Search Results (302)

Background/Objectives: The prevalence of metabolic syndrome (MetS) is steadily increasing worldwide, driven by complex genetic, nutritional, and environmental factors. Caffeine metabolism, primarily mediated by CYP1A2 (though other enzymes such as CYP1A1 may also be involved), and the status of micronutrients such as vitamin B12 and folate have each been linked to MetS components. This study investigates the interaction between CYP1A2 genetic variants and vitamin B12/folate levels in patients with MetS, aiming to identify a novel biomarker axis with potential implications for personalized interventions. Methods: This cross-sectional observational study included 356 adults diagnosed with MetS, recruited from Western Romania. Genotyping for CYP1A2 rs762551 was performed using TaqMan PCR assays. Daily caffeine intake was assessed via validated dietary questionnaires. Serum levels of folate and vitamin B12 were measured using chemiluminescence immunoassays. Results: AA genotype patients with a moderate coffee intake (1–2 cups/day) had significantly higher folate and B12 levels than AC or CC carriers. These nutritional advantages were associated with more favorable BMI and triglyceride profiles. The interaction between CYP1A2 genotype and coffee intake was significant for both micronutrient levels and metabolic parameters, particularly in the AA group. No significant associations were found in high-coffee-intake subgroups (≥3 cups/day). Conclusions: The interplay between CYP1A2 polymorphisms and B-vitamin status may represent a clinically relevant biomarker axis in MetS. Moderate caffeine intake in slow metabolizers (AA genotype) may boost micronutrient status and metabolic health, supporting personalized nutrition. Full article

Background: The early detection of type 1 diabetes (T1D) through screening for major islet autoantibodies is receiving increasing attention as a public health strategy, exemplified by the recent implementation of a pilot pediatric screening program in Italy. The transition from research-based screening to large-scale population initiatives needs automated and standardized assays that are capable of processing extensive sample volumes. Hence, this study aimed to evaluate the analytical performance and comparability of a fully automated chemiluminescence immunoassay (CLIA) compared to a conventional enzyme-linked immunosorbent assay (ELISA) for the detection of three classes of major islet antibodies—anti-GAD (GADA), anti-IA-2 (IA-2A), and anti-ZnT8 (ZnT8A). Methods: A total of 104 serum specimens were analyzed for each autoantibody using both ELISA (RSR and Medyzim, DYNES, DSX) and CLIA (MAGLUMI 800). Assay precision and linearity were assessed through intra-assay variability studies and dilution protocols. Methods agreement was evaluated with Passing–Bablok regression, Spearman’s correlation, Bland–Altman analysis, and Cohen’s kappa statistics. Results: The CLIA showed good precision and excellent linearity across clinically relevant concentration ranges of all islet antibodies. Correlation coefficients and categorical agreement between CLIA and ELISA were high (r > 0.96 and Cohen’s kappa >0.8 for all), with ZnT8A exhibiting the highest concordance. However, proportional biases were found, as CLIA systematically underestimated GADA and ZnT8A levels, while overestimated IA-2A compared to the ELISA. Conclusions: The CLIA displayed satisfactory precision and agreement with ELISA for GADA, IA-2A, and ZnT8A detection. Our findings support the use of these automated immunoassays in large-scale population initiatives for diagnosing T1D, but we also highlight the need for further efforts to achieve better inter-assay harmonization. Full article

Hydrocortisone is a typical glucocorticoid commonly used in livestock production; however, its overuse can result in hormone residues in milk. Long-term consumption of such milk may lead to a series of health issues. Therefore, the timely and rapid detection of hydrocortisone in milk is crucial for protecting human health. In this study, a magnetic particle-based direct chemiluminescence immunoassay (MP-DCLIA) incorporating a streptavidin–biotin signal amplification system was developed for the rapid and high-throughput detection of hydrocortisone in milk. Automated operations reduce human error and enhance the accuracy and repeatability of tests. The assay can be completed in 12 min with a linear detection range of 13.09–261.71 μg/L, a limit of detection (LOD) of 4.94 μg/L, a limit of quantification (LOQ) of 14.84 μg/L, and intra- and inter-batch variations of less than 5%. The method demonstrated stability and exhibited no cross-reactivity with structural analogues. Spiked recoveries of milk samples ranged from 85.85% to 100.30%, with results strongly correlating with those obtained from LC-MS/MS. The MP-DCLIA offers rapidity, high efficiency, stability, and precision, making it a promising tool for practical testing applications. Full article

Background: Immune checkpoint inhibitors (ICIs) used for the treatment of advanced melanoma have yielded significant results, with long-term responses and improved survival rates, but not for all treated patients. Therefore, predictive biomarkers of response to ICI therapy have been intensively explored. Our study aimed to evaluate the dynamics of peripheral blood lymphocyte variation and their correlation with a set of related inflammatory factors in Nivolumab-treated advanced melanoma patients. Methods: The immunophenotypic assessment of peripheral blood immune cell subpopulations (CD3⁺, CD4⁺, and CD8⁺ T cells; CD19⁺ B cells; CD16⁺CD56⁺ NK cells; and CD4⁺/CD8⁺ ratio) was performed by the flow cytometry technique, concomitantly with a complete blood count; levels of S100, IL-6, and TNF-α proteins were quantified in serum by immunoassays, and lactate dehydrogenase (LDH) by a chemiluminescence assay. Results: Approximately 85% and 79% of patients recorded a trend of increasing levels of CD8⁺ lymphocytes and NK cells, respectively, during therapy. The percentage of NK cells negatively correlated with CD3⁺, CD4⁺, and CD19⁺ cells; the last three cell populations also established negative correlations with the inflammatory neutrophile/lymphocyte ratio (NLR). Furthermore, CD19⁺ cells were negatively correlated with the systemic inflammatory response index (SIRI) and systemic immune-inflammation index (SII). The evaluation of progression biomarkers showed that LDH levels directly correlated with IL-6 and S100 proteins, but no correlation was found with TNFα; IL-6 levels negatively correlated with percentages of CD3⁺, CD4⁺, and CD8⁺ lymphocytes. Conclusions: Variation in lymphocyte subpopulations during immunotherapy of advanced melanoma patients, associated with other cellular and/or molecular inflammatory markers, might provide insights about immune system response, but additional prospective studies are needed. Full article

Oxidative stress (OS) contributes to poor sperm parameters and increased sperm DNA fragmentation (sDF), yet effective therapeutic strategies remain limited. This study aimed to evaluate the in vitro efficacy of pentoxifylline (PTX) in improving sperm motility and reducing OS and sDF in men with isolated asthenozoospermia. Thirty semen samples from patients with asthenozoospermia were processed using density gradient centrifugation. Each sample was divided into two aliquots: one treated with PTX at a dose of 3.6 mM and the other without PTX treatment. The sperm viability and motility were assessed at 30 min, 1 h, 2 h, and 24 h post-treatment. OS was evaluated using nitro blue tetrazolium staining and a chemiluminescence assay. sDF was assessed using the alkaline Comet assay. The sperm samples treated with PTX, compared to the controls, exhibited a significant increase in total sperm motility (71.8 ± 23.03% versus 47.47 ± 4.88%, respectively; p < 0.0001). However, no significant difference was observed in the sperm viability. PTX treatment significantly reduced ROS production and sDF levels compared to controls (p < 0.01). These findings suggest that in vitro PTX supplementation enhances sperm motility and reduces the nuclear sperm injury associated with seminal ROS production. Therefore, PTX supplementation in vitro may be beneficial in assisted reproductive technology procedures involving men with asthenozoospermia. Full article

Background/Objectives: Patients suffering from inflammatory bowel diseases (IBDs) undergoing treatment with anti-TNF antibodies mount a diminished humoral immune response to vaccination against SARS-CoV-2 compared to healthy controls. The characterization of variant-specific immune responses is particularly warranted among immunosuppressed patients, where reduced responses may necessitate further medical interventions. Methods: This pilot study investigated the humoral immune response of vaccinated IBD patients on anti-TNF medication and a comparable group of healthy individuals against the viral variants Alpha, Beta, Gamma, Delta, and Omicron BA.1 and BA.5. While total IgG antibodies targeting the receptor binding site of the spike protein of SARS-CoV-2 were quantified using a chemiluminescence microparticle immunoassay (CMIA), their potential neutralizing capacity was determined using commercial and variant-specific in-house surrogate virus neutralization tests (sVNTs) against a variant-specific in-house VSV-pseudotyped virus neutralization test (pVNT) as the gold standard. Results: Employing variant-specific assays recapitulated the immune escape functions of virus variants. Conspicuously, antibody reactivity against Alpha and Omicron BA.1 and BA.5 was strikingly poor in IBD patient sera post-initial vaccination compared to healthy individuals. A comparison of the diagnostic performance of assays with the pVNT revealed that identification of patients with inadequate humoral responses by CMIA and sVNT may require adjustments to cut-off values and end-point titration of sera. Following adaptation of cut-off values, patient sera exhibited reduced reactivity against all tested variants. The assay panel used substantiated the impact of anti-TNF therapy in IBD patients as to reduced strength, function, and breadth of the immune response to several SARS-CoV-2 variants. The immune response measured following the second vaccination was comparable to the antibody response observed in healthy individuals following the first vaccination. Conclusion: Variant-specific sVNTs and pVNTs have the potential to serve as valuable tools for evaluating the efficacy of adapted vaccines and to inform clinical interventions in the care of immunosuppressed patients. Anti-TNF-treated individuals with antibody levels below the optimized CMIA threshold should be considered for early booster vaccination and/or close immunological monitoring. Full article

Background/Objectives: Chronic low back pain (LBP) remains a leading cause of disability and healthcare utilization globally, with complex, multifactorial pathophysiology. Despite advances in imaging, diagnosis often remains challenging due to poor correlation between structural findings and clinical symptoms. Recent evidence suggests inflammatory mechanisms may underlie persistent pain. This study investigated whether systemic inflammatory cytokines are altered in military personnel with chronic LBP and examined their relationships with clinical manifestations, psychological factors, and radiological findings. Methods: In this cross-sectional study, we enrolled 50 patients with chronic non-specific LBP (duration ≥ 3 months) and 50 age-, sex-, and BMI-matched healthy controls. All patients underwent a comprehensive clinical assessment, which included evaluation of pain intensity (VAS), neuropathic pain screening (DN4), psychological assessment (HADS), fibromyalgia screening (FIRST), and assessment of functional disability (Oswestry Disability Index and Roland-Morris Disability Questionnaire, EIFEL). Serum levels of IL-6, IL-8, IL-1β, TNF-α, and IL-10 were measured using chemiluminescence and enzyme-linked immunosorbent assay (ELISA) techniques. Radiological findings were documented through MRI and CT imaging of the lumbar spine. Results: Serum IL-8 levels were significantly elevated in patients with chronic LBP compared to healthy controls (8.52 ± 6.7 vs. 4.8 ± 0.56 pg/mL, p < 0.001). Weak positive correlations were observed between IL-8 levels and anxiety scores (r = 0.3, p = 0.02) and functional disability, as measured by the EIFEL questionnaire (r = 0.3, p = 0.04); however, these associations did not remain significant after Bonferroni correction for multiple testing. Similarly, IL-6 showed a weak positive correlation with BMI (r = 0.21, p = 0.03) and a weak negative correlation with lumbar mobility, as assessed by Schober’s test (r = −0.38, p = 0.03), which also did not survive correction for multiple comparisons. Conclusions: This study identified serum IL-8 as a potential biomarker for chronic LBP. While we observed associations between specific inflammatory markers and psychological distress and functional disability, these correlations were weak and did not remain significant after correction for multiple testing. These preliminary findings suggest possible connections between inflammation and the psychophysiological aspects of chronic LBP that warrant further investigation in larger cohorts. Full article

Background/Objectives: Anti-cyclic citrullinated peptide (anti-CCP) antibodies are highly specific markers for rheumatoid arthritis (RA). Over the past decade, novel automating detection systems have been developed for anti-CCP detection. The present study aimed to evaluate the diagnostic performances of three fully automated anti-CCP assays in comparison to a conventional manual enzyme-linked immunosorbent assay (ELISA). Methods: One hundred ninety-nine patients with rheumatic symptoms (100 with RA and 99 without RA) were tested for anti-CCP autoantibodies using four assays: a manual-ELISA (EUROIMMUN^®), two chemiluminescence immunoassays (CLIAs) performed on the MAGLUMI X3^® and iFlash 1800^® platforms, and an enzyme immunoassay (EIA) run on the UNI^® analyzer. Results: The Kappa statistic indicated a moderate qualitative agreement among the EUROIMMUN, iFlash, and UNI assays (0.734 ≤ ĸ ≤ 0.778), while the MAGLUMI anti-CCP assay showed only weak-to-moderate agreement with the others (0.510 ≤ ĸ ≤ 0.628). A strong positive correlation was observed between anti-CCP levels measured by the four assays (0.747 ≤ rho ≤ 0.839). At the manufacturers’ cut-off values, sensitivities ranged from 76% to 99% and specificities from 69.7% to 99%, depending on the assay. However, at a fixed specificity of 95%, all the four assays showed good diagnostic performances for RA, with sensitivities ranging from 80% to 89% and positive likelihood ratios (LRs+) from 16 to 17.8. Conclusions: Our results revealed that at the manufacturers’ cut-offs, the UNI anti-CCP assay was the most valuable alternative to the conventional ELISA for diagnosing RA in our cohort. Nevertheless, after an appropriate adjustment of the thresholds, all the evaluated assays showed good diagnostic performances for RA. Full article

Objectives: To establish age-adjusted cut-off values for glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and assess their impact on the diagnostic performance of the mild traumatic brain injury (mTBI) assay. Methods: The study included 175 adult mTBI patients presenting at the emergency department (ED) within 12 h from head trauma in whom head CT scan was performed. GFAP and UCH-L1 were measured using chemiluminescence immunoassays on an Abbott analyzer (Abbott Laboratories, USA). Results: Using manufacturer’s defined cut-offs (GFAP < 35 ng/L, UCH-L1 < 400 ng/L), the mTBI assay exhibited diagnostic sensitivity (Se) of 93.1%, specificity (Sp) of 28.8%, negative predictive value (NPV) of 95.5% and a positive predictive value (PPV) of 20.6%. In the subgroup of patients aged under 50, Se and NPV were below 100% (i.e., 75.0% and 92.3%), due to two false negative mTBI results. Age-adjusted cut-offs were defined for three patient groups, ≤49 years, 50–69 years and ≥70 years, and were set to 22.4, 37.0 and 62.3 ng/L for GFAP, and 349.3, 351.6 and 369.0 ng/L for UCH-L1. Using these cut-offs, in all patient groups Se and NPV were 100%, while increased Sp was obtained in patients older than 50 years. Conclusions: Diagnostic Se and NPV can be improved by the use of age-adjusted cut-offs. In this way, the triage protocol for mTBI and head CT scan can be refined, further contributing to the optimization of the diagnostic management of mTBI patients at the ED. Full article

Background: International guidelines recommend the use of high-sensitivity cardiac troponin (hs-cTn) I and T methods for the detection of myocardial injury as a pre-requisite for the diagnosis of acute myocardial infarction (AMI) in patients admitted to the emergency department. Recently, Mindray (Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) has introduced a new chemiluminescence immunoassay (CLIA) for the detection of the cTn complex. The present study aims to verify and validate the hs-cTnI Mindray assay on the new automated CL2600i analyzer compared to the routine Alinity-i series instrument by Abbott (Abbott, Chicago, IL, USA). Methods: This study evaluated linearity, precision through the 5 × 5 protocol, methodological comparison on plasma and serum matrices, hs-cTnI 99th percentile imprecision, and the hs-cTnI detection rate in a healthy population. Results: The results obtained proved that the performance of the Mindray hs-cTnI test on the CL2600i platform was closely comparable to the Abbott Alinity-i system (plasma R²: 0.974; serum R²: 0.995). The CVs were consistently low, and no significant differences were reported. Excellent analytical performance, with high sensitivity, was also observed in the healthy population (overall detection rate: 79%), as well as good linearity within the measuring range (R²: 0.994). Conclusions: The Mindray hs-cTnI test confirms its robustness and utility in routine practice as an advanced assay. The new technology, with more sensitive detection methods, may improve the accuracy and reliability of cardiac biomarker testing, ultimately leading to better outcomes in the management of patients with AMI and other cardiac conditions. Full article

Background: Vitamin D intake and synthesis are essential. Vitamin D deficiency is increasing across all age groups, raising concerns regarding public health. Serum 25(OH)D is measured to define vitamin D deficiency. However, its quantification in non-invasively collected biological matrices is still poorly studied. This study aimed to assess 25(OH)D levels in unconventional matrices using cost-effective analytical methods. Methods: Serum, urine, and saliva were collected from 62 healthy, non-smoking volunteers, 25–44 years of age. Biological samples were analysed using the Enzyme-Linked Immunosorbent Assay (ELISA). The serum was additionally analysed via the chemiluminescent microparticle immunoassay (CMIA), which was used as a benchmark. Results: We observed a linear correlation (Pearson r = 0.44; p = 0.05) between the benchmark and ELISA-measured 25(OH)D urinary levels. After stratification by sex, the correlation was stronger and significant only in females (Pearson r = 0.62; p = 0.04). Salivary 25(OH)D levels did not correlate with serum levels for both ELISA and CMIA measures. Subjects with a CMIA serum-based deficiency showed lower urinary 25(OH)D levels (p = 0.04). Conclusion: Our study opens up the possibility of using urinary 25(OH)D levels as a proxy measurement of vitamin D. Such an approach may allow future investigations on the association between environmental factors and vitamin D assessed in non-invasively collected biological matrices via cost-effective analytical methods. Full article

Many countries have established regulatory frameworks to monitor and mitigate Salmonella contamination in poultry products. The ability to rapidly quantify Salmonella is critical for poultry processors to facilitate early detection, implement corrective measures, and enhance product safety. This study aimed to develop an Immunomagnetic Chemiluminescent Assay (IMCA) for the quantification of Salmonella Typhimurium in ground chicken. Immunomagnetic microbeads functionalized with monoclonal antibodies were employed to selectively capture and concentrate Salmonella from ground chicken samples. A biotin-labeled monoclonal antibody, followed by an avidin-horseradish peroxidase conjugate, was used to bind the captured bacteria and initiate a chemiluminescent reaction catalyzed by peroxidase. Light emission was quantified in relative light units (RLUs) using two luminometers. Ground chicken samples were inoculated with a four-strain S. Typhimurium cocktail ranging from 0 to 3.5 Log CFU/g. Bacterial concentrations were confirmed using the Most Probable Number (MPN) method. Samples underwent enrichment in Buffered Peptone Water (BPW) supplemented with BAX MP Supplement at 42 °C for 6 and 8 h before analysis via IMCA. A linear regression analysis demonstrated that the optimal quantification of Salmonella was achieved at the 8 h enrichment period (R² ≥ 0.89), as compared to the 6 h enrichment. The limit of quantification (LOQ) was determined to be below 1 CFU/g. A strong positive correlation (R² ≥ 0.88) was observed between IMCA and MPN results, indicating methodological consistency. These findings support the application of IMCA as a rapid and reliable method for the detection and quantification of Salmonella in ground chicken. Full article

Lateral flow tests (LFTs) had a pivotal role in combating the spread of the SARS-CoV-2 virus throughout the COVID-19 pandemic thanks to their affordability and ease of use. Most of LFT devices were based on nitrocellulose membrane strips whose industrial upscaling to billions of devices has already been extensively demonstrated. Nevertheless, the assay option in an LFT format is largely restricted to qualitative detection of the target antigens. In this research, we surveyed the potential of UV nanoimprint lithography (UV-NIL) and extrusion coating (EC) for the high-throughput production of disposable capillary-driven, foil-based tests that allow multistep assays to be implemented for quantitative readout to address the inherent lack of on-demand fluid control and sensitivity of paper-based devices. Both manufacturing technologies operate on the principle of imprinting that enables high-volume, continuous structuring of microfluidic patterns in a roll-to-roll (R2R) production scheme. To demonstrate the feasibility of R2R-fabricated foil chips in a point-of-care biosensing application, we adapted a commercial chemiluminescence multiplex test for COVID-19 antibody detection originally developed for a capillary-driven microfluidic chip manufactured with injection molding (IM). In an effort to build a complete ecosystem for the R2R manufacturing of foil chips, we also recruited additional processes to streamline chip production: R2R biofunctionalization and R2R lamination. Compared to conventional fabrication techniques for microfluidic devices, the R2R techniques highlighted in this work offer unparalleled advantages concerning improved scalability, dexterity of seamless handling, and significant cost reduction. Our preliminary evaluation indicated that the foil chips exhibited comparable performance characteristics to the original IM-fabricated devices. This early success in assay translation highlights the promise of implementing biochemical assays on R2R-manufactured foil chips. Most importantly, it underscores the potential utilization of UV-NIL and EC as an alternative to conventional technologies for the future development in vitro diagnostics (IVD) in response to emerging point-of-care testing demands. Full article

The inhibitory effects of two novel lophine derivatives were unexpectedly discovered during the development of a chemiluminescent monoacylglycerol lipase (MAGL) assay. The proposed lophine derivatives were found to exhibit concentration-dependent inhibitory effects on MAGL with the octanoic and palmitic acid esters of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole showing the strongest activity. Reversibility assays using a fluorometric method confirmed that these compounds interact with MAGL in a stable, irreversible manner. To further investigate their mode of interaction, docking studies were performed, supporting the hypothesis that compounds 3 and 4 may act as competitive and irreversible inhibitors. Lophine derivatives were initially designed and synthesized as potential chemiluminescence pro-enhancers. However, assay optimization revealed no signal production upon MAGL hydrolysis, precluding their use as chemiluminescent probes. These findings suggest that lophine is a promising candidate for the development of MAGL inhibitors, although further optimization is needed to enhance binding affinity and selectivity. Full article

Recently, 2-Coumaranones have emerged as a highly promising class of chemiluminescent compounds, distinguished by their unique structural properties that facilitate efficient light emission. This review provides a comprehensive analysis of their synthesis, structural characteristics, and chemiluminescence mechanisms, integrating historical perspectives with the latest advancements in the field. Beyond their intrinsic photophysical and chemical properties, 2-coumaranones have demonstrated broad utility across bioanalytical and material sciences. Notable applications include enzyme-catalyzed chemiluminescence in aqueous systems, glucose and urease-triggered detection assays, and mechano-base-responsive luminescence for stress sensing. Additionally, recent developments in chemiluminescent protective groups and their incorporation into advanced functional materials underscore the versatility of these compounds. Despite significant progress, key challenges remain, particularly in optimizing quantum yield, emission properties, and solvent compatibility for practical applications. Future research should prioritize the development of highly tunable 2-coumaranone derivatives with enhanced spectral and kinetic properties, further expanding their potential in diagnostics, bioimaging, and mechanoluminescent sensing. By addressing these challenges, 2-coumaranones could pave the way for next-generation chemiluminescent technologies with unprecedented sensitivity and adaptability. Full article