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Application of the small-angle cleavage technique to thickness measurement of TEM samples.

In modelling transmission electron microscopy (TEM) images with the dynamical theory of electron diffraction, the sample thickness at the region of interest must be accurately known. A technique of sample preparation for cross-sectional single-crystal samples has been developed to provide this information. This technique, based on the small-angle cleavage technique, can allow a direct measurement of the sample thickness at the region where cross-sectional TEM analysis has been undertaken. Several cross-sectional samples of GaAs-based multiple quantum wells were prepared with parallel cleaved faces and thicknesses in the range of 50 to 200 nm. The samples were initially mounted so that cross-sectional images were obtained, then on suitable samples the mount was adjusted to obtain a plan view image of the same area. With suitable care, this technique provided an accurate thickness measurement of a cross-sectional region of a sample, which allowed subsequent modelling and analysis.

Microscopy, Electron↗

c-erbB-2 over-expression in amplified and non-amplified breast carcinoma samples.

We investigated c-erbB-2 oncogene amplification and over-expression in 79 invasive breast carcinoma samples using fluorescence in situ hybridization (FISH) and immunohistochemistry, with the aim of studying relationships between neoplasms over-expressing c-erbB-2 with or without amplification and bio-pathological parameters used in clinical breast cancer. Nineteen samples showed amplification, and all of these were positive by immuno-histochemistry. Moderate or intense immunostaining was present in a further 22 samples without c-erbB-2 amplification and was not related to any increased number of c-erbB-2 signals: 15 samples exhibited chromosome 17 polysomy, 3 monosomy and 4 no FISH abnormalities. Thirty-eight samples were immunonegative: 18 exhibited chromosome 17 polysomy, 9 monosomy and 11 no alterations. Samples having c-erbB-2 over-expression associated with amplification showed DNA aneuploidy and hormonal receptor loss to a greater extent than those expressing c-erbB-2 without amplification or immunonegative samples (chi2 test, p = 0.007, 0.008 and 0.008, respectively). The proliferation rate, detected by Ag-NOR staining, was highest in amplified samples (Kruskal Wallis test, p = 0.009). Our results indicate that tumours showing both c-erbB-2 over-expression and amplification exhibit more aggressive biological characteristics than those with only over-expression or immunonegative tumours. Since both c-erbB-2 amplification and over-expression have been related to negative responses to chemotherapy and poor prognosis, these differences might have clinical implications. The combination of FISH and immuno-histochemistry may be helpful to achieve this aim.

Breast Neoplasms↗

Chiral assay of atenolol present in microdialysis and plasma samples of rats using chiral CBH as stationary phase.

Two different enantioselective chiral chromatographic methods were developed and validated to investigate the disposition of the beta 1-receptor antagonist atenolol in blood and in brain extracellular fluid of rats (tissue dialysates). System A for the plasma samples was a one-column chromatographic system with a chiral CBH column with an aqueous buffer as mobile phase into which cellobiose was added for selective regulation of the retention of the internal standard, (S)-metoprolol. The plasma samples were analysed after a simple extraction procedure. The limit of quantitation was 0.2 micrograms/ml for the atenolol enantiomers. The repeatability of the medium concentration quality control plasma sample (6.0 micrograms rac-atenolol/ml) was 11-18% for the enantiomers. The dynamic linear range of the plasma samples was 0.5-20 micrograms/ml. For system B, since atenolol is an extremely hydrophilic drug, the tissue dialysate sample required a much more sensitive system as compared to the plasma samples. A coupled column system was used for peak compression of the enantiomers in the eluate after the separation on the Chiral CBH column, hence increasing the detection sensitivity. The limit of quantification was 0.045 micrograms/ml for the atenolol enantiomers in artificial CSF. The repeatability of the medium concentration quality control samples (0.1 and 4.0 micrograms rac-atenolol/ml in artificial CSF and Hepes Ringer, respectively) was 2.8-9.3% for the two enantiomers. The dynamic linear range of the brain samples was 0.05-1.0 and 0.5-20 micrograms/ml in artificial CSF and Hepes Ringer, respectively.

Adrenergic beta-Antagonists↗

Detection of bacterial DNA in serial synovial samples obtained during antibiotic treatment from patients with septic arthritis.

OBJECTIVE: The management of septic arthritis could benefit from sensitive tests that detect the persistence of microorganisms in the joint. The aim of this study was to determine the feasibility of monitoring the presence of bacterial DNA in synovial samples from septic arthritis patients during antibiotic treatment. METHODS: Synovial fluid (SF) and synovial tissue (ST) samples were collected serially from 6 patients with septic arthritis before and during antibiotic therapy. In addition, peripheral blood (PB) samples were available for polymerase chain reaction (PCR) analysis from 5 of the 6 patients before treatment. All samples were analyzed for the presence of bacterial DNA with the use of a PCR with universal 16S ribosomal RNA primers. Automated sequencing and comparative data analysis were performed to identify the species. These data were compared with Gram staining and culture results. RESULTS: The bacterial species cultured from the synovium could be identified in all 6 patients using PCR and subsequent sequence analysis of the amplicons. In virtually all cases, positive Gram stain and culture findings in the synovial samples became negative after 2-3 days of antibiotic treatment. Bacterial DNA persisted in the SF and/or ST after culture conversion; in 2 patients, bacterial DNA was still detected at day 10, in 1 patient, at day 20, and in another patient, at day 22 after the initiation of treatment. Synovial samples were available for PCR analysis from 2 patients at day 26. At this time point, bacterial DNA could not be detected anymore. All PB samples were negative by both culture and PCR analysis. CONCLUSION: PCR analysis can be used to monitor the presence of bacterial DNA in synovial samples from patients with septic arthritis during antibiotic treatment. The absence of bacterial DNA could help in the decision to discontinue antibiotic treatment.

Adult↗

Nutritional status by socioeconomic level in an urban sample from Bilbao (Basque Country).

Anthropometry, records of food intake, and bioelectric impedance were gathered from a sample of 297 adults of both sexes who lived in marginal districts in the town of Bilbao and benefited from a program of social assistance. This survey aimed to determine present nutritional status and to ascertain the relationship between food intake and nutritional conditions. A comparative evaluation of anthropometry and food intake was then carried out using a reference from the Basque Country and a control sample from the same area but from better-off nutritional and socioeconomic conditions. Males from low socioeconomic status (SES) displayed lower heights and weights compared to the local reference and control sample. They showed higher skinfold thickness than males in the local reference but lower thicknesses than those in the control sample. All anthropometric variables, except height, were higher in women of low socioeconomic status compared to the local reference and control sample. Low socioeconomic males had lower estimated percentage of body fat than the control sample, while females showed the opposite pattern. On the other hand, estimates of food intake in males did not reveal great differences among samples from different socioeconomic backgrounds, while low SES females had greater intakes of food than the better-off control sample. Estimated zinc intake showed differences by SES in both sexes.

Adult↗

Haplotype association study between DRD1 gene and bipolar type I affective disorder in two samples from Canada and Sardinia.

Based on the dopaminergic hypothesis, the dopamine D(1) receptor gene (DRD1) is considered to be a good candidate gene involved in the susceptibility of bipolar disorder (BP). Genetic association between three DRD1 single nucleotide polymorphisms (SNPs) (-800T/C, -48A/G, and 1403T/C) and bipolar type I (BP I) disorder was performed in a case-control sample of Sardinian origin (170 BP I and 209 controls) and in an enlarged sample (229 families) of BP I trios from Toronto. The haplotype analyses generated significant global chi-square in both samples (P-value 0.024 in Toronto and 0.00042 in Sardinian). The main representative haplotypes in both samples were the -800T/-48A/1403C and the -800C/-48G/1403T. Considering each group individually, the -800C/-48G/1403T was transmitted more frequently from parents to BP I probands in Toronto sample (nominally P-value = 0.047) and was more frequent in cases than in control subjects in Sardinian sample although showing no significant evidence of association (nominally P-value = 0.16) When the estimated haplotype counts of both samples were combined, the global chi(2) was significant (P-value = 0.00085) and the nominal P-value for the haplotype -800C/-48G/1403T was 0.01. The fact that the same haplotype shows a similar trend for association in samples originating from different ethnic backgrounds seems to imply that the -800C/-48G/1403T haplotype may be considered as a risk factor for BP I disorder.

Adult↗

Analysis of the Ft. Laurens, Ohio, skeletal sample.

This report describes the analysis of the Colonial Period (1779) skeletal sample (N = 21) from Ft. Laurens, Ohio. The determination of age, sex, and ancestry reveals a relatively young sample (mean = 23.5 years) of males of northern European ancestry. Morphometric analysis shows little difference in cranial and postcranial size and shape measures between this sample and a modern sample of Euro-Americans and previously described Colonial to Civil War samples. The analysis of skeletal pathologies indicates that the individuals in the Ft. Laurens sample were active and subject to at least moderate amounts of stress. Dental pathologies were frequent, with a caries incidence higher than would be expected for a Euro-American Colonial period sample. Numerous traumatic pathologies, in the form of cut and hack marks, are present in this sample. These marks were expected, since historical records indicate a violent death and scalping for virtually all individuals lost at Ft. Laurens. The results of the various analyses are interpreted in light of available historical information. In most cases the results are consistent with historically based expectations.

Adolescent↗

Millimeter wave power density in aqueous biological samples.

Power density distribution inside a water sample placed between two parallel lossy dielectric plates (Polystyrene) was calculated using Fresnel equations for the frequency range of 42.25-53.57 GHz. Due to the multiple internal reflections from the sample boundaries, the distribution of the power density within the thin sample is more uniform than that within a semi-infinite medium. The power density in a sample depends on the thicknesses of the sample and the adjacent dielectric plates. For the given frequency range the sample thickness optimal for power density uniformity varies between 0.28 and 0.33 mm. The front plate has a significant effect on the magnitude of the power density within the sample but little effect on the power density distribution. The thicker the rear plate, the greater is the non uniformity of the power density distribution within the sample. Based on the calculated data, we determined the dimension of an exposure chamber providing the optimal power density distribution uniformity for mm-wave irradiation.

Air↗

Sample size in fine needle aspiration biopsy.

The weights of biopsy specimens from bovine liver taken for fine needle aspiration cytology (FNAC) by three groups of physicians, were compared. The groups differed from each other in their extent or degree of experience in FNAC. When the physicians used their normal technique, the mean sample weight increased significantly with experience, from 4.6 mg in the least experienced group to 17.2 mg in the group with the longest experience. Thirty-six per cent of the samples taken by the group with the least experience were in the weight class 0-2 mg, whereas no such samples were encountered for the other groups. When all the physicians were advised to use the same, standard technique there were no statistically significant differences in mean sample weight between the groups or in the proportion of samples in class 0-2 mg. Sample weights correlated significantly (P less than 0.001) with cell counts. This study shows that differences in the technique of FNAC are responsible for differences in sample weights and cell counts, including the number of the acellular samples obtained. This could explain in some part the wide variation in the reported accuracy rates of FNAC.

Animals↗

Cytodiagnosis of nipple discharge: a study of 602 samples from 484 cases.

To find out the gross and microscopic differentiating features between nipple discharges (ND) due to various breast lesions, smears of 602 ND samples from 484 cases were reviewed by one of the investigators (D.K.D.). The reviewed cytodiagnoses were as follows: benign nipple discharge (59.1%), inflammatory ND (6.5%), ?papillary lesions (2.5%), papillary lesions (20.6%), papillary lesions with atypia (3.8%), duct cells with atypia (0.2%), suspicious for malignancy (0.5%), malignant ND (1.2%), and inadequate (5.6%). Following review, samples with epithelial abnormalities (?papillary lesion, papillary lesion with and without atypia, duct cells with atypia, suspicious for malignancy, and malignancy) increased from 16.6% to 30.4% of adequate samples (P < 0.0001). 37.9% unilateral ND samples showed epithelial abnormalities, as opposed to 18.9% of bilateral ND samples (P < 0.0001). Bloodstained ND showed epithelial abnormalities in 41.5% samples, as compared to 22.1% of ND with other specified gross characteristics (P < 0.0001). The samples with epithelial abnormalities differed significantly from benign and inflammatory ND in respect of frequency of benign duct cells, duct cells with atypia, papillary clusters with or without atypia, malignant cells, columnar cells, red blood cells, inflammatory cells, and background lipid vacuoles (P < 0.01 to < 0.0001). The ND samples with suspicious and malignant cytology, besides the presence of malignant cells (P < 0.0001), differed significantly from rest of the lesions in respect of foam cells (P < 0.0001), red blood cells (P < 0.01), and inflammatory cells (P < 0.05). When compared with histopathological diagnosis in 20 cases, the benign or malignant nature of the lesion was correctly identified in ND in 80% cases. The ND cytologies in 7 histologically proved malignant cases were malignancy (3 cases), suspicious for malignancy (1 case), papillary lesion with atypia (1 case), papillary lesion (1 case), and benign ND (1 case).

Breast Diseases↗

Dual stacking of unbuffered saline samples, transient isotachophoresis plus induced pH junction focusing.

A dual stacking mechanism based on transient isotachophoresis (TITP) and induced pH junction focusing is demonstrated as a means to increase the concentration sensitivity in capillary electrophoresis of highly saline samples. When stacking was carried out with an unbuffered saline sample of fluorescein between two zones of low mobility background electrolyte at high pH under an electric field of reverse polarity, two transient peaks at both boundaries of the sample zone were observed. One peak at the rear boundary could be inferred as a transient isotachophoretic stacked zone. Through computer simulations of an unbuffered sample with a high concentration of sodium chloride, we showed that the fast moving zones of sodium and chloride ions induced pH changes at both boundaries to satisfy the electroneutrality condition and that the peak at the front boundary was due to the induced pH junction. To verify the pH changes, an indicator, thymol blue, was added to an NaCl solution and the color changes under an electric field were observed. The proposed mechanism was supported by observing the dual stacking procedure for an unbuffered sample of 4-nitrophenol and measuring additional sensitivity enhancements by dual stacking for ten weakly acidic compounds. For the ten analytes including nucleoside phosphates, every dual stacking of an unbuffered sample exhibited an additional enhancement up to 86% larger than that of usual transient isotachophoresis of the corresponding buffered sample without loss of separation efficiency and reproducibility. Therefore, it would be useful to skip over buffering in sample preparation for TITP, contrary to the general recommendation.

Electrophoresis↗

Strategies for the determination of cefazolin in plasma and microdialysis samples by short-end capillary zone electrophoresis.

Capillary zone electrophoresis was employed to determine cefazolin, a first-generation cephalosporin antibiotic, in plasma and microdialysis samples from patients. To shorten the analysis, the samples were injected from the short end of the capillary, resulting in a separation time of < 3 min. Due to a high ionic strength of the biological matrices it was necessary to optimize the stacking conditions. For microdialysis samples a 1:10 dilution with water before injection was sufficient to obtain good peak shape. For plasma samples a protein removal step was required to obtain clean electropherograms and a good peak shape. Acetonitrile was used as precipitant resulting in an enhanced sample stacking in comparison to water dilution. The disadvantage of using acetonitrile was severe evaporation loss making quantitation impossible. A self-sealing film was used to seal each individual sample vial to suppress evaporation during long-term sequences. The calibration curves for spiked plasma and cefazolin in Ringer's solutions were linear in the range from 2-500 and 2.5-100 microg/mL, respectively. Limits of detection were 1.0 and 2.0 microg/mL in plasma and microdialysis samples, respectively. The assay was successfully applied to plasma and microdialysis samples obtained in vivo from the interstial space fluid of subcutaneous adipose and muscle tissue of patients undergoing cardiac surgery.

Acetonitriles↗

Nonrandom sampling in human genetics: skewness and kurtosis.

When a multivariate normal sample is chosen from a truncated space of one of its components one can no longer make use of the normality assumption for the sample observations or for the estimates derived from them. In this paper, skewness and kurtosis for each component are derived analytically under a broad class of nonrandom sampling. It is shown that the distortions in skewness and kurtosis produced by nonrandomness are negligible, except those for the component with respect to which the selection of sampling regions is based. The usual tests of normality from sample values of skewness and kurtosis measures remain valid under nonrandom sampling, except for the selection variable. The implications of these analytical results in the context of commingling analysis in genetic epidemiology are discussed. It is recommended that when samples of families are obtained through nonrandomly ascertained probands, a commingling analysis should treat each relative class separately, since such analyses based on the pooled sample of individuals may involve unspecified bias in the levels of the test procedure.

Genetics, Medical↗

Methylation profile in tumor and sputum samples of lung cancer patients detected by spiral computed tomography: a nested case-control study.

We evaluated the aberrant promoter methylation profile of a panel of 3 genes in DNA from tumor and sputum samples, in view of a complementary approach to spiral computed tomography (CT) for early diagnosis of lung cancer. The aberrant promoter methylation of RARbeta2, p16(INK4A) and RASSF1A genes was evaluated by methylation-specific PCR in tumor samples of 29 CT-detected lung cancer patients, of which 18 had tumor-sputum pairs available for the analysis, and in the sputum samples from 112 cancer-free heavy smokers enrolled in a spiral CT trial. In tumor samples from 29 spiral CT-detected patients, promoter hypermethylation was identified in 19/29 (65.5%) cases for RARbeta2, 12/29 (41.4%) for p16(INK4A) and 15/29 (51.7%) for RASSF1A. Twenty-three of twenty-nine (79.3%) samples of the tumors exhibited methylation in at least 1 gene. In the sputum samples of 18 patients, methylation was detected in 8/18 (44.4%) for RARbeta2 and 1/18 (5%) for both RASSF1A and p16(INK4A). At least 1 gene was methylated in 9/18 (50%) sputum samples. Promoter hypermethylation in sputum from 112 cancer-free smokers was observed in 58/112 (51.7%) for RARbeta2 and 20/112 (17.8%) for p16, whereas methylation of the RASSF1A gene was found in only 1/112 (0.9%) sputum sample. Our study indicates that a high frequency of hypermethylation for RARbeta2, p16(INK4A) and RASSF1A promoters is present in spiral CT-detected tumors, whereas promoter hypermethylation of this panel of genes in uninduced sputum has a limited diagnostic value in early lung cancer detection.

Aged↗

Clonal heterogeneity in breast cancer: karyotypic comparisons of multiple intra- and extra-tumorous samples from 3 patients.

Intratumor phenotypic heterogeneity is one of the characteristics of breast carcinomas, and genetic mechanisms are likely to contribute to it. We have studied breast cancer clonal heterogeneity by cytogenetic analysis of multiple tumor samples (one from each tumor quadrant) as well as samples of macroscopically normal surrounding breast tissue from 3 patients with this disease. Clonal chromosome aberrations were found in all 8 successfully analyzed samples from the carcinomas. Two to 6 cytogenetically unrelated clones were detected in each case, unevenly distributed among the tumor quadrants. Karyotypic abnormalities were also found in 4 out of 9 macroscopically tumor-free samples from the surrounding tissue; in 2 of these samples, a ductal carcinoma in situ was detected histologically, and the cytogenetic evidence suggests that the remaining 2 samples also contained neoplastic cells. Quantitative analysis of the findings revealed a statistically significant higher frequency of karyotypically abnormal cells in samples with a histologic diagnosis of carcinoma vs. samples without any detected malignancy. That cells bearing cytogenetic evidence that they belong to the tumor parenchyma are left behind during breast-conserving surgery for carcinoma of the breast may account for the relatively high long-term local relapse rates seen in this disease.

Adult↗

Effects of pond water, sediment and sediment extract samples from New Hampshire, USA on early Xenopus development and metamorphosis: comparison to native species.

In an effort to assess potential ecological hazards to amphibian species in selected regions within New Hampshire, the traditional Frog Embryo Teratogenesis Assay-Xenopus (FETAX), a 14-/21 day tail resorption thyroid disruption assay and >30 day limb development tests were conducted with representative surface water and sediment samples. Two separate sets of samples collected from five sites were evaluated. The primary objectives of the study were to determine if samples were capable of inducing early embryo-larval maldevelopment, to determine if maldevelopment included limb defects, to determine if thyroxine co-administration altered the rates of limb malformation and to evaluate the impact of the samples on growth rates, developmental progress and metamorphic climax. Results from these studies suggested that pond water and sediment extract samples, but not whole sediment samples, from B2, FW, LP and W ponds were capable of inducing abnormal early embryo-larval development. In addition, water samples from B2 and W ponds induced significant abnormal hindlimb development. Some abnormal forelimb development was noted in the tail resorption studies, but not to the same extent as the hindlimbs. Each of the water samples induced appreciable developmental delay, including the paired reference site B1, which could be reversed by the addition of exogenous thyroxine.

Animals↗

A transmission electron microscope (TEM) calibration standard sample for all magnification, camera constant, and image/diffraction pattern rotation calibrations.

A calibration sample for transmission electron microscopy (TEM) has been developed that performs the three major instrument calibrations for a transmission electron microscope: the image magnification calibration for measurements of images, the camera constant calibration for indexing diffraction patterns, and the image/diffraction pattern rotation calibration for relating crystal directions to features in the image. This offers an improvement over commercially available calibration standards, where up to five different samples are required to perform these three calibrations. The new calibration sample consists of an electron-transparent cross-sectional TEM sample made from a molecular beam epitaxy (MBE)-grown, single-crystal semiconductor wafer. When the calibration structure is viewed in a TEM, it appears as a series of light and dark layers where the layer thicknesses are very accurately known. The calibrated thickness measurements of these light (silicon) and dark (SiGe alloy) layers are based on careful TEM measurements of the [111] lattice spacing of silicon which is visible on the calibration sample itself, and are supported by X-ray diffraction measurements. Furthermore, the layer thickness variation across the entire silicon wafer has been verified to be less than 1%, allowing all samples prepared from the same wafer to have errors in the given layer thickness values of less than 1%. As the sample is a single crystal of silicon, the calibrations requiring electron diffraction information such as the camera constant calibration and the image/diffraction pattern rotation calibration can also be performed easily and unambiguously. One single calibration sample can therefore be used to provide all three of the major TEM instrument calibrations at all magnifications and all camera lengths.

Calibration↗

Determination of sample time for T1 measurement.

In spin-lattice relaxation time measurements, the relaxation time T1 is estimated from measuring the longitudinal magnetization during its return to thermal equilibrium from an initial (nonequilibrium) state. T1 estimation error depends on a number of factors, including the sample spacing, number of sample points, target T1 range, etc. We describe here a sample-time determination method based on the principles of optimal experimental design. A two-parameter model and a more general three-parameter model of the general T1 measurement experiment are used in this study. Both linear and power-law sample spacing strategies are evaluated. The proposed method formulates the sample-time determination problem in closed form expressions that allow for easy calculation of optimum sample times, if a prior T1 estimate or a probable T1 distribution over the target range is given. Valuable insights can be gained from evaluation of these expressions concerning the relationship of T1 estimation error and the sample spacing, number of sample points, target T1 range, etc.

Humans↗