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June 1, 2005
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The utilization of accurate and sensitive methods for the measurement of cytokines in body fluids is prerequisite for the proper use of these mediators in clinical practice. Many factors contribute to the complexity of cytokine quantitation. Bioassays historically preceded immunoassays, which are now very popular, but there is a need for standardization. Nevertheless, due to the local effects of cytokines, the study of their blood levels is of limited value for an understanding of the pathophysiology of these mediators. This explains the development of alternative approaches to assess the ability of cells to produce cytokines. These include the Enzyme-Linked Immuno Spot Assay (ELISPOT), the measurement of cell-associated cytokines by flow cytometry, and the study of cytokine secretion by isolated peripheral blood mononuclear cells or by whole blood test. All these techniques, associated with a local detection of cytokines by immunohistochemistry or in situ hybridization and reverse transcriptase polymerase chain reaction, appear to be complementary tools for a better understanding of the biology of cytokines. Selected examples of possible clinical applications related to infectious diseases, cancer, autoimmune diseases, allergy, transplantation and preclinical evaluation of drugs and biotechnology products are given.
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June 1, 2005
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Measurements of lipids and lipoproteins in the clinical laboratory have become increasingly important because of their predictive association with cardiovascular diseases, especially coronary artery disease. The US National Institutes of Health-sponsored National Cholesterol Education Program and counterparts in other countries have developed national consensus guidelines for diagnosis and treatment of coronary artery disease which provide risk cut-points and define use of the lipid/lipoprotein analytes in case finding and therapy. Total and low density lipoprotein cholesterol and triglycerides are measured as positive risk factors and high density lipoprotein cholesterol as an inverse risk factor for coronary artery disease. A National Cholesterol Education Program-sponsored expert laboratory panel has developed guidelines for measurements with requisite analytical performance targets for total error and corresponding precision and bias. The US Centers for Disease Control and Prevention have established reference methods for total and high density lipoprotein cholesterol and for triglycerides, with a method for low density lipoprotein cholesterol in development. Standardization programs for research laboratories and a Cholesterol Reference Method Laboratory Network for diagnostic manufacturers and clinical laboratories provide reliable access and documentation of traceability to accepted reference methods. Methods for the lipid/lipoprotein analytes have improved dramatically in recent years and, coupled with improved chemistry analyzer systems and more attention to standardization by manufacturers, offer considerable improvement in analytical performance. Fully automated homogeneous assays for high density lipoprotein cholesterol and newer similar assays for low-density lipoprotein cholesterol have potential for better precision as well as more convenient and cost-effective measurements. Attention to pre-analytical sources of variation is also important in making reliable classification of patients.
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June 1, 2005
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Certain subsets of the population are especially sensitive to carcinogens, and this can be determined using molecular biological methods. In the literature there has been evidence presented for the use of p21ras (ras) as a tumor marker for human carcinogenic substances such as asbestos, polycyclic aromatic hydrocarbons, and vinyl chloride in the workplace. In this study we have examined whether serum ras could serve as a biomarker for the early detection of occupationally derived lung cancer, with an emphasis on Schneeberger (radon-induced) lung cancer. Sera were taken from 65 male tumor patients. Fifty-nine patients suffered from primary lung cancer (including 18 patients with Schneeberger lung cancer and 12 patients with asbestos-related lung cancer). Additionally, 29 patients with non-malignant lung disease, and a healthy control group (44) including 32 former uranium miners of SDAG Wismut exposed to ionizing radiation (radon and its decay products) were examined. Ras protein was determined via three different methods: 1) immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting; 2) SDS-PAGE using 5–17% gradient gels followed by Western blotting; 3) pre-incubation with Blue Sepharose, SDS-PAGE on 5–17% gradient gels, and Western blotting. The results show that 1 ng ras protein was measurable in serum standards. This protein could not be detected in patient sera or in sera from any of the study groups. Thus, ras cannot be considered useful as a marker for the early detection of asbestos-induced or Schneeberger lung
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June 1, 2005
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Elevated level of plasma lactate dehydrogenase activity in a patient with myocarditis was found to be due to the presence of lactate dehydrogenase-immunoglobulin complexes in circulation. The complexes were demonstrated by counter-immunoelectrophoresis. In this case, three immunoglobulins (IgG, IgA and IgM) and two types of light chains (λ and κ) combined with lactate dehydrogenase
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June 1, 2005
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Total prostate-specific antigen (PSA) and complexed PSA were determined in venous blood from 12 patients with prostate cancer before and after radical prostatectomy by using Immuno 1 PSA assays. The elimination kinetics of complexed PSA were compared with that of total PSA. Nearly constant concentrations of complexed PSA were found during the first six hours after surgery, in contrast to the rapid elimination of free PSA and the significant decrease of total PSA. From day one to ten there was a continuous and nearly identical decrease of complexed PSA compared to total PSA. Our findings suggest that the initial rapid decrease of free PSA immediately after operation could be caused by formation of new PSA-complex.
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June 1, 2005
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In this study, we present significant changes occurring in serum drug concentrations while using blood collection tubes that contain a barrier gel. This report also contains results with antidepressant drugs, which have not been studied before with human samples. The drug concentrations were measured either with high performance liquid chromatography (HPLC) or fluorescence polarization immunoassay (FPIA). The results show that gel tubes are suitable for blood collection for antiepileptic, antibiotic, asthma and cardioactive drug measurements, since only slight adsorption was seen (0–5%). However, the studied tubes are not suitable for blood collection of antidepressants nor benzodiazepines, because the adsorption can be 5–30%. The adsorption was even higher (up to 40%) when samples were stored for 24 h after centrifugation in gel tubes. When the centrifugation step was performed after storage the effect of the barrier gel was lower (only 0–13%). Antidepressant drug measurements performed from patient specimens collected in the studied gel tubes and stored for 3 h showed <10% adsorption of the studied drugs. After 24 h storage time, concentrations of all analysed drugs decreased even more: adsorbed amount of drugs were about 5–20%. The studied gel tubes are proposed to be satisfactory for blood collection for antidepressant drug measurements if separation step is performed within 3 h after blood clotting. With the spiked samples the adsorption to barrier gel was higher, so it seems that adsorption is faster when drugs are not highly bound to serum proteins.
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June 1, 2005
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Increased carbamylated hemoglobin formed in erythrocytes during uremia may interfere with Hba 1c assays, but few studies compared directly both parameters. We measured carbamylated hemoglobin by HPLC in 45 non-diabetic uremic patients (16 with acute and two with chronic renal failure, 27 with transplant recipients) as 57.8 ± 22.3 μg carbamylvaline/g Hb (mean ± standard deviation) vs. 31.6 ± 5.1 in 15 controls (+83%, p < 0.001). In these samples, HbA 1c was evaluated by three ion-exchange HPLC methods, 1: Diamat (BioRad), 2: A1c2.2 (Tosoh) and 3: HA8140 (Menarini), and one immunoassay method (Tinaquant II Roche). Whichever the method, mean HbA 1c values obtained increased in patients with high (> 60 μg carbamylvaline/g Hb) vs. low (< 45) carbamylated hemoglobin values (+0.08 to 0.25% of total Hb), but differences were not significant. Minor peaks on the chromatograms were however increased in parallel to carbamylated hemoglobin. HbA 1c values over 6% were found in 4, 1, 2 and 0 samples, with HPLC 1, 2, 3 and immunoassay, respectively. Fructosamine values were not significantly altered. Our results show that Hb adducts, whether due to carbamylation or to other chemical reactions, interfere to a variable extent with different HbA 1c assay methods, and confirm that HbA 1c values should be interpreted with caution in uremic patients.
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June 1, 2005
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The determination of the total concentration of plasma homocysteine is of interest in a variety of clinical circumstances, especially, in the evaluation of the risk of cardiovascular disease. However, most of the methods available to date, many of them chromatographic, are not well suited for the majority of clinical laboratories. Several automated methods are now or will be, shortly, commercially available. We have compared one of them, the fluorescence polarization immunoassay (FPIA) adapted to the IMx ® analyzer (Abbott Laboratories), with the high-performance liquid chromatography (HPLC) method with fluorescent detection currently used in our laboratory. The results show that the FPIA-IMx ® method is less imprecise and slightly more sensitive than the HPLC. The comparison of 67 clinical plasma specimens indicated that there is a proportional error disagreement between FPIA-IMx ® and HPLC (FPIA=1.19 HPLC + 0.92; confidence region for slope and y- intercept were, respectively, from 1.06 to 1.31 and from−0.06 to 2.32). The nature of this error is not explained by the experiments performed to study the inaccuracy of both methods, which included the investigation of dilution parallelism, analytical recovery and cross-reactivity. The different results of homocysteine concentration obtained with FPIA-IMx ® and HPLC must be taken into account when a change of methodology is under consideration.
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June 1, 2005
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An analytical evaluation of the thyroid stimulating hormone (TSH-3) assay on the Bayer ADVIA ® Centaur TM immunoassay system was performed. General analytical requirements (linearity, resistance to typical interferences, absence of a carry-over effect) were fulfilled and reproducibility was satisfactory. Inter-assay coefficient of variation (CV) of a human serum pool with a concentration of 0.014 mU/l was 22.3%; at concentrations between 0.26 and 83 mU/l CV was below 6%. Method comparison study demonstrated close agreement of TSH results compared to those obtained with the Roche Elecsys ® 2010 TSH assay (ADVIA Centaur = 1.08 × Elecsys−0.18 mU/l; r=0.987; n=324). Handling and practicability of the ADVIA Centaur system proved to be convenient with a very high sample throughput. We conclude that the ADVIA Centaur TSH-3 assay meets requirements for clinical use.
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June 1, 2005
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The biochemical composition of "unstimulated" whole saliva was determined in healthy adult subjects. Based on their relative concentration, salivary analytes could be classified into three arbitrary categories: concentration lower than in serum (saliva/serum ratio < 0.5; 12 analytes), similar to serum (ratio = 0.5–1.5; five analytes), and higher than in serum (ratio > 1.5; five analytes). Consistent with local production, an elevated lactate dehydrogenase (LDH) activity in the saliva was associated with a non-serum like LDH isoenzyme pattern: LDH5 >> LDH4 > LDH3 >> LDH2 > LDH1. Compared with serum, the concentrations of hydrogen (as reflected in the pH), potassium and inorganic phosphorus were much higher (saliva/serum ratio ≥ 3), whereas that of sodium, total magnesium, chloride, and total carbon dioxide were lower (saliva/serum ratio ≤ 0.3). The concentration of ionized calcium was similar in saliva and serum (saliva/serum ratio = 0.8), while ionized magnesium was unmeasurable in saliva. The salivary ionized calcium fraction was higher (0.76) than previously suggested (0.51). The difference between the main salivary cations (potassium, sodium), and anions (phosphate, chloride) was similar to serum (anion gap: 4 vs. 11 meq/l). Highly significant (p ≤ 0.012) correlations occured among salivary pH, dihydrophosphate, total calcium, and potassium. Our data suggest that calcium, potassium, chloride and phosphates are the major salivary complex-forming ions. The major compositional differences between serum and saliva show that saliva is not a passive "ultrafiltrate" of serum and salivary constituents may play a distinct physiological role.
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June 1, 2005
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A new commercially available enzyme-linked immunosorbent assay (ELISA) kit has been evaluated for the measurement of neopterin concentrations in serum, plasma and urine. This competitive ELISA is technically simple, requires only small sample volume and is rapid to perform. The assay procedure consists of sequential 1.5 h and 10 min room temperature incubation steps. The ELISA is accurate, sensitive, specific, and precise. Linear regression analysis of neopterin concentrations measured with the new ELISA and with an established method yielded a highly singificant correlation (r = 0.99). The new assay is applicable to ELISA workstations, thus enabling determination of neopterin in large series of samples. The neopterin ELISA kit has been used in routine laboratory testing of blood donations in a blood bank.
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June 1, 2005
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The evaluation of cardiac troponin I (cTnI) on the Dimension RxL ® -HM analyzer is presented. The one-step enzyme immunoassay is based on two cTnI specific monoclonal antibodies. Performed on a separate module of the analyzer, assay-time is 17 minutes. Using as criterion a between-run impression CV <20% the functional limit of detection was set at 0.1 μg/l. Cutoff level for minor myocardial damage of 0.1 μg/l was found. In Duchenne's dystrophy, patients showed increased cardiac Troponin T (cTnT) but no increased cTnI. In patients with a history of coronary heart disease undergoing chronic hemodialysis, cTnT and cTnI were increased. In different patients with submassive pulmonary embolism, increased cTnI was determined. In coronary artery bypass surgery without perioperative myocardial infarction, patients with extracorporeal circulation showed significantly higher cTnI at 24 h after surgery than those with minimal cardiac surgery. In patients with unstable angina, increased cTnI was found more often than on Stratus analyzer. In conclusion, the new assay is a very sensitive cTnI assay, fast and easy to perform in parallel to enzyme and substrate assays.
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June 1, 2005
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This paper will familiarize the reader with the terms used to describe the behavior of ion-selective electrodes, particularly in relation to their use in clinical chemistry for determination of blood electrolyte cations. It serves as an introduction to a series of papers dealing with important cations in blood, namely calcium, sodium, and potassium. The detailed relationships between the ion activity determined by means of ion-selective electrode potentiometry in undiluted specimens, and the total substance concentration measured by flame atomic-emission spectrometry are described by flow chart and equations. Adoption of a convention for reporting results is recommended. The Working Group on Selective Electrodes has taken into account recent revisions of IUPAC recommendations on nomenclature and selectivity coefficient determinations for ion-selective electrodes, and benefited from the experience of a member of the WG, who was also involved in the IUPAC discussions. Nomenclature for determined quantities follows previous IUPAC/IFCC joint recommendations.
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June 1, 2005
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Promotion of the professional growth and development of specialists in the field of clinical chemistry in European countries, and harmonisation of quality assessment and accreditation procedures are listed among the main goals and activities of Federation of European Societies of Clinical Chemistry (FESCC), according to its 1999–2000 strategic plan. The European countries that are members of the European Union are in the process of establishing the "European Register for Clinical Chemists", based on minimum standards of education, training and experience as defined by the European Communities Confederation of Clinical Chemists (EC4). Many other European countries would like to adapt their system of professional education to this model. Data on postgraduate training in EC4 FESCC members have already been gathered in 1998. However, at the present time, there is no detailed knowledge of pre- and post-graduate professional education of specialists in clinical chemistry in the non-EC4 European countries. FESCC launched a survey in July 1998 in order to gather this information with the hope to start a database about existing systems. All FESCC members received the same questionnaire on accreditation (seven questions) and non-EC4 FESCC members received an additional questionnaire with 11 questions related to post-graduate training in clinical chemistry. The response rate of the 35 FESCC member countries was 93% from the 15 EC4 members (14 responses/15 countries) and 80% from the 20-non-EC4 (16 responses/20 countries). The heterogeneity of the data on post-graduate training in clinical chemistry indicates that a great effort will be needed before harmonisation is reached. These results, however, will provide an interesting basis for further discussion and promotion of post-graduate training in clinical chemistry. The data provided on accreditation show that the total number of accredited laboratories was relatively low in EC4 countries and even lower in non-EC4 members. It was not surprising to see that the number of accredited laboratories was the highest in the two countries which started accreditation the earliest ( i.e. Sweden and UK, 1992). This situation, however, is changing at a fast rate in most countries and the number of the accredited sites is expected to increase rapidly in the next few years.
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June 1, 2005
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June 1, 2005
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July 27, 2005
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July 27, 2005