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At least 613 records · Page 34Linked to original sources
PubMed1989

An undetectable source of technical error that could lead to false negative results in enzyme linked immunosorbent assay of antibodies to HIV-1.

Since the institution of routine testing for antibodies to Human Immunodeficiency Virus (HIV) using the enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of this assay system has received significant scrutiny. During previous use of this methodology, we have quantified rates of false biological positive results using commercial kit assays in a normal donor population. In this study, we have identified a potential source for false negative results. Using multiple lots of two different commercial ELISA kits, the absorbance readings at the test end point could not differentiate between normal non-reactive donor samples and blanks containing no sample. These results occur using normal donor samples, even though the assays could distinguish between blank wells and the manufacturers' "normal controls", provided with the assay. Our findings suggest that a technical pipetting error is presently undetectable, either visually or by statistical methods, and could permit an untested, potentially HIV-1 positive, unit to be released into the transfusable blood supply. A possible solution is suggested.

Enzyme-Linked Immunosorbent Assay↗
PubMed2000

Antibiotic-labeled probes and microvolume fluorimetry for the rapid detection of bacterial contamination in platelet components: a preliminary report.

BACKGROUND: Approximately 1 platelet in 2000 components is bacterially contaminated. Most commonly, contaminating organisms are gram positive skin saprophytes (such as Staphylococcus sp. or Bacillus sp.). A novel approach to the rapid diagnosis of gram positive contamination by the use of a fluorescence-labeled antibiotic probe with affinity for the gram positive cell was investigated. STUDY DESIGN AND METHODS: Two isolates of Staphylococcus epidermidis were inoculated into bags of Day 0 platelets. Quantitative cultures along with a semi-automated screening assay on a microvolume fluorimeter employing a fluorescence-conjugated vancomycin probe was performed for each day of storage. In addition, serial dilutions of the bacteria were added to sterile platelets to achieve a range spanning 10(1) to 10(8) CFUs per mL. RESULTS: All samples with a bacterial contamination of > or =10(5) CFU per mL were detected. Sterile samples were nonreactive. The entire procedure requires three pipetting steps and took less than 1 hour to perform. CONCLUSION: These preliminary results with the use of fluorescence-labeled antibiotics as probes combined with microvolume fluorimetry for the rapid detection of bacterial contamination of platelet components suggest that this is a promising approach. Further studies with additional organisms and alternative conjugates, bacteria, and antibiotics are underway.

Anti-Bacterial Agents↗
PubMed2001

A new flow cytometric method for simultaneous measurement of residual platelets and RBCs in plasma: validation and application for QC.

BACKGROUND: Guidelines for the preparation of FFP in Austria and Germany require the quantification of residual RBCs in plasma. However, currently there is no validated method for enumeration of RBC counts as low as 1 x 10(9) per L. STUDY DESIGN AND METHODS: A new flow cytometric method was developed for QC, to simultaneously determine if fresh unfrozen plasma contains residual platelets and RBCs. This method uses test tubes (TruCount, Becton Dickinson Immunocytometry Systems) that contain a known number of brightly fluorescent polystyrene beads. Plasma is pipetted into these tubes and mixed with FITC-labeled anti-CD41 and PE-conjugated anti-glycophorin A MoAbs. Acquisition can be performed on a flow cytometer after an incubation period of 15 minutes. RESULTS: Individual standard curves for platelet counts revealed excellent correlation coefficients (r>0.998). Platelet counts were validated against simultaneous measurements by using a cell counter and a hemocytometer. While the flow cytometric method slightly overestimated platelet counts of >40 x 10(9) per L by 10 percent, its precision in the critical range was very good-that is, a deviation of platelets < or = 1 x 10(9) per L from target values, which was even better than microscopic enumeration. The limit of sensitivity was <0.5 x 10(9) per L of platelets. RBC counts were also validated against simultaneous measurements made with a second cell counter. Standard curves for RBC counts also revealed excellent correlation coefficients (r>0.997). The limit of sensitivity was <0.25 x 10(9) RBCs per L. Platelet counts in plasma obtained by plateletpheresis or plasmapheresis ranged from 3 to 60 x 10(9) per L. About 25 percent of all plasma samples had platelet counts greater than the allowed upper limit of 25 x 10(9) per L, while all plasma samples contained fewer RBCs than the upper limit of 6 x 10(9) per L. CONCLUSION: This newly developed method provides a simple, quick, precise, and easily reproducible tool for simultaneous measurement of residual platelets and RBCs in fresh plasma.

Erythrocyte Count↗
PubMed2002

Yield of HCV and HIV-1 NAT after screening of 3.6 million blood donations in central Europe.

BACKGROUND: HCV and HIV-1 NAT of all blood donations was initiated at our institutions in January 1997 to reduce the residual risk of transfusion-transmitted virus infections. The yield of NAT after testing more than 3.6 million donations in central Europe is reported. STUDY DESIGN AND METHODS: Automated pipetting instruments were used to pool up to 96 donor samples including those that were antibody reactive. To compensate for dilution of the individual donor samples by pooling, viruses were enriched from the pools by centrifugation at 48,000 x g. A commercial PCR (Cobas Amplicor, Roche) and an in-house PCR were applied for HCV and HIV-1 amplification, respectively. RESULTS: Six HCV and 2 HIV-1 PCR confirmed-positive, antibody-negative donations (yield, 1 in 600,000 and 1 in 1.8 million, respectively) were identified. Thirty-nine and 11 multiple-time donors seroconverted for HCV and HIV, respectively, and look-back procedures were initiated. Archived samples from preseroconversion donations were thawed and retested by single-sample PCR and remained negative. The recipients of the blood components were traced and tested. All traced recipients were negative for HCV and HIV antibodies. CONCLUSION: The yield of NAT in central European Red Cross blood donors was less than expected from theoretical calculations for American and German multiple-time donors. Look-back procedures for HCV and HIV indicated that no donation given before seroconversion of the donor was missed by minipool PCR. Sensitivity of minipool PCR testing after virus enrichment seems to be sufficiently high to close the diagnostic window almost completely.

Antibodies, Viral↗
PubMed2000

[The effect of a nasal spray consisting of a standardized mixture of citrus limon (succus) and an aqueous extract of Cydonia oblongata (fructus) on nasal mucociliary clearance].

In a three-way-crossover study in 18 healthy male and female subjects aged from 20 to 49 years the influence of a 1% and 3% solution of a standardized composition of Citrus limon, succus, and extract from Cydonia oblonga, fructus (Gencydo) on the intranasal mucociliar clearance was investigated after multiple administration. The pH of the solution was about pH 2.3-3.2. The dose regimen consisted of 20 puffs (0.13 ml per puff) in each nostril within 24 h, which was by factor 3-10 higher than the usual therapeutic dosage of 2-6 puffs per nostril and 24 h. The mucociliar transport time was measured by a modified saccharin test, where 1 microliter of a 3-molar aqueous sodium saccharinate solution was applicated at the inferior nasal turbinate 1 cm from its anterior end using a glass capillary microliter pipet. This test was performed at screening examination, before each administration period, directly after each 24 h treatment period and 24 h after the end of each treatment. The time of initial taste perception could be defined with high precision by the volunteer since it appeared very spontaneously. Neither after intranasal administration of the 1% and 3% Citrus/Cydonia solution nor after placebo solution a prolongation of the perception time was found. It could be concluded that there is no measurable influence of the test products on the intranasal ciliar function.

Administration, Intranasal↗
PubMed1992

[Hemostatic disorders following polytrauma--the role of physiologic coagulation inhibitors during the preclinical phase].

Coagulation changes due to polytrauma are considered to be an important determinant for the outcome. In this context, physiological inhibitors of activated coagulation are highlighted with special reference to antithrombin-III (AT-III). Blood samples of 20 randomly selected adults with polytrauma (Injury Severity Score mean = 36.7 +/- 8.6) were investigated. To investigate the very early onset of coagulation changes, samples were taken as early as possible at the site of emergency (mean = 18.3 +/- 5.5 min. after trauma) as well as at hospital admission (mean = 78.0 +/- 10.4 min.). By means of a specially designed "mini-lab", basic processing of the samples harvested (centrifugation, pipetting, freezing) was done on the spot to obtain haemostaseological results that agree as closely as possible with the subsequent analyses. Due to the activation of intravascular coagulation as well as the consumption of physiological inhibitors, comprehensive coagulation disturbances become obvious at the time of hospital admission. These are intensified by haemodilution as a consequence of high-dose fluid replacement. However, significantly elevated levels of specific coagulation reaction products (thrombin-antithrombin-III-complex) give evidence of the consumption of inhibitory potential exceeding haemodilution.

Adult↗
PubMed1988

[Experiences and successes with intravaginal fertilization and culture of human oocytes].

We present in this study our first experience with intravaginal culture (IVC) of human oocytes as a modified technique for in-vitro fertilization. Up to 4 oocytes and washed, pre-incubated spermatozoa (final concentration: 0.1-0.2 x 10(5)/ml) are pipetted into a plastic capsule. The capsule is closed, avoiding any air inclusion. It is placed into the maternal vagina for up to 50 hours, held in place by a diaphragm. The first 15 patients treated by IVC achieved a fertilization rate of 58% (35/60). Following transfer of the cleavage stages, 3 intact clinical pregnancies were established: 2 in the group with tubal sterility factors and 1 in the group of patients with endometriosis. In the group with andrological causes of sterility, one beta-hCG-positive reaction was detected. However, no intact clinical pregnancy was achieved. Our preliminary results show IVC to be a promising alternative in particular cases of sterility treatment. In contrast to the conventional technique, IVC minimizes exposure of gametes to factors which may affect their early development, such as light or low temperature. Furthermore, there is an important psychological factor to be considered, as the patient actively participates in the incubation period of in-vitro fertilization.

Embryo Transfer↗
PubMed1984

Salt taste sensitivity and stimulus volume: sips and drops. Some implications for the Henkin taste test.

Various presentation procedures and stimulus volumes were compared in their effect on salt taste sensitivity. For sodium chloride (NaCl) stimuli pipetted onto the tongue, subjects had comparable measured sensitivity to 10 ml and 1 ml volumes but a reduced sensitivity to 0.1 ml volumes. The greatest sensitivity was achieved with 10 ml sipped volumes. Modification of the Henkin dropwise triangular taste test, by increasing the stimulus volumes, increased subjects' sensitivity. Only the 1 ml pipette presentation increased the sensitivity of the Henkin procedure, albeit slightly, to the detection of changes in taste sensitivity, the purpose for which the procedure was actually designed. Such modification did not perturb salivary NaCl levels more than the regular Henkin procedure, so it would not be expected to introduce sensitivity drift.

Adolescent↗
PubMed2000

A dipstick immunoassay to rapidly measure serum oestrone sulfate concentrations in horses.

A dipstick, competitive immunoassay for rapidly measuring serum oestrone sulfate (OS) concentrations in horses was developed to distinguish mares 100 or more days pregnant from non-pregnant animals. 6-Ketoestrone 6-carboxymethyloxime conjugated to bovine serum albumin (oestrone CMO-BSA) was 'dotted' 25 mm from the bottom edge of 45 x 5 mm strips of polyester-film-supported cellulose nitrate membrane, pore size 3 microm. The strips were blocked, dried and a 15 x 5-mm cellulose absorbent sink attached 10 mm from the top of each strip. The manufactured dipsticks were stored with desiccant at room temperature until used. A monoclonal antibody recognizing OS was coated onto uniform, blue-dyed polystyrene microspheres (mean diameter, 0.31 microm) by adsorption. After blocking, several washes and resuspension by sonication, the antibody-coated microspheres were stored at 4 degrees C. The concentrations of oestrone CMO-BSA dotted onto the dipsticks and OS antibody coated onto the microspheres were optimized to produce a test that allowed maximum discrimination between the concentrations of OS found in serum of mares 100 or more days pregnant (i.e. >30 ng OS mL(-1)) relative to those found in non-pregnant mares (i.e. <10 ng OS mL(-1)). To perform the dipstick test, 30 microL of carrier buffer, 10 microL of OS antibody-coated microspheres and 10 microl of OS standard or serum sample were pipetted into a microwell and mixed. A dipstick was placed in the solution. All the liquid migrated up the dipstick into the absorbent sink within 15-20 min leaving a blue dot where the OCMO-BSA had been placed. The intensity of colour of the blue dot, which correlated inversely with the concentration of OS in the standard or serum sample, was assessed visually and by computer image analysis. An OS concentration less than 5 ng mL(-1) produced a deep blue dot, 20 ng/ml a light blue dot and a concentration greater than 50 ng mL(-1) a very faint blue dot, or none at all. Serum samples from 42 non-pregnant mares and 40 mares over 100 days pregnant were analysed by the dipstick test. All the serum samples from non-pregnant mares produced dipsticks with deep blue dots that ranged in intensity from 20 to 38 colour intensity units, equivalent to OS concentrations less than 7 ng mL(-1). Sera from all the pregnant mares generated dipsticks with either faint blue dots or none at all (i.e. < or =5 colour intensity units, equivalent to OS concentrations >40 ng mL(-1). It is concluded that this novel, rapid dipstick immunoassay offers a practical, alternative means of analysing serum OS concentrations in horses, and enables mares that are 100 or more days pregnant to be distinguished from those that are not pregnant.

Animals↗
PubMed2003

Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size.

The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus-oocyte complexes (COCs) were collected from 1-<2, 2-<3, 3-<4, 4-<6 and 6-<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL(-1) hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 microg mL(-1) follicle-stimulating hormone, 10 microg mL(-1) luteinizing hormone, 1.5 microg mL(-1) oestradiol, 75 microg mL(-1) streptomycin, 100 IU mL(-1) penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1-<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1-<2 mm (107.7 +/- 1.6 microm), 2-<3 mm (108 +/- 1.1 microm) and 3-<4 mm (114.6 +/- 1.3 microm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4-<6 mm (124.4 +/- 1.3 microm) and 6-<8 mm (131.9 +/- 1.4 microm) in diameter. A majority of oocytes (P< 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4-<6- and 6-<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2-<3- and 3-<4-mm follicles, respectively, whereas significantly (P< 0.05) more oocytes from 4-<6- and 6-<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.

Animals↗
PubMed2000

Automated parallel DNA sequencing on multiple channel microchips.

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Automation↗
PubMed1970

A new approach to isoelectric focusing and fractionation of proteins in a pH gradient.

This paper describes a new method of condensation (focusing) of extended volumes of mixtures of proteins (or other ampholytes) into an isoelectric spectrum of discrete zones located at points of a pH gradient corresponding to the pI value of the individual proteins. In contradistinction to the currently practiced isoelectric focusing in "natural" pH gradients which may require as much as 96 hours for completion, the present method yields clear-cut condensations marking the isoelectric pH within about five minutes and complete fractionation within about 15 minutes. No "Ampholines" or other special buffers are required and establishment of the pH gradient requires only about 10 seconds rather than days as is common in natural pH gradient focusing. The pH gradient is generated by utilization of the temperature dependence of pH. By establishing a temperature gradient between 0 degrees and 50 degrees C in an electrophoretic column, a stable pH gradient extending over 1 pH unit can be maintained in the absence as well as the presence of a current. The pH gradient can be easily controlled by variation of the temperature limits so that high resolving power can be achieved in shallow pH gradients. The pI of the focused fractions is determined by measurement (by a thermistor or resistance thermometer) of the local temperature within each of the isoelectric zones that determine the local pH. The individual zones can be pipetted out. The method is illustrated by simultaneous condensation and evacuation of hemoglobin in two pH gradients traversed by opposite currents and by separation of hemoglobins A and S in 16 minutes in a pH gradient where the current passes in the direction of increasing pH in Tris buffer solutions stabilized by a sucrose density gradient.

Electrophoresis↗
PubMed1978

"Panning" for lymphocytes: a method for cell selection.

We have developed a simple method for the fractionation of T and B lymphocytes. Plastic dishes coated with antibodies specific for mouse Ig selectively bind splenic B lymphocytes. The adherent cells are easily removed by gentle pipetting; both adherent and nonadherent populations retain immunologic function. In a typical experiment, when 3 X 10(7) splenic lymphocytes were added to a 100 X 15 mm plastic dish coated with microgram quantities of anti-Ig, 98 % of the nonadherent cells were Ig negative and 97% of the adherent cells were Ig positive. The method is sufficiently sensitive to allow detection and separation of cell types comprising as little as 2% of the total population and can be modified to allow the selection of cells by a double-antibody procedure. We believe that the plastic dish method will be generally useful for fractionating cells on the basis of their cell surface antigens.

Animals↗
PubMed1980

Autonomous movements of cytoplasmic fragments.

Tiny fragments from the cytoplasm of human skin fibroblasts with about 2% of the original cell volume ("microplasts") were prepared by treatment with cytochalasin B, vigorous pipetting, and trypsinization of the attached fragments. They remained alive for 8 hr or longer. Some of the microplasts were able to produce and move filopodia, ruffle, or both; others blebbed continuously. Slow flattening was observed in the larger microplasts. In all cases tested, microplasts avoided contact with other cells or microplasts. The observations suggest that the cytoplasmic matrix and the membranes of animal cells are so constructed as to express locally and autonomously any one of the elementary amoeboid movements listed above. More importantly, whatever types of motile surface projections a microplast expressed, it continued to produce and move them in a stereotypical way as if there were long-lived structural or material determinants for each type. The microplasts were unable to locomote autonomously. Therefore, it is conceivable that directional movement of whole cells may require a supervising mechanism that confers a certain coordination and strategy on its component cytoplasmic bits. Otherwise they would continue to move in stereotypical and autonomous ways without ever displacing themselves, as suggested by the behavior of the microplasts.

Cell Movement↗
PubMed1982

Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells.

We have studied the effects of batrachotoxin (BTX) on sodium channels in hybrid mouse neuroblastoma cells NG108-15 by using the suction pipet voltage clamp method. BTX-modified sodium channels activate with first-order kinetics and, over most of the potential range, activate more slowly than normal sodium channels. The peak conductance-voltage curve and the time constant of activation-versus-voltage curve for BTX-modified sodium channels are shifted about 50 mV in the hyperpolarizing direction compared to the corresponding curves for normal sodium channels. There is no change in the slope of the conductance-voltage curve. These results suggest that BTX slows down one of the steps leading to channel opening, which consequently becomes rate-limiting. In addition, BTX eliminates both fast and slow inactivation.

Animals↗
PubMed1984

Movement of scallop myosin on Nitella actin filaments: regulation by calcium.

In order to determine if Ca2+ regulates scallop myosin movement on actin, we have measured motility of scallop myosin along actin filaments using a direct visual assay. This procedure consists of covalently linking myosin to 1-micron beads and pipetting them onto a parallel array of actin filaments located on the cytoplasmic face of a Nitella internodal cell. In the absence of Ca2+, scallop myosin-coated beads exhibit no directed motion; however, in the presence of pCa2+ of greater than 5.84, these beads undergo linear translocations with average velocities of 2.0 micron/s. This Ca2+ -sensitive motility requires the presence of regulatory light chains on the scallop myosin. Removal of regulatory light chains with 10 mM EDTA produces a "desensitized" myosin, no longer sensitive to Ca2+, which moves at rates of 0.09-0.3 micron in the presence or absence of Ca2+. Readdition of regulatory light chains to preparations of desensitized myosin once again confers Ca2+-sensitive motility. The Ca2+ dependence of scallop-myosin motility shows a sharp transition, consistent with the Ca2+ activation sensitivity of the actin-activated ATPase. Furthermore, relative rates of movement of calcium-regulated myosins from various molluscan species are consistent with their respective rates of ATP hydrolysis. Thus, myosin motility along actin filaments provides a sensitive and direct assay of myosin activity and is suitable for studying myosin regulation.

Actins↗
PubMed1985

Direct measurement of the electrogenic activity of o-type cytochrome oxidase from Escherichia coli reconstituted into planar lipid bilayers.

Turnover of o-type cytochrome oxidase purified from Escherichia coli and reconstituted into proteoliposomes leads to the generation of a transmembrane electrical potential (interior negative) by means of vectorial electron flow. In the experiments reported here, purified oxidase is reconstituted in planar lipid bilayers formed at the tip of patch pipets, and open-circuit membrane potentials generated by electron transfer are measured directly. Potentials of up to 4 mV (substrate side positive) are generated in the presence of reduced phenazine methosulfate or ubiquinol-1, and with both substrates, electrogenic activity is inhibited by cyanide. Furthermore, the membrane potential generated during oxidase turnover is inhibited progressively with applied voltages (substrate side positive), decreasing almost to zero at an applied voltage of 150 mV.

Electrochemistry↗
PubMed1986

Role of calcium in regulating the cyclic GMP cascade of phototransduction in retinal rods.

Both cGMP and Ca2+ appear to be involved in the process of phototransduction in vertebrate rods, but their precise roles have been the subject of debate. To investigate the role of Ca2+ we have artificially increased the calcium buffering capacity of the rod by using a patch pipet to incorporate the calcium buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the rod cytoplasm. In the presence of buffer the Na+-Ca2+ exchange current became greatly slowed, suggesting that the cytoplasmic calcium concentration (Cai) had indeed been buffered substantially. Although the presence of buffer had negligible effect on the rising phase of the light response, it profoundly altered the later behavior. Responses to brief flashes became prolonged and exhibited an overshoot, apparently because the shut-off process was modified. The normal acceleration of time-to-peak with brighter flashes (an early sign of light adaptation) disappeared. Responses to steady adapting illumination took much longer than normal to settle to a steady level, although the final level represented a similar fractional suppression of current. With superimposed test flashes the presence of such adapting illumination caused a more rapid recovery, whereas the presence of calcium buffer slowed the recovery. The results are consistent with the idea that the rapid drop in Cai, which has recently been shown to accompany the light response, is involved in terminating the light response, and that Cai is thereby involved in setting the operating point and sensitivity of phototransduction. From comparison with other work we infer that Cai appears to act, at least in part, by means of control of cGMP phosphodiesterase activity.

3',5'-Cyclic-GMP Phosphodiesterases↗