European journal of clinical chemistry and clinical biochemistry : journal of the Forum of European Clinical Chemistry Societies最新文献

We describe a new dip- and read dipstick that detects urine albumin at concentrations of 10 mg/l and above and urine creatinine at concentrations of 300 mg/l and above. The albumin assay is based on a high-affinity, dye-binding technique while the creatinine assay is based on the peroxidase-like activity of copper creatinine complexes. With these two-test dipsticks, urines from normal adults supplemented with albumin and creatinine were correctly identified to within +/- 15% of the expected value for both analytes; the between-day coefficients of variation ranged from 7.1% to 16.1%. We tested 275 patients' unmodified urines by the Bayer and Boehringer Mannheim Micral-Test albumin dipsticks and for albumin with the Beckman Array on the same specimens. We also analyzed 42 selected urines from the group of 275 for albumin by another quantitative immunochemical method and by electrophoresis plus a total protein method to estimate the albumin concentration. The quantitative immunochemical methods appear to underestimate the urine albumin concentrations; in these 42 urines measured as negative, i.e., < ca. 16-20 mg/l, by one of the quantitative method but positive by the Bayer dipstick, 33 of these were positive by the electrophoresis/total protein assay combination. The Bayer albumin dipstick correctly identified urines as having < 16 mg/l or > or = 16 mg/l at an 80% rate. At a cutoff of 20 mg/l, the rate increased to 87%. We also determined the urinary albumin/creatinine ratios on the 275 patients using the Bayer two-pad dipstick and found agreement 84% of the time with the same ratio obtained from a quantitative immunochemical method for albumin and a rate-Jaffe method for creatinine; an albumin/creatinine ratio (mg/g) of 30 was used as the discrimination point. Albumin stability studies performed on the Beckman Array patients with six fresh urines showed small but consistent decreases at -20 degrees C but not at 4 degrees C after one month of storage. The albumin in contrived urines, as estimated by electrophoreses/total protein and by the dipsticks did not change at these storage conditions. Boric acid at 1 g/l as a urine preservative had no effect on the measurement of albumin by any of the methods described here nor of the assay of creatinine. Other urinary proteins present at abnormal excretion rates did not interfere with the Bayer albumin dipstick. Abnormal concentrations of bilirubin, citrate, creatine, ascorbic acid, albumin, hemoglobin and myoglobin in urine did not interfere with the creatinine dipstick measurements. The first four of the above did not affect the Bayer dipstick results for albumin.

We investigated the influence of cyclosporine A on the concentration of tissue factor pathway inhibitor and von Willebrand factor antigen in plasma of heart transplant outpatients. Tissue factor pathway inhibitor was quantified in plasma of blood donors (n = 50) and heart transplant outpatients (n = 50) by a chromogenic substrate assay with a mean of 32.4 micrograms/l and 98.2 micrograms/l, respectively. Von Willebrand factor antigen was determined with an enzyme-linked immunoassay with a mean of 90.9% for blood donors and 184.5% in plasma of heart transplant recipients. In addition, we investigated the effect of cyclosporine A on endothelial cell cultures over an incubation period of four days. A dose-dependent effect of cyclosporine A on the release of endothelial tissue factor pathway inhibitor and von Willebrand factor antigen was determined in a concentration range from 100 to 200 micrograms/l cyclosporine A. The tissue factor pathway inhibitor and von Willebrand factor antigen concentrations in the cell culture supernatant increased during the incubation time according to the cyclosporine A concentration 2-3 fold and 2 fold, respectively. For a further elucidation of the cyclosporine A effect we investigated the influence of cremophor EL, the vehicle of cyclosporine A. Cremophor EL alone did not increase the tissue factor pathway inhibitor release. However, the release was enhanced 2-4 fold after co-stimulation with the calcium ionophore A 23187 (10(-4) mol/l) in a concentration-dependent mode. We conclude that a generalized endothelial damage or activation is most probably caused by cyclosporine A and its vehicle cremophor EL. This process probably depends upon the increase of cytosolic free calcium, as described for the liberation of von Willebrand factor by endothelial cells.

Recent studies suggest that advanced glycation endproducts play an important role in cardiovascular complications of ageing, diabetes and end-stage renal failure. Since highly elevated levels of advanced glycation endproducts are present in serum of patients on maintenance haemodialysis, an accurate and rapid assay for their determination would be useful. This would be particularly valuable for monitoring the removal of advanced glycation endproducts by novel dialysis membranes, as well as the effect of new drugs for the inhibition of their formation. Measurement of advanced glycation endproducts in serum was performed by two competitive ELISAs, using a monoclonal antibody directed against imidazolone, an advanced glycation endproduct formed by the reaction of arginine with 3-deoxyglucosone, and a polyclonal antibody directed against keyhole limpet haemocyanin-advanced glycation endproduct, as well as by quantitative fluorescence spectroscopy. Each of the assays showed significant differences between the controls and the maintenance haemodialysis patients. Advanced glycation endproduct levels determined by each of the ELISAs correlated with total and protein-bound fluorescence, but not with each other, suggesting a variable distribution of advanced glycation endproducts on serum proteins among the maintenance haemodialysis patients.

A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 mumol/l and proved to be sensitive with a detection limit of 0.1 mumol/l and the lowest limit of reliable quantification of 0.5 mumol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both < 4% at a concentration of 10 mumol/l homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 mumol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.

The validation of a clinical laboratory report is a process that guarantees the results contained in the report have been obtained under satisfactory metrological conditions and that they are compatible with the information available on the patient. This validation is generally carried out manually by a clinical laboratory professional, but also may be done by an expert system properly programmed, such as the VALAB system. The evaluation presented in this article consists of comparing human and system decisions of validation for 500 randomly selected clinical laboratory reports from hospitalized patients. In this evaluation, 84.8% of the reports examined by the VALAB are accepted directly without any human aid, and only 15.2% require examination by clinical biochemists.

We present a standardization model for the measurement of specific polypeptides and proteins, which is based on an integrated development of all important elements of a reference measurement system. Generally, the model is in line with other current recommendations. However, it puts special emphasis on the definition of the analyte and on the role of reference methods for verification of the standardization process by measurement of patient specimens. Further, we discuss the needs for its implementation in the routine laboratory. In the light of this model, we investigate the current stage of standardization of routine methods for enzymes, peptide hormones, proteins, apolipoproteins, glycohaemoglobin, and tumour markers.

A method for the determination of inorganic sulphate based on high performance ion chromatography is presented. The separation was performed on an anion-exchange column with a 1.8 mmol/l sodium carbonate/ 1.7 mmol/l sodium hydrogen carbonate-buffer, pH 10.35. Conductivity of the eluate was monitored after suppression of the background conductivity caused by the eluent-buffer. Serum and synovial fluid samples were prepared by ultrafiltration through membranes with a molecular mass cutoff of M(r) 10,000. The viscosity of the synovial fluids was reduced by treatment with hyaluronate lyase before ultrafiltration. The method showed a linear response for sulphate concentrations between 0.5 and 1000 mumol/l. The limit of detection was 1 mumol/l for aqueous standards. For serum the coefficient of variation within-run was 2.3%-2.4%, the coefficient of variation between days 2.9%-3.1%. For synovial fluids the coefficient of variation within-run was 3.1%-3.4%, the coefficient of variation between days 4.6%-5.7%. Standard recovery experiments performed by spiking pools of human sera containing low sulphate concentrations with sulphate concentrations between 5 mumol/l and 40 mumol/l showed recoveries between 98.9% and 100.6%. The corresponding experiments with pools of synovial fluids showed recoveries of 98.3% to 100.9%. As determined from 127 serum samples the reference range for sulphate was 262 mumol/l-420 mumol/l, with a mean value of 314 mumol/l. No dependence on age or sex was observed. The sulphate concentration in 36 synovial fluids from knees affected by inflammatory processes showed a mean value of 424 mumol/l and a standard deviation of 70 mumol/l. In 41 synovial fluids from knees affected by chronic degeneration joint disease, the sulphate concentrations were statistically significantly lower, with a mean of 374 mumol/l and a standard deviation of 58 mumol/l. The concentrations of sulphate in the synovial fluids were statistically significantly higher than those in the serum samples used for determination of the reference range. Following the oral application of a subtoxic single dose of acetaminophen (32.5 mg/kg body weight-62.5 mg/kg body weight) to 4 healthy volunteers, there was a significant decrease in the concentration of sulphate in serum with a minimum at 4-5 h after application of the drug. The cumulative concentration decrease of sulphate in serum and the kinetic constant of the sulphate depletion were not correlated with the applied acetaminophen dose normalized for body weight.

Clinical management of ovarian cancer patients is facilitated by CA 125 determinations in serum. Presently, several assay systems based on different concepts and different methodologies are available to measure CA 125. Method comparison analysis of such assay systems is usually performed through (linear) regression analysis, which requires assumptions about the distribution of experimental data and its measurement error. The aim of the present study was to compare four newly developed second generation assay systems for quantitation of CA 125 by utilizing an alternative simple approach to method comparison analysis. This alternative comprises the construction of relative difference plots and mountain plots, previously described by Krouwer et al. (Eur J Clin Chem Clin Biochem 1995; 33:525-7). In addition, the diagnostic value of the assays was illustrated through receiver-operating-characteristic (ROC) curves. Sera obtained from 300 women were assayed for CA 125 using the Abbott IMx CA 125 assay (Abbott), the Centocor CA 125 II RIA assay (Centocor), the Berilux Ov testing kit for CA 125 (Behringwerke), and the CA 125 TR-FIA assay (Wallac Oy). Both the relative difference plots and the mountain plots revealed higher serum concentrations with the Centocor RIA II (Median +33%, P2.5 to P97.5: -25% to 161%) and Berilux (Median +28%, P2.5 to P97.5: -17% to 108%) compared to the Abbott IMx system. The TR-FIA assay system showed lower serum concentrations (Median - 17%, P2.5 to P97.5: -74% to 229%). The combination of relative difference plots and mountain plots demonstrated clearly the wide range of differences between CA 125 assays measuring the same analyte. The relative difference plots provided insight into the distribution of the differences over the range of measurement as well as the identification of outliers. A simple quantitative assessment of the median differences could be made from the overlaying mountain plots. The close correspondence observed between the ROC curves illustrated that assay systems for CA 125 differing in design (type of antibodies used) and format can produce similar results on group level. However, the results of the clinical evaluation underline the importance of the application of assay specific cut-off values.