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Tuberculosis in Kenya: follow-up of the second (1974) national sampling survey and a comparison with the follow-up data from the first (1964) national sampling survey. An East African and British Medical Research Council co-operative investigation.

A total of 1,490 newly diagnosed patients admitted to a national sampling survey of tuberculosis conducted in 11 of 30 districts in Kenya in 1974 have been followed up after a year under the routine tuberculosis treatment services. The results are reported and compared with those observed in the follow-up of a similar survey in the same districts 10 years previously. The main follow-up concerns 1,351 patients with pulmonary tuberculosis of whom 803 had a positive culture at the start of treatment, 283 a negative culture, and 265 had no specimen examined. At 1 year or later, 57% of the 1,351 patients were culture negative, 8% were culture positive, 16% had died in the year of follow-up, 5% were permanently lost from observation and 14%, although known to be alive, had no bacteriological results. Of the 803 patients with a positive culture initially, 11% had a positive culture at 1 year or later, as had 3% of the 283 with a negative culture initially. The policies of therapy practised in the routine treatment services were studied in 1,207 patients; 95% were admitted to hospital at the start of chemotherapy for an average duration of 39 days, 95% were prescribed isoniazid and thiacetazone supplemented initially by a course of streptomycin, the average duration of which was 36 days. There was little variation in the policies applied in different district. Only 24% of the patients received or attended to collect a 12 months' supply of chemotherapy and 72% a 6 months' supply. The therapeutic results achieved in 1974 compare favourably with those observed in the 1964 survey. The estimated culture negativity rate at 1 year or later for the patients who had bacteriologically confirmed disease initially was 63% in the earlier survey and 71% in the 1974 survey. The proportion of patients lost from observation was 9% in 1964 and 5% in 1974. The policies of therapy were much more standardised in the second survey. In 1964 hospital admission, its duration and the usage of streptomycin varied considerably from district to district, but in the second survey nearly all of the patients were admitted to hospital and received the nationally recommended regimen of isoniazid and thiacetazone supplemented initially by streptomycin. Even so, the duration of chemotherapy received did not improve, only 28% in 1964 and 24% in 1974 receiving a 12 months' and 74% in 1964 and 72% in 1974 receiving a 6 months' supply. The transfer of patients from one district to another was more often successful in 1974 than 1964.

Adolescent↗

A simple and inexpensive sample-handling method for the semi-preparative RP-HPLC of polypeptides and non-polar peptide derivatives: pre-adsorption of samples.

A simple method is described for the application onto HPLC columns of very crude or alternatively poorly soluble polypeptide samples prior to their chromatographic purification. The procedure involves the batch pre-adsorption of the crude polypeptide mixture from a dilute solution onto an appropriate preparative-grade chromatographic adsorbent, removal of the solvent by rotary evaporation or lyophilisation and then dry-packing the pre-adsorbed chromatographic material into guard column cartridges of suitable dimensions. The polypeptide products can then be eluted either by isocratic or gradient elution methods through the cartridge coupled in tandem with prepacked semi-preparative HPLC columns. This method has been successfully utilised for the routine RP-HPLC purification of polar and hydrophobic polypeptides prepared by solid phase peptide synthesis (SPPS) methods as well as peptide derivatives and intermediates used as part of SPPS procedures.

Adsorption↗

Fully automated high-performance liquid chromatographic analysis of whole blood and plasma samples using on-line dialysis as sample preparation. Determination of oxytetracycline in bovine and salmon whole blood and plasma.

A fully automated technique for high-performance liquid chromatographic analysis of whole blood and plasma is described. Samples are automatically injected into a dialyser where proteins and blood cells are removed. The dialysates are concentrated on a small column prior to analysis. This technique is used for the determination of oxytetracycline in whole blood and plasma. After dialysis oxytetracycline and the internal standard, tetracycline, are retained on a polystyrene enrichment column and subsequently separated on a polystyrene analytical column by ion-pair chromatography. Using ultraviolet detection 50 ng/ml can be detected. Validation showed good within-day and between-day accuracy and precision. Different oxytetracycline concentrations were found in plasma and whole blood. This difference varied between the species.

Animals↗

Effect of sample handling on analysis of cytokine responses to Mycobacterium tuberculosis in clinical samples using ELISA, ELISPOT and quantitative PCR.

Measuring cytokine responses to infection has proven to be invaluable for the understanding of immunity to tuberculosis in the laboratory. However, far less data are available from studies in humans and these have often produced conflicting results. Here we describe a comprehensive multi-center comparison of the most commonly used protocols for cytokine analysis: ELISA, ELISPOT and RT-PCR, in cohorts of TB patients, their household contacts and community controls. In particular, we have studied the effect on these protocols of conditions that commonly prevail in field studies, such as delays between sample collection and analysis, or different source material, such as whole blood or frozen PBMC. The results clearly show that while there is good correlation between the methods under optimal conditions, each method has strengths and weaknesses that render them more or less suitable for particular types of analyses. Researchers should carefully consider these factors when planning human field studies.

Adolescent↗

A new type of sample tube for reducing convection effects in PGSE-NMR measurements of self-diffusion coefficients of liquid samples.

Pulsed gradient spin-echo (PGSE) NMR measurements of the self-diffusion coefficients of low viscosity liquids are greatly hampered by the effects of convection especially away from ambient temperature. Here we report on a new NMR tube designed to minimize the deleterious effects of convection. In this tube, which derives from a Shigemi symmetrical NMR tube, the sample is contained in an annulus formed from a concentric cylinder of susceptibility matched glass. The performance of this tube was demonstrated by conducting measurements on the electrochemically important LiN(SO3CF3)2 (LiTFSI)-diglyme (DG) system. Calibrations were first made using DG at column heights of 2, 3, and 4-mm in the temperature range between -40 and 100 degrees C. Measurements of the diffusion coefficients of the lithium, anion, and DG were then performed to probe the solvent-ion and ion-ion interactions in the DG doped with LiTFSI. Changes in the 1H, 7Li, and 19F PGSE-NMR attenuation curves at -40 degrees C provided clear evidence of interactions between the DG and lithium ion.

Convection↗

The effects of oil exploration and production in the Fladen Ground: composition and concentration of hydrocarbons in sediment samples collected during 2001 and their comparison with sediment samples collected in 1989.

Due to the potentially accumulative nature of the Fladen Ground, an area of intense oil activity in the North Sea, a survey was carried out in 1989 to map the distribution of contamination in relation to these oil activities. All the sediments collected were screened by ultraviolet fluorescence (UVF) for polycyclic aromatic hydrocarbons (PAHs) and selected samples were analysed for n-alkanes (by GC-FID), PAHs and biomarkers (by GC-MSD). This survey was repeated in 2001, with all the 1989 sites being resampled. All of these sediments were analysed for UVF oil equivalents, PAHs, n-alkanes and biomarkers. The concentrations of these parameters decreased between 1989 and 2001, with average decreases ranging from 43% to 88%. In addition, no significant difference was found, for all the parameters, between near field (<5 km from an oil installation) and far field (>5 km from an oil installation) sites in 2001 indicating that the Fladen Ground is approaching a 'steady state' or background concentration for contamination.

Carbon↗

Causes and effects of sample loss using a dry capsule injection method for the automated gas chromatographic analysis of pesticide samples.

The automatic capsule sampling system is said to have many advantages for accurate gas chromatographic analysis. One is that the solvent may be removed by evaporation before injection, so that no solvent peak is obtained to interfere with the separation. However, working with aldrin and dieldrin, we have shown that up to 50% of the material can be lost from the capsules by air-drying at normal ambient temperature before injection. These losses occurred only with aldrin and it is suggested that they are connected with its relatively high vapour pressure. Significant amounts of material enter the top third of the capsule which is removed during the sealing process. The losses appear to be due to a combination of adsorption to the capsules, creep-up the capsule walls, co-distillation with the solvent, and direct volatilisation. Silylation of the capsules was quite effective in reducing these losses.

Absorption↗

Field-based random sampling without a sampling frame: control selection for a case-control study in rural Africa.

Selection bias, particularly of controls, is common in case-control studies and may materially affect the results. Methods of control selection should be tailored both for the risk factors and disease under investigation and for the population being studied. We present here a control selection method devised for a case-control study of tuberculosis in rural Africa (Karonga, northern Malawi) that selects an age/sex frequency-matched random sample of the population, with a geographical distribution in proportion to the population density. We also present an audit of the selection process, and discuss the potential of this method in other settings.

Bias↗

Liquid chromatography coupled with multi-channel electrochemical detection for the determination of daidzin in rat blood sampled by an automated blood sampling system.

Daidzin, a soy-derived biologically active natural product, has been reported to inhibit mitochondrial aldehyde dehydrogenase and suppress ethanol intake. This paper describes a method for the determination of daidzin in rat blood. After administration of daidzin, blood samples were periodically collected from awake, freely moving animals by a Culex automated blood sampler. Daidzin was extracted from 50 microl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 12 min using a microbore C(18) (100 x 1.0 mm) 3 microm column with a mobile phase containing 20 mM sodium acetate, 0.25 mM EDTA, pH 4.3, 4% methanol and 11% acetonitrile at a flow-rate of 90 microl/min. Detection was attained using a four-channel electrochemical detector with glassy carbon electrodes using oxidation potentials of +1100, 950, 850, 750 mV vs. Ag/AgCl. The limit of detection for daidzin in rat plasma was 5 ng/ml at a signal-to-noise ratio of 3:1. The extraction recovery of daidzin from rat plasma was over 74%. Linearity was obtained for the range of 25-1000 ng/ml. The intra- and inter-assay precisions were in the ranges of 2.7-6.6 and 1.9-3.7%, respectively. This method is suitable to routine in vivo monitoring of daidzin in rat plasma.

Animals↗

Characterization of interlayer Cs+ in clay samples using secondary ion mass spectrometry with laser sample modification.

Ultraviolet laser irradiation was used to greatly enhance the secondary ion mass spectrometry (SIMS) detection of Cs(+) adsorbed to soil consisting of clay and quartz. Imaging SIMS showed that the enhancement of the Cs(+) signal was spatially heterogeneous: the intensity of the Cs(+) peak was increased by factors up to 100 for some particles but not at all for others. Analysis of standard clay samples exposed to Cs(+) showed a variable response to laser irradiation depending on the type of clay analyzed. The Cs(+) abundance was significantly enhanced when Cs(+)-exposed montmorillonite was irradiated and then analyzed using SIMS, which contrasted with the behavior of Cs(+)-exposed kaolinite, which displayed no Cs(+) enhancement. Exposed illitic clays displayed modest enhancement of Cs(+) upon laser irradiation, intermediate between that of kaolinite and montmorillonite. The results for Cs(+) were rationalized in terms of adsorption to interlayer sites within the montmorillonite, which is an expandable phyllosilicate. In these locations, Cs(+) was not initially detectable using SIMS. Upon irradiation, Cs(+) was thermally redistributed, which enabled detection using SIMS. Since neither the illite nor the kaolinite is an expandable clay, adsorption to inner-layer sites does not occur, and either modest or no laser enhancement of the Cs(+) signal is observed. Laser irradiation also produced unexpected enhancement of Ti(+) from illite and kaolinite clays that contained small quantities of Ti, which indicates the presence of microscopic titanium oxide phases in the clay materials.

Journal Article↗

A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples.

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.

DNA↗

Bilayer sample for fast or slow magic angle oriented sample spinning solid-state NMR spectroscopy.

An alternative setup for Magic Angle Oriented Spinning Spectroscopy is proposed. Samples were prepared by orienting lipid bilayers onto polymer films, which were wrapped into a spiral so as to fit into 4 or 7 mm MAS rotors. This geometry resulted in narrow line widths and a higher upper spinning limit when compared to the conventional MAOSS setup with stacked glass plates. Whereas orientational information was extracted from low spinning spectra, fast spinning will be applicable to high-resolution multidimensional NMR pulse sequences.

Lipid Bilayers↗

Photographic sampling: a photographic sampling method for mites on plants.

A photographic sampling method for mites on plants was evaluated using Tetranychus urticae and Phytoseiulus persimilis on pepper plants. It was found to be 92% accurate for T. urticae eggs and 98% accurate for P. persimilis eggs at densities up to 45 eggs per cm2 for T. urticae, and up to 3 eggs per cm2 for P. persimilis. The motiles of the two species were not confused, nor were they confused with exuviae or other matter.

Animals↗

Comparison of methods for sample preparation of individual rat cerebrospinal fluid samples prior to two-dimensional polyacrylamide gel electrophoresis.

Different pre-treatment methods have been compared for two-dimensional mapping of individual rat cerebrospinal fluid samples based on acetone, trichloroacetic acid/acetone and methanol/acetone precipitation of proteins. Acetone precipitation following incubation with DTT gave the highest protein recovery (72%) and the largest number of protein spots (92 +/- 4) as well as minimizing the time taken.

Acetone↗

Microdialysis sampling of carbamazepine, phenytoin and phenobarbital in subcutaneous extracellular fluid and subdural cerebrospinal fluid in humans: an in vitro and in vivo study of adsorption to the sampling device.

The purpose of the study was to determine if binding of the drugs to the sampling equipment during microdialysis would influence the results for carbamazepine, phenytoin and phenobarbital. In vitro experiments with microdialysis catheters and separate parts of catheters were performed to estimate the degree of drug binding to the dialysis equipment. A mathematical model to calculate drug binding and recovery is proposed. In vivo protein unbound carbamazepine concentrations in subcutaneous extracellular fluid at different flow rates (6 patients), unbound carbamazepine (1 patient) and unbound phenobarbital (I patient) in subdural cerebrospinal fluid and subcutaneous extracellular fluid were estimated and the in vivo data were compared to the in vitro results and data generated by the mathematical model. Binding to the soft outlet polyurethane tubing was extensive and variable for phenytoin, which precluded in vivo testing, but limited and more predictable for carbamazepine and phenobarbital. None of the three compounds bound to the hard internaltubing. Phenytoin and phenobarbital did not bind to the dialysis membrane, while a small degree of binding may be present for carbamazepine. In vivo estimates of carbamazepine protein unbound subcutaneous extracellular concentrations by microdialysis, adjusted for binding to the plastic tubing, were 81% of protein unbound plasma concentrations. In single case studies, subdural cerebrospinal fluid and subcutaneous extracellular levels of carbamazepine and phenobarbital were similar and when corrected for binding to the plastic tubings they were also close to protein unbound plasma concentrations. Microdialysis can be used for reliable estimations of protein unbound carbamazepine and possibly phenobarbital concentrations when drug binding to the plastic tubing is considered. Reliable estimation of unbound phenytoin is not possible at present.

Adult↗

Application of recent advances in aerosol sampling science towards the development of improved sampling devices: the way ahead.

This paper reviews the framework that underpins the development of a new generation of personal samplers capable of operating at much lower flowrates that those of the current generation and so capable of being used for exposure assessment not only for 'traditional' occupational populations (i.e., industrial workers) but also for people exposed to aerosols in the ambient atmosphere (including children). The opportunity for this new generation of samplers stems from the availability of very light and compact low-flowrate pumps. The development and deployment of such instruments presents: (a) physical challenges in terms of how to collect particle size fractions in a manner which is consistent with the new particle size-selective sampling criteria, and (b) analytical challenges in terms of how to quantitate the much smaller amounts of collected material that need to be analysed. The paper lays out the physical and analytical scenarios, and points the way forward to how such challenges can be overcome. Work is already in progress in several countries to develop prototype instruments for applications like those described.

Adult↗

No delayed temporal response to sample concentration changes during enhanced microdialysis sampling using cyclodextrins and antibody-immobilized microspheres.

The temporal response to concentration changes external to a microdialysis probe containing trapping agents in the perfusion fluid was studied. Native beta-cyclodextrin and a water-soluble beta-cyclodextrin polymer were used as trapping agents in the microdialysis perfusion fluid to study the temporal concentration response to carbamazepine, a hydrophobic analyte. The temporal response of microdialysis probes containing antibody-immobilized microspheres against five different cytokines (tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5) to concentration changes outside of the probe was also determined. In both cases, no delayed temporal response of enhanced microdialysis was observed for either carbamazepine or the cytokines as compared to standard microdialysis sampling procedures.

Animals↗