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At least 847 records · Page 47Linked to original sources
PubMed1992

Research note: effects of agitation and temperature changes on turkey sperm viability after twenty-four hour storage.

The effects of agitation and temperature changes on turkey sperm viability were estimated before and after 24 h storage at 7 C using the ethidium bromide (EB) procedure and a sperm stress test. The stress test is identical to the EB procedure except the buffer is hypotonic. Labile sperm lyse and are then stained with the EB. Treatments consisted of semen diluted 1:1 and subjected to one of the following procedures: agitation (pipetting up and down 25 times, repeated 4 times at 15-min intervals); temperature changes (semen moved from 7 to 26 C and back to 7 C 3 times at 30-min intervals); or control semen procedures (diluted semen is placed on an orbital shaker at 7 C). Each semen treatment was evaluated within 3 h of dilution and after 24 h storage. Agitation and temperature changes did not affect viability of sperm. Regardless of treatment, the EB procedure revealed no differences in sperm viability before or after 24 h storage. In contrast, the same samples subjected to the stress test revealed significant increases in nonviable sperm after 24 h storage. However, there was no treatment by test interaction. These data indicate that mixing semen with diluent and mild changes in semen temperature associated with the transport of semen between the interval of semen collection to insemination, even up to 24 h storage, have minor effects on sperm viability.

Animals↗
PubMed2002

IFN-gamma-induced apoptosis and microbicidal activity in monocytes harboring the intracellular bacterium Coxiella burnetii require membrane TNF and homotypic cell adherence.

IFN-gamma is critical for the protection against intracellular bacteria through activation of the antimicrobial machinery of phagocytes. Coxiella burnetii, the etiological agent of Q fever, is a strictly intracellular bacterium that inhabits monocytes/macrophages. We previously showed that IFN-gamma induced C. burnetii killing by promoting the apoptosis of infected monocytes. We show in this study that IFN-gamma-induced apoptosis of infected monocytes was characterized by a time- and dose-dependent activation of caspase-3. IFN-gamma-mediated caspase-3 activation and C. burnetii killing depend on the expression of membrane TNF. Indeed, TNF was transiently expressed on the cell surface of infected monocytes a few hours after IFN-gamma treatment. In addition, anti-TNF Abs inhibited IFN-gamma-mediated caspase-3 activation whereas soluble TNF had no effect on infected cells. Concomitantly, IFN-gamma induced homotypic adherence of C. burnetii-infected monocytes. The latter required the interaction of beta(2) integrins with CD54. When adherence was disrupted by pipetting, by a combination of Abs specific for CD11b, CD18, and CD54, or by an antisense oligonucleotide targeting CD18 mRNA, both cell apoptosis and bacterial killing induced by IFN-gamma were inhibited. Thus, adherence via CD54/beta(2) integrins together with membrane TNF are required to eliminate C. burnetii-infected cells through cell contact-dependent apoptosis. Our results reveal a new component of the antimicrobial arsenal mobilized by IFN-gamma against infection by intracellular bacteria.

Apoptosis↗
PubMedDate not supplied

[Comparison of the MTP immunoassay with EMIT in blood screening for drugs].

We compared the MTP immunoassay with EMIT for the screening of drugs of abuse (opiates, cannabinoids, cocaine metabolites and amphetamines) in whole blood samples. These blood samples were obtained from the German police, when driving under the influence of drugs of abuse was suspected. For screening with the MTP immunoassay 25 microliters of serum or blood (without any pretreatment) was pipetted into the wells of the microtiter plates and the procedure was followed as described. Prior to screening with a Cobas Mira and EMIT reagents, the samples were treated with acetone to precipitate serum proteins. The cutoff for all drugs of abuse was set at 10 ng per ml of serum or blood. In most cases there was a good agreement between the negative and positive results of the two screening assays. The agreement between the two assays in the detection of opiates and cocaine was 91% and 93%, respectively, and for cannabinoids and amphetamines approximately 80%. The MTP immunoassay was more sensitive than EMIT for the detection of cannabinoids--but at the same time the MTP immunoassay was less specific. Both screening assays have a sensitivity of 100% for the detection of opiates and cocaine, but the specificity of the EMIT--also for opiates--was substantially lower. The MTP immunoassay has in respect to amphetamines a very high sensitivity, whereas the sensitivity of EMIT for amphetamines is inacceptable due to losses during sample preparation. The specificity of MTP immunoassay for amphetamines is not optimal, because a relatively large amount of samples tested false-positive for amphetamines at the cutoff of 10 ng/ml. In summary the MTP immunoassay, although not automated, performs well in comparison with EMIT, especially if the sample preparation for EMIT testing ist considered.

Enzyme Multiplied Immunoassay Technique↗
PubMedDate not supplied

Use of a modified nominal group process for improving laboratory performance in human immunodeficiency virus type 1 antibody testing. Reaching consensus on three questions concerning HIV-1 testing.

Using expert panels of medical technologists and public health microbiologists, a modified nominal group process was used to reach a consensus on three questions concerning current human immunodeficiency virus type 1 (HIV-1) testing methods. The questions related to important sources of error, improving the testing process, and improving proficiency testing. The modified nominal group process proved to be effective in developing lists of errors in laboratory testing; it provided a fast, economic means of identifying possible areas for improving laboratory quality assurance and training programs. For the HIV testing model, the focus group panelists indicated laboratory pipetting errors, labeling, and specimen identification as the most important sources of error. To improve the quality of this testing, the panel recommended standardizing test interpretation and restricting testing to laboratories licensed to perform HIV-1 testing. To improve proficiency testing, the use of blind specimens and establishing minimum standards of performance were suggested.

California↗
PubMedDate not supplied

Stepwise gradient elution of capillary electrochromatography in the analysis of environmental samples.

Two modes of stepwise gradient elution of capillary electrochromatography were developed for the analysis of environmental samples on both a laboratory-made and a commercial CE instrument. Using the laboratory-made apparatus, another mobile phase was dropped directly into the inlet vial with a pipet. By this means, nine 2,4-dinitrophenylhydrazine (DNPH)-derivatized ketones and aldehydes were separated with high resolution. Not only was analysis time shortened by more than one half, but the detection limit was greatly improved compared to that in isocratic elution. The latter was achieved using the P/ACE 5510 system (Beckman Instruments, Fullerton, CA, U.S.A.), which moved the mobile phase vials automatically. The stop-flow technique was utilized during transfer. It had no effect on solute retention. With this method, the concentration and type of organic modifier in the mobile phase could be changed. A mixture of 13 aromatic hydrocarbons was separated completely in 14 min by changing the mobile phase from 80% methanol to 80% acetonitrile and 90% acetonitrile in a sequential step mode. The RSD of the retention time of each component in six consecutive injections was below 0.374%. All of these results demonstrate that stepwise gradient elution of capillary electrochromatography may be useful in the analysis of complex environmental samples.

Electrophoresis, Capillary↗
PubMed1999

[Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures].

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.

Animals↗
PubMedDate not supplied

Health and safety in clinical laboratory practice in Ibadan, Nigeria.

The economic implications of safety precautions in laboratory practice make compliance difficult especially for laboratories in countries with poor economies. However, economic factors are not the only determinants of a safe work environment. Worker's perception of health and safety also influence compliance with safety guidelines. This study was conducted to determine the attitude and practice of laboratory workers with regard to safety. A structured questionnaire was administered to laboratory workers in 10 clinical laboratories. The questionnaire inquired about the use of protective equipment, safe work practices and immunization status of workers. Another questionnaire sought information on safety codes, accident records and first aid facilities in each laboratory. Questionnaires were coded and analysed. Ninety out of 106 laboratory workers (85%) responded to the inquiry. Unsafe work practices such as eating or drinking in laboratories and mouth pipetting of biological samples were practised by 41% and 10% of workers, respectively. Ninety percent of workers used white coats while 64% used gloves when handling biological samples. None of these workers used goggles or face shields in the course of their work. Fifty-one percent had received immunisation against tuberculosis, 73% against tetanus and 16% against hepatitis B. Thirty-seven percent of senior staff and 72% of junior staff did not receive any formal safety training. Accident records and first aid facilities were absent in most laboratories. The commonest health problem reported by workers was low back pain. The poor coverage of hepatitis B immunization among health care workers in an endemic area is currently a cause for concern among medical and paramedical staff. There is a need for education programmes to increase awareness on safety. Occupational health workers in this setting face the challenge of promoting safe work practices among workers in the face of the lack of funding and apathy among poorly paid junior workers.

Adult↗
PubMed1999

An enzyme-linked immunosorbent assay applicable to screen blood donors for IgA deficiency.

BACKGROUND AND OBJECTIVE: In order to build panels of IgA deficient blood donors, an assay is described that is sensitive, inexpensive and easily adaptable to the automated sample processors and turnaround times of blood banks. DESIGN AND METHODS: We developed a two-step enzyme-linked immunosorbent assay (ELISA) carried out in microwell plates coated with rabbit anti-human IgA antibody. Captured IgA was revealed with the same antibody conjugated to horseradish-peroxidase. The assay was adapted to the automatic pipetting system and ELISA processors used in routine blood donor screening. RESULTS: The assay sensitivity was 0.1 microg/mL. Intra-assay coefficient of variation (CV) for IgA concentrations between 0.1 and 100 microg/mL ranged from 0.69% to 3.80%. The median interassay CV was 3.05% (range: 1.2-7.9%). Coated plates can be stored frozen for at least 3 months without any loss in performance. The assay takes around 80 min to be performed. By using this ELISA we found 32 IgA-deficient individuals among 20,000 blood donors (prevalence 1:625). INTERPRETATION AND CONCLUSIONS: The ELISA has a good sensitivity, is reproducible, precise and timesaving. It is easily adaptable to the automated sample processors and operating procedures used in blood banks. This facilitates the building of panels of IgA-deficient blood donors.

Automation↗
PubMed1999

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34 + lin hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well can receive a single hematopoietic stem cell candidate. Sequential movement of each cell-containing well to a microscopic imaging system, serially over a several-day to several-week experiment, is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Bio-box built on the microscope stage. New image analysis software facilitates in the tracking of cell movement, recording of the time of cell division, and immunophenotyping of each of multiple individual or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and also may be applicable to a wide range of other systems of interest in tissue engineering.

Biotechnology↗
PubMed2000

Evaluation of an alkaline lysis method for the extraction of DNA from whole blood and forensic stains for STR analysis.

A modified alkaline lysis protocol for extracting DNA from forensically relevant specimens is evaluated and compared with the chelex 100 method. For whole blood, bloodstains and sperm stains, both methods yielded comparable results after amplification for a pentameric STR locus (HumCD4). The main advantages of the new method are: only approximately ten minutes and two pipetting steps are necessary and the expenses for the extraction are extremely low as only NaOH, TrisHCl buffer and a single microcentrifuge tube are required. Alkaline lysis also proved to yield DNA suitable for typing longer STRs by using dye-labeled primers and capillary electrophoresis. These advantages should render this protocol especially interesting for high-throughput laboratories in combination with multiplex PCR and fluorescent dye technology, although the storability of the extracts proved to be problematic.

Blood Chemical Analysis↗
PubMed1999

Hematology laboratory standardization: a plan for harmonization in Asia.

Hematology laboratory is generally required in the hospital. At the macroscale, hematology laboratories have served a large number of population. In Asia, more than 3,000 million people are potentially to use the hematology laboratory service, particularly the complete blood count. Since 1970s, automated technology has been introduced to Asia and as years passed by, technology diversity is increasing. However, there are considerable number of hematology laboratories that have no automated machine. They are still relied on manual technology which is still variable in spectrophotometer for hemoglobin determination, centrifuge for hematocrit and diluting pipet for cell counting. In particular, blood smear preparation and interpretation are very difficult to control for standardization from person to person and laboratory to laboratory. Different methodology and a large population in the huge geographical area in Asia, the agreement of standard criteria is greatly important. This report has shown strategy and action plan to reach the goal of hematology laboratory standardization in Asia.

Asia↗
PubMed1975

[Comparative studies of methods of salmonella enrichment (author's transl)].

Eight different methods of salmonella enrichment were compared in two series of experiments involving 100 samples of whole-egg powder and 80 samples of frozen whole liquid egg, respectively. 66 out of a total of 100 samples of whole-egg powder had been artificially infected with varying numbers of S. typhi-murium; 60 out of 80 samples of frozen whole liquid egg were found to be naturally infected with various salmonella species. 3 of the 8 methods (Table 1) were compared within an international collaborative study with 14 laboratories in 11 countries participating. A reduction of the pre-enrichment period from 18 to 6 hours and of volumes used in pre-enrichment and selective enrichment from 10 and 100 ml, respectively to 1 and 10 ml, respectively were found to have adverse influence upon the result of isolations, in particular in the case of weakly infected samples. In contrast, extended incubation over 48 hours as well as preparation of two sub-cultures on solid selective media following incubation of enrichment cultures over 18-24 hours and 42-48 hours, respectively always resulted in a certain increase of salmonella yield which, however, exhibited gradual differences for the individual methods examined. Preparation of a 2nd sub-culture meant, in particular, a decisive improvement of the result of isolations from artificially infected samples if selenite-cystine enrichment volumes were 10 and 100 ml, respectively. The best results could be obtained by means of the following methods of enrichment: Pre-enrichment of material in buffered peptone water at 37 degrees C over 18 hours; pipetting of 10 ml inoculated and incubated pre-enriched material into 100 ml selenite-cystine or tetrathionate enrichment medium according to MULLER-KAUFFMANN; onward incubation of the enrichment culture at 43 degrees C over 48 hours; and preparation of sub-cultures on solid selective media after 24 and 48 hours. The method using tetrathionate enrichment medium was found to be most expensive, results, however, were the most consistent ones.

Bacteriological Techniques↗
PubMed1975

Improved radioimmunoassay of plasm cortisol.

We report a simplified radioimmunoassay procedure for determination of plasma cortisol. The method offers the advantage that tracer, antibody, and assay buffer are added in a single step with the use of semi-automated equipment. Thus, critical pipetting procedures are minimized, and assay time is reduced to 4 h. No prior extraction or chromatographic purification of cortisol is required. The procedure is simple, reliable, and accurate. For either between-assay or within-assay determinations, the coefficients of variation are about 6%. Our results compare well with those obtained from a procedure that included extraction and chromatographic purification of cortisol in several plasma samples. Conditions for optimizing the assay are also discussed.

Autoanalysis↗
PubMed2001

[Is the diffusion method useful for testing the effectiveness of biocides?].

The authors tested the fungicide effect of different biocides by the diffusion method. Its principle involves pipeting samples of the tested fungicide preparations into pits in an agar medium on Petri dishes. The agar is inoculated over the whole surface with spores of fungi. It is incubated at 28 degrees C for 2 to 3 days. The size of the inhibition zones in the increments of fungi round the pits with the preparation are recorded. The authors discuss the suitability of this method for different types of preparations with regard to the results they assembled.

Antifungal Agents↗
PubMed1975

Computer-controlled automation of radioimmunoassay based on gel entrapment of antibody.

We describe a new, broadly applicable approach to radioimmunoassay. Solid-phase binding reagent is prepared by entrapping antibody in polyacrylamide gel. This gel is then fragmented, sieved, and dehydrated in 95 percent ethanol. Upon evaporation of the ethanol, the dry antibody-gel particles are dispensed into miniature disposable plastic columns. A unidirectional flowing stream configuration is used to construct a standard curve for the polypeptide hormone, angiotensin I, used here to exemplify the technique. Good reproducibility of this standard curve is demonstrated. The analytical system includes a computer-controlled sample turntable, digital pipet, and liquid-switching assembly.

Acrylamides↗
PubMed2001

Use of microtiter plates for latex agglutination immunoassay of serum alpha 2-macroglobulin.

A simple, sensitive turbidimetric immunoassay is described for analysis of alpha 2-macroglobulin (AMG) in serum, using microtiter plates and a commercial kit that features a sensitive latex reagent. Using microwells as reaction vessels and the latex reagent made it possible to minimize the sample size and the amounts of reagents. The procedure involves two pipetting steps and a brief incubation (10 min) at room temperature; the assay measures AMG concentrations ranging from 0.12 to 5 g/L and is unaffected by icterus, hemolysis, lipemia, or rheumatoid factor, even at high concentrations. Recovery of AMG added to three pooled serum samples averaged 95-105%. CVs for replicate analyses ranged from 1.9 to 4.8% (within-run) and 6.1 to 9.3% (between-run). Based on paired analyses of 70 serum specimens, AMG concentrations obtained by this turbidimetric microtiter immunoassay were in close agreement (correlation coefficient = 0.99) with results obtained by an enzyme-linked immunosorbent assay (ELISA). Serum AMG concentrations in 48 healthy adults (mean +/- 2SD) were 1.87 +/- 1.00 g/L.

Adult↗
PubMed1975

Evaluation of the Beckman "System TR enzyme Analyzer".

We evaluated 16 claims made by Beckman Instruments, Inc. for its Enzyme Analyzer (System TR), under a rigid written protocol for the Product Evaluation Subcommittee of the Standards Committee of the College of American Pathologists. We found the following to be within the company's specifications: (a) accuracy and precision of the temperature control; (b) accuracy and precision of the sample and reagent pipets; (c) instrument precision, both within-run and between-day; (d) carry-over from a sample with activity greater than 1000 U/liter; (e) instrument-to-instrument variation; (f) analytical linearity; (g) analysis time; (h) correlation of the instrument-printed answer with the activity calculated manually from a strip-chart recorder; (i) precision of the instrument's built-in electronic "standard"; (j) effectiveness of the over-range indicators; and (k) correlation between results of these enzyme assay methods and those for kinetic methods used in our laboratory. The instrument performed well.

Alanine Transaminase↗
PubMed1975

Modification of a commercially available digoxin radioimmunoassay kit for higher precision.

The procedure prescribed for the Schwarz/Mann 125-l-digoxin kit was modified with regard to pipetting and counting procedure. Incubation time with dextran-coated charcoal was also investigated. The modifications resulted in a significantly higher precision wihout introduction of a systematic error and the analysis time per eight-tube batch was decreased by 20 min.

Digoxin↗