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June 1, 2005
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The identification of circulating autoantibodies contributes to the correct diagnosis as well as to the follow-up of rheumatic diseases. Some autoantibodies are even included in diagnostic and classification criteria for these types of autoimmune diseases. There are several relatively specific screening and identification methods for the measurement of autoantibodies available. The type of assay crucially influences the diagnostic value of the parameters. In general, routine laboratories should prefer enzyme immunoassays (ELISA) using well characterized antigens, although ELISA tests tend to produce more false-positive and true weakly positive results, which reduce their positive predictive value. Therefore one should be aware that laboratory results can only be properly interpreted when there is a correlation with the clinical situation and when the limitations of the technologies used for autoantibody identification have been taken into consideration. A diagnostic algorithm consisting of screening and identification steps should be established by each laboratory in order to create a rational, evidence-based and cost-effective basis for the diagnosis of rheumatic diseases.
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June 1, 2005
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Protein arrays hold a promise in basic and clinical applications. As the first step to develop such array system, I used an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL) to demonstrate the feasibility of simultaneous detection of multiple proteins. In the direct ELISA system, different known immunoglobulin Gs (IgGs) were immobilized onto polyvinylidine difloride (PDVF) membrane through 96-well format Bio-Dot unit. The antigens were then individually and collectively detected by incubation of membranes with different antibodies coupled with ECL. In the sandwich ELISA system, the cytokine capture antibodies were immobilized onto PDVF membranes. The membranes were then incubated with single cytokine or a combination of different cytokines. The captured cytokines were detected by biotin-conjugated antibodies coupled with ECL system. Experiments demonstrated that multiple IgGs and cytokines could be simultaneously detected using this approach with high specificity and sensitivity. More importantly, cytokines from biological samples were detected using this approach, which can be used in any general laboratory setting without any sophisticated equipment. This concept could be extended to develop a protein-based array system.
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June 1, 2005
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This article describes a 13 C-labelling and nuclear magnetic resonance approach for hippuric acid test which is potentially useful for evaluating liver reserve. In this approach, urine samples collected after ingestion of 13 C-labelled benzoic acid were directly analysed by 13 C nuclear magnetic resonance spectroscopy, and the excreted 13 C-labelled hippuric acid formed from the administered benzoic acid was quantitated. The amount of labelled hippuric acid excreted in a specified time can be a useful index of liver reserve. In this study, the feasibility of the nuclear magnetic resonance approach has been investigated in several healthy subjects. This approach is simple and convenient compared with conventional analytical procedures, because no chromatographic separation is required. The approach could give new insights into the liver reserve, because the benzoic acid conversion to hippuric acid intimately relates to the hepatic energy metabolism. This measurement can be conducted at a wide range of dosages without interference from endogenous hippuric acid.
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June 1, 2005
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Hippuric acid (HA) originating from the conjugation of benzoic acid with glycine is a physiological component of human urine. Findings suggest that HA inhibits calcium oxalate (CaOx) growth and considerably enhances the CaOx solubility in artificial urine. Thus, it is assumed that HA is a major modifier of CaOx formation. However, only a slight CaOx growth inhibition of 1–8 ％ was also reported. These values were also derived from artificial urine. The key mechanism, which led HA to be of interest in urolithiasis research is the fact that in presence of Ca 2+ ions HA can form a hippurate complex. By forming such a complex, Ca 2+ concentration in urine decreases, and as a consequence, CaOx formation is inhibited. This study was performed in order to clarify the role of HA in native and artificial urine. Biochemical analyses to calculate the relative CaOx supersaturations and crystallisation experiments using an in-line laser probe were examined. BONN Risk Indices indicating the risk of CaOx crystallisation were calculated from the results of the crystallisation experiments. The results obtained from artificial as well as from native urines showed that HA has no significant effects on CaOx formation. We suggest that HA plays only a minor role as a crystallisation modifier in human urine.
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June 1, 2005
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Convenient techniques for measuring rates of bone turnover have been developed in recent years with the advent of biochemical markers of bone metabolism. One recent of these techniques is a collection method and quantitative enzyme immunoassay for free pyridinoline crosslinks in human sweat. The concentrations of pyridinoline crosslinks in 5-day sweat collections and first morning void and 24-hour urine collections from healthy subjects and subjects with established metabolic bone disorders were determined. T-scores were higher in the sweat system than in the urine system by up to 10-fold in postmenopausal subjects, women with hyperparathyroidism, and subjects with postmenopausal osteoporosis. For subjects with postmenopausal osteoporosis, receiver-operating characteristic curve analysis yielded areas under the curve of 0.699, 0.629, and 0.520 for sweat pyridinoline, first morning void urine pyridinoline, and 24 hour urine pyridinoline respectively. The areas under the curve of the sweat and first morning void urine measurements were significantly greater (p﹤0.05) than the 24-hour pyridinoline measurements. Healthy postmenopausal subjects and subjects with postmenopausal osteoporosis were monitored before and during estrogen replacement therapy or alendronate therapy. Sweat pyridinoline values declined by 49.0 ±12.4％ and 19.4 ±19.9％ for estrogen and alendronate subjects respectively. We conclude that this non-invasive technique is a sensitive and specific measure of bone resorption and is appropriate as an adjunct to techniques such as bone density and may also be useful in monitoring of response to anti-resorptive therapies.
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June 1, 2005
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Reduced and total homocysteine, cysteine, glutathione and cysteinylglycine in plasma were investigated in 19 patients with chronic obstructive pulmonary disease and in 29 healthy subjects. The purpose was to examine the influence of pro-oxidant activity caused by the lung disease on the metabolism of homocysteine and other plasma thiols. We observed a decreased concentration of reduced glutathione and a decreased ratio of reduced/total glutathione in the patients compared to the healthy individuals, which supports the hypothesis of an association between free radicals and pathogenesis in some lung diseases. We also observed an increased total plasma homocysteine. The influence of a possible extracellular pro-oxidant activity on the concentration of total plasma homocysteine is discussed.
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June 1, 2005
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The impact of exercise tolerance test on oxidative stress was assessed by thiobarbituric acid reactive substances and markers of antioxidant status, namely Cu Zn superoxide dismutase, glutathione peroxidase, glutathione and vitamin E in blood samples of patients with exertional angina. The study was aimed to differentiate patients with positive exercise test (coronary heart disease patients) from patients with negative exercise test, at rest and peak exercise with respect to the investigated variables. Significantly lower values for both glutathione peroxidase activity and glutathione level were observed in patients after exercise test (p﹤0.01 and p﹤0.05, respectively). Only the patients with positive exercise test had significantly lower values for Cu Zn superoxide dismutase, glutathione peroxidase and glutathione, and a significantly higher ratio of thiobarbituric acid reactive substances/glutathione after exercise, as compared to before (p﹤0.05, p﹤0.05, p﹤0.05, p﹤0.01, respectively). Our findings indicate that the exercise test applied to patients with exertional angina oxidatively stresses the erythrocytes to a greater extent in exercise test (+) patients than in exercise test (−) patients.
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June 1, 2005
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In patients suffering from a variety of severe diseases the detection of erythroblasts in peripheral blood is associated with poor prognosis. However, as yet the prognostic significance of erythroblasts in the blood of patients after cardiothoracic surgery has not been assessed. In a retrospective study we analyzed the database of 2074 patients, of whom 87 died in hospital during the postoperative period. All patients underwent cardiothoracic surgery using a heart-lung machine. Together with erythroblasts in blood, age, sex, body mass index, preoperative ejection fraction, smoking, diabetes mellitus, type of operation, emergency surgery, renal deficiency, pulmonary hypertension, and endocarditis were considered. The postoperative mortality of patients with erythroblasts in peripheral blood (n=57) was 45.6％ (n=26), being significantly higher (p﹤0.001) than the mortality of patients without erythroblasts (3.0％). None of six patients with more than 2000 erythroblasts x 106/l survived. The postoperative detection of erythroblasts is highly predictive of death, the odds ratio after adjustment for the other known prognostic factors being 7.2 (95％ confidence interval 3.4–15.1). Erythroblasts were detected for the first time on average 11 ±2 days (median: 7 days; n=57) after surgery and 8 ±2 days (median: 6 days; n=26) before death. The detection of erythroblasts in blood after cardiothoracic surgery has a high prognostic significance in terms of in-hospital mortality, helping physicians to identify patients at high risk of death. This finding has to be confirmed by a prospective study with the use of a more sensitive and reliable technology and prospectively defined time intervals for counting blood cells.
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June 1, 2005
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The occurrence of acne in women with hyperandrogenemia is well known; a question remains, however, as to whether a further positive relationship can be detected between the intensity of acne and the levels of testosterone, androgen precursors and sex hormone binding globulin (SHBG). A procedure of interactive data analysis extracting relevant information from original data was applied. Exploratory data analysis (EDA) identifies basic statistical features and patterns of data using a variety of diagnostic displays. The need for this step is particularly acute in biochemical and clinical data, the distribution of which is mostly non-Gaussian and often corrupted by the outliers. The omission of EDA can lead to incorrect results and false conclusions. In the EDA (i) several graphical tools for summarizing data are applied, (ii) the peculiarities of a sample distribution are investigated, (iii) a construction of distribution is carried out, (iv) a graphical comparison of the sample distribution with selected theoretical distributions is employed. The proposed procedure is illustrated by typical case study in the evaluation of differences between mean values of serum levels of testosterone, androgen precursors and SHBG in a group of patients with mild and severe forms of acne. A knowledge of the interval estimate of the mean value in both groups enables their comparison at the chosen probability level. As will be apparent from the evaluation of inter-group SHBG differences, an incorrect approach to the determination of group mean values could result in a complete misinterpretation of the data. The results indicate that androgens are not significantly related to the intensity of acne, and that SHBG is higher in patients with more severe forms of acne.
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June 1, 2005
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Standardization of 22 clinical chemistry analytes and five serum protein constituents has been performed in the Fukuoka Prefecture, which has a population of approximately five million. The standardization project was established to determine reference intervals for these analytes by educating physicians, medical technologists and staff of medical institutions, and by daily or monthly monitoring the use of common control samples through e-mail. Standardization extended to 97％ of the institutions in the prefecture. Results for 14 of the 22 clinical chemistry analytes have become highly reliable and differences between institutions decreased. Standardization of other analytes is now in progress. Regional collaboration based on international guidelines led to a significant improvement in interlaboratory comparability. Areas where further improvements are needed have been identified.
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June 1, 2005
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Measurement of lipoprotein lipase activity in postheparin plasma is generally accompanied by moderate within-run variation Cv w-R (﹤10％) and higher between-run variation Cv b-R (5–25％). A calibration system was introduced in order to improve the reproducibility of measurements and to compare lipoprotein lipase activities from different days. Every day a calibration curve for lipoprotein lipase activity was constructed. Fifteen calibration curves designed over 2 years, show linearity over the whole biological spectrum and a considerable reduction of between-run variation in lipoprotein lipase activity, from 42％ to 5.3％ as estimated from two control postheparin plasma samples. The lipoprotein lipase calibration system is an easy and very cheap arrangement, which makes it possible to compare lipoprotein lipase activities achieved over years. When the lipoprotein lipase control values are compared with reference lipoprotein lipase samples determined in other lipase laboratories, the calibration-control system becomes an important tool for reducing analytical bias. The article reviews the original analytical criteria of catalytic measurement of lipoprotein lipase activity and describes the implementation of the calibrationcontrol system. We describe a model for reduction of the analytical variability in the measurement of lipoprotein lipase activity. Other standardization efforts need to be made in the future, especially to define the reference material for calibration.
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June 1, 2005
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We evaluated a direct assay for the determination of LDL-cholesterol (LDL-C) L-Type assay, Wako Pure Chemicals in two laboratories. This assay is applicable to most random access clinical chemistry analyzers, allowing full automation. Between-run coefficient of variation (NCCLS EP5) varied between 1.29 ％ and 3.13 ％ and thus met the National Cholesterol Education Program (NCEP) goal. The assay was considered linear over a physiologically relevant range of LDL-C, 2.22 to 7.04 mmol/l (NCCLS EP6). Method comparison yielded identical results at both evaluation sites for LDL-C when assayed with the direct method. LDL-C results obtained with the homogeneous method under investigation (y) differed significantly from values from density-gradient ultracentrifugation (x) according to Chung (y = 0.87x + 0.43 mmol/l, s yx = 0.38 mmol/l, r = 0.91). With the latter method as a reference method, mean bias was 3.16 ％ meeting the NCEP criteria. Diagnostic performance was excellent at a clinically relevant cut-off level of 3.37 mmol/l. Results of the direct method (y) and the commonly used Friedewald formula (x) were highly correlated (s yx = 0.22 mmol/l, r = 0.97), but both slope and intercept differed significantly from one and zero respectively (y = 0.90x + 0.37 mmol/l). Bilirubin, hemolysis and ascorbate did not interfere; triglycerides did not cause clinically relevant interference below 11.3 mmol/l. The direct method we investigated is user-friendly and provides an improvement in the determination of LDL-C in routine laboratories.
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June 1, 2005
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July 27, 2005
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A reference measurement procedure for substance concentration determination of total CO 2 in blood, plasma (the anticoagulant is usually heparin) or serum is described. The document covers the principle of the method, the materials and equipment needed and essential aspects of the procedure. The substance concentration of total CO 2 in blood, plasma or serum is accurately determined and therefore this procedure is recommended for assigning reference values to reference materials and for blood gas quality assurance in laboratories and by manufacturers of blood gas equipment.
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July 27, 2005
