Search Results (13)

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

Background and aims: The aim of this study was to investigate the possible preanalytical effect of incomplete filling of blood tubes on molecular biology assays. Materials and methods: The study population consisted of 13 healthy volunteers from whom 11 mL of whole blood was collected and then distributed in different volumes (1.5, 3.0, and 6.0 mL, respectively) into three 6.0 mL spray-dried and evacuated K₂EDTA blood tubes. Automated RNA extraction was performed using the Maxwell^® CSC RNA Blood Kit. DNA was extracted with a MagCorePlusII, with concomitant measurement of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) gene expression. The nucleic acid concentration was calculated using the NanoDrop 1000 spectrophotometer, and purity was assessed using A260/280 and A260/230 absorbance ratios. Results: The RNA concentration was higher in the tubes filled with 1.5 and 3.0 mL of blood than in the reference 6 mL filled tube. The RNA 260/280 and RNA 260/230 ratios did not differ significantly between the differently filled blood tubes. The DNA concentration remained constant in the differently filled tubes. Compared to the 6.0 mL reference filled tube, the 1.5 mL and 3.0 mL filled blood tubes displayed a lower DNA 260/280 nm ratio. The DNA 260/230 ratio did not differ significantly in any of the variously filled tubes. Compared to the 6.0 mL reference filled blood tube, the 1.5 mL and 3.0 mL filled blood tubes showed a significant increase in the GAPDHcycle threshold. Conclusions: Our results suggest that underfilling of K₂EDTA blood tubes may be a modest but analytically significant source of bias in molecular biology testing. Full article

Studies investigating the potential role of circulating bile acids (BAs) as diagnostic biomarkers for cholangiocarcinoma (CCA) are sparse and existing data do not adjust for confounding variables. Furthermore, the mechanism by which BAs affect the expression of the oncogenic mucin 5AC (MUC5AC) has never been investigated. We performed a case–control study to characterise the profile of circulating BAs in patients with CCA (n = 68) and benign biliary disease (BBD, n = 48) with a validated liquid chromatography–tandem mass spectrometry technique. Odd ratios (OR) for CCA associations were calculated with multivariable logistic regression models based on a directed acyclic graph structure learning algorithm. The most promising BAs were then tested in an in vitro study to investigate their interplay in modulating MUC5AC expression. The total concentration of BAs was markedly higher in patients with CCA compared with BBD controls and accompanied by a shift in BAs profile toward a higher proportion of primary conjugated BAs (OR = 1.50, CI: 1.14 to 1.96, p = 0.003), especially taurochenodeoxycholic acid (TCDCA, OR = 42.29, CI: 3.54 to 504.63, p = 0.003) after multiple adjustments. Western blot analysis of secreted MUC5AC in human primary cholangiocytes treated with primary conjugated BAs or with TCDCA alone allowed us to identify a novel 230 kDa isoform, possibly representing a post-translationally modified MUC5AC specie. Full article

This study investigated the biological effects on circulating monocytes after challenge with SARS-CoV-2 recombinant spike protein. Whole blood collected from seven ostensibly healthy healthcare workers was incubated for 15 min with 2 and 20 ng/mL final concentration of recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Samples were analyzed with Sysmex XN and DI-60 analyzers. Cellular complexity (i.e., the presence of granules, vacuoles and other cytoplasmic inclusions) increased in all samples challenged with the recombinant spike protein of the Ancestral, Alpha, and Delta variants, but not in those containing Omicron. The cellular content of nucleic acids was constantly decreased in most samples, achieving statistical significance in those containing 20 ng/mL of Alpha and Delta recombinant spike proteins. The heterogeneity of monocyte volumes significantly increased in all samples, achieving statistical significance in those containing 20 ng/mL of recombinant spike protein of the Ancestral, Alpha and Delta variants. The monocyte morphological abnormalities after spike protein challenge included dysmorphia, granulation, intense vacuolization, platelet phagocytosis, development of aberrant nuclei, and cytoplasmic extrusions. The SARS-CoV-2 spike protein triggers important monocyte morphological abnormalities, more evident in cells challenged with recombinant spike protein of the more clinically severe Alpha and Delta variants. Full article

Type 2 diabetes (T2D) is a multisystem disease that is the subject of many studies, but the earliest cause of the disease has yet to be elucidated. Mitochondrial impairment has been associated with diabetes in several tissues. To extend the association between T2D and mitochondrial impairment to blood cells, we investigated T2D-related changes in peripheral mononucleated blood cells’ (PBMCs) mitochondrial function in two groups of women (CTRL vs. T2D; mean age: 54.1 ± 3.8 vs. 60.9 ± 4.8; mean BMI 25.6 ± 5.2 vs. 30.0 ± 5), together with a panel of blood biomarkers, anthropometric measurements and physiological parameters (VO_2max and strength tests). Dual-energy X-ray absorptiometry (DXA) scan analysis, cardio-pulmonary exercise test and blood biomarkers confirmed hallmarks of diabetes in the T2D group. Mitochondrial function assays performed with high resolution respirometry highlighted a significant reduction of mitochondrial respiration in the ADP-stimulated state (OXPHOS; −30%, p = 0.006) and maximal non-coupled respiration (ET; −30%, p = 0.004) in PBMCs samples from the T2D group. The total glutathione antioxidant pool (GSHt) was significantly reduced (−38%: p = 0.04) in plasma samples from the T2D group. The fraction of glycated hemoglobin (Hb1Ac) was positively associated with markers of inflammation (C-reactive protein-CRP r = 0.618; p = 0.006) and of dyslipidemia (triglycerides-TG r = 0.815; p < 0.0001). The same marker (Hb1Ac) was negatively associated with mitochondrial activity levels (OXPHOS r = −0.502; p = 0.034; ET r = −0.529; p = 0.024). The results obtained in overweight postmenopausal women from analysis of PBMCs mitochondrial respiration and their association with anthropometric and physiological parameters indicate that PBMC could represent a reliable model for studying T2D-related metabolic impairment and could be useful for testing the effectiveness of interventions targeting mitochondria. Full article

Despite intense investigation, the pathogenesis of COVID-19 and the newly defined long COVID-19 syndrome are not fully understood. Increasing evidence has been provided of metabolic alterations characterizing this group of disorders, with particular relevance of an activated tryptophan/kynurenine pathway as described in this review. Recent histological studies have documented that, in COVID-19 patients, indoleamine 2,3-dioxygenase (IDO) enzymes are differentially expressed in the pulmonary blood vessels, i.e., IDO1 prevails in early/mild pneumonia and in lung tissues from patients suffering from long COVID-19, whereas IDO2 is predominant in severe/fatal cases. We hypothesize that IDO1 is necessary for a correct control of the vascular tone of pulmonary vessels, and its deficiency in COVID-19 might be related to the syndrome’s evolution toward vascular dysfunction. The complexity of this scenario is discussed in light of possible therapeutic manipulations of the tryptophan/kynurenine pathway in COVID-19 and post-acute COVID-19 syndromes. Full article

Objective: In this work we monitored both total and IgG anti-SARS-CoV-2 antibodies responses after BNT162b2 vaccine booster immunization in a cohort of ostensibly healthy healthcare workers. Methods: The study population consisted of 266 subjects (median age, 46 years and interquartile range (IQR), 35–52 years; 168 females) undergoing homologous 30-µg BNT162b2 booster administration. Serum samples were collected immediately before the booster dose and 1 month after. Results: The concentration of anti-SARS-CoV-2 RBD total antibodies and anti-SARS-CoV-2 spike trimeric IgG increased by 31 (IQR, 16–53) and 22 (IQR, 11–43) folds, respectively, after receiving the BNT162b2 vaccine booster. A highly significant Spearman′s correlation was found between the relative increase (i.e., ratio of post-booster and pre-booster serum values) of anti-SARS-CoV-2 RBD total antibodies and anti-SARS-CoV-2 spike trimeric IgG (r = 0.86; p < 0.001). Conclusion: These results suggest that monitoring of post-BNT162b2 booster immunization for purposes of identifying “low responders” could be conducted almost interchangeably with either total or IgG anti-SARS-CoV-2 antibodies. Full article

A paucity of information currently exists on plasma bile acid (BA) profiles in patients with and without type 2 diabetes mellitus (T2DM). We assayed 14 plasma BA species in 224 patients with T2DM and in 102 nondiabetic individuals with metabolic syndrome. Plasma BA levels were measured with ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) technique. Multivariable linear regression analyses were undertaken to assess associations between measured plasma BA species and T2DM status after adjustment for confounding factors. The presence of T2DM was significantly associated with higher plasma concentrations of both primary BAs (adjusted-standardized β coefficient: 0.279, p = 0.005) and secondary BAs (standardized β coefficient: 0.508, p < 0.001) after adjustment for age, sex, adiposity measures, serum alanine aminotransferase and use of statins or metformin. More specifically, the presence of T2DM was significantly associated with higher levels of plasma taurochenodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, hyodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, deoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid and glycodeoxycholic acid (adjusted-standardized β coefficients ranging from 0.315 to 0.600; p < 0.01 or less), as well as with lower plasma levels of cholic acid (adjusted-standardized β coefficient: −0.250, p = 0.013) and taurocholic acid (adjusted-standardized β coefficient: −0.309, p = 0.001). This study shows that there are marked differences in plasma BA profiles between patients with and without T2DM. Further research will be needed to better understand how these differences in plasma BA profiles may interplay with the pathophysiology of T2DM. Full article

Background: This study monitored total anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RBD (receptor-binding domain) antibodies levels in a large population of healthcare workers undergoing mRNA COVID-19 vaccination. Methods. The study population consisted of employees of Pederzoli Hospital of Peschiera del Garda (Verona, Italy), who underwent voluntary vaccination with two doses of COVID-19 mRNA BNT162b2 (Comirnaty; Pfizer Inc). Venous blood was drawn immediately before the first vaccine dose, as well as 21 days (immediately before second vaccine dose) and 50 days afterwards. Humoral response was assessed with Roche Elecsys Anti-SARS-CoV-2 S total antibodies, on Roche Cobas 6000 (Roche Diagnostics). Results: The final study population consisted of 925 subjects (mean age, 44 ± 13 years; 457 women), 206 (22.3%) anti-SARS-CoV-2 baseline seropositive. The increase of total anti-SARS-CoV-2 RBD antibodies levels 21 days after the first vaccine dose was ~3 orders of magnitude higher in seropositive than in seronegative individuals (11782 vs. 42 U/mL; p < 0.001). Total anti-SARS-CoV-2 RBD antibodies levels further increased by over 30-fold after the second vaccine dose in baseline seronegative subjects, while such increase was only ~1.3-fold in baseline seropositive subjects. In multivariate analysis, total anti-SARS-CoV-2 RBD antibodies level was inversely associated with age after both vaccine doses and male sex after the second vaccine dose in baseline seronegative subjects, while baseline antibodies value significantly predicted immune response after both vaccine doses in baseline seropositive recipients. Conclusion: Significant difference exists in post-mRNA COVID-19 vaccine immune response in baseline seronegative and seropositive subjects, which seems dependent on age and sex in seronegative subjects, as well as on baseline anti-SARS-CoV-2 antibodies level in seropositive patients. Full article

Background. Childhood obesity is becoming a major health issue and contributes to increasing the risk of cardiovascular disease in adulthood. Since dysregulated metabolism of bile acids (BAs) plays a role in progression of obesity-related disorders, including steatosis and hypertension, this study aimed to investigate BAs profiles in obese children with and without steatosis and hypertension, as well as exploring the interplay between BAs profile and vascular function. Methods. BAs concentrations were quantified with liquid chromatography-tandem mass spectrometry in 69 overweight/obese children and adolescents (mean age, 11.6 ± 2.5 years; 30 females). Liver steatosis was defined with abdomen ultrasonography, whilst hypertension was defined according to the current European guidelines. Vascular function was assessed with ultrasound technique, by measuring carotid intima media thickness (cIMT) and common carotid artery distensibility (cDC). Results. Total and individual glycine-conjugated BAs concentrations were found to be significantly higher in males compared to females, as well as in pre-pubertal compared to pubertal stage (p < 0.05 for both). No difference in BAs concentration was observed between hypertensive and normotensive subjects. Total BAs and glycine conjugated BAs were significantly higher in participants with steatosis compared to those without (p = 0.004 for both). The values of total glycine-conjugate acids were positively correlated with cDC and this association remained significant in linear regression after adjusting for sex, age, pubertal stage, body mass index and aspartate aminotransferase. Conclusion. The results suggest a possible role of BAs in the pathogenesis of liver and/or vascular damage in children and adolescent. Further studies are hence needed to validate these preliminary findings. Full article

Bile acids (BAs) assessments are garnering increasing interest for their potential involvement in development and progression of cholangiocarcinoma (CCA). Since machine learning (ML) algorithms are increasingly used for exploring metabolomic profiles, we evaluated performance of some ML models for dissecting patients with CCA or benign biliary diseases according to their plasma BAs profiles. We used ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for assessing plasma BAs profile in 112 patients (70 CCA, 42 benign biliary diseases). Twelve normalisation procedures were applied, and performance of six ML algorithms were evaluated (logistic regression, k-nearest neighbors, naïve bayes, RBF SVM, random forest, extreme gradient boosting). Naïve bayes, using direct bilirubin concentration for normalisation of BAs, was the ML model displaying better performance in the holdout set, with an Area Under Curve (AUC) of 0.95, 0.79 sensitivity, 1.00 specificity. This model, also characterised by 1.00 positive predictive value and 0.73 negative predictive value, displayed a globally excellent accuracy (86.4%). The accuracy of the other five models was lower, and AUCs ranged 0.75–0.95. Preliminary results of this study show that application of ML to BAs profile analysis can provide a valuable contribution for characterising bile duct diseases and identifying patients with higher likelihood of having malignant pathologies. Full article

Bile acids (BA) play a pivotal role in cholesterol metabolism. Their blood concentration has also been proposed as new prognostic and diagnostic indicator of hepatobiliary, intestinal, and cardiovascular disease. Liquid chromatography tandem mass spectrometry (LC–MS/MS) currently represents the gold standard for analysis of BA profile in biological samples. We report here development and validation of a LC–MS/MS technique for simultaneously quantifying 15 BA species in serum samples. We also established a reference range for adult healthy subjects (n = 130) and performed a preliminary evaluation of in vitro and in vivo interference. The method displayed good linearity, with high regression coefficients (>0.99) over a range of 5 ng/mL (lower limit of quantification, LLOQ) and 5000 ng/mL for all analytes tested. The accuracies were between 85–115%. Both intra- and inter-assay imprecision was <10%. The recoveries ranged between 92–110%. Each of the tested BA species (assessed on three concentrations) were stable for 15 days at room temperature, 4 °C, and −20 °C. The in vitro study did not reveal any interference from triglycerides, bilirubin, or cell-free hemoglobin. The in vivo interference study showed that pools obtained from hyper-cholesterolemic patients and hyper-bilirubinemic patients due to post-hepatic jaundice for benign cholestasis, cholangiocarcinoma and pancreatic head tumors had clearly distinct patterns of BA concentrations compared with a pool obtained from samples of healthy subjects. In conclusion, this study proposes a new suitable candidate method for identification and quantitation of BA in biological samples and provides new insight into a number of variables that should be taken into account when investigating pathophysiological changes of BA in human diseases. Full article

This study was designed to evaluate the analytical performance of the recently commercialized Beckman Coulter Access procalcitonin (PCT) chemiluminescent test on the Access immunoassay system. The analytical assessment encompassed the estimation of limit of blank (LoB), limit of detection (LoD), functional sensitivity (i.e., PCT value with ≤10% imprecision), linearity, imprecision and comparability of values with BRAHMS PCT-sensitive Kryptor. LoB, LoD and functional sensitivity were 0.002 μg/L, 0.003 μg/L and 0.003 μg/L, respectively. Intra-assay, inter-assay and total imprecision for plasma pools with low, medium and high PCT values were 1.8–2.1%, 2.4–3.7% and 3.1–4.3%, respectively. The assay exhibited excellent linearity between 0.02 and 84.0 μg/L. Excellent correlation (r = 0.999; p < 0.001) and negligible bias (3.2%) were found by comparing values obtained in paired plasma samples with BRAHMS PCT-sensitive Kryptor. Diagnostic agreement at 0.5, 2.0 and 10 μg/L PCT values ranged between 98%-100%. The results of this study confirm that Access PCT displays excellent analytical performance and high comparability with BRAHMS PCT-sensitive Kryptor. Full article