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A simplified method for determining plasma glycerol is described. This assay utilizes the fluorometric measurement of the reduced adenine dinucleotide, NADH(2) which is formed when glycerol is oxidized by glycerol dehydrogenase. Only three pipettings are necessary for each reaction tube, and a large number of samples can be included in each assay.
A series of experiments was conducted to assess the performance of technologists in the blood bank, with particular reference to precision and accuracy in the technical and mechanical aspects of routine work, including preparation of erythrocyte suspensions, pipetting, interpretation of hemagglutination, detection of mixed-field agglutination, detection of weak alloantibodies, and antibody titration and scoring. The authors conclude that use of semiquantitative technics contributes significantly to imprecision in blood bank work. A plea is made for expediture of increased effort in quality assurance programs directed toward the technical staff.
The present paper summarizes the experience of the authors in the setting up of the radioimmunoassay (RIA) for human follicle simulating hormone (H-FSH), with the purified antigen for radioiodination, the F-FSH standard and the specific antibodies kindly donated by the National Pituitary Agency of the National Institutes for Arthritis, Metabolic and Digestive Diseases of the National Institutes of Health, Bethesda, MD. U.S.A. The conditions for the RIA have modified somewhat and simplified with respect to the suggested instructions accompanying the reagents. Thus, the amount of Chloramine T and the time of exposure of the labeled H-FSH (H-FSH) has been studied. It is always purified on Sephadex G-100 immediately before addition to the RIA and in this manner it may be used for up to 2 month after labeling when kept at --20 degrees C. Curves obtained at different dilutions of the H-FSH Standard, carried out with phosphosaline buffer, pH 7.4-7.8 (PBS) containing 1 % human serum albumin, or with horse plasma, of with PBS containing 0,25 % serum from non-immune rabbits (RIA Buffer) have been compared iwth those abtained by serial dilutions of sera from post-menopausal with these diluents. The most consistent results were obtained with the RIA buffer as diluent. The redisual error was smaller, and serial of dilution curves of the H-FSH standard were parallel to those of plasma and acetone precipitates of urine from post-menopausal women. Parallelism was not god using those serum. Results using PBS contianing human seum albumin were poor. PBS containing bovine serum albumin was avoided as some batches were found to interfere with the binding of the F-FSH to the antibody. The stability of the different dilutions of the H-FSH standard prepared in RIA buffer was tested. It was found that the standard curves could be prepared, pipetted into the RIA tubes and kept ready, frozen at --20 degrees C for one to two months. This shortens the actual setting up of a given RIA and decreases interassay variation of results. Parallelism of the H-FSH standard curve with serial dilutions (in RIA-buffer) of sera from women on the day of the preovulatory was confirmed. The data obtained in men and women, during stimulation with LH-RH are also given. No cross reactivity was found the HCG or sera from women, in agreement with the fact that the antiserum had been absorbed with HCG. There is, however, a considerable degree of cross reactivity with H-TSH; Thus, sera containing 15 muU/ml H-TSH or more, would give false H-FSH results. Such H-TSH values are not only found in hypothyroid patients, but might be reached during TRH responses to TRH.
An eight-channel programmed flow system for automatic lipid extraction and TLC application is described. Each channel has a container for lipid extraction connected by Acidflex tubing through an AutoAnalyzer pump to a TLC applicator needle. Extraction containers are prepared from disposable Oxford sampler pipet tips by inserting a small cotton filter into their lower, narrower end, which is connected to the pump tubing. The applicator needles are supported vertically in a manifold, and their tips rest on a TLC plate placed on a hot plate. Serum is added to isopropanol in each extraction container, and proteins are completely precipitated in 2 min and retained in the extraction chambers by the cotton filters; lipid extracts are then transferred on to the heated TLC plate by intermittent pumping at a rate allowing for continuous evaporation of isopropanol under streams of warmed air or nitrogen. The lipids accumulate on the plate in eight small spots, one for each channel. Solvent is proportionally added into the extraction chambers from a common reservoir through Acidflex tubing in a second AutoAnalyzer pump. During the extraction procedure, both pump motors are automatically operated by a programmed timer with a solid-state switch. Of several different solvents tested, isopropanol is the fastest for protein precipitation and lipid extraction and does not extract substances from the Acidflex tubing which interfere with chromatographic separation.
The sweep co-distillation technique of Storherr et al. was investigated and simplified. Six organochlorine pesticides in animal fats were cleaned up under various distillation conditions as follows: No solvent was used, or solvent injection rates were 1 mL/min or 2 mL/3 min. Distillation tubes of 6.7 and 9.0 mm id diameter were compared. Distillation temperatures were varied from 150 to 300 degrees C. The complex condenser of Storherr et al. was compared with a simpler pipet condenser. A U-tube condenser which allows direct introduction onto a Florisil column for secondary cleanup was evaluated. The following modifications to the sweep co-distillation technique resulted: no solvent introduction, distillation temperature 230 degrees C, nitrogen flow rate 600 mL/min, 6.7 mm id distillation tubes with simplified packing, and incorporation of the U-tube condenser. The new technique gave recoveries comparable to those of Storherr et al., but it is faster than most bisolvent partition methods and no large volumes of solvent are required.
Experience with exposure to, or infection with pathogenic agents at the National Animal Disease Center is summarized. A total of 60 laboratory-associated exposures to infectious disease agents were reported. Forty-nine exposures resulted from known accidents, but the other 11 were identified only after the development of clinical or serological manifestations of infection. Eighteen cases of laboratory-acquired infections were reviewed. Brucellosis, the most frequently reported laboratory-acquired infection, accounted for one-half of the cases summarized. Three cases of leptospirosis, two cases of Newcastle disease, two cases of ringworm, and a single infection with Mycobacterium bovis and with Salmonella arizonae were also encountered. The most frequently reported causes of exposure were: auto-inoculation or spray exposure associated with the use of the hypodermic syringe, cuts or lacerations, direct contact with infected animals, and mouth pipetting. Although the infecting event could not be identified in 11 infections, presumptive evidence suggests aerogenic transmission as a probable route of exposure in a number of such cases.
Over 4,000 cases of laboratory-associated infection have been recorded. Some of the agents involved often in the past have been less frequently the cause of such infection in recent years, and some agents are more likely to infect those working with them than others. Pipetting, the use of a needle and syringe, and spills have been most frequently involved in accidents resulting in infection, but in the majority of cases no recognized accident occurred. In these instances, infectious aerosols, produced in various ways, are probably the most frequent causes of laboratory-associated infection. The introduction of protective devices and emphasis on safe procedures seem to be reducing the risk of accidental infection. Although work with tumor viruses and recombinant DNA research may not be as hazardous as was originally feared, continued caution and surveillance is advised.
A device that lowers a micropipet into position just above a thin-layer plate is described. Use of a self-filling pipet and foot-operated bellows allows one to apply many portions of a sample to the same point at high speed without damaging the adsorbent coating.
During endolymphatic sac surgery on patients with Meniere's disease, very small amounts (1 microliter) of the intra-sac endolymph were obtained with micro-glass pipets. An immunological assay of microanalysis of the protein by the single radial immunodiffusion (SRID) method was applied to 2 patients, one with bilateral involvement and the other unilateral. Among the protein fractions Alb, IgG, IgM, IgA, C3, C4, transferrin and alpha 2M, the concentrations of IgG were relatively higher, especially in the Meniere patient in the active stage. From 2 other patients and a control deaf patient with temporal bone fracture, the same amounts of endolymph were obtained, accompanied by a tiny specimen of the outer wall of the sac. When a radio-immunological procedure was applied to the endolymph and the homogenized sac, the specific concentration of IgG was higher in the Meniere patients than in the control.
We have facilitated radiommunoassay of testosterone by mechanizing every pipetting step and separating bound from free hormone by shaking assay tubes with activated magnesium silicate (Florisil) in a multi-tube Vortex mixer. Compared with dextran-charcoal separation, Florisil separation has the advantage that testosterone is only negligibly adsorbed except during active shaking, so that the interval for adding adsorbent does not produce discrepancies in bound hormone estimates between first and last assay tubes in a series. Our method gave CVs of 5% (within assay) and 6% (between assay) for samples containing 4.0 mug of testosterone per liter, considerably better than those obtained with charcoal (respectively 12 and 15%). The assay also had low blank values and demonstrated excellent accuracy.
125I-iodohydroxybenzylpindolol (IHYP) is a beta-adrenergic antagonist used as a ligand for the characterization of beta-adrenergic receptors in various cell preparations. Since IHYP may be adsorbed to surfaces such as test tubes and pipet tips, it is necessary to measure the actual free concentration of IHYP in the incubation solution. With this modification, the number of beta-adrenergic receptors in human peripheral PMNs can be determined with greater precision.
The MAST Immunodiagnostic Test System was developed to provide a comprehensive, simple means for the in vitro measurement of multiple antigens or antibodies. The first commercial application of the MAST system incorporates several novel features for cost-effective diagnosis of IgE-mediated allergy in a clinical laboratory or a physician's office. The basis of the MAST system is a unique analytical test chamber, which contains cellulose thread as the solid-phase matrix and allows multiple test results from a single assay. This test chamber incorporates both positive and negative controls and requires no volume-dependent pipetting steps. Immunographic exposure onto high-speed Polaroid instant film allows for quantifying results with an automatic recording infrared-transmittance densitometer. Test results are easily interpreted by using a patient test record provided with the system. The MAST system greatly simplifies testing for allergen-specific IgE, while retaining specificity and sensitivity. Currently, with the MAST system one can simultaneously measure picomoles of allergen-specific IgE in up to 35 different allergen classes. In addition to allergy testing, the MAST technology is applicable to other immunodiagnostic profiles.
Electrochemical detector offers a rapid, accurate, precise and inexpensive micromethod for measuring iron in serum, in practise 50 microliters of serum is pipetted into the electrochemical cell and 60 seconds after the serum iron concentration is displayed digitally in mcg/dl. Day to day precision is 1.2%, the linearity is +/- 2% over range 0-1000 mcg/dl. Normal value in pediatric patients are 42-94 mcg/dl.
The different effects of homogenisation of four commonly used homogenisating apparatus, such as Stomacher, Ultraturrax, Waring-Blender, Bühler-homogenisator) were compared with each other by applying 1 artificially and 6 naturally moulded, non-fluid food products. Furthermore the influence of sedimentation in the homogenisate as well as the time of homogenisation was examined. One part of the examination was the direct microscopic observation on stained Breed-smears, the other part the cultural methods (number of mould plate count on malt extract agar). The homogenisation effect of the Stomacher was as good as with all other mentioned apparatus with the food products applied, except for the home-made smoked sausage. With this home made smoked sausage, much better results were obtained by these homogenisating apparatus, especially by the Bühler Homogenisator. This is due to the fact that the sausage was minced to a much higher degree and thus the inner mould flora was set free.--A more tender effect of the Stomacher on hyphal elements could not be achieved.--Prolonging the time of homogenisation from 1 minute to 2 up to 4 minutes did not give any better results.--A short sedimentation of coarser food particles facilitates the pipetting of the homogenisates without influencing the mould counts.--The naturally moulded foods, which were kept at room temperature for several weeks, showed a significantly smaller number of plate count than of total mould count in contrary to those foods moulded just before experimental beginning.
Fertilization checks in dairy cattle performed by estimation of progesterone in milk require a simple technique, the use of a sample taken independently of milking procedure and a simple management of the results. The laboratory equipped with a sample processor and pipetting device, high throughput counter for 125iodine and a tabletop computer can analyse about 1000 samples of fat-free milk in one day. The results are used for computer management of reproduction and veterinary service in farms with 600 to 2000 cows.
With the ultramicroanalytical system described here we can measure glucose in 1 microL of plasma or serum. The sample is placed on a dry, multilayer film element (Eastman Kodak), where a colored spot about 3.5 mm in diameter develops. The reflectance of these spots is measured with a reflectance digital matrix photometer that was conceived, designed, and constructed in our laboratory. The spot is illuminated with monochromatic light and its image is projected by a camera lens onto the photosensitive surface of a linear photodiode array containing 512 individual photodetectors. The photodetector signals are processed by a computer to obtain the reflectance and diameter of the spot. The latter is proportional to sample volume. Because the reflectance of the spot does not depend greatly on sample volume, accurate pipetting is not required. The coefficients of variation of repeatable glucose analyses were, for 400, 3000, and 5120 mg/L, 1.7, 2.3, and 2.8%, respectively. The correlation coefficient (r) between glucose analyses by our method (y) and with the Ektachem 400 (x) was 0.9918; the regression equation was y = 1.07x - 94.3 mg/L.
A simple technique was used for administration of powdered material directly into the stomach of rats. A commerically available positive displacement pipetting device was filled by tamping the cannula tip into the powder. The contents of the cannula could then be discharged into the stomach by insertion of the cannula via the esophagus. The device required only minor modifications prior to its use in this manner. The technique provided a means of accurate dosing of insoluble materials and eliminated the difficulties encountered in the oral administration of suspensions.
Platelets reportedly inhibit lactate dehydrogenase activity in plasma under reaction conditions of low osmolality. We describe observations inconsistent with these reports, and we attribute this "inhibition" to optical interference by platelets during the course of a reaction. We conclude that when platelet lysis is prevented and the optical interference of platelets corrected, platelet-rich plasma, platelet-poor plasma, and serum show essentially the same lactate dehydrogenase activity. Furthermore, platelet contamination can cause unexpected problems when lactate dehydrogenase is assayed with centrifugal analyzers. Results can be high or low, depending on the volume of diluent pipetted with the sample, and extreme within-run variations in activity are possible. When plasma is used instead of serum for routine analyses, regular checks for platelet contamination should be performed as a quality-control procedure, especially by laboratories separating plasma with bench-top centrifuges. Platelets can also interfere optically with assay of other enzymes and metabolites.