Identification of DAGLA as an autoantibody target in cerebellar ataxia.

BACKGROUND: We aimed to investigate the clinical, imaging and fluid biomarker characteristics in patients with antidiacylglycerol lipase alpha (DAGLA)-autoantibody-associated cerebellitis. METHODS: Serum and cerebrospinal fliud (CSF) samples from four index patients were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IIFA). Immunoprecipitation, mass spectrometry and recombinant protein assays were used to identify the autoantigen. Sera from 101 patients with various neurological symptoms and a similar tissue staining pattern as the index patient samples, and 102 healthy donors were analysed in recombinant cell-based IIFA (RC-IIFA) with the identified protein. Epitope characterisation of all positive samples was performed via ELISA, immunoblot, immunoprecipitation and RC-IIFA using different DAGLA fragments. RESULTS: All index patients were relatively young (age: 18-34) and suffered from pronounced gait ataxia, dysarthria and visual impairments. Paraclinical hallmarks in early-stage disease were inflammatory CSF changes and cerebellar cortex hyperintensity in MRI. Severe cerebellar atrophy developed in three of four patients within 6 months. All patient samples showed the same unclassified IgG reactivity with the cerebellar molecular layer. DAGLA was identified as the target antigen and confirmed by competitive inhibition experiments and DAGLA-specific RC-IIFA. In RC-IIFA, serum reactivity against DAGLA was also found in 17/101 disease controls, including patients with different clinical phenotypes than the one of the index patients, and in 1/102 healthy donors. Epitope characterisation revealed that 17/18 anti-DAGLA-positive control sera reacted with a C-terminal intracellular DAGLA 583-1042 fragment, while the CSF samples of the index patients targeted a conformational epitope between amino acid 1 and 157. CONCLUSIONS: We propose that anti-DAGLA autoantibodies detected in CSF, with a characteristic tissue IIFA pattern, represent novel biomarkers for rapidly progressive cerebellitis.

Longevity of the humoral and cellular responses after SARS-CoV-2 booster vaccinations in immunocompromised patients.

We assessed the humoral and cellular immune responses after two booster mRNA vaccine administrations [BNT162b2 (Pfizer-BioNTech vaccine)] in cohorts of immunocompromised patients (n = 199) and healthy controls (HC) (n = 54). All patients living with HIV (PLWH) and chronic kidney disease (CKD) patients and almost all (98.2%) of the primary immunodeficiency (PID) patients had measurable antibodies 3 and 6 months after administration of the third and fourth vaccine dose, comparable to the HCs. In contrast, only 53.3% and 83.3% of the multiple sclerosis (MS) and rheumatologic patients, respectively, developed a humoral immune response. Cellular immune response was observed in all PLWH after administration of four vaccine doses. In addition, cellular immune response was positive in 89.6%, 97.8%, 73.3% and 96.9% of the PID, MS, rheumatologic and CKD patients, respectively. Unlike the other groups, only the MS patients had a significantly higher cellular immune response compared to the HC group. Administration of additional vaccine doses results in retained or increased humoral and cellular immune response in patients with acquired or inherited immune disorders.

The EFLM European Urinalysis Guideline 2023.

BACKGROUND: The EFLM Task and Finish Group Urinalysis has updated the ECLM European Urinalysis Guidelines (2000) on urinalysis and urine bacterial culture, to improve accuracy of these examinations in European clinical laboratories, and to support diagnostic industry to develop new technologies. RECOMMENDATIONS: Graded recommendations were built in the following areas. MEDICAL NEEDS AND TEST REQUISITION: Strategies of urine testing are described to patients with complicated or uncomplicated urinary tract infection (UTI), and high or low-risk to kidney disease. SPECIMEN COLLECTION: Patient preparation, and urine collection are supported with two quality indicators: contamination rate (cultures), and density of urine (chemistry, particles). CHEMISTRY: Measurements of both urine albumin and α1-microglobulin are recommended for sensitive detection of kidney disease in high-risk patients. Performance specifications are given for urine protein measurements and quality control of multiproperty strip tests. PARTICLES: Procedures for microscopy are reviewed for diagnostic urine particles, including urine bacteria. Technologies in automated particle counting and visual microscopy are updated with advice how to verify new instruments with the reference microscopy. BACTERIOLOGY: Chromogenic agar is recommended as primary medium in urine cultures. Limits of significant growth are reviewed, with an optimised workflow for routine specimens, using leukocyturia to reduce less important antimicrobial susceptibility testing. Automation in bacteriology is encouraged to shorten turn-around times. Matrix assisted laser desorption ionization time-of-flight mass spectrometry is applicable for rapid identification of uropathogens. Aerococcus urinae, A. sanguinicola and Actinotignum schaalii are taken into the list of uropathogens. A reference examination procedure was developed for urine bacterial cultures.

Infrared Spectroscopy in Gynecological Oncology: A Comprehensive Review of Diagnostic Potentials and Challenges.

The early detection of gynecological cancers, which is critical for improving patient survival rates, is challenging because of the vague early symptoms and the diagnostic limitations of current approaches. This comprehensive review delves into the game-changing potential of infrared (IR) spectroscopy, a noninvasive technology used to transform the landscape of cancer diagnosis in gynecology. By collecting the distinctive vibrational frequencies of chemical bonds inside tissue samples, Fourier-transform infrared (FTIR) spectroscopy provides a 'molecular fingerprint' that outperforms existing diagnostic approaches. We highlight significant advances in this field, particularly the identification of discrete biomarker bands in the mid- and near-IR spectra. Proteins, lipids, carbohydrates, and nucleic acids exhibited different absorption patterns. These spectral signatures not only serve to distinguish between malignant and benign diseases, but also provide additional information regarding the cellular changes associated with cancer. To underscore the practical consequences of these findings, we examined studies in which IR spectroscopy demonstrated exceptional diagnostic accuracy. This review supports the use of IR spectroscopy in normal clinical practice, emphasizing its capacity to detect and comprehend the intricate molecular underpinnings of gynecological cancers.

Applications of Artificial Intelligence in Urinalysis: Is the Future Already Here?

BACKGROUND: Artificial intelligence (AI) has emerged as a promising and transformative tool in the field of urinalysis, offering substantial potential for advancements in disease diagnosis and the development of predictive models for monitoring medical treatment responses. CONTENT: Through an extensive examination of relevant literature, this narrative review illustrates the significance and applicability of AI models across the diverse application area of urinalysis. It encompasses automated urine test strip and sediment analysis, urinary tract infection screening, and the interpretation of complex biochemical signatures in urine, including the utilization of cutting-edge techniques such as mass spectrometry and molecular-based profiles. SUMMARY: Retrospective studies consistently demonstrate good performance of AI models in urinalysis, showcasing their potential to revolutionize clinical practice. However, to comprehensively evaluate the real clinical value and efficacy of AI models, large-scale prospective studies are essential. Such studies hold the potential to enhance diagnostic accuracy, improve patient outcomes, and optimize medical treatment strategies. By bridging the gap between research and clinical implementation, AI can reshape the landscape of urinalysis, paving the way for more personalized and effective patient care.

Urine transfer devices may impact urinary particle results: a pre-analytical study.

OBJECTIVES: Well-standardized procedures in the pre-analytical phase of urine diagnostics is of utmost importance to obtain reliable results. We investigated the effect of different urine collection methods and the associated urine transfer tubes on urine test strip and particle results. METHODS: In total, 146 selected urine samples were subdivided into three different collection containers and subsequently transferred into its accompanying transfer tube (BD, Greiner, Sarstedt vacuum and Sarstedt aspiration). As reference, the original urine sample was directly measured on the analyser. Both chemical test strip analysis (Sysmex UC-3500) and fluorescence flow cytometry particle analysis (Sysmex UF-5000) were performed on all samples. RESULTS: No statistically significant differences in test strip results were found between the studied transfer methods. On the contrary, transfer of urine samples to the secondary tubes affected their particle counts. Clinically significant reductions in counts of renal tubular epithelial cells and hyaline casts were observed using the BD and Greiner transfer tubes and in counts of pathological casts using the BD, Greiner and Sarstedt vacuum tubes. CONCLUSIONS: The results of this study indicate that the use of urine transfer tubes may impact counts of fragile urine particles. Clinical laboratories need to be aware about the variation that urine collection methods can induce on urine particle counts.

Pitfalls in the diagnosis of hematuria.

Detection of hemoglobin (Hb) and red blood cells in urine (hematuria) is characterized by a large number of pitfalls. Clinicians and laboratory specialists must be aware of these pitfalls since they often lead to medical overconsumption or incorrect diagnosis. Pre-analytical issues (use of vacuum tubes or urine tubes containing preservatives) can affect test results. In routine clinical laboratories, hematuria can be assayed using either chemical (test strips) or particle-counting techniques. In cases of doubtful results, Munchausen syndrome or adulteration of the urine specimen should be excluded. Pigmenturia (caused by the presence of dyes, urinary metabolites such as porphyrins and homogentisic acid, and certain drugs in the urine) can be easily confused with hematuria. The peroxidase activity (test strip) can be positively affected by the presence of non-Hb peroxidases (e.g. myoglobin, semen peroxidases, bacterial, and vegetable peroxidases). Urinary pH, haptoglobin concentration, and urine osmolality may affect specific peroxidase activity. The implementation of expert systems may be helpful in detecting preanalytical and analytical errors in the assessment of hematuria. Correcting for dilution using osmolality, density, or conductivity may be useful for heavily concentrated or diluted urine samples.

Infrared Spectroscopy: A New Frontier in Hematological Disease Diagnosis.

Hematological diseases, due to their complex nature and diverse manifestations, pose significant diagnostic challenges in healthcare. The pressing need for early and accurate diagnosis has driven the exploration of novel diagnostic techniques. Infrared (IR) spectroscopy, renowned for its noninvasive, rapid, and cost-effective characteristics, has emerged as a promising adjunct in hematological diagnostics. This review delves into the transformative role of IR spectroscopy and highlights its applications in detecting and diagnosing various blood-related ailments. We discuss groundbreaking research findings and real-world applications while providing a balanced view of the potential and limitations of the technique. By integrating advanced technology with clinical needs, we offer insights into how IR spectroscopy may herald a new era of hematological disease diagnosis.

Automation in Busulfan Therapeutic Drug Monitoring: Evaluation of an Immunoassay on two Routine Chemistry Analyzers.

BACKGROUND: Therapeutic drug monitoring (TDM) of busulfan is recommended for hematopoietic stem cell transplant recipients. Timely reporting of these TDM results is essential given the short administration period and the planned dose adjustments on day 2. The authors evaluated the performance of a new nanoparticle-based competitive immunoassay on two routine clinical chemistry analyzers and compared its performance to that of an in-house high-resolution mass spectrometry (HRMS) method. METHODS: The MyCare Oncology Busulfan Assay Kit (Saladax Biomedical) was applied on two routine clinical chemistry analyzers (Abbott Architect c8000 and Roche Cobas c502) with a linearity range of 187-2000 ng/mL. The study evaluation measured imprecision and accuracy, sample probe carry-over, and dilution integrity. Method comparison with liquid chromatography (LC)-HRMS was performed using samples from patients undergoing busulfan treatment. RESULTS: Within- and between-run coefficient of variations for both analyzers were ≤5.23% and ≤8.45%, respectively, across the busulfan concentration range. The obtained biases were ≤10.3%. Both analyzers met the acceptance criteria for sample probe carry-over and dilution integrity. Agreement between the immunoassay and LC-HRMS was high: 92% and 89% of the samples measured on Architect and Cobas, respectively, were within the ±15% limit compared to the corresponding LC-HRMS results. CONCLUSIONS: Overall, good analytical performance and high agreement with LC-HRMS results were obtained for the immunoassay installed on both routine clinical chemistry analyzers. Therefore, this assay could be implemented as a valid alternative for LC methods in clinical laboratories on different open-channel clinical chemistry analyzers, resulting in shorter turn-around times for reporting busulfan TDM results with subsequent faster dosage adjustments.

Total lab automation: sample stability of clinical chemistry parameters in an automated storage and retrieval module.

OBJECTIVES: Automated storage and retrieval modules (SRM), as part of total lab automation (TLA) systems, offer tremendous practical and economic benefits. In contrast to manual storage systems, SRMs indicate continuous motion of samples and may leave samples prone to temperature fluctuations. This study investigates analyte stability in serum and heparin plasma within an automated storage module. METHODS: The stability of 28 common biochemistry analytes was investigated using 57 freshly obtained routine serum samples and 42 lithium-heparin plasma samples. Following baseline measurement, samples were stored at 2-8 °C in the automated SRM of the Accelerator a3600 TLA and reanalyzed at fixed time points (2, 4, 8, 12, 24, 48 and 72 h) on the Abbott Architect c16000 chemistry analyzer. The concentration at each time point was expressed as %-difference to the baseline value and mean results were compared to the criteria for desirable bias derived from the biological variation database. RESULTS: Nine of the analytes exceeded the bias criterion within 72 h after initial measurement in either serum samples, plasma samples or both. Lithium-heparin plasma samples showed increasing values for phosphor, potassium and lactate dehydrogenase (LDH), which were only considered stable for respectively 24, 12 and 4 h, glucose was considered stable for 8 h. Electrolyte concentrations and LDH activity significantly increased in serum samples beyond 48 h. Bicarbonate should not be performed as add-on test at all. CONCLUSIONS: The presented data indicate that the conditions within an SRM have no clinical impact on sample stability and allow stable measurement of routine analytes within 72 h, comparable to manual storage facilities.

Improving clinical performance of urine sediment analysis by implementation of intelligent verification criteria.

OBJECTIVES: Urinary test strip and sediment analysis integrated with intelligent verification criteria can help to select samples that need manual review. This study aimed to evaluate the improvement in the diagnostic performance of combined urinary test strip and urinary sediment analysis using intelligent verification criteria on the latest generation automated test strip and urinary fluoresce flow cytometry (UFFC) analysers. METHODS: Urine test strip and sediment analysis were performed using the Sysmex UC-3500 and UF-5000 (Kobe, Japan) on 828 urinary samples at the clinical laboratory of the Ghent University Hospital. The results were compared to manual microscopy using phase-contrast microscopy as a reference. After the application of the intelligent verification criteria, we determined whether the diagnostic performance of urine sediment analysis could be improved. RESULTS: Application of intelligent verification criteria resulted in an increase in specificity from 88.5 to 96.8% and from 88.2 to 94.9% for red blood cells and white blood cells, respectively. Implementing review rules for renal tubular epithelial cells and pathological casts increased the specificity from 66.7 to 74.2% and from 96.2 to 100.0%, respectively; and improved the diagnostic performance of urinary crystals and atypical cells. CONCLUSIONS: The implementation of review rules improved the diagnostic performance of UFFC, thereby increasing the reliability and quality of urine sediment results.

Comparison of the Quantitative DiaSorin Liaison Antigen Test to Reverse Transcription-PCR for the Diagnosis of COVID-19 in Symptomatic and Asymptomatic Outpatients.

We evaluated the quantitative DiaSorin Liaison severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test in symptomatic and asymptomatic individuals consulting their general practitioners (GPs) during a period of stable intense virus circulation (213/100,000 habitants per day). Leftover reverse transcription-PCR (RT-PCR) positive (n = 204) and negative (n = 210) nasopharyngeal samples were randomly selected among fresh routine samples collected from patients consulting their GPs. Samples were tested on Liaison XL according to the manufacturer's instructions. Equivocal results were considered negative. The overall sensitivity and specificity of the Liaison antigen test compared to RT-PCR were 65.7% (95% confidence interval [CI], 58.9% to 71.9%) and 100% (CI, 97.8% to 100%). Sensitivity in samples with viral loads of ≥105, ≥104, and ≥103 copies/ml were 100% (CI, 96.3% to 100.0%), 96.5% (CI, 91.8% to 98.7%), and 87.4% (CI, 81.3% to 91.5%), respectively. All samples with ≤103 copies/ml were antigen negative. The ratio of antigen concentration to viral load in samples with ≥103 copies/ml was comparable in symptomatic and asymptomatic individuals (P = 0.58). The proportion of RT-PCR-positive participants with a high viral load (≥105 copies/ml) was not significantly higher in symptomatic than in asymptomatic participants (63.9% [CI, 54.9% to 72.0%] versus 51.9% [CI, 41.1% to 62.6%]; P = 0.11), but the proportion of participants with a low viral load (<103 copies/ml) was significantly higher in asymptomatic than in symptomatic RT-PCR-positive participants (35.4% [CI, 25.8% to 46.4%] versus 14.3% [CI, 9.0% to 21.8%]; P < 0.01). Sensitivity and specificity in samples with a viral load of ≥104 copies/ml were 96.5% and 100%. The correlation of antigen concentration with viral load was comparable in symptomatic and asymptomatic individuals.

Renal tubular epithelial cells add value in the diagnosis of upper urinary tract pathology.

Background Diagnosis of upper urinary tract infections (UTI) is challenging. We evaluated the analytical and diagnostic performance characteristics of renal tubular epithelial cells (RTECs) and transitional epithelial cells (TECs) on the Sysmex UF-5000 urine sediment analyzer. Methods Urinary samples from 506 patients presenting with symptoms of a UTI were collected. Only samples for which a urinary culture was available were included. Analytical (imprecision, accuracy, stability and correlation with manual microscopy) and diagnostic performance (sensitivity and specificity) were evaluated. Results The Sysmex UF-5000 demonstrated a good analytical performance. Depending on the storage time, storage conditions (2-8 °C or 20-25 °C) and urinary pH, RTECs and TECs were stable in urine for at least 4 h. Using Passing-Bablok and Bland-Altman analysis, an acceptable agreement was observed between the manual and automated methods. Compared to TECs, RTECs demonstrated an acceptable diagnostic performance for the diagnosis of upper UTI. Conclusions While TECs do not seem to serve as a helpful marker, increased urinary levels of RTECs add value in the diagnosis of upper UTI and may be helpful in the discrimination between upper and lower UTIs.

Exploring the possibilities of infrared spectroscopy for urine sediment examination and detection of pathogenic bacteria in urinary tract infections.

Objectives In this study, the possibilities of Fourier-transformed infrared spectroscopy (FTIR) for analysis of urine sediments and for detection of bacteria causing urinary tract infections (UTIs) were investigated. Methods Dried urine specimens of control subjects and patients presenting with various nephrological and urological conditions were analysed using mid-infrared spectroscopy (4,000-400 cm-1). Urine samples from patients with a UTI were inoculated on a blood agar plate. After drying of the pure bacterial colonies, FTIR was applied and compared with the results obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Chemometric data analysis was used to classify the different species. Results Due to the typical molecular assignments of lipids, proteins, nucleic acids and carbohydrates, FTIR was able to identify bacteria and showed promising results in the detection of proteins, lipids, white and red blood cells, as well as in the identification of crystals. Principal component analysis (PCA) allowed to differentiate between Gram-negative and Gram-positive species and soft independent modelling of class analogy (SIMCA) revealed promising classification ratios between the different pathogens. Conclusions FTIR can be considered as a supplementary method for urine sediment examination and for detection of pathogenic bacteria in UTI.

Estimated urinary osmolality based on combined urinalysis parameters: a critical evaluation.

Background Urinary conductivity allows a coarse prediction of urinary osmolality in most cases but is insensitive to the osmolal contribution of uncharged particles and the presence of roentgen contrast media. Urinary osmolality can be estimated on the recently introduced Sysmex UF-5000 urine analyzer using conductivity. In this study, we evaluated the analytical performance of this research parameter. Secondly, we aimed to improve the manufacturer's algorithm for estimating urinary osmolality, based on standard urinalysis parameters (creatinine, glucose, relative density). Methods The analytical performance was determined and a prediction model to estimate urinary osmolality based on urinalysis parameters was developed. We further developed and validated a prediction model using another set of routine urine samples. In addition, the influence of roentgen contrast media on urinary osmolality was studied. Results The within-run and between imprecision for osmolality and conductivity measured on the Sysmex UF-5000 ranged from 1.1% to 4.9% and 0.7% to 4.8%, respectively. Multiple regression analysis revealed urinary creatinine, conductivity and relative density to be the strongest predictors to estimate urinary osmolality. A mean difference of 1.3 mOsm/kg between measured and predicted osmolality demonstrated that the predictive performance of our model was favorable. An excellent correlation between the relative density and % contrast media was demonstrated. Conclusions Urinary osmolality is an important parameter for assessing specimen dilution in urinalysis. Urinary conductivity, along with relative density and urinary creatinine allows a coarse prediction of urinary osmolality and is insensitive to the osmolal contribution of uncharged particles and the presence of roentgen contrast media.

Semiquantitative, fully automated urine test strip analysis.

OBJECTIVES: Urinalysis is one of the most frequently ordered diagnostic laboratory tests. In order to reduce workload and costs, rapid screening tests such as urine test strip analyses are applied. The aim of this study was to evaluate the analytical performance of the UC-3500 as well as the diagnostic performance in comparison with reference methods. DESIGN AND METHODS: We measured within-run and between-run imprecision based on quantitative reflectance values. 347 prospectively included urine specimens were investigated for the presence of glucose, protein, albumin, leukocyte esterase, and hemoglobin peroxidase activity, and ordinal scale results were compared to an automated urine particle analyzer (UF-5000, Sysmex, Kobe, Japan) and wet chemistry (Roche Cobas 8000, Mannheim, Germany). RESULTS: Within-run and between-run imprecision results based on reflectance data for both the 9 and 11 parameter test strips ranged from 0.07% to 1.36% for the low-level control and from 0.37% to 6.13% for the high-level control, depending on the parameter. Regarding diagnostic performance, the sensitivity/specificity for glucose, protein, albumin, leukocyte esterase, and hemoglobin peroxidase was 100/60%, 94.2/88.2%, 81.8/89.2%, 81.7/92.8%, and 85.1/88.6%, respectively; the negative predictive value was 100%, 83.3%, 89.1%, 94.6%, and 96.1%. The Spearman correlation coefficients of the UC-3500 vs reference methods ranged from 0.915 to 0.967, depending on the parameter. CONCLUSION: This fully automated urine test strip analyzer overall shows a satisfying performance and can reliably screen out negative urine samples in order to focus on further characterization of positive samples in the following steps of the workflow.

Factors impacting unbound vancomycin concentrations in neonates and young infants.

Vancomycin pharmacokinetic (PK) and pharmacodynamic (PD) data in neonates are based on total concentrations. However, only unbound vancomycin is pharmacologically active. The objective was to determine vancomycin protein binding and the covariates impacting unbound vancomycin concentration in neonates and young infants. In neonates and young infants to whom vancomycin was administered intermittently for medical indications, total and unbound vancomycin plasma concentrations were determined using LC-MS/MS. Sampling occurred randomly during vancomycin exposure, covering a broad range of concentrations. Impact of covariates on unbound vancomycin concentration was determined using linear regression. Significant results of the univariate regressions were entered in a stepwise multiple regression. Passing-Bablok regression and Bland-Altman were used to assess the difference between measured and calculated unbound vancomycin concentration. Thirty-seven samples in 33 patients (median (interquartile range) gestational age 35 (29-39) weeks) were collected. Median total and unbound vancomycin concentrations were 14.2 (7.4-20.6) and 13.6 (7.2-22.5) mg/L, respectively. Median unbound fraction was 0.90 (0.77-0.98). Multiple regression revealed total vancomycin concentration (ß = 0.884, p < 0.001) and albumin (ß = - 0.323, p = 0.007) as most important covariates of unbound vancomycin concentrations, with an R2 adjusted of 0.953 (p < 0.0001). Mean absolute difference between calculated and measured unbound vancomycin was - 0.008 (95% CI - 0.92-0.91) mg/L. The unbound vancomycin fraction in neonates is higher compared to that in children and adults, and total vancomycin concentration and albumin were the most important covariates of unbound vancomycin concentration. Integration of protein binding in future PK/PD analyses is appropriate to optimize vancomycin dosing and to determine population-specific vancomycin PD targets for neonates.