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PubMedDate not supplied

[Comparison of the determination of cyclosporin-A in blood samples collected on filter paper and by the ordinary technique].

Monitoring cyclosporin-A (CsA) blood levels is of utmost importance for the rational use of this drug. Although many centers perform transplants, in Brazil there are few laboratories able to measure CsA blood levels. Therefore making blood samples reach the laboratory emerged as a problem. Collection of blood on filter paper has been a technique used for a long time in special cases. PURPOSE--To confirm the usefulness of measuring CsA blood levels in blood samples collected on filter paper and in the usual way. METHOD--We studied twenty renal cadaver kidney recipients who were receiving CsA, azathioprine and prednisone. Ninety five blood samples were collected and divided into two aliquots. One of them was sent routinely to one laboratory to perform whole blood CsA measurements. From the other aliquot, 20 microliters were pipetted on filter paper. When dried they were mailed to the other laboratory, where, after elution, CsA was measured. In both cases radioimmunoassay with polyclonal antibody was used. RESULTS--Linear correlation between both measurements revealed r = 0.81 with no statistical difference. CONCLUSION--The technique showed to be useful in clinical practice. In countries with continental size, as Brazil, it may be very helpful.

Blood Chemical Analysis↗
PubMed1995

Enhanced interleukin 8 secretion in circulation of patients with Behçet's disease.

OBJECTIVE: The pathogenesis of Behçet's disease (BD) has not yet been determined. Several hypotheses have been postulated and cytokines that control growth proliferation and hematopoiesis of progenitor cells play a role in relation to immune response and inflammatory dysfunction. We investigated whether cytokines play a role in pathogenesis of BD. METHODS: We employed the quantitative sandwich enzyme immunoassay technique, in which antibody is already coated on the microtiter plate standard and samples are pipetted into the wells. Antigen present is bound by immobilized antibody. After incubation and washing steps, conjugate, which is enzyme linked polyclonal antibody specific for the antigen is added to the wells. Following a wash to remove any unbound antibody enzyme reagent, a substrate solution is added to the wells, and color develops in proportion to the antigen bound in the initial steps, which can be read in terms of optical density present in the standards and in the samples. RESULTS: Of a total of 53 samples with BD, 33 (64%) had detectable levels of interleukin 8 (IL-8). Levels of IL-6, tumor necrosis factor alpha and interferon-gamma were not significantly elevated in patients with BD. CONCLUSION: We found that IL-8 levels are higher in patients with active BD, and since IL-8 has a potent effect on the neutrophil, this cytokine most likely participates in the inflammatory response of this disease.

Behcet Syndrome↗
PubMed1996

Development and maintenance of outer segments by isolated chick embryo photoreceptor cells in culture.

PURPOSE: To investigate the capacity of isolated chick embryo photoreceptors to develop and maintain outer-segment processes in dissociated cell cultures, in the absence of pigment epithelial and glial cells. METHODS: Cells were obtained from the retinas of embryonic day (ED) 17 chick embryos, after the onset of outer-segment formation in vivo. After 5 to 12-minute incubation in Ca++ and Mg++-free Hank's balanced salt solution, neural retinas were freed from other optical tissues, including the pigment epithelium. Retinal cell suspensions were prepared by repeated pipetting after mild trypsinization and were grown in serum-containing medium on a polyornithine-coated substratum. Cell differentiation was evaluated using phase-contrast and transmission electron microscopes and by autoradiographic analysis of the uptake of putative amino acid neurotransmitters, lectin cytochemical analysis, and immunocytochemical analysis with rod and cone-specific antibodies. Cells isolated from ED 8 retinas, before the onset of outer-segment formation in vivo, were also studied. RESULTS: At culture onset, ED 17 cells appeared morphologically undifferentiated and devoid of processes; differentiated features could be detected after 24 to 48 hours in vitro. Photoreceptor cells were the most abundant cell type after 6 days in vitro, followed by nonphotoreceptor multipolar neurons and morphologically undifferentiated cells. Autoradiographic analysis showed extensive Na+ -dependent uptake of (2,3,4-(3)H)gamma- aminobutyric acid in nonphotoreceptor neurons, whereas photoreceptors were labeled predominantly with 3H-glutamate. Most of the photoreceptors were labeled with fluorescent peanut lectin and with a sheep polyclonal antibody against bovine rhodopsin. Subsets of photoreceptors, on the other hand, were immunoreactive with cone- or rod-specific monoclonal antibodies COS-1, OS-2, 50-1B11, or Rho-4D2. Approximately 50% to 65% of the photoreceptors positive with these monoclonal antibodies showed a remarkable polarization of immunoreactive materials, which accumulated predominantly, or even exclusively, in an outer-segment-like apical process. When viewed on the transmission electron microscope, these outer-segment-like processes appeared as distal expansions of the photoreceptor cilium and contained disc-like membranous profiles. Outer-segment-like processes also could be detected using the electron microscope and by immunocytochemical analysis of cultures of ED 8 retinal cells. CONCLUSIONS: After undergoing morphologic dedifferentiation as a result of tissue dissociation, isolated retinal photoreceptors, grown in the absence of contact-mediated cell interactions and of pigment epithelial and glial cells, can regenerate and maintain a highly polarized pattern of structural and molecular organization, including the formation of outer-segment-like processes. The cultures provide an experimental system for the investigation of cellular and molecular mechanisms regulating further development and maturation of these photoreceptor structures.

Animals↗
PubMed1996

Automated HPLC screening of newborns for sickle cell anemia and other hemoglobinopathies.

Automated HPLC is used to test dried blood-spot specimens from newborns for hemoglobins (Hb) F, A, S, C, E, and D. We present the method and report on its performance determined during >4 years of testing 2.5 x 10(6) newborns. The method features automated derivation of presumptive phenotypes; quantitative quality control and proficiency testing; throughput of one specimen per minute; small sample volume; hemoglobin concentrations quantified with an interlaboratory CV of 14-18%; retention times with interlaboratory CV of <2% and matching, within +/- 0.03 min, of laboratories and reagent lots; control of peak resolution; 0.5% detection limit for Hb S and C, and 1.0% for Hb F, A, E, and D; few interferences; and negligible background and carryover. Shortcomings of the method are the absence of microplate barcode identification and the need for manually pipetting the sample eluate into the microplate.

Anemia, Sickle Cell↗
PubMed1977

[Investigations of amino acid metabolism by thin layer chromatography for differentiation of Brucella (author's transl)].

The amino acid metabolism of 23 different Brucella strains was investigated for differentiation purposes. The results were evaluated by thin layer chromatography, after enzymatic incubation. The organisms (Tab. 1) were grown on Tryptose blood agar at 37 degree C for 24 or 48h. Two mg wet weight of bacteria in 0.2 ml PBS, 0.01 M, were incubated with 12.5 microng (0.025 ml) amino acid in small tubes for 16h at 37 degree C, and centrifuged for 15 min at 7500 X g. For controls, bacterial suspensions were heated for 15 min at 100 degree C to destroy enzymatic activity, and also contrifuged for 15 min at 7500 X g. Usually 4 micronl of the supernatant fluids (6 micronl for L-asparagine, and 10 micronl for L-proline) were pipetted on the thin layer plate. The tests were run in n-butanol acetic acid water, 20:5:5, with a distance of 8 cm. Amino acids were stained with ninhydrine. The tests were repeated 3-5 times with identical results. Amino acid metabolism was indicated by different staining intesities (+ to ++) in comparison to control preparations. All species could be exactly differentiated from each other, with the exception of B. suis, biotype 2, and B canis, which could not be differentiated by their amino acid metabolism. Biotyps of the same species were mostly identical. The results of these investigations could be reproduced qualitatively as well as quantitatively. The method described is recommended for routine investigations.

Amino Acids↗
PubMed1996

[Fluorescence polarization immunoassay and microparticle enzyme immunoassay].

We describe a new clinical laboratory instrument, the Abbott AxSYM, which provides random- and continuous-access testing for immunoassays, 20 onboard reagents, primary tube sampling, and a throughput of 80 to 120 tests per hour. The AxSYM incorporates three separate analytical technologies for processing immunoassays: microparticle enzyme immunoassay, fluorescence polarization immunoassay, and a novel technology known as ion-capture immunoassay. The system incorporates both common and technology-specific subsystems controlled by a real-time software scheduling processor. Tests can be processed in one- or two-step sandwich or competitive formats, with variable pipetting steps, incubation periods, optical read formats, and wash sequences. Menu capabilities include test for hepatitis, retrovirus, tumor markers, fertility markers, thyroid functions, and therapeutic drugs. The time to first result is 15 approximately 25 min for most routine assay and < or = 15 min for stat assays (i.e., creatine kinase MB isoenzyme, human chorionic gonadotropin beta subunit, and therapeutic drugs). AxSYM assay performance for 23 assays was comparable with that of the Abbott IMx and TDx analyzers; specimen correlation data had correlation from 0.99 to 1.10. Within-run imprecision (CV) was 1.5% to 11.4%, with most assays (19 of 23) demonstrating CVs < or = 8.0%.

Fluorescence Polarization Immunoassay↗
PubMed1996

COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR.

The COBAS AMPLICOR system automates amplification and detection of target nucleic acids, making diagnostic PCR routine for a variety of infectious diseases. The system contains a single thermal cycler with two independently regulated heating/cooling blocks, an incubator, a magnetic particle washer, a pipettor, and a photometer. Amplified products are captured on oligonucleotide-coated paramagnetic microparticles and detected with use of an avidin-horseradish peroxidase (HRP) conjugate. Concentrated solutions of amplicon or HRP were pipetted without detectable carryover. Amplified DNA was detected with an intraassay CV of < 4.5%; the combined intraassay CV for amplification and detection was < 15%. No cross-reactivity was observed when three different target nucleic acids were amplified in a single reaction and detected with three target-specific capture probes. The initial COBAS AMPLICOR menu includes qualitative tests for diagnosing infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and hepatitis C virus. All tests include an optional Internal Control to provide assurance that specimens are successfully amplified and detected.

Autoanalysis↗
PubMed1977

Erythrocyte osmotic fragility: micromethod based on resistive-particle counting.

A new procedure has been developed to measure the osmotic fragility of human erythrocytes. It is based on the resistive-particle counter and the finding that lysed erythrocytes have a lower electrical resistance than intact cells after the current in the sensing orifice reaches a critical value. The most suitable test conditions involve a 19 mu orifice, a high current setting, and the simple use of size discriminators to exclude lysed cells from being counted. Since only 1 to 5 microliter of blood are needed per salt concentration, a complete fragility test can be carried out with less than 50 mu of blood. Results with the new procedure were comparable to the classic spectrophotometric one for normal blood, in clinical conditions of increased or decreased erythrocyt osmotic fragility, and for different anticoagulants. It has a specific advantage in that ratio counting makes accurate pipetting of blood unnecessary.

Computers↗
PubMed1997

Accurate and precise isotope dilution mass spectrometry method for determining glucose in whole blood.

An accurate and precise method to determine glucose concentration in whole blood is presented. The method, based on isotope dilution gas chromatography-mass spectrometry (ID GC-MS), was developed to be used as a Reference Method for determining glucose concentration in capillary or venous whole blood. Blood samples and standards are pipetted manually with "microcap" micropipettes, which makes it possible to collect samples even at the patient's bedside. Glucose is quantified as its aldononitrile pentaacetate. [13C6]Glucose is used as an internal standard. Assay of Seronorm and Pathonorm L and H controls by ID GC-MS gave within-run CVs of 0.66%, 0.96%, and 0.92%, respectively. For whole blood with glucose concentrations in the low, normal, and high ranges, the within-run CVs were 1.27%, 0.91%, and 0.78%, respectively. The between-run CV for glucose calculated from 36 separate single analyses of Seronorm was 1.44%. In an accuracy assessment test of the HemoCue blood glucose analyzer, 140 capillary blood samples were measured in parallel after split-sampling. For all samples the HemoCue analyzer results had a mean bias of +2.0% compared with the ID GC-MS results.

Blood Glucose↗
PubMed1976

Rapid radioimmunoassay of total urinary estriol.

We describe a radioimmunoassay for total urinary estriol in pregnancy. A 25-mul aliquot of the urine specimen is acid hydrolyzed, neutralized, and diluted before assay. We use rabbit antisera against estriol-6-(O-carboxymethyl)oxime/bovine serum albumin and ammonium sulfate precipitation at room temperature. Results are unaffected by glucose or methenamine mandelate, a urinary tract antiseptic. Using semi-automatic pipetting equipment, one laboratory technologist can complete 50 assays within 8 h. This technique is both reliable and convenient and should decrease the expense of routine estriol assays.

Colorimetry↗
PubMed1976

Modifications and evaluation of double antibody radioimmunoassay of human carcinoembryonic antigen.

Double antibody radioimmunoassay of carcinoembryonic antigen (CEA), a cancer-associated antigen of the human digestive system, was subjected to certain modifications and critically evaluated. Modifications pertained to: (a) the production of a high titer goat anti-CEA antiserum that was rendered highly specific by solid phase immunoabsorption with cyanogen bromide-activated Sepharose conjugates of normal plasma liver, and colon perchloric acid-soluble glycoprotein antigens: (b) the introduction of suitable alterations in the experimental conditions of radioiodination procedure to minimize and to prevent breakdown of the antigen, thus prolonging the storage of the labeled antigen; (c) the extended incubation period of CEA-anti-CEA immune reaction; and (d) the use of sodium acetate buffer, pH 6.1. Furthermore, the use of an automatic pipetting station for accurate and rapid reagent dispensation and statistical analysis of the radioimmunoassay data on a modern computer to ensure strict quality control of the assay provided some definite improvement over the existing assay.

Absorption↗
PubMed1976

Toward absolute methods in clinical chemistry: application of mass fragmentography to high-accuracy analyses.

We review recently developed reference methods based on mass fragmentography (specific ion monitoring) with use of isotope-labeled internal standard, for determination of cholesterol, triglycerides, urea, glucose, cortisol, progesterone, and testosterone. With an optimal ratio between standard and material to be determined, the relative standard deviation of these methods, based on duplicate determinations, is between 1.3 and 2.7%. The very high specificity of the methods, in combination with the fact that the ratio between labeled and unlabeled molecules is determined with a high degree of accuracy, makes it likely that the most significant errors in the methods are related to errors in pipetting. The possibility is discussed that these methods might be developed into "absolute" or "definitive" methods by subjecting each individual step in the determination to a detailed analysis with respect to possible error. We also discuss some of the consequences of the different reference methods on the choice of routine methods.

Blood Glucose↗
PubMed1998

[Rhesus factor determination of the genome of stereomicroscopy selected amniocytes].

This study intended to develop a molecular biological method for the determination of the rhesus blood type through the genome of amniocytes. Using micropipettes amniocytes were isolated under a stereomicroscope from amniotic fluid samples. Single or several (> or = 10) amniocytes, respectively, were pipetted into 0.2 ml vials each, and were lysated at 65 degrees C for 15 min using an alkaline solution making the DNA accessible to polymerase chain reaction. Thereafter, the rhesus D gene as well as the CE gene were amplified with nested primers in two subsequent PCR. Finally, the amplification product was electrophoretically analyzed using agarose gel. When single aminocytes were analysed the amplification rate was 64% for the rhesus D gene, and 80% for the rhesus CE gene; "allele specific non amplification" was observed in 24% of cases. With 10 or more amniocytes per vial the amplification rate was 100%. This method permits to reliably predict the fetal rh-type at low costs within about 4 hours.

Amniocentesis↗
PubMed1998

Changes of nuclear glycoproteins in normal and simian virus 40-transformed WI-38 human lung fibroblasts.

To investigate changes in nuclear glycoproteins after transformation, nuclei and nuclear extracts were prepared from the normal and SV40-transformed WI-38 human lung fibroblasts grown in vitro. The nuclei of the normal and transformed cells were isolated by a vigorous pipetting method, and the isolated nuclei were verified by morphological and biochemical examinations. The nuclear glycocomponents, which were labelled with fluorescein isothiocyanate-lectins, were evenly found all through the isolated nuclei. Lectin blotting analysis showed that dozens of nuclear proteins contain both Man and GlcNAc moieties. The glycoproteins bearing the terminal N-acetylglucosamine sugar moieties were also demonstrated by in vitro galactosylation with [3H]-galactose. The result showed that nine glycoproteins specifically appeared or disappeared upon viral transformation or as SV40 replication products, indicating that there are significant differences in nuclear glycoproteins between the normal and SV40-transformed cells. These results suggest that the modification of Man and GlcNAc moieties are dynamic changes.

Cell Line↗