Prediction of relative glomerular filtration rate in adults: new improved equations based on Swedish Caucasians and standardized plasma-creatinine assays.

OBJECTIVE: To evaluate newly developed equations predicting relative glomerular filtration rate(GFR) in adult Swedish Caucasians and to compare with the Modification of Diet in Renal Disease(MDRD) and Mayo Clinic equations using enzymatic and zero-calibrated plasma creatinine assays. MATERIAL AND METHODS: GFR was measured with iohexol clearance adjusted to 1.73 m(2). One population sample (n=436/Lund) was used to derive an equation based on plasma-creatinine/age/gender, and a second with the addition of lean body mass (LBM). Both equations were validated in a separate sample (n=414/Malmö). The coefficients of the equations were eventually fine-tuned using all 850 patients and yielding Lund-Malmö equations without (LM) and with LBM-term (LM(LBM)). Their performance was compared with the MDRD(CC) (conventional creatinine calibration), MDRD(IDMS) (isotope dilution mass spectroscopy traceable calibration) and Mayo Clinic equations. RESULTS: The Lund equations performed similarly in both samples. In the combined set, the Mayo Clinic/MDRD(CC) resulted in +19.0/+10.2 % median bias, while bias for the other equations was < 10 %. LM(LBM) had the highest accuracy (86 % of estimates within 30 % of measured GFR), significantly (p < 0.001) better than for MDRD(IDMS) (80 %). In men with BMI < 20 kg/m(2), MDRD(IDMS)/LM had +46 %/+19 % median bias. MDRD(IDMS) also overestimated GFR by 22 %/14 % in men/women above 80 years of age. The LM(LBM) equation had < 10 % bias irrespective of BMI, age or GFR except for a 15 % negative bias at GFR > 90 mL/min/1.73 m(2). CONCLUSION: The newly developed Lund-Malmö equations for GFR estimation performed better than the MDRD(IDMS) and Mayo Clinic equations in a Swedish Caucasian sample. Inclusion of an LBM term improved performance markedly in certain subgroups.

The Nordic Reference Interval Project 2000: recommended reference intervals for 25 common biochemical properties.

Each of 102 Nordic routine clinical biochemistry laboratories collected blood samples from at least 25 healthy reference individuals evenly distributed for gender and age, and analysed 25 of the most commonly requested serum/plasma components from each reference individual. A reference material (control) consisting of a fresh frozen liquid pool of serum with values traceable to reference methods (used as the project "calibrator" for non-enzymes to correct reference values) was analysed together with other serum pool controls in the same series as the project samples. Analytical data, method data and data describing the reference individuals were submitted to a central database for evaluation and calculation of reference intervals intended for common use in the Nordic countries. In parallel to the main project, measurements of commonly requested haematology properties on EDTA samples were also carried out, mainly by laboratories in Finland and Sweden. Aliquots from reference samples were submitted to storage in a central bio-bank for future establishment of reference intervals for other properties. The 25 components were, in alphabetical order: alanine transaminase, albumin, alkaline phosphatase, amylase, amylase pancreatic, aspartate transaminase, bilirubins, calcium, carbamide, cholesterol, creatine kinase, creatininium, gamma-glutamyltransferase, glucose, HDL-cholesterol, iron, iron binding capacity, lactate dehydrogenase, magnesium, phosphate, potassium, protein, sodium, triglyceride and urate.

Nordic Reference Interval Project Bio-bank and Database (NOBIDA): a source for future estimation and retrospective evaluation of reference intervals.

In the Nordic Reference Interval Project 2000 (NORIP) serum, Li-heparin plasma and EDTA buffy coat were collected at 102 laboratories in 5 Nordic countries from healthy individuals aged 18 years or more and evenly distributed for laboratory, gender and age. Multiple aliquots of these samples from each of about 3000 persons are now stored at the Nordic Reference Interval Project Bio-bank and Database (NOBIDA) at a temperature of below -80 degrees C. The commutable NFKK Reference Serum X with certified values traceable to reference methods and measured in NORIP in the same series as the samples is also available from NOBIDA. Data describing the person and the sample conditions are stored together with analytical results and data describing the measurement systems. The bio-bank along with material and data is administered by the NOBIDA committee on behalf of the NFKK (Scandinavian Society of Clinical Chemistry) to be used by Nordic laboratories for any purpose beneficial to the development of clinical biochemistry in general and particularly for creating reference intervals for other biochemical properties than those established by NORIP. Furthermore, research on the already stored information alone is encouraged. Thus colleagues are now welcome to use this extensive material for research and development in clinical biochemistry.

Differential counting of blood leukocytes using automated microscopy and a decision support system based on artificial neural networks--evaluation of DiffMaster Octavia.

The morphological appearance of blood cells has an established association to clinical conditions. A novel system, DiffMaster Octavia for differential counting of blood leukocytes, has been evaluated. The system consisted of a microscope, 3-chip color charge coupled device (CCD) camera, automated motorized stage holder, electronic hardware for motor and light control and software for automatic cell location and image processing for preclassification of blood cells using artificial neural networks. The DiffMaster test method, was evaluated on 322 routine blood samples (400 cells per sample) using manual microscopy as reference method. The results showed a correlation of determination (r(2)) of 0.8-0.98 for the normal cell classes and blast cells. The DiffMaster correctly preclassified 89% of all leukocytes with a good reproducibility. After verification of the cell classes, the agreement between the test and reference method was 91% whether the sample was abnormal or normal. The clinical sensitivity was 98% and specificity 82%. The sensitivity to identify blast cells was slightly higher with the DiffMaster than manual microscopy. Similar levels of short-term imprecision for the two methods were found for all cell classes. In conclusion this study shows that the DiffMaster can provide a decision support system which, together with a qualified morphologist, can generate leukocyte differential count reports of high quality.

Computer-supported detection of M-components and evaluation of immunoglobulins after capillary electrophoresis.

BACKGROUND: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. METHODS: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the gamma- and ss-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. RESULTS: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. CONCLUSIONS: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.

[Four new instrument for blood gas analysis are tested: handy cassettes for easier use]. / Fyra nya instrument för analys av blodgaser testade: behändiga kassetter skall ge bättre användarvänlighet.

An entirely new type of blood gas analyser has made its way into the marketplace, to be used, for example, in emergency rooms, intensive care units, ambulances, and bedside with quarantined patients in infectious diseases units. The instruments reviewed here employ new miniaturised analysis circuitry, integrated into the cassette on which the blood sample is applied. These instruments are designed for use by care-givers without specific laboratory training. Four point-of-care blood gas analysers are tested: OPTI 1 (AVL), I-STAT (HP), IRMA (Infiniti) och ABL 70 (Radiometer).

Effects of ethanol on muscarinic receptor-stimulated c-fos expression in human neuroblastoma cells.

The effect of ethanol exposure on muscarinic receptor-stimulated expression of c-fos was investigated in SH-SY5Y cells. Four days of ethanol exposure enhanced carbachol-stimulated c-fos mRNA expression, analyzed with Northern blot, and Fos/AP-1 binding activity, measured with gel mobility super shift assay. Pre-incubation with muscarinic antagonists or the protein kinase C inhibitor GF109203X demonstrated that, in both control and ethanol-treated cells, carbachol-induced c-fos expression was mediated via muscarinic M1 receptors and to a large extent through protein kinase C. However, phorbol ester-induced c-fos expression was unaffected in ethanol-treated cells. Acute exposure to ethanol caused a suppression of both carbachol- and phorbol ester-stimulated c-fos expression. These results demonstrate that muscarinic receptor-stimulated gene expression is sensitive to both acute and long-term ethanol exposure.

Long-term exposure to ethanol increases the number and function of muscarinic M1 receptors in human neuroblastoma cells.

The effect of long-term ethanol exposure on muscarinic receptors was investigated in human neuroblastoma SH-SY5Y cells. Exposure to 100 mM ethanol for 4 days enhanced both peak and steady-state levels of carbachol-stimulated inositol 1,4,5-bisphosphate increase. An ethanol concentration of 50 mM was sufficient for an enhancement of this event. The carbachol-stimulated decrease in [3H]inositol-labeled [3H]phosphatidylnositol 4,5-bisphosphate and increase [3H]inositol trisphosphate and [3H]inositol bisphosphate were also potentiated in ethanol-treated cells, which demonstrated that the receptor-stimulated activation of phospholipase C is augmented. Experiments with pirenzepine showed that carbachol-stimulated inositol 1,4,5-trisphosphate increase is mediated via M1 receptors both in ethanol-treated and control cells. Ethanol exposure for 2 or 4 days also caused an increase in [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate binding sites and elevation of [3H]pirenzepine binding, which indicated that the number of muscarinic M1 receptors is increased in ethanol-treated SH-SY5Y cells. These results demonstrate that long-term exposure to ethanol potentiates muscarinic M1 receptor-stimulated activation of phospholipase C in SH-SY5Y cells. This is likely to be explained by an increased number of muscarinic M1 receptors.

Carbohydrate-deficient transferrin measured by high-performance liquid chromatography and CDTect immunoassay.

Carbohydrate-deficient transferrin (CDT) was measured in three populations using anion-exchange chromatography followed by radioimmunoassay (CDTectTM) and a new high-performance liquid chromatography (HPLC) method. The correlation between the methods in 50 consecutive clinical samples was good (r = 0.87). However, in a set of 49 samples with CDT concentrations close to the reference value of the methods, the correlation was low (r = 0.51). In addition, among controls, no correlation between the methods was found (r = 0.10). Abnormal isoforms of transferrin were noted in 5% of the clinical samples. HPLC determination of CDT offers the advantage of identifying these isoforms of transferrin and thus reducing analytical pitfalls associated with the CDTectTM method.

Ethanol inhibits the peak of muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate in neuroblastoma SH-SY5Y cells.

The effect of ethanol on muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate was studied in human neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced a biphasic increase of inositol 1,4,5-triphosphate with an initial peak after 10 sec declining to a plateau phase of elevation above basal levels, which was sustained for at least 5 min in the presence of agonist. The peak, but not the plateau phase, was concentration-dependently decreased by exposure to ethanol. Maximal inhibition was obtained within 30 sec of exposure to ethanol. Ethanol caused an increase in the EC50 value of carbachol for the initial rate of inositol 1,4,5-trisphosphate formation, measured after 10 sec of stimulation, from 98 microM in the absence to 196 microM in the presence of 100 mM ethanol. The potencies of pirenzepine and hexahydro-sila-difenidol hydrochloride for inhibiting [3H]quinuclidinyl benzilate binding and inositol 1,4,5-trisphosphate formation suggest that both phases are mediated via the muscarinic M1 receptor. Phorbol 12-myristate 13-acetate inhibited both phases of inositol 1,4,5-trisphosphate formation, whereas okadaic acid and modulators of cAMP-dependent protein kinase were without any effect. There was no inhibitory effect of ethanol when protein kinase C was inhibited by H7 and calphostin C, indicating that the ethanol effect is dependent on protein kinase C activity.

Effect of beta-glucuronidase on urinary benzodiazepine concentrations determined by fluorescence polarization immunoassay.

In samples from patients treated with oxazepam, beta-glucuronidase increased the immunoreactivity of urinary benzodiazepines analyzed by fluorescence polarization immunoassay (FPIA). Increasing concentrations of beta-glucuronidase added to samples from drug-free controls did not influence the results. In the absence of beta-glucuronidase, 22 of 35 samples from patients undergoing detoxification gave positive results at a cutoff concentration of 200 micrograms/L. Pretreatment with beta-glucuronidase increased the number of drug-positive samples to 33. The drug-negative samples were obtained from two patients who had been oxazepam-free for at least 1 week. Thus, beta-glucuronidase can be used to increase the sensitivity of the urinary benzodiazepine FPIA without reducing the specificity of the method.

Mechanisms of muscarinic receptor-stimulated expression of c-fos in SH-SY5Y cells.

In this study, the signal cascade transducing carbachol stimulation into c-fos expression in SH-SY5Y neuroblastoma cells was investigated. 1,2-Diacylglycerol formation and c-fos expression were mediated via stimulation of muscarinic M1 receptors and the first 5 min of receptor stimulation were critical for these events. Application of 1,2-dioctanoylglycerol induced c-fos expression and this, as well as carbachol-stimulated c-fos expression, was inhibited by protein kinase C inhibitors. Increasing the intracellular Ca2+ concentration had only small effects on c-fos expression. There was a dependency on extracellular Ca2+ for maximal c-fos expression and 1,2-diacylglycerol formation. The carbachol-stimulated c-fos expression was potentiated by application of the protein phosphatase inhibitor okadaic acid. These results demonstrate the importance of 1,2-diacylglycerol formation for muscarinic receptor-stimulated, protein kinase C-mediated c-fos expression in the SH-SY5Y cells and that this cascade is counteracted by an okadaic acid-sensitive protein phosphatase.

Adaptation of signal transduction in brain.

Cell culture models were used to study the effects of long-term ethanol exposure on neuronal cells. Effects on phospholipase C and phospholipase D mediated signal transduction were investigated by assaying receptor-binding, G protein function, activities of lipases, formation of second messengers and c-fos mRNA. The signal transduction cascades displayed abnormal activities from 2 to 7 days of exposure which differed from the acute effects. Phosphatidylethanol formed by phospholipase D is an abnormal lipid that may harmfully affect nerve cell function.

An okadaic acid-sensitive protein phosphatase counteracts protein kinase C-induced phosphorylation in SH-SY5Y cells.

Protein phosphorylation and subsequent dephosphorylation was studied in digitonin-permeabilized neuroblastoma SH-SY5Y cells by measuring the incorporation of [32P]phosphate into myelin basic protein (MBP). 1,2-Dioctanoyl-sn-glycerol (DOG) and calcium synergistically induced phosphorylation of MBP, which was inhibited by the protein kinase C (PKC) pseudosubstrate peptide (PKC19-36). The phosphorylation increased for 10 min when a net dephosphorylation started to appear. The dephosphorylation was inhibited by okadaic acid. Regardless of calcium concentration, the presence of DOG was necessary for significant effects of okadaic acid on MBP phosphorylation. H7 and staurosporine dose-dependently inhibited the phosphorylation of MBP, induced by DOG and calcium in the presence of okadaic acid. Different PKC pseudosubstrate peptides were applied and all showed an inhibitory effect on the phosphorylation of MBP under these conditions. These results demonstrate the presence, in SH-SY5Y cells, of a protein phosphatase, possibly protein phosphatase 2A, with a high basal activity that counteracts PKC-induced phosphorylation.

Phosphatidylethanol affects inositol 1,4,5-trisphosphate levels in NG108-15 neuroblastoma x glioma hybrid cells.

Phosphatidylethanol is formed by phospholipase D in animal cells exposed to ethanol. Previous reports have demonstrated that the degradation of phosphatidylethanol is slow, indicating that this lipid may be present in the cells after ethanol itself has disappeared. Accumulation of an abnormal alcohol metabolite may influence cellular functions. In the present study, cultivation of NG108-15 neuroblastoma x glioma hybrid cells in the presence of ethanol resulted in an accumulation of phosphatidylethanol and a simultaneous increase in basal inositol 1,4,5-trisphosphate levels. The direct effects of phosphatidylethanol on the phosphoinositide signal transduction system were examined through incorporation of exogenous phosphatidylethanol into membranes of ethanol-naive cells. An incorporation amounting to 2.8% of cellular phospholipids was achieved after a 5-h incubation with 30 microM phosphatidylethanol. Phosphatidylethanol was found to cause a time- and dose-dependent increase in the basal levels of inositol 1,4,5-trisphosphate. The effects on inositol 1,4,5-trisphosphate levels of exogenously added phosphatidylethanol and ethanol exposure for 2 days were not additive. No effect on bradykinin-stimulated inositol 1,4,5-trisphosphate production could be detected. However, the increase in basal inositol 1,4,5-trisphosphate levels indicates that phosphatidylethanol affects inositol 1,4,5-trisphosphate turnover and emphasizes the importance of considering phosphatidylethanol as a possible mediator of ethanol-induced effects on cellular processes.

Desensitization of acetylcholine induced inositol 1,4,5-trisphosphate formation in neuroblastoma SH-SY5Y cells following repetitive acetylcholine stimulations.

In this study, the desensitization of acetylcholine-induced inositol 1,4,5-trisphosphate [I(1,4,5)P3] formation, upon short-time prestimulations, was investigated in cultures of human neuroblastoma SH-SY5Y cells. Four repeated stimulations for 10 seconds with 10 microM acetylcholine were necessary to induce a desensitization of the I(1,4,5)P3 formation. The desensitization was observed 4 hours after the initiation of repetitive stimulations. The same effect was obtained by a single prestimulation with 1 mM acetylcholine. Preincubation of the cells with phorbol 12-myristate 13-acetate (PMA) markedly down-regulated the acetylcholine-induced I(1,4,5)P3 formation. However, the protein kinase C (PKC) inhibitors H7 and staurosporine did not influence the desensitization induced by four repeated stimulations with 20 microM acetylcholine. These results indicate that the signal transduction can be desensitized following repeated stimulations with sub-maximal concentrations of receptor agonist and although activation of PKC can induce the same down-regulation, PKC is most likely not involved in the desensitization induced by repetitive acetylcholine-stimulations.

Evaluation of ethanol effects on PLC signal transduction pathways using cell lines of neuronal origin.

Human neuroblastoma cells SH-SY5Y and neuroblastoma-glioma cells NG 108-15 have been used as models for the elucidation of the effects of ethanol on receptor-mediated phospholipase C activity, c-fos mRNA expression and protein kinase C activity. Cells were exposed to ethanol (0-200 mM) for varying periods up to seven days. Agonist stimulated events were obtained in NG 108-15 cells with bradykinin and in SH-SY5Y cells with carbachol. Chronic ethanol exposure reduced the agonist-stimulated formation of inositol 1,4,5-trisphosphate in NG 108-15 cells and in SH-SY5Y cells. 100 mM ethanol for seven days increased the membrane bound and cytosolic forms of protein kinase C activity in SH-SY5Y cells. Carbachol (1 mM) induced a maximal c-fos mRNA response after 40 minutes in SH-SY5Y cells, an effect that could be mimicked through protein kinase C stimulation by phorbol esters.