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Journal: Scandinavian journal of clinical and laboratory investigation

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While von Willebrand factor (vWF) has been reported to be elevated in smokers, there are no reports on the effects of smoking on its cleaving protease ADAMTS-13, particularly in subjects of Arab ethnicity. This study was conducted to determine the effects of smoking on vWF and ADAMTS-13 antigen and activity levels in Arab males. Venous blood samples from 80 smoking (at rest) and 80 non-smoking healthy males were collected after asking subjects to fast and refrain from smoking for 8 hours. Similar sampling was done for 40 smokers (acute smokers), who were asked to smoke one cigarette immediately before blood collection. Plasma was used to measure ADAMTS-13 antigen and activity levels, as well as vWF antigen and collagen binding activity levels using commercial ELISA kits. Compared to non-smokers, ADAMTS-13 and vWF activities were significantly lower in smokers at rest (p < 0.05). Acute smokers had significantly higher levels of vWF activity and ADAMTS-13 antigen and activity levels (p < 0.01), compared to smokers at rest. Our results suggest that high vWF activity is accompanied by an increase in ADAMTS-13 activity as a natural physiological mechanism to degrade the elevated vWF molecules. If not followed by a subsequent smoke, the activities of both proteins subside. It is possible that the repeated increase in vWF and constant degradation by ADAMTS-13 results in lower overall levels of both proteins in smokers (at rest) compared to nonsmokers who do not experience a similar (repeated) injury to the endothelium.

Concepts: Blood, Platelet, Hematology, Von Willebrand factor, Von Willebrand disease, ABO blood group system, Heyde's syndrome, Weibel-Palade body

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Introduction. Besides hypogammaglobulinemia and recurrent infections, abnormalities of T-cells might contribute to lung damage in common variable immunodeficiency disorders (CVID). Materials and methods. In 16 adult patients, the majority of whom had pulmonary abnormalities, we studied T-cell subsets and markers of inflammation in bronchoalveolar lavage fluid (BALF) and blood and their relations with pulmonary function and high resolution computed tomography (HRCT). Results. We demonstrated that some of the lymphocyte abnormalities previously demonstrated in peripheral blood from CVID patients, such as low CD4/CD8 T-cell ratio, were also present in BALF. Moreover, low BALF CD4/CD8 ratio (≤ 1), found in seven patients, was significantly associated with higher blood CD8(+) cell count and to lower values of the lung function variables; forced expiratory volume (FVC), total lung capacity (TLC), vital capacity (VC) and residual volume (RV) in % of predicted. The expression of the inflammatory markers HLA-DR and CCR5 on T-cells was significantly higher, and the expression of CCR7 significantly lower, in BALF compared to blood, possibly reflecting an inflammatory/cytotoxic T-cell phenotype within pulmonary tissue in CVID. Furthermore, patients with bronchiectasis had higher concentrations of the pro-inflammatory cytokine TNFα in plasma, compared to those without. Conclusion. Our findings suggest that inflammation and T-cell activation may be involved in the immunopathogenesis of pulmonary complications in CVID.

Concepts: Immune system, Pulmonology, Asthma, Lung, Respiratory physiology, Bronchoalveolar lavage, Lung volumes, Spirometer

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Background. Unbound iron binding capacity (UIBC) in serum, which is s-total iron binding capacity (2 times s- transferrin) minus s-iron, may be a more accurate marker of empty iron stores than serum transferrin saturation. Previously we have shown this for healthy females of childbearing age. Methods. Now we used receiver operating characteristic (ROC) curve analysis to compare the diagnostic accuracy of s-iron, s-transferrin, s-transferrin saturation and s-UIBC in diagnosing empty iron stores in 29,251 female and 19,652 male outpatients. Empty iron stores were defined as s-ferritin less than 10, 15 or 20 μg/L. Results. At all definitions of empty iron stores s-UIBC had a better diagnostic accuracy than the other tests in both male and female outpatients, with an area under the ROC curve of 0.85-0.97. Also in subpopulations with elevated s-CRP or low b-hemoglobin s-UIBC was more accurate than the other tests. All tests performed better in males than in females, and generally they were more accurate in adults than in children. Conclusion. When diagnosing empty iron stores calculation of s-UIBC is a better way to utilize the information in s-iron and s-transferrin than the calculation of s-transferrin saturation.

Concepts: Male, Female, Receiver operating characteristic, Transferrin, Transferrin saturation, Serum iron, Total iron-binding capacity

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Objectives. The aim of this study was to determine the normal values of serum IGF-1 and IGFBP-3 in Turkish children and adults (1-79 years). Material and methods. The study included 571 healthy children and 625 healthy adults from the West Black Sea region of Turkey. Serum IGF-1 and IGFBP-3 concentrations were determined using a chemiluminescent immunometric assay on an Immulite 1000 analyzer. Results. IGF-1 and IGFBP-3 levels tended to be higher in girls compared to boys among the children. The differences were statistically significant in puberty from age 12-14 years for IGF-1 and prepubertally from age 9-10 years for IGFBP-3. Peaks of serum IGF-1 levels were observed 2 years earlier in girls (14 years) than boys (16 years). The general pattern of IGFBP-3 was similar to IGF-1 during puberty. In adults, IGF-1 and IGFBP-3 levels decreased by age. There was no significant difference in IGF-1 and IGFBP3 values between men and women in any age group. Conclusions. This study established age- and sex-specific reference values for serum IGF-1 and IGFBP-3 in healthy Turkish children and adults.

Concepts: Statistical significance, Insulin-like growth factor 1, Difference, Growth factors, Turkey, Insulin-like growth factor, IGFBP3, Black Sea

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To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3).

Concepts: DNA, Histone, Nucleosome, Chromatin

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Oxidative stress biomarkers of oral and systemic diseases can be found in saliva. However, there is no uniformity for the saliva collection time in these kinds of analyses and saliva composition may change because of mechanical stimulation. Therefore, the aim of this work was to determine the effect of mechanical stimulation for 10 min on the concentrations of vitamin C, vitamin E, total antioxidants and total oxidants in saliva. Saliva samples from individuals of both sexes, aged between 18 and 38 years, were collected for 10 min at 2 minintervals. Saliva flow rate in each 2 min period was measured, as well the total oxidant state, the total antioxidant capacity, vitamin C and vitamin E concentrations. All analyses were performed in triplicate and were determined using colorimetric tests. The results were analysed using t-test, Friedman’s test and analysis of variance (ANOVA) for repeated measures. Mauchly’s sphericity test was applied and, if necessary, technical corrections were made using the Greenhouse-Geisser test. We found no significant difference between the amounts of saliva produced across the collection times. Total oxidant status, total antioxidant capacity, vitamin C and vitamin E concentrations remained stable. Based on our findings, saliva can be collected for 10 min or less with masticatory stimulation without any variations in the concentration of the variables analysed. However, we suggest using saliva samples after two minutes of mechanical stimulation.

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Cytokines are biological response modifiers involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). This study investigated the potential use of cytokines as disease severity biomarkers in COPD patients and the possible effect of statin therapy on cytokine expression. Possible associations between cytokines, body mass index (BMI) and smoking have also been studied. Cytokines IFN-γ, IL-2, IL-12 p70, TNF-α, TNF-β, IL-4, IL-5, IL-6, IL-10, IL-1β and IL-8 were measured in the plasma of 100 clinically stable COPD patients using a fluorescent bead immunoassay on a flow cytometer. When patients were grouped according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage (A-D), no significant differences in cytokine concentrations were found (p > .05). Significantly decreased concentrations of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12p70 and TNF-α were found in COPD patients receiving statin therapy in comparison with COPD patients not receiving statin therapy (p < .05). COPD patients with increased BMI (>25) had decreased IL-2 (p=.038), IL-8 (p = .039) and IL-10 (p = .005) concentrations compared to normal BMI (20-25) patients. Current COPD smokers had increased concentrations of IL-5 (p = .037) compared to former COPD smokers. Hierarchical cluster analysis showed several patterns of measured cytokines in serum of patients with stable COPD. Statin therapy is associated with decreased expression of selected Th1 and Th2 cytokines in COPD, and this effect could be of relevance in COPD patients with increased cardiovascular risk. Concentrations of Th1 and Th2 cytokines in plasma cannot be used as biomarkers of disease severity or progression of COPD.

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Edoxaban is an oral direct factor Xa inhibitor for prophylaxis and treatment of thromboembolic disorders. The effects on common coagulation assays are clinically valuable information and in certain clinical situations a quick assessment of the anticoagulant is wanted. Our aim was to investigate the effect of edoxaban on routine coagulation methods and evaluate anti-Xa assays, commonly used for other direct factor Xa inhibitors, for estimation of the drug concentration. Edoxaban was spiked to plasma samples from healthy subjects in the concentration range 0-742 µg/L and analyzed using different reagents for activated partial thromboplastin time (APTT) and prothrombin time (PT). Assays for antithrombin, activated protein C resistance, lupus anticoagulant (LA) and chromogenic anti-Xa assays were also included. Edoxaban displayed similar effects in vitro to other oral direct Xa inhibitors. The concentration needed to double the coagulation time varied between assays and reagents; 539-758 µg/L for the APTT and between 329 and 2505 µg/L for the PT. Edoxaban gave false high antithrombin activities in assays based on Xa-inhibition. Two integrated assays for LA, both based on activation with dilute Russell’s viper venom, displayed different results. Chromogenic anti-Xa assays displayed linear dose-response curves with edoxaban up to approximately 500 µg/L. In conclusion, therapeutic concentrations of edoxaban variably affect different coagulation assays, and even different reagents within an assay group. In comparison with other oral Xa-inhibitors, the in vitro effects of edoxaban were more similar to rivaroxaban than apixaban. For measurement of edoxaban concentration in plasma, it is possible to use the chromogenic anti-Xa assays.

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The effect of omega-3 fatty acids on platelet aggregation and coagulation is highly unclear. Studies both support and refute the impacts of omega-3 fatty acids on prolonged bleeding time and platelet inhibition as well as its purported positive effects on cardiovascular disease. In a previous pilot study we suggested an inhibition of platelet aggregation measured with multiple electrode aggregometry. Following on that, the aim of the present study was to investigate the effects of supplementary high doses of omega-3 fatty acids on platelet aggregation and coagulation in a sample-size calculated number of healthy volunteers using Sonoclot, multiple electrode aggregometry, and flow-based Cellix instruments after 10 days of omega-3 fatty acid intake. Twelve healthy human volunteers ingested 2520 mg of supplementary omega-3 fatty acids per day for 10 days. Venous blood was sampled and platelet aggregation and coagulation were measured before and after the treatment period. The viscoelastic test instrument Sonoclot, multiple electrode aggregometry, and flow-based Cellix instruments with collagen-coated channels were used to evaluate platelet aggregation and coagulation. There were no differences in any of the measured variables after the treatment period as compared to before. In this well-powered study on healthy volunteers, no effects of high doses of omega-3 fatty acids after 10 days of intake could be demonstrated, either on coagulation or platelet function. Further studies are needed to clarify whether omega-3 fatty acids have a role in the regulation of the putative complex processes in vivo.

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Therapeutic drug monitoring of tacrolimus and cyclosporine is crucial to the success of organ transplantation. We evaluated the analytical performances and accuracy of two commercially available tacrolimus and cyclosporine assays (Roche ISD and Siemens) in comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 342 leftover whole blood samples requested for tacrolimus or cyclosporine assays were stored at -20 °C until analysis. Repeatability and between-run imprecision were evaluated using quality control materials provided by the manufacturer. Ring trial samples were used for the assessment of recovery. The results of the Roche ISD assay were compared with those of Siemens tacrolimus and cyclosporine assays and LC-MS/MS. Repeatability and between-run imprecision were 2.1-5.3% and 2.6-7.5%, respectively. Recovery of Roche ISD was 85.7 - 90.6% for cyclosporine and 96.2-98.5% for tacrolimus. The two immunoassays showed slight positive biases relative to LC-MS/MS for cyclosporine. For tacrolimus, Roche ISD produced virtually identical results to those of LC-MS/MS, whereas Siemens showed proportional differences, especially in patients receiving kidney transplantation. The analytical performances of Roche ISD were generally acceptable, especially regarding accuracy. Clinical laboratory staff should be aware of the strengths and weaknesses of commercial immunoassays in order to ensure accurate results.