achieved using a 50-mm by 2.1-mm, 1.9-µm, polar Hypersil Gold C18 column and isocratic mobile phase consisting of 0.1% formic acid-acetonitrile (60%-40%, vol/vol) at a flow rate of 330 µl/min. The analyte was detected using a triple-stage quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode with positive heated electrospray ionization (HESI+) within a single runtime of 2.00 min. The present LC-MS/MS method was validated according to the international guidelines of the International Conference on Harmonisation (ICH) and the U.S. Food and Drug Administration (FDA). Linearity of F901318 concentration ranges was verified by the Mandel test. The calibration curve was tested linear across the range and fitted using least-squares regression with a weighting factor of the reciprocal concentration. The limit of detection was 0.0011 mg/liter, and the lower limit of quantitation was 0.0033 mg/liter. Intraday and interday precisions ranged from 1.17% to 3.23% for F901318, and intraday and interday accuracies (percent bias) ranged from 0.75% to 5.01%. In conclusion, a method was established for the rapid quantitation of F901318 concentrations in serum and plasma samples in patient trials, and it optimizes therapeutic drug monitoring in applying an easy-to-use single method.

Identification of antibodies against extracellular matrix proteins in human osteoarthritis.

We investigated the presence of autoantibodies against the extracellular matrix proteins thrombospondin-4 (TSP-4), cartilage oligomeric matrix protein (COMP), C-type lectin domain family 3 member A (CLEC3A), collagen II, collagen VI, matrilin-3, and fibrillin-2 in the serum of osteoarthritis (OA) patients. We compared those results with the presence of such antibodies in rheumatoid arthritis (RA) patients and in healthy donors (HD). Our study examines whether antibodies against extracellular proteins can be used as potential biomarkers to support the clinical diagnosis of OA. 10 OA, 10 RA patients and 10 HD were enrolled in this explorative cross-sectional study. SDS-PAGE and immunoblot were used to investigate the presence of antibodies against extracellular matrix proteins. The serum of 5/10 OA patients but 0/10 HD exhibited TSP-4 IgG isotype antibodies (P = 0.033). The serum of 8/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4 or COMP (P = 0.005). The serum of 9/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4, COMP or CLEC3A (P = 0.005). We found strong evidence for the presence of IgG isotype autoantibodies against the cartilage extracellular matrix proteins TSP-4, COMP and CLEC3A in OA. The detection of IgG isotype autoantibodies against TSP-4, COMP and CLEC3A may support the clinical diagnosis of OA. OA with autoantibodies against cartilage extracellular matrix proteins defines a new OA subgroup suggesting that patients with high concentrations of autoantibodies may benefit from an immune suppressive therapy.

Indirect methods for reference interval determination - review and recommendations.

Reference intervals are a vital part of the information supplied by clinical laboratories to support interpretation of numerical pathology results such as are produced in clinical chemistry and hematology laboratories. The traditional method for establishing reference intervals, known as the direct approach, is based on collecting samples from members of a preselected reference population, making the measurements and then determining the intervals. An alternative approach is to perform analysis of results generated as part of routine pathology testing and using appropriate statistical techniques to determine reference intervals. This is known as the indirect approach. This paper from a working group of the International Federation of Clinical Chemistry (IFCC) Committee on Reference Intervals and Decision Limits (C-RIDL) aims to summarize current thinking on indirect approaches to reference intervals. The indirect approach has some major potential advantages compared with direct methods. The processes are faster, cheaper and do not involve patient inconvenience, discomfort or the risks associated with generating new patient health information. Indirect methods also use the same preanalytical and analytical techniques used for patient management and can provide very large numbers for assessment. Limitations to the indirect methods include possible effects of diseased subpopulations on the derived interval. The IFCC C-RIDL aims to encourage the use of indirect methods to establish and verify reference intervals, to promote publication of such intervals with clear explanation of the process used and also to support the development of improved statistical techniques for these studies.

Residual rivaroxaban exposure after discontinuation of anticoagulant therapy in patients undergoing cardiac catheterization.

PURPOSE: Patients treated with direct oral anticoagulants (DOACs) frequently undergo interventional procedures requiring temporary discontinuation of anticoagulant therapy. Little is known about remaining peri-procedural exposure to rivaroxaban in real-world patients. METHODS: Fifty-six patients with rivaroxaban treatment and scheduled cardiac catheterization were included in this prospective, observational, and single-center study. Rivaroxaban concentrations were determined by LC-MS/MS and a chromogenic anti-Xa assay. Population pharmacokinetic modeling was carried out on LC-MS/MS concentration data using NONMEM software, and results were applied to Monte Carlo simulations to predict appropriate rivaroxaban discontinuation intervals. RESULTS: Rivaroxaban concentrations ranged from

Reliable and Easy-To-Use Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Analysis of Fluconazole, Isavuconazole, Itraconazole, Hydroxy-Itraconazole, Posaconazole, and Voriconazole in Human Plasma and Serum.

BACKGROUND: A fast and easy-to-use liquid chromatography-tandem mass spectrometry method for the determination and quantification of 6 triazoles [fluconazole (FLZ), isavuconazole (ISZ), itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), posaconazole (PSZ), and voriconazole (VRZ)] in human plasma and serum was developed and validated for therapeutic drug monitoring. METHODS: Sample preparation was based on protein precipitation with acetonitrile and subsequent centrifugation. Isotope-labeled analogues for each analyte were used as internal standards. Chromatographic separation was achieved using a 50 × 2.1 mm, 1.9 µm polar Hypersil Gold C18 column and mobile phase consisting of 0.1% formic acid/acetonitrile (45%/55%, vol/vol) at a flow rate of 340 µL/min. The triazoles were simultaneously detected using a triple-stage quadrupole mass spectrometer operated in selected reaction monitoring mode with positive heated electrospray ionization within a single runtime of t = 3.00 minutes. RESULTS: Linearity of all azole concentration ranges was verified by the Mandel test and demonstrated for all azoles. All calibration curves were linear and fitted using least squares regression with a weighting factor of the reciprocal concentration. Limits of detection (µg/L/L) were FLZ, 9.3; ISZ, 0.3; ITZ, 0.6; OH-ITZ, 8.6; PSZ, 3.4; and VRZ, 2.1. The lower limits of quantitation (µg/L/liter) were FLZ, 28.3; ISZ, 1.0; ITZ, 1.7; OH-ITZ, 26.2; PSZ, 10.3; and VRZ, 6.3. Intraday and interday precisions ranged from 0.6% to 6.6% for all azoles. Intraday and interday accuracies (%bias) of all analytes were within 10.5%. In addition, we report on a 29-year-old white woman (94 kg body weight) with a history of acute myeloid leukemia who underwent stem cell transplantation. Because of diagnosis of aspergillus pneumonia, antifungal pharmacotherapy was initiated with different application modes and dosages of ISZ, and plasma concentrations were monitored over a time period of 6 months. CONCLUSIONS: A precise and highly sensitive liquid chromatography-tandem mass spectrometry method was developed that enables quantification of triazoles in plasma and serum matrix across therapeutically relevant concentration ranges. It was successfully implemented in our therapeutic drug monitoring routine service and is suitable for routine monitoring of antifungal therapy and in severely ill patients.

Critical comments to a recent EFLM recommendation for the review of reference intervals.

In a recent EFLM recommendation on reference intervals by Henny et al., the direct approach for determining reference intervals was proposed as the only presently accepted "gold" standard. Some essential drawbacks of the direct approach were not sufficiently emphasized, such as unacceptably wide confidence limits due to the limited number of observations claimed and the practical usability for only a limited age range. Indirect procedures avoid these disadvantages of the direct approach. Furthermore, indirect approaches are well suited for reference limits with large variations during lifetime and for common reference limits.