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PubMedDate not supplied

Improved microbiological assay for folic acid based on microtiter plating with Streptococcus faecalis.

We have developed an improved method, using 96-well microtiter plates, for the microbiological assay of folic acid. With this method, the tedium of conventional microbiological analysis is substantially decreased. Culture volumes have been reduced 33-fold, and pipetting procedures have been simplified. Assay time has been reduced to 14 h, and sensitivity has increased 10-fold (0.1 ng/mL). Analytical recoveries range from 98 to 104%. Intra-assay and interassay variabilities are less than 11%. The assay does not require extensive manipulation of inoculum. Day-to-day variability has been minimized by using saline aliquots of the bacterial suspension stored at 4 degrees C. The procedure is accurate, selective, and useful for direct measurement of folic acid in multivitamin formulations.

Biological Assay↗
PubMed1995

[Clinical biochemistry and laboratory medicine in the 21st century].

The author gives an outline of the assumed development of clinical biochemical examinations in future decades based on the development during the past 40 years. The development of biochemical analyzers will probably mean more perfect robotization; the increase in capacity (number of tests performed per hour) probably will not result from more rapid individual analyses (the rate of 900 tests/hour is probably optimal) but the capacity will be increased by doubling and trebling of critical elements (photometry or technique of measurements, pipetting etc.). Probably the volume of blood samples will not be reduces (to less than 1-2 microliters); however, amplification techniques will be used far more (as at present the polymerase chain reaction and others (for replication of cellular substances such as DNA). There will be a shift and greater use of "in vivo" techniques such as magnetic resonance spectroscopy and examinations using biosensors (because of the possibility and in particular expedience of examining some analytes continuously). The examined spectrum will be extended by further substances (in particular those with a short-term existence such as e.g. cytokines, selectins, adhesive molecules etc.; manifestations of circadian dynamics will be followed up much more e.g. in tumours, or chronopharmacokinetics of some drugs. A great development may be foreseen in the sphere of the use of computer technique for evaluation and communication of laboratory data and other data on the patient. For consultations global (or at least regional) computer networks will be much more widely used.

Chemistry, Clinical↗
PubMedDate not supplied

[The complement fixation reaction: a traditional, updated method for the detection of antibodies in the diagnosis of infections].

Complement-fixation (CF) is still an important basic serologic test for the diagnosis of infectious diseases. In several areas of microbiology (viral, bacterial, parasitic and fungal) it has served as a reference standard against which other methods have been compared. Its partial displacement by other techniques, as advocated in some recent literature, is often unfounded and uncritical; displacement is mostly due not to problems inherent in the method, but rather to the lack of reagents of satisfactory quality. The CF technique has been greatly improved in recent years. Improvements include the replacement of reaction tubes with microtiter 96-well plate systems, the availability of semi-automated and automated pipetting devices, the wide range of commercially offered antigens (over 60, by far larger than the range of antigens available for other test systems), an extreme stability of freeze-dried reagents with shelf-lives over decades and, last but not least, low reagent costs (particularly if compared with some other methods). For some diseases CF is still the method of choice (Campylobacter jejuni, Neisseria gonorrhoeae, Mycoplasma pneumoniae, Influenza A and B and some other respiratory viruses). CF has been recognized as a confirmatory test for Lyme disease (B. burgdorferi) and, probably, for Legionellosis. It has been used routinely for the follow-up of antibiotic treatment in syphilis and, recently, in Helicobacter pylori infections. A positive CF result is also indicative for the treatment of complications in Entamoeba histolytica carriers.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement Fixation Tests↗
PubMed1993

Microinjection of cells: basic considerations.

At least four types of technological advances have made cell microinjection procedures precise and practical, while minimizing negative impacts on cell viability: 1) hydraulic and electromechanical (stepper) micromanipulators, which provide operators with precise spatial control; 2) high-precision syringe and automated pneumatic microinjectors, with high reproducibility in the injected volume; 3) regulated pipet-pulling devices that yield highly reproducible tip diameters, down to 0.1 microns; and 4) specially adapted microscopes in both upright and inverted configurations, which ensure high-quality observation of cellular features in brightfield and all modern contrasting techniques, while reducing movement-induced vibrations at the contact point with the cell.

Cells↗
PubMed1993

Abbott AxSYM random and continuous access immunoassay system for improved workflow in the clinical laboratory.

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 tests for hepatitis, retrovirus, tumor markers, fertility markers, thyroid functions, and therapeutic drugs. The time to first result is approximately 15-25 min for most routine assays 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 coefficients ranging from 0.97 to 0.99 and slopes ranging 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%.

Autoanalysis↗
PubMed1993

[(Partially) automated gel techniques for determining erythrocyte antigens and antibodies].

Today, in addition to the classical test tube technology to determine red blood cell antigens and antibodies, not only the microtitration plate is increasingly used, but also the gel card. Since this new technology is easy to practice and since it further provides a higher specificity and sensitivity, we investigated whether, and if so to what extent, the gel card can be automated. On the basis of our own experience spanning many years, we were able to show that antigen determinations and antibody screening can be automated. Once the samples to be tested and the tests to be performed have been recorded by electronic data processing and combined in a job list, any sampler can distribute liquids, such as reagents and serum and/or plasma as well as red blood cell suspensions, on the basis of these data. A positive sample identification is made feasible by bar coding sample tubes and gel cards; the pipetting action is organized via the bar codes. Incubation and centrifugation are of secondary importance for automation. At this time, an automatic evaluation of gel cards is not yet possible so that result interpretation must be visual and the input of results must be manual. For documentation, however, laboratory books and result interpretation reports can be printed out automatically.

Blood Grouping and Crossmatching↗
PubMed1994

Humoral and cell mediated immune responses against Schistosoma spindale in BALB/c mice.

(BALB/c mice were infected with cercariae of Schistosoma spindale by tail immersion technique and by dropping some cercariae from a pipet onto the outer surface of the pinna of the ears. Groups of mice were removed on Days 10, 20 and 30 and tested for humoral and cell mediated immune responses using either adult worm or cercarial antigen. On Day 50 the mice were sacrificed and the worm burden was determined for each mouse. This method resulted in an infectivity rate of 89.7%. There was a significant increase in antibody titer to the adult worm antigen while no significant increase was observed for cercarial antigen over the period of the study. Results obtained for cell mediated immunity were more dramatic. There was a significant increase in foot pad swelling for adult worm antigen compared to a significant decrease for cercarial antigen during the course of the infection.

Animals↗
PubMed1994

Automation and quality control in the coagulation laboratory.

Hemostasis is a balance between complex interactions of directly opposing systems (coagulation and fibrinolysis) with seemingly unrelated systems at both the enzymatic and cellular levels (platelets, endothelium, leukocytes). It should not be surprising that coagulation disorders often accompany many different disease states. Because the function of each protein involved in coagulation is now better defined, newer methodologies have been developed to assay them. In this regard, the hemostasis laboratory can take a two-step approach to diagnosis: global screening and targeted analysis. Several new global test systems provide more detailed, quantitative, and more physiologically relevant evaluations than earlier assays allowed. In addition, individual enzymes, inhibitors, cellular release products, and low molecular weight products of activation reactions (molecular markers) can now be measured in sensitive, specific assays. With this new perspective, genetic predisposition to pathologic hemostatic conditions can be identified through molecular biology and can be identified during the early stages of disease (i.e., at subclinical stages before major pathologic complications are established), and more specifically targeted prophylactic, as well as therapeutic drug interventions, can be administered. The molecular markers of hemostatic activation that can be assessed by various immunochemical methods provide very early evidence of thrombotic, fibrinolytic, or platelet-involved aberrations. Technological advances in methodology and instrumentation have changed the scope of all clinical laboratories but, in particular, that of the coagulation laboratory. The dramatic growth and development have resulted from the influences of clinical chemistry, clinical immunology, pharmacology, biochemistry, and biotechnology. Analytical instruments for use in hemostatic testing go beyond plasma or whole blood clot-based readers, platelet aggregometers, and microscopes to a range of automated, discrete, chemistrylike analyzers, spectrophotometers, microliter ELISA systems, RIA systems, and multiprobe instruments designed to measure simultaneously the different assay end-points of colorimetric and clotting assays and flow cytometers. Instruments are designed for batch processing of single tests on multiple samples or multiple test panels on a single sample. Versatility ranges from instruments that measure only the final reaction solution, by end-point or kinetic analysis, to instruments that automatically pipet reagents and sample, incubate, and analyze the reaction for truly walk-away assay performance. Typically, a wider range of assays are available in automated laboratories as opposed to laboratories performing manual assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Automation of the Maxam-Gilbert chemical sequencing reactions.

A practical automated method of Maxam-Gilbert chemical sequencing reactions that uses solid-phase chromatography methods to purify DNA following chemical modification and cleavage is described in this report. The automation has primarily been made possible by using specially designed BioPak mini-columns, compatible with the Biomek 1000 automated workstation, which can be utilized in a manner similar to that of standard pipet tips. This automated chromatographic sequencing method produces rapid and reliable data as verified by sequencing a known human factor IX exon VIII gene fragment. The procedure presented in this report is a prototype for a single-fragment reaction and can easily be expanded to perform reactions on as many as 8 fragments at a time. The automation eliminates the tedious and time-consuming steps in the original method and increases the rate of sequence acquisition. This technology makes the Maxam-Gilbert chemical sequencing protocol more accessible, especially in large-scale, automated sequencing projects.

Autoanalysis↗
PubMed1993

Identification and use of constitutive proteins for the normalization of high resolution electrophoretograms.

Quantitative inter-gel comparisons of proteins separated by high resolution two-dimensional protein electrophoresis present a number of problems. These problems may arise from: variations in pipetting and other mechanical manipulations of samples, protein loss during transfer from the first to the second gel dimension, variations in staining, and/or variations in film development during autoradiography, in the case of radioactively labeled proteins. This study presents a discussion of these issues and a normalization algorithm to deal with variations, which relies on a class of proteins present in most biological samples which by their nature may be considered internal standards. This class consists of proteins which are controlled by constitutive genes. Constitutive genes are genes that are expressed constantly. We have developed an algorithm which is currently available as a subroutine, 'FINDCONS', in the computerized densitometry and protein comparison analysis program, developed by Olson & Miller (1988). This algorithm identifies potentially 'constitutive' proteins. A normalization method employing these potentially 'constitutive' proteins was compared to several others by examining 2D-electrophoretograms of proteins from developing gypsy moths (Lymantria dispar L.) insect tissue. Following normalization, inter-gel comparisons of spots, which were 'identified' as 'constitutive', were observed to vary less in density than when no normalization method was used, or when normalization based on total integrated spot density was used. In addition to its use as a normalization tool, this algorithm and the subroutine FINDCONS may be useful as an aid in biological studies to identify 'constitutive' proteins.

Algorithms↗
PubMed1993

[Gel technique, a new sensitive method for the determination of erythrocyte antibodies. Advantages and disadvantages with introduction of a new method].

A gel technique for the detection of red blood cell antibodies was evaluated for antibody screening. A total of 1872 samples were screened using both gel technique and the conventional serological method, and later 18,292 samples were analysed by the gel technique and results confirmed by conventional methods. In both methods, Anti Human Globulin technique and enzyme technique were applied. The gel system gave false-negative results in 0.2% and false-positive results in 0.9% of the samples. The gel system was more sensitive than conventional technique in the detection of antibodies of clinical importance and less sensitive in detecting antibodies with low incidence of haemolytic transfusion reactions and haemolytic disease of the newborn. The gel technique seems suitable for laboratories with equipment for automatic pipetting of samples and reagents.

Antibodies↗
PubMedDate not supplied

Ochratoxin A in cow's milk and in human milk with corresponding human blood samples.

A method for determining ochratoxin A in milk has been elaborated in which the sample was subjected to a liquid-liquid extraction step and then purified on a silica gel column packed in a Pasteur pipet. The purified samples were analyzed by ion-pair liquid chromatography with fluorescence detection. The detection and quantitation limits for determination of ochratoxin A in cow's milk were 10 and 40 ng ochratoxin A/L milk, respectively. The same limits were valid for the analysis of human milk. A total of 36 cow's milk and 40 human milk samples were analyzed. All samples were collected in Sweden. Ochratoxin A was found in 5 (14%) of the cow's milk samples (range 10-40 ng/mL) and in 23 (58%) of the human milk samples (range 10-40 ng/L). Blood samples were collected from the mothers who gave milk samples. A total of 39 samples were analyzed. All blood samples contained ochratoxin A in concentrations exceeding the quantitation limit (60 ng/L blood). The mean concentration of ochratoxin A in the samples was 167 ng/L blood (range 90-940 ng/L). The concentration of ochratoxin A in human milk was < or = 0.1 of that in the human blood.

Animals↗
PubMed1977

Determination of cadmium in blood by use of atomic absorption spectroscopy with crucibles--and a rational procedure for dry-ashing.

A simple, minimal-reagent procedure for analysis of cadmium in blood is introduced. Blood samples from workers occupationally exposed to cadmium were collected with heparin. Titon X-100 surfactant was added, and 15-mul aliquots of the blood were pipetted into small nickel sampling cups and placed in a holder for 100 cups, which was enclosed in a stainless steel box. After the samples were dried and ashed in a muffle oven at various times and temperatures, cadmium was determined in an air/acetylene flame in an atomic absorption instrument with a deuterium arc background corrector. Absorbance measurements were made by signal-averaging facilities in the instrument. The conditions for analysis are specified. The method appears to be accurate and reproducible, with a CV of about 6% in the range 35-160 nmol/liter. The detection limit was 3.0 nmol/liter of blood. With the new procedure, many blood samples can be dried and ashed in parallel, which favors both simplicity and precision.

Cadmium↗
PubMed1993

Ambient-temperature-stable molecular biology reagents.

We have processed biological materials to generate several reagents that are ambient temperature stable and ready to use. Stabilized biomolecules in a glassy matrix of carbohydrate polymers offer water-soluble reagents for complex molecular biology applications. This approach is particularly useful for reagent systems composed of enzymes, nucleotides and other components dispensed in single-use aliquots. Reconstitution of the glassy matrix delivers buffered enzymes and/or nucleotides for restriction, modification, sequencing and/or amplification of nucleic acids. These ambient-temperature-stable reagents allow a high level of reproducibility as they minimize the potential for pipetting errors. They also provide advantages in shipping, storage and subsequent handling. Added convenience includes elimination of setup time, cross contamination and refrigeration. Applications of ambient-temperature-stable biological reagents for routine molecular biology methods are presented.

Biotechnology↗
PubMed1993

Robotic chromatography: development and evaluation of automated instrumentation for assay of glycohemoglobin.

The measurement of glycohemoglobin (GHb) by boronate affinity chromatography is useful in monitoring long-term glucose control in diabetic subjects. The inherent disadvantage of this method is the hands-on time required because the hemoglobin fractions are separated on individual disposable columns. To overcome this disadvantage, we have programmed a Hamilton Microlab 2200 automated pipetting cartesian robot to complete the procedure, from the aspiration of blood from the sample-collection tube to the transfer of the separated hemoglobin fractions to a microtiter plate for absorbance measurement. This automated robotic system can analyze 96 specimens, including patients' samples and control material, in approximately 3 h. The precision (CV) of the method ranged from 1.6% to 3.5% within-run and from 2.7% to 3.5% day-to-day. The results correlated with those obtained with the Accuflex semiautomated robot, which used the identical disposable column, and those obtained with a Primus high-performance liquid chromatograph, which used a regenerated microparticle column. Automation of the GHb procedure allowed improved throughput, reduced labor cost, improved precision, and offered greater laboratory safety.

Autoanalysis↗
PubMed1977

Life history and host specificity of Mediorhynchus centurorum Nickol 1969 (Acanthocephala: Gigantorhynchidae).

Examination of 1905 arthropods from a swamp and surrounding pasture in sothern Louisiana revealed larval specimens of Mediorhynchus centurorum in 8 of 228 woodroaches, Parcoblatta pensylvanica, but in no other species. The life cycle was confirmed by feeding eggs of M. centurorum to laboratory-reared woodroaches. Cystacanths later recovered from the woodroaches developed into mature worms when pipetted into esophaguses of red-bellied woodpeckers, Centurus carolinius: red-headed woodpeckers, Melanerpes erythrocephalus; yellow-shafted flickers, Colaptes auratus; and a hairy woodpecker, Dendrocopos villosus. Infection was achieved in all woodpeckers fed cystacanths at least 47 days old and the mean prepatent period was 35 days. Cystacanths fed starlings, Sturnus vaulgaris, and red-winged blackbirds, Agelaius phoeniceus, did not produce infections although cystacanths from the same pool were infective to control woodpeckers. Larval development of Mediorhynchus centurorum in woodroaches did not differ significantly from that of M. grandis in grasshoppers. M. centurorum is the only species of the Gigantorhynchidae for which the life cycle has been confirmed by laboratory infections using an intermediate host species known infected nature.

Acanthocephala↗
PubMed1995

Use of Multiscreen plates for the preparation of bacterial DNA suitable for PCR.

A rapid and inexpensive method for isolating bacterial DNA for use in PCR is described. The method is based on the guanidinium thiocyanate (GuSCN)-lysis method of Boom et al. (J. Clin. Microbiol. 28:495-503, 1990) and enables a multiple of 96 samples to be prepared in only one hour. We use Multiscreen plates and a vacuum manifold from Millipore. Clinical samples are lysed and washed in the wells of a Multiscreen plate, and DNA is eluted in a standard microplate. Purified DNA was recovered with high yields (over 25%). The method allows multichannel or robotic pipetting for both the sample preparation as well as for the PCR step. The method has been applied successfully to detect pathogenic Streptococcus suis type 2 in nasal and tonsil swab specimens of pigs.

Animals↗
PubMed1977

Some problems associated with assay of 25-hydroxycalciferol in human serum.

Methods available for the assay of 25-hydroxycalciferol in human serum are evaluated and compared to one another. Ethanol was chosen for use in the initial extraction procedure and rat-kidney cytosol as the binding protein, although good alternative methods are also available. We used silicic acid for chromatography and found this an essential step. Reproducibility was increased when, after "bound" and "free" material were separated, an aliquot of the supernate was pipetted into the counting vial instead of the entire supernatant fluid being decanted. Beta-lipo-protein added to the assay system was of no advantage; added bovine serum albumin interfered with the assay by giving rise to high blank values. With ethanol extraction, silicic acid chromatography, rat kidney cytosol and separation on dextran-coated charcoal, sera from normal subjects showed a mean 25-hydroxycalciferol concentration of 28.5 microng/liter (range, 13.1 to 43.9) during the fall season. Coefficients of variation for a control serum were 4.9% (intra-assay) and 10.9% (interassay).

Animals↗