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Abstract
Total quality management involves the consideration of many quality subjects as part of the management, such as quality processes, quality education, quality assurance, quality planning, quality results and quality document management. But crucial quality elements are also communication, data management and information sharing. Web applications and other associated computer communication applications such as E-mail and newsgroups, for example, offer to the laboratory environment the best tools to achieve proper communication and data management/sharing. These applications, enabling the set-up of Internet and Intranet sites, are used to share the information in the form of simple text pages or of completely interactive pages, which could comprise audio and video files, web page formulae and web data management applications. These applications are being associated to several applications and also being integrated into the laboratory information system (LIS).
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Abstract
There is increasing evidence that atherosclerosis is a chronic inflammatory disorder resulting from a combination of processes, and that acute exacerbations of this inflammation are associated with the acute coronary syndromes such as myocardial infarction and unstable angina. Measurement of the serum level of acute phase proteins, such as C-reactive protein and serum amyloid A protein, has been used to predict the risk of acute events in patients with atherosclerosis. Prospective studies have shown that higher serum acute phase protein levels, often within the reference range, are associated with increased risk of myocardial infarction (MI), stroke or peripheral vascular disease and predict risk of infarction and death among high-risk patients. These observations have important implications for the assessment of patients and for treatment.
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Tissue homeostasis is fundamentally influenced by the functional integrity and state of endothelial cells. Survival and death of endothelial cells are encountered in cardiovascular disease and may, moreover, affect and determine the development of atherosclerosis and restenosis following intracoronary therapeutical interventions. Apoptosis was studied in cultured human umbilical vein endothelial cells (HUVEC) to investigate the regulation of endothelial cell death following serum/growth factor depletion as well as incubation with actinomycin-D. Apoptosis was verified by DNA fragmentation and quantified by fluorescence activated cell sorting (FACS) analysis after TdT-mediated deoxyuridine-triphosphate nick end-labeling (TUNEL). An ELISA was used for detecting intracytoplasmatic nucleosomes. Untreated HUVEC showed 16 ± 6 % TUNEL positive cells after 24 hours as analyzed by FACS. Serum/growth factor depletion increased apoptosis by 79 ± 7%, while 50 ng/ml of the pro-apoptotic drug actinomycin-D induced comparable effects (72 ± 11% ). Apoptosis by serum/growth factor depletion could be blocked completely by the anti-apoptotic agent cycloheximide (2 μg/ml), but was ineffective in blocking actinomycin-D-induced apoptosis. Pyrrolidine dithiocarbamate (PDTC) also acted as an anti-apoptotic agent by blocking apoptosis induced by actinomycin-D, but had no effect on apoptosis induced by factor depletion. Thus, two independent mechanisms for regulation of apoptosis are suggested to be present in human vascular endothelial cells.
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Abstract
Lipid peroxidation biomarkers and antioxidant status were measured in 76 cystic fibrosis (CF) patients and compared to 40 control subjects. Univariate and multivariate statistics were performed in this study. Results showed that indicators of lipid peroxidation were higher in CF patients than in controls; thiobarbituric acid reactants and autoantibodies against oxidized low-density lipoproteins were significantly increased in CF patients. Red blood cells and whole blood glutathione peroxidase activities were lower in CF patients than in controls. No difference in red blood cell superoxide dismutase activity was observed. Measured concentration of glutathione peroxidase in plasma showed a higher mean value of this protein in CF patients than in controls. Retinol, α-tocopherol and β-carotene concentrations were all reduced in CF patients as compared to controls; this was particularly pronounced for β-carotene. The decreased α-tocopherol concentration was associated with higher percent hemolysis in CF patients. The results of this study indicate that both lipid peroxidation biomarkers and antioxidant status were disturbed in CF patients, despite medical assistance. Measures of oxidative stress parameters, such as thiobarbituric acid reactants, glutathione peroxidase, and β-carotene concentrations can be considered as significant indicators to discriminate CF patients and control subjects.
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Abstract
Autoantibodies against oxidised low-density lipoprotein (OxLDL-Abs) have been proposed to be an indicator of endothelial dysfunction and a novel tool for finding individuals with a high cardiovascular risk. In a cross-sectional study, OxLDL-Abs were measured in 297 patients with obstructive sleep apnoea (OSA) and 54 controls using an enzyme-linked immunosorbent assay. The autoantibodies were increased in patients with OSA when compared to controls (age, body mass index (BMI) and gender adjusted, p = 0.001). However, within the OSA patients, OxLDL-Abs were not related to smoking, hypertension or BMI, and there was a weak negative correlation (r = −0.16, P = 0.007) between age and levels of OxLDL-Abs. In conclusion, at present the measurement OxLDL-Abs still remains a method for basic research and is not applicable for screening of at-risk patients with OSA.
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Human inter-α-inhibitor (IαI) has been shown to exert a beneficial therapeutic effect in a porcine model of endotoxin shock. It is therefore useful to have a better understanding of IαI metabolism during severe inflammatory syndromes. Experimental bacterial pneumonia was induced in pigs. The acute phase response was highlighted by an increase in pig major acute phase protein (pig-MAP) and haptoglobin concentrations in plasma collected daily over 4 days. In the same samples, the IαI levels remained unchanged. Moreover, crossed-immunoelectrophoretic and immunoblot analyses did not show any qualitative modification of IαI throughout the experiment. IαI has been reported to be a negative acute phase protein in both humans and rats. Here we demonstrated that IαI behavior clearly differs in humans and pigs and is definitively species specific.
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Abstract
The prosclerotic transforming growth factor β1 (TGF-β1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-β1 mRNA levels, the comparative kinetic reverse transcription-polymerase chain reaction strategy was used. In this method, cellular mRNA levels of the target and a house-keeping gene are reverse transcribed, amplified by the polymerase chain reaction (PCR) and the kinetics of PCR amplification are compared. Since the current determination of the PCR products, using electrophoretic separation in polyacrylamide gel, staining and scanning of the gel, is timeconsuming (≥ 5 hours) and inaccurate, we have developed a method using capillary gel electrophoresis (CGE) in combination with laser-induced fluorescence (LIF) detection for quantification of PCR-products. Using the CGE-LIF method, a minute aliquot of the PCR reaction mixture is separated and quantified within 10 min. Comparison of the values with those obtained by polyacrylamide gel electrophoresis demonstrates the improved sensitivity (> 1000 fold) and accuracy of the proposed method. The CGE-LIF procedure offers a convenient way of automated, comparative analysis of low levels of mRNA via reverse transcription PCR in low cell numbers or small amounts of tissue samples.
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A reliable simple reversed-phase liquid chromatographic method for the routine determination of ascorbic acid in plasma and urine with ultraviolet detection is described. This method enables the complete separation of the ascorbic acid peak from others with a recovery of above 95 % within 8 minutes. The method can be used for analysing multiple samples within a day. In addition, the storage conditions and stability of ascorbic acid in plasma and urine were investigated. Samples of plasma and urine can be stored on ice in darkness for at least 60 min without reduction of ascorbic acid concentration. Prepared samples can be stored in darkness at 4 °C for at least 120 min and in liquid nitrogen for 42 days.
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We evaluated a new ready to use liquid assay for the homogeneous determination of HDL-cholesterol (HDL-C; Merck, Darmstadt, Germany) in comparison to phosphotungstic acid precipitation and a homogeneous assay, based on sulfated α-cyclodextrin and polyethylene glycol-modified enzymes (Roche Diagnostics/Boehringer Mannheim, Germany). The new liquid homogeneous HDL-C assay had inter-assay coefficients' of variation of less than 2.1 %. The method is linear up to at least 3.11 mmol/l HDL-C, but even at 4.40 mmol/l the deviation from the expected value is less than 5%. Spinking experiments with low density lipoproteins and very low density lipoproteins proved that the new assay was specific for high density lipoproteins up to cholesterol associated with low density lipoproteins (LDL-C) and very low density lipoproteins (VLDL)-triglyceride concentrations of 18.13 and 22.60 mmol/l, respectively. Free fatty acids above 2 mmol/l did not interfere. Icteric samples with bilirubin concentrations between 170 and 400 μ mol/l did not show any systematic deviation compared to the precipitation procedure. In addition, serum hemoglobin concentrations up to 7.0 mmol/l and ascorbic acid up to 3000 μ mol/l did not interfere with the HDL-C assay. An intermethod comparison including 120 samples revealed good agreement of the liquid HDL-C assay and the precipitation procedure (y = 0.943x + 0.074 mmol/l; r = 0.992). The new homogeneous HDL-C assay is thus precise, comparable and robust. Due to its ease of handling this assay will significantly facilitate attempts to include the differentiation between HDL-C and LDL-C in the routine screening for cardiovascular risk factors and in the monitoring of lipid lowering therapy.
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In this study, 112 serum samples were analyzed for total prostate-specific antigen with three well-established assays i.e. Tandem R and Tandem E (both from Hybritech Inc., San Diego, USA) and Prostatus Free/Total from Wallac Oy, Turku, Finland. Thirty-two samples were collected from prostate cancer patients, 32 from patients with benign prostate hyperplasia and 48 from men participating in a screening study for prostate cancer. The aim of the study was to compare the results before and after recalculation with the data obtained with two reference preparations for total prostate-specific antigen: Stanford 90:10 PSA Calibrator and Certified Reference Material 613 Prostate-Specific Antigen. Comparing the actual results revealed almost perfect correlations between Tandem R and Tandem E and between both Tandem assays and Prostatus. We observed statistically significant differences in accuracy between Tandem R and Tandem E: y(Tandem E)= 1.05 × (Tandem R)+0.07, and between Tandem E and Prostatus: y(Prostatus)= 0.94 × (Tandem E)+0.02 In both comparisons prostate-specific antigen values ranged from 0–40 μg/l. Recalculation with both reference preparations did not solve these discrepancies. One exception was the combination Tandem R and Tandem E. The application of either reference preparation solved the differences in accuracy here. In conclusion, even after recalibration, assays for total prostate-specific antigen are still not completely interchangeable.
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The aim of the study was to deduce analytical quality specifications for the determination of catalytic concentration of serum lactate dehydrogenase isoenzyme 1 (S-LD-1) according to clinical goals (the clinical utility model). We defined clinical goals for false positive and false negative S-LD-1 measurements in the monitoring of patients with testicular germ cell tumors (TGCT), clinical stage I, on a surveillance only program. The absolute S-LD-1 catalytic concentrations were routinely corrected for contamination from preanalytical hemolysis. A reference group of 37 men had a near ln-Gaussian distribution for the absolute S-LD-1 catalytic concentration. The geometric mean was 76 U/l and an S-LD-1 > 128 U/l (99.72 percentile, the decision limit) indicated a high risk of a relapse of TGCT. We have previously shown that an S-LD-1 > 160 U/l (treatment limit) was associated with a suboptimal outcome from the treatment of metastatic TGCT. The maximum allowable analytical positive bias was 5 U/l, and the maximum allowable analytical negative bias was −32 U/l. The maximum allowable analytical coefficient of variation, CV A , was 11 % (≈14 U/l) at a bias = −5 U/l. For S-LD-1 measurements not corrected for hemolysis, the decision limit was 145 U/l, the maximum allowable negative bias −19 U/l, and CV A 8 % (≈12 U/l). A routine correction for hemolysis had a large impact on the analytical quality specifications.
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N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, known as HEPES buffer, with p K in the physiological range was studied for use as an alternative to conventional phosphate buffer for the calibration of pH in modern clinical analyzers. In different series of aqueous equimolar HEPES buffer, pH was measured at 37 °C with a capillary glass electrode standardized previously using phosphate, and variations due to changes in total HEPES buffer concentration (0.025 to 0.320 mol/l), and NaCl (0 to 0.250 mol/l) were monitored. For 0.05 equimolar HEPES buffer without NaCl, the pH of 7.362 ± 0.003 (n = 15) obtained coincided well with the reference pH (7.364) from the National Institute of Standards and Technology (NIST). In particular, in the preferred 0.05 equimolar HEPES buffer/0.110 mol/l NaCl, which is isotonic to human plasma (0.160 mol/l), and termed physiological HEPES buffer (PHB), the pH of 7.346 ± 0.003 (n = 84) can be related to the calculated corresponding reference pH from NIST without liquid junction (7.374), and is also compatible with the pH measured in normal arterial blood, pH = 7.403 ± 0.003 (n = 20). Hence, in the two-point calibration of clinical analyzers, PHB, which is defined operationally with respect to the glass electrode and to phosphate buffer, may be useful as a calibrator in the range of buffer adjustment control to meet the correct values for pH when measuring in blood. Whereas Na-HEPES salt is hygroscopic and does not meet the declared purity grade (> 99 %), pure HEPES acid is non-hygroscopic and conforms to the manufacturer's purity grade (≥ 99 %). Therefore, for easy preparation of PHB, HEPES acid is the preferred starting material.
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The population sample of the Kristianstad survey, a reference intervals survey in the county of Kristanstad, was used to establish new reference intervals in clinical chemistry at the laboratories of the Central Hospital in Kristianstad, the University Hospital in Lund and the University Hospital in Malmö. Three-hundred and fifty nine subjects, male and female, aged 20–80+ years, were invited to participate in the study, with a participation rate of 70 %. Up to 70 analyses were performed on each subject, general clinical chemistry parameters in all three laboratories, specialized analyses where available. Separate a priori exclusion criteria were defined for each test. In addition, the test pattern of each individual was evaluated for signs of preclinical disease. Twelve cases of preclinical disease were discovered and clinically confirmed. Details on all test methods are presented along with information concerning instruments used, calibration procedures, methods of calculation and obtained reference intervals. Although the methods were in general calibrated against acknowledged reference materials, in some instances differences were found that made common reference intervals across all laboratories impossible. Problems relating to the practical use of international recommendations and the establishment of reliable reference intervals are discussed.
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Cytokines are key mediators in cell regulation and communication. The concentration of these proteins can rapidly and importantly increase during severe clinical situations. However, current techniques are not adapted to stat measurement, thus making their clinical use limited. In this context, the commercialization of five new kits for cytokine measurement interleukin ((IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-α and IL-2R) on an automated immunoanalyzer, the Immulite ® , seems to be a new approach for the determination of these markers. We report here the evaluation of the performance of these tests. The technique is based on a solid phase (bead) two site chemiluminescent enzyme immunometric assay. The analysis is performed within 60 to 90 minutes and the calibration is stable for 15 days. The values of the between-run imprecision study were similar to those from the within-run study with coefficients of variation (CV) ranging from 2% (low values of IL-8) to 11.5 % for intermediate concentrations of IL-6 (500 pg/ml). CVs were usually around 5%. The accuracy was determined by a linearity study using standards (except for IL-2R) provided by the National Institute for Biological Standards and Control (NIBSC). Slopes obtained during this study were close to 1 (r 2 = 0.99), except for IL-6, for which the slope was 1.55. TNF-α values were close to those expected. IL-1 results were about 20 % higher. IL-6 values were over estimated above 100 pg/ml and under estimated below this value. IL-8 study seemed to be impaired by the poor stability of this molecule in the NIBSC preparation. Correlation study with standard laboratory techniques gave variable results : for IL-1 (n = 43) the slope was 0.77 (study carried out using cell culture media), for IL-6 (n = 54) the slope was 0.78, for IL-8 (n = 37) the slope was 1.64, for TNF-α (n = 40) the slope was 0.33 and the slope for IL-2R (n = 51) was 5.1. For the last cytokine, the unit in Immulite assay was different from the one used in our comparison technique. Cross-calibration results were consistent with these data and show that the bias is probably linked to a calibration problem. The study demonstrated excellent practicality of the system, and good stability of the calibration curve (15 days). However, the sample volume required (350 μl for the IL-6 and the TNF-α) could constitute a limitation for pediatric measurements.
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