Standardization of creatine kinase-MB (CK-MB) mass assays: the use of recombinant CK-MB as a reference material.
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BACKGROUND: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays. METHODS: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples. RESULTS: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias. CONCLUSION: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays. (+info)
Measurement of urea in human serum by isotope dilution mass spectrometry: a reference procedure.
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BACKGROUND: A reference measurement procedure is needed to demonstrate the traceability of results of urea measurements in human serum. We developed a measurement procedure using the principle of isotope dilution gas chromatography/mass spectrometry. METHODS: [(13)C,(15)N(2)]Urea as internal standard was added to a serum sample and equilibrated with endogenous nonlabeled urea. For the preparation of calibrators, the same amount of labeled urea was mixed with known amounts of nonlabeled urea. The serum samples were treated with ethanol to remove proteins by precipitation. The labeled and nonlabeled urea of the samples was converted into a trimethylsilyl derivative of 2-hydroxypyrimidine. The gas chromatography/mass spectrometry system was adjusted to monitor m/z 153 and 168 for the nonlabeled urea derivative and m/z 156 and 171 for the isotopically labeled analogs. The results of the determination were calculated from peak ratios by a hyperbolic calculation function based on the theory of isotope dilution analysis. RESULTS: The procedure was applied to control samples and patient samples and evaluated with respect to its trueness and precision. The standard uncertainty of the results was 0.47-1.72%. CONCLUSIONS: This reference measurement procedure allows values to be assigned to controls and calibrators that are traceable to the primary urea reference material of NIST and, therefore, to the Systeme International unit "mole" with a low degree of uncertainty. This procedure provides a tool for the highly accurate determination of urea in control materials as well as in patient sera. (+info)
Analytical characterization of electrochemical biosensor test strips for measurement of glucose in low-volume interstitial fluid samples.
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BACKGROUND: Minimally invasive interstitial fluid (ISF) sampling and glucose measurement technologies were integrated into a hand-held device for diabetic glucose monitoring investigations. METHODS: Conventional electrochemical test strip technology (Bayer Glucometer Elite) was adapted to measure glucose in small (0.5-2.0 microL) samples of ISF. Test strip glucose measurements were performed on a commercial potentiostat and were compared to various reference glucose methodologies (YSI 2300 analyzer, microhexokinase procedure, Bayer Glucometer Elite). Characterizations of the integrated ISF sampling-glucose test strip design included accuracy and precision in various sample media (saline, ISF surrogates, diabetic ISF samples), sample volume dependence, test strip sterilization studies (electron beam, gamma irradiation), and diabetic ISF sampling and glucose measurements. RESULTS: Glucose measurements were free from significant media effects. Sample volume variations (0.6-3.2 microL) revealed only modest dependence of glucose measurement bias on sample volume (-1.5% per microliter). Sterilization treatments had only a minor impact on glucose response and test strip aging and no significant impact on interferent responses of the glucose test strips. Diabetic subject testing under minimum fasting conditions of at least 2 h with integrated ISF sampling and glucose measurement gave low ISF glucose measurement imprecision (CV, 4%) and mean glucose results that were indistinguishable from reference (microhexokinase) ISF glucose measurements and from capillary blood glucose measurements (Glucometer Elite). CONCLUSIONS: Conventional single-use, electrochemical glucose test strip and ISF collection technologies can be readily integrated to provide real-time ISF sampling and glucose measurements for diabetic monitoring applications. (+info)
A new method with general diagnostic utility for the calculation of immunoglobulin G avidity.
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The reference method for immunoglobulin G (IgG) avidity determination includes reagent-consuming serum titration. Aiming at better IgG avidity diagnostics, we applied a logistic model for the reproduction of antibody titration curves. This method was tested with well-characterized serum panels for cytomegalovirus, Epstein-Barr virus, rubella virus, parvovirus B19, and Toxoplasma gondii. This approach for IgG avidity calculation is generally applicable and attains the diagnostic performance of the reference method while being less laborious and twice as cost-effective. (+info)
International standards for the assessment of the risk of thermal strain on clothed workers in hot environments.
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The International Standards Organisation (ISO) has produced an integrated series of international standards for the assessment of human responses to thermal environments. They include standards for the assessment of thermal comfort, heat stress and cold stress and many have been adopted as European and British standards. This paper describes the series of standards and in particular those concerned with the assessment of risk in hot environments. A three tier approach is taken which involves a simple thermal index that can be used for monitoring and control of hot environments (ISO 7243), a rational approach which involves an analysis of the heat exchange between a worker and his or her environment (ISO 7933) and a standard that describes the principles of physiological measurement which can be used in the establishment of personal monitoring systems of workers exposed to hot environments (ISO 9886). The standards are self-contained and can be used independently. In any comprehensive assessment however they would be used in conjunction. The simple index provides a first stage analysis and can confirm whether or not there is likely to be unacceptable thermal strain. Where a more detailed analysis is required then ISO 7933 provides an analytical method that can provide a more extensive assessment and interpretation leading to recommendations for improvement to the working environment. Where a method needs to be confirmed, or conditions are beyond the scope of ISO 7243 and ISO 7933, then ISO 9886 provides guidance on physiological measurement and interpretation. This would be used in extreme environments where individual responses are required to ensure health and safety or, in the case where personal protective equipment (PPE) is worn, which is beyond the scope of ISO 7243 and ISO 7933. The ISO system therefore covers almost all exposures to hot environments. It would be useful however to extend the scope of the standards that provide a simple index or analytical approach. This paper describes the current standards and their scope and forms the basis and background for descriptions of proposed extensions to the scope of the standards described in other papers in this special issue. (+info)
Development of a draft British standard: the assessment of heat strain for workers wearing personal protective equipment.
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Existing methods for estimating heat stress, enshrined in British/International Standards (the Wet Bulb Globe Temperature (WBGT) index [BS EN 27243] and the Required Sweat Rate equation [BS EN 12515; ISO 7933 modified]), assume that the clothing worn by the individual is water vapour permeable; the WBGT index also assumes that the clothing is relatively light. Because most forms of personal protective equipment (PPE) either have a higher insulative value than that assumed or are water vapour impermeable, the Standards cannot be accurately applied to workers wearing PPE. There was, therefore, a need to develop a British Standard which would allow interpretation of these existing Standards for workers wearing PPE. Relevant information was obtained through reviewing the literature and consulting experts. Two questionnaire surveys of potential users of the Standards were conducted, and physiological data collected both experimentally and in work situations were considered. The information collected was used to develop the draft British Standard. It provides information and data on: The general effect of PPE on heat balance of the body (the ability of the body to maintain its 'core' temperature within an acceptable range). The effect of specific forms of PPE on metabolic heat production rate. The thermal insulation and evaporative resistance of types of PPE. The effect of the closure of the garments to the body on heat transfer. The effect of the PPE on the proportion of the body covered. The effect of an air supply (for example, Breathing Apparatus [BA]) to the wearer. Guidance is given on conducting an analysis of the work situation, taking account of the impact of PPE. Detailed methods of interpreting both BS EN 27243 and BS EN 12515 for workers wearing PPE are given, taking account of the factors listed above. Three worked examples using BS EN 27243 and BS EN 12515 are given in the Annex of the draft Standard. (+info)
Analysis of gamma-hydroxybutyrate (GHB) in urine by gas chromatography-mass spectrometry.
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A simple method for the direct analysis of gamma-hydroxybutyrate (GHB) from human urine is described. The method uses solid-phase extraction, liquid-liquid extraction, and silyl-derivatization, then gas chromatographic-mass spectrometric analysis using GHB-d6 as the internal standard. The method was linear from 5 to 500 mg/L, and coefficients of variation were less than 10%. Twenty-six urine specimens previously analyzed by an existing method were analyzed and yielded GHB concentrations ranging from 0 to 6100 mg/L; the results correlated between the two methods. Compared with existing methods, the method described here is superior because it is specific to GHB and can discriminate between GHB and gamma-butyrolactone. (+info)
Quantitative determination of LSD and a major metabolite, 2-oxo-3-hydroxy-LSD, in human urine by solid-phase extraction and gas chromatography-tandem mass spectrometry.
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An assay has been developed for quantitative determination of lysergic acid diethylamide (LSD) and a major metabolite of LSD in human urine at concentrations as low as 10 pg/mL. In most LSD-positive urine samples the metabolite, 2-oxo-3-hydroxy-LSD, is present at higher concentrations than LSD and can be detected for a longer time than LSD after ingestion of the drug. Urine samples are extracted using Varian Bond Elut Certify extraction cartridges. Confirmatory identification is accomplished by trimethylsilylation of LSD and 2-oxo-3-hydroxy-LSD, followed by gas chromatography-tandem mass spectrometry analysis using positive ion chemical ionization and selected reaction monitoring. Commercially available lysergic acid methylpropylamide and 2-oxo-3-hydroxy-LAMPA are used as internal standards. With selected reaction monitoring, both compounds gave linear calibration curves from 10 pg/mL to 5000 pg/mL. Forty-nine human urine samples that had previously been shown to contain LSD were reanalyzed by the new method. These samples showed an average LSD concentration of 357 pg/mL and an average 2-oxo-3-hydroxy-LSD concentration of 3470 pg/mL. Additional experiments using clinical samples in which two subjects were dosed with LSD support the conclusion that analysis for 2-oxo-3-hydroxy-LSD can permit identification of LSD users for a longer period following ingestion than analysis for the parent drug. (+info)