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Laboratory evaluation of oil spill bioremediation products in salt and freshwater systems.

Ten oil spill bioremediation products were tested in the laboratory for their ability to enhance biodegradation of weathered Alaskan North Slope crude oil in both freshwater and saltwater media. The products included nutrients to stimulate inoculated microorganisms, nutrients plus an oil-degrading inoculum, nutrients plus compounds intended to stimulate oil-degrading activity, or other compounds intended to enhance microbial activity. The product tests were undertaken to evaluate significant modifications in the existing official United States Environmental Protection Agency (EPA) protocol used for qualifying commercial bioremediation agents for use in oil spills. The EPA protocol was modified to include defined formulas for the exposure waters (freshwater, saltwater), a positive control using a known inoculum and nutrients, two negative controls (one sterile, the other inoculated but nutrient-limited), and simplified oil chemical analysis. Three analysts conducted the product test independently in each type of exposure water in round-robin fashion. Statistical tests were performed on analyst variability, reproducibility, and repeatability, and the performance of the various products was quantified in both exposure media. Analysis of variance showed that the analyst error at each time-point was highly significant (P values ranged from 0.0001 to 0.008, depending on water type and oil fraction). In the saltwater tests, six products demonstrated various degrees of biodegradative activity against the alkane fraction of the crude oil and three degraded the aromatic hydrocarbons by >10%. In the freshwater tests, eight products caused >20% loss of alkane hydrocarbons, of which five degraded the alkanes by >50%. Only four products were able to degrade polycyclic aromatic hydrocarbons (PAHs) by >20%, one of which caused 88% removal. However, when the variability of the analysts was taken into consideration, only one of the ten products was found to yield significant percent removals of the PAH fraction and only in freshwater. Viable microorganism population analysis (most-probable-number method) was also performed on every sample by each operator to measure the changes in aromatic and alkane hydrocarbon-degrading organism numbers. In general, little evidence of significant growth of either alkane- or PAH-degraders occurred among any of the ten products in either the saltwater or freshwater testing.

Alkanes↗

REP-PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India.

Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was used to characterize 67 field isolates of Pasteurella multocida originating from different animal species and geographical regions of India. REP-PCR was found to be rapid and reproducible (three repeats were done). These isolates yielded different 23 profiles which were clustered into eight groups. The discrimination index was moderate (D value 0.83). Somatic and antigenic typing of the isolates did not reveal any correlation with REP-PCR profiles. There was no host-specific, type-specific, region-specific or pathenogenicity-specific pattern. The REP profiles of isolates obtained from wild animals were similar to those obtained from domestic animals. Two common bands were present in all the isolates irrespective of somatic or antigenic types. The results were not comparable with earlier findings, which had shown high discrimination index and correlation with disease presentation.

Animal Diseases↗

Blood pressure, heart rate, pressure-rate product and electrocardiographic changes in healthy children during treadmill exercise.

Heart rate, systolic and diastolic blood pressure, rate-pressure product and electrocardiographic changes were measured in 288 healthy children during treadmill exercise. A constant speed (3.5 miles/hour) protocol was used to facilitate the measurements of physiologic data during exercise. Because responses to exercise vary with size, the children were separated into four groups according to body surface area. Comparisons were also made between male and female and between black and white children. The values for heart rate and systolic pressure showed excellent reproducibility in repeated tests. These data establish for clinical use reference values for children during treadmill exercise.

Adolescent↗

Leukotriene D4 elicits a non-sustained contraction of the guinea pig trachea in calcium-free buffer.

The contraction of the isolated guinea pig trachea elicited by leukotriene D4 (LTD4) in Ca2+-free buffer (containing 10(-4) M EGTA) achieved a maximum at 6-8 min and relaxed back to baseline approximately 25 min after challenge with LTD4. In contrast, LTD4 elicited a sustained contraction in the presence of 1.8 mM calcium. This sustained contraction in the presence of calcium was reproduced upon repeated LTD4 challenge, whereas in Ca2+-free buffer, only one LTD4-induced contraction could be obtained. The amplitude of the LTD4-induced contraction in Ca2+-free buffer decreased in a time-dependent manner which was also dependent upon the concentration of LTD4. At 10(-7) and 10(-6) M LTD4, small contractions (11% and 20% of control, respectively) were measured after 30 min in Ca2+-free buffer, whereas 10(-8) M LTD4 elicited a contraction at 15 min but not after 30 min in Ca2+-free buffer. Whereas washing the trachea for 5 min with LaCl3 (1.8 mM) only partially suppressed the LTD4-induced contraction in the presence of calcium, the contraction elicited by LTD4 in Ca2+-free buffer was not affected by LaCl3. The LTD4-induced contraction in Ca2+-free buffer was not affected by verapamil (10(-6) M); in contrast, the putative intracellular calcium antagonist, TMB-8 (10(-4) M), blocked the LTD4-induced contraction. These results provide evidence that the release of an intracellular calcium store plays an important role in the initiation of the LTD4-induced contraction of the guinea pig trachea. In addition, these results suggest that an extracellular calcium source may account for a small part of the LTD4-induced contraction.

Airway Resistance↗

Retinal blood flow and its autoregulation measured by intraocular hydrogen clearance.

Retinal blood flow (RBF) was measured with polarographic electrodes at the inner retinal surface using the hydrogen clearance technique. This new intraocular technique allowed repeated and reproducible measurements of retinal blood flow. Usually the clearance curves were monoexponential, indicating that hydrogen clearance in only one compartment, the retina, was being measured. In order to quantitatively describe the autoregulation of retinal blood flow with oxygen tension, the retinal blood flow was measured at levels of arterial blood oxygen tension ranging from 20 to 250 mmHg. The retinal blood flow was found to decrease progressively with increased arterial blood oxygen tension. Linear regression analysis showed a statistical fit (P less than 0.01) between retinal blood flow and arterial blood oxygen tension (PaO2): RBF (ml min-1 100 g-1)-1 = 79.2-0.17 x PaO2 (mmHg)-1. The average blood flow in the cat was found to be 56 +/- 10 ml min-1 100 g-1 (mean +/- 1 S.D., n = 11) at an average arterial blood oxygen tension of 128 mmHg.

Animals↗

Single cell microspectroscopy reveals that erythrocytes containing hemoglobin S retain a 'memory' of previous sickling cycles.

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer-controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a 'memory' of its previous cycle(s), possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.

Anemia, Sickle Cell↗

Multimycotoxin detection and clean-up method for aflatoxins, ochratoxin and zearalenone in animal feed ingredients using high-performance liquid chromatography and gel permeation chromatography.

A sensitive and reliable method is described for the determination of aflatoxins B1, B2, G1 and G2, ochratoxin A and zearalenone in animal feed ingredients. A multi-toxin extraction and clean-up procedure is used, with dichloromethane-1 M hydrochloric acid (10:1) being used for the extraction and gel permeation chromatography being used for the clean-up. The liquid chromatographic method developed for the separation of the six mycotoxins involves gradient elution with a reversed-phase C18 column and fluorescence detection. Recoveries, repeatability and reproducibility have been determined on maize, palm and wheat. The detection limits varied depending on the type of feed.

Aflatoxins↗

Simple and sensitive high-performance liquid chromatography--fluorescence method for the determination of citrinin application to the analysis of fungal cultures and cheese extracts.

A new and highly sensitive method for the detection of the important mycotoxin, citrinin, has been developed. Spectroscopic studies demonstrate that the fluorescence of this metabolite is influenced by the pH of the environment. This fact was exploited in the chromatographic determination of citrinin with fluorescence detection. The proposed method, based on the addition of 1 M hydrochloric acid as an acidic post-column reagent, has a limit of detection of 0.9 center dot 10(-7) M. Analytical validation shows that linearity can be assumed from 2 center dot 10(-7) to 10(-4) M citrinin. The repeatability and reproducibility are satisfactory, with R.S.D. = 5.1% (n = 9, c = 10(-5) M) and R.S.D. = 7.2% (n = 9, c = 10(-5) M). The method was also applied to the determination of this mycotoxin produced by mould cultures isolated from soft cheese and also from soft cheese and also from cheese extracts spiked with citrinin. The specificity of the method is demonstrated and the necessity for post-column acidification is illustrated on real samples.

Cheese↗

Determination of gentiopicroside, mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in the traditional Chinese medicinal preparation sann-joong-kuey-jian-tang by high-performance liquid chromatography.

High-performance liquid chromatography was employed to determine the contents of several marker substances such as gentiopicroside, mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in Sann-Joong-Kuey-Jian-Tang. The separation was performed on a Cosmosil 5C18-AR column by gradient elution with 0.03% (v/v) phosphoric acid-acetonitrile (0 min, 90:10; 10 min, 87:13; 17-27 min, 77:23; 40 min, 62:38; 50 min, 55:45) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 254 nm. n-Propylparaben was used as the internal standard and seven regression equations revealed linear relationships between the peak-area ratios (marker substances/internal standard) and concentrations. The repeatability and reproducibility (relative standard deviation) of the method were in the ranges 0.02-1.78% and 1.44-4.95%, respectively.

Berberine↗

A simple in vitro method of screening panels of monoclonal antibodies for tumor binding.

We have developed a simple in vitro method of evaluating the relative binding properties of anti-tumor antibodies to human tumor and normal tissues. Cryopreserved surgical explants of tissues as 1 mm cubes are incubated in microtiter plate wells containing media and radiolabeled antibody. We show that the accumulation of antibody in tumor tissue is a specific process which may be reduced by preincubation with saturating levels of unlabeled specific antibody. Evaluation of 7 anti-breast and 4 anti-colorectal tumor antibodies against their respective tumor tissues showed good reproducibility of repeat measurements and up to a 100-fold difference in accumulation among different antibodies to the same tissue. Equivalent results were obtained with the same tissues employed fresh and after cryopreservation. Because of the simplicity of the assay, panels of antibodies may be screened against the large numbers of tumor and normal tissues required to identify superior antibodies for human trials.

Acid Phosphatase↗

Preliminary investigations into factors that affect plasma aldosterone concentrations in horses.

The effect of feeding diets with low, adequate and high sodium contents on plasma aldosterone concentrations in horses and ponies was evaluated using human immunoassay kits. The effect of moderate to high intensity exercise of up to six minutes duration on plasma aldosterone concentrations in three thoroughbred horses was also investigated. On an adequate sodium diet plasma aldosterone concentrations increased to a peak around four hours after feeding. Little daily variation was found in the pre-feeding aldosterone concentrations over three days. Feeding additional salt resulted initially in no increase in plasma aldosterone concentrations in three out of four animals. After five days all four animals had lower pre-feeding concentrations, an increase in the magnitude of the response to feeding but a decreased rise in absolute concentration. Feeding a diet with a decreased sodium content for several months did not result in a consistent change in the pre-feeding aldosterone concentrations although there were times when all three animals showed an increase in the magnitude of the aldosterone response to feeding. No correlation between changes in the fractional electrolyte excretion values determined and alterations in aldosterone response was found. Exercise resulted in a marked increase in aldosterone concentrations. The expected biological response to feeding and exercise was demonstrated with an acceptable level of reproducibility and repeatability. Samples had similar values when assayed by either of the kits evaluated.

Aldosterone↗

Mechanisms of autonomic control of carotid chemoreceptor activity.

Single chemoreceptor fibres dissected from the cut carotid sinus nerves of cats were studied when carotid body blood flow was normal, and when it had been abruptly halted by reducing the local perfusion pressure to zero. Ten chemoreceptor fibres which, when normally supplied with blood, increased their discharge by at least 25% in response to sympathetic stimulation, and 7 fibres which, when normally supplied with blood, decreased their discharge by at least 10% in response to carotid sinus nerve stimulation, were chosen for study. The development of discharge during the period of stagnant asphyxia following stoppage of flow was reproducible in repeated control trials for each fibre investigated. Neither sympathetic nor carotid sinus nerve stimulation, commencing at the stoppage of flow and continued throughout the period of asphyxia, produced any significant alteration from the control pattern of developing chemoreceptor discharge. These experiments provide evidence that the effects of sympathetic and carotid sinus nerve stimulation on carotid chemoreceptor discharges are mediated through alterations in carotid body blood flow. When there is no flow there are no effects on discharge.

Animals↗

Frequency and volume thresholds for inhibition of inspiratory motor output during mechanical ventilation.

We quantified volume and frequency thresholds necessary for the inhibition of respiratory motor output during prolonged normocapnic mechanical ventilation in healthy subjects during wakefulness (n = 7) and NREM sleep (n = 5). Subjects were ventilated at eupneic frequency (fR) with 3 min step-wise increases in tidal volume (VT), or at eupneic VT with step-wise increases in fR, or by combinations of these two parameters. Inhibition of respiratory motor output was determined using mask pressure and, when available, esophageal pressure and diaphragmatic EMG. During wakefulness, the volume threshold (at eupneic fR) averaged 969 +/- 94 ml or 1.3-1.4 times the average eupneic tidal volume; the frequency threshold (at eupneic VT was 14.1 +/- 0.7 min-1 or 1.2 times the average eupneic frequency. The volume threshold was reduced when MV was provided at an fR above the eupneic value, and the frequency threshold was decreased when MV was provided at a VT above the eupneic level. During NREM sleep (n = 5) the volume threshold for inhibition was 835 +/- 108 ml or 1.4-1.5 times eupneic VT. The inhibitory thresholds for VT and fR were reproducible upon repeat trials within subjects. We conclude that inhibition of respiratory motor output during prolonged normocapnic mechanical ventilation in wakefulness or NREM sleep is highly sensitive to changes in ventilator VT, fR and their combination.

Adult↗

The measurement of radiocaesium in sheep by an in vivo technique.

This paper discusses the use of a large sodium iodide detector to determine gamma-ray emitting radionuclides in living animals, and in particular the application of the technique to investigations that have followed the Chernobyl reactor accident. A series of experiments to validate the technique is presented. The detector and its associated electronics and data collection equipment are sufficiently robust for use in the field, and ancillary equipment to immobilize subjects such as sheep and cattle are readily available. Although the in vivo procedure underestimates activity concentrations in muscle tissue compared to results from samples obtained post mortem, the advantage is that the same animal can be measured repeatedly and reproducibly.

Animals↗

Filter bleeding time: a new in vitro test of hemostasis. I. Evaluation in normal and thrombocytopenic subjects.

Disadvantages of current bleeding time methods include poor reproducibility, limited repeatability, and at times scar formation. We report a new, in vitro technique which circumvents these problems. Venous blood is collected in sodium citrate, stored capped in a siliconized tube at 37 degrees C until use, and made to flow under constant pressure through a filter of Woven Dacron. Flow rate progressively falls as platelet aggregates occlude the filter, as verified by scanning electron microscopy and 111Indium-labeled platelet radioactivity of the filter. Filter bleeding time (FBT) was 4.33 +/- 1.79 (means x +/- SD) min in 23 healthy human volunteers and 3.08 +/- 1.08 min in 14 normal dogs. Bleeding volume (BV) (number of drops) was 29 +/- 12 in the humans and 16 +/- 6 in the dogs. Initial bleeding rate (IBR) (number of drops during first minute) was 14 +/- 3 in citrated blood and 25 +/- 5 drops/min in EDTA blood of the humans (P = 9 X 10(-10)), 8 +/- 2 in citrated blood and 14 +/- 6 drops/min in EDTA blood of the dogs (P = .0004). Percent platelet reduction during passage of blood through the filter (PCR) was 27.5 +/- 7.2 in citrated blood and 4.9 +/- 2.6 in EDTA blood of the humans (P = 2 X 10(-12)), while in dogs it was 25.7 +/- 8.3 in citrated blood and 5.3 +/- 4.2 in EDTA blood (P = 8 X 10(-7)). The results of these parameters using human platelet rich plasmas (PRPs) were similar to those with human whole blood, but significant decrease of drop rate was not observed in canine PRPs during passage of PRP through the filter. FBT, BV, and IBR were correlated (r = -0.91, -0.84 and -0.62 respectively) with platelet count in 12 specimens obtained from 4 dogs in which thrombocytopenia (107 to 4 X 10(3) platelets/microliter) was induced by estradiol. This new test is unaffected by specimen transport by pneumatic tube, is sensitive to platelets, and should be useful for analysis of both quantitative and qualitative platelet abnormalities in man.

Animals↗

Immunomodulatory properties of cimetidine in ARC patients.

The immunomodulatory potency of cimetidine, a histamine H2 receptor antagonist, was investigated in 33 AIDS-related complex (ARC) patients performing detailed immunological and clinical evaluations. Cimetidine was administered orally in daily doses of 1200 mg for a period of 5 months with an interruption of therapy after the first 3 months for an interval of 3 weeks. Significant (P less than 0.05) elevations of immunoglobulins (IgG, IgA), complement C4, B-lymphocytes, and OKT4+ (helper/inducer) cells were found after cimetidine intake. The in vitro lymphocyte proliferative response to plant mitogens was significantly increased, and the in vivo cell-mediated hypersensitivity reaction assessed by intradermal application of seven recall antigens improved significantly. These effects were both reversible with the discontinuation of cimetidine and reproducible with repeated administration of the drug. Clinical data such as performance status, body weight, and fever were influenced favorably (P less than 0.05) by cimetidine. The frequency of diarrhea and the lymph node size were also diminished significantly. The data suggest that cimetidine may at least partially restore immunofunctions in AIDS-related complex.

AIDS-Related Complex↗

An adapter for computed tomography-guided stereotaxis.

A new, versatile adapter for computed tomography-guided stereotaxis is presented. The instrument consists of a light aluminum frame, which by means of a nasion support and two ear plugs is fixed to the patient's head. Reproducibility of repeated fixations is very high. The adapter is fitted to most stereotactic frames and can be used in all adult patients. Comparison between computed tomography- and ventriculography-guided determinations of thalamic targets showed a mean difference of 0.6 and 0.7 mm for the x and y coordinates, respectively. The z coordinates seldom showed any measurable difference. We now perform all types of stereotactic neurosurgery (tumor biopsy, implantation of depth electrodes, thalamotomy, dentatotomy, cingulotomy, etc.) with computed tomography guidance only. The short-term results in a small number of patients have been at least as good as after conventional ventriculography-guided surgery. In addition to open stereotactic surgery, the adapter is also suitable for external stereotactic irradiation of intracranial targets with a linear accelerator.

Brain↗

A microassay of O2 concentrations based on oxygen-induced chemiluminescence in anaerobic lipoxygenase reactions.

The preparation of chemiluminescence probes for assaying O2 concentrations in microsamples is described. This probe is based on an anaerobic lipoxygenase-linoleate system continuously generating reactive intermediates which in a spontaneous reaction with added O2 yield an excited species. The resulting chemiluminescence signals are highly reproducible upon repeated sample application and unaffected by even large variations in the contents of lipoxygenase-1 and linoleic acid. The linear assay range is between 0.25 and 25 nmol of O2. The assay system described is stable for 90 +/- 10 min, irrespective of the number of samples added, and the probe can be regenerated thereafter by adding linoleic acid.

Anaerobiosis↗