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PubMed2006

Evaluation of 31P high-resolution magic angle spinning of intact tissue samples.

The first detailed evaluation is presented of high-resolution (31)P MRS using magic angle spinning (MAS) of intact tissue samples and comparison with the conventional method of studying tissue extracts. The main motivation is that MAS leaves the sample intact at the end of the study for histopathological evaluation. While MAS of tissue samples has previously been demonstrated for (1)H MRS, (31)P MRS is better suited to study of the phospholipid metabolites of importance in cancer. Samples of rhabdomyosarcoma and RIF-1 experimental tumours were maintained at 4 degrees C, spun at 3 kHz and measured in 28-min acquisitions at 11.7 and 14 T. Metabolite stability was evaluated using four sequential 28-min acquisitions. High-resolution MRS was performed on extracts of the same tissue samples. (31)P HR-MAS yielded well-resolved high-resolution spectra, showing peaks from phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate, glycerophosphoethanolamine and glycerophosphocholine, with linewidths in the range 3-20 Hz. In tumour samples there was no significant change in peak areas over a 2-h period, while peaks sensitive to pH (inorganic phosphate, PE and PC) showed a small change in chemical shift, corresponding to a change of 0.13 +/- 0.06 pH units. Tissue metabolite concentrations showed good agreement with concentrations measured from extracts of the same pieces of tissue. For calculation of metabolite concentrations, the measurement of a reference compound in a separate measurement is more robust than using the signal from a reference compound in the rotor with the sample. Compared with performing tissue extracts, use of MAS of intact tissue samples requires less preparation, is quicker and permits the same sample to be used for subsequent histopathology. The methodology has particular application in studying phospholipid metabolism in cancer and in monitoring tumour response to treatment, where concentrations of phospholipid-related metabolites are found to alter following response to a wide range of anti-cancer therapies.

Animals↗
PubMed2003

HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR.

OBJECTIVE: To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin. METHODS: Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR. RESULTS: All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers. CONCLUSIONS: These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.

Adult↗
PubMed1992

Comparing sample size formulae for trials with unbalanced allocation using the logrank test.

This paper compares the sample size formulae given by Schoenfeld, Freedman, Hsieh and Shuster for unbalanced designs. Freedman's formula predicts the highest power for the logrank test when the sample size ratio of the two groups equals the reciprocal of the hazard ratio. The other three formulae predict highest powers when sample sizes in the two groups are equal. Results of Monte Carlo simulations performed for the power of the logrank test with various sample size ratios show that the power curve of the logrank test is almost flat between a sample size ratio of one and a sample size ratio close to the reciprocal of the hazard ratio. An equal sample-size allocation may not maximize the power of the logrank test. Monte Carlo simulations also show that, under an exponential model, when the sample size ratio is toward the reciprocal of the hazard ratio, Freedman's formula predicts more accurate powers. Schoenfeld's formula, however, seems best for predicting powers with equal sample size.

Clinical Trials as Topic↗
PubMed2006

Middle cerebral artery pulsatility index: reliability at different sampling sites.

OBJECTIVES: To analyze the interobserver reliability of measurement of the middle cerebral artery (MCA) pulsatility index (PI) at two different sampling sites. METHODS: This study included 100 consecutive singleton pregnancies between 24 and 40 weeks with normal fetal growth. The PI was calculated by two independent operators at proximal and distal sampling sites of the near-field MCA. Reliability analyses were performed between observers at each sampling site by means of the intraclass correlation coefficient (ICC) for agreement. Differences between observers were explored and agreement limits calculated by means of the Bland-Altman test. RESULTS: Satisfactory flow velocity waveforms were obtained successfully in each fetus at both sampling sites. Peak systolic, end-diastolic and time-averaged maximum velocities were significantly higher at the proximal compared with the distal sampling site. Conversely, PI was significantly higher at the distal compared with the proximal site. ICCs for PI were 0.3 and 0.33 at the proximal and the distal sampling sites, respectively. The 95% interval of the PI differences between observers were +0.91 and -1.14 at the proximal and +1.03 and -1.08 at the distal sampling sites. In about 30% of the cases the PI difference between observers was greater than 0.5 at both sampling sites. CONCLUSIONS: Moderate interobserver reliability in the measurement of end-diastolic and time-averaged maximum MCA flow velocities results in limited agreement of the PI calculation at both proximal and distal sampling sites. These results may preclude its clinical applicability.

Adolescent↗
PubMed1999

Immobilized sample amplification for quantitative determination of retroviruses.

Immobilized sample amplification (ISA) is a novel method for amplification, detection, monitoring, and quantitative determination of nucleic acids from a minute amount of sample. We present here a novel quantitative ISA assay for retroviruses using a replication-defective recombinant retrovirus as a model retrovirus. Samples, as small as 5 to 10 microl or as large as 1 ml or more in volume, are readily immobilized on a nylon or polyester matrix. Retroviral RNA is directly amplified following the rehydration of the immobilized samples, thus eliminating the needs for retroviral RNA extraction. An ISA assay of a 10-microl viral sample generates results equal to or better than that of RT-PCR on equivalent amount RNA isolated from larger sample volumes. Recovery of RNA from small volumes, such as 10 microl, is almost impossible, whereas ISA assay detects retroviruses from as small as 1 to 5 microl of viral samples containing 10(4) cfu/ml determined by colony-forming assay. Extraction of RNA from a small amount of infectious viral samples not only is a difficult, biohazardous procedure, but also introduces random errors which contribute to variability in viral quantitation. Since the ISA method eliminates the isolation/extraction of the nucleic acids, it significantly shortens the handling time for the biohazardous materials and simplifies the procedure for analyzing small quantities of biological samples. This method detects less than 10 infectious retroviral particles as determined by both colony-forming assay and electron microscope studies. The format and protocol of this quantitative ISA assay can be easily automated to fit into numerous platforms, thus making it attractive for laboratory automation.

3T3 Cells↗
PubMed2002

Are 24-hour urine samples and creatinine adjustment required for analysis of inorganic arsenic in urine in population studies?

Compliance with 24-h urine samples can be low in population-based studies and first morning void urine samples are often collected for convenience. Interpretation of arsenic concentrations in urine is influenced by a range of factors unrelated to exposure. To reduce the influence of such factors, creatinine adjustment is routinely used. This study aimed to determine whether first morning void urinary inorganic arsenic concentrations approximate 24-h urinary arsenic concentrations and whether creatinine adjustment improved the correlation between environmental arsenic concentrations and urinary inorganic arsenic concentrations. One hundred sixty spot samples and corresponding 24-h urine samples were collected from people living in areas with a range of environmental arsenic concentrations and analyzed for inorganic arsenic using borohydride arsine generation followed by atomic absorption spectrophotometry. There were no significant differences between the urinary inorganic arsenic concentrations for the different sample types or whether creatinine adjusted or not. Significant correlations were observed between adjusted and unadjusted samples. The data set was highly skewed and when only detectable arsenic samples were considered, the relationship between sample types became nonsignificant. The results of this study suggest that spot samples may be adequate for measuring short-term exposure, using inorganic arsenic as the outcome variable; however, additional work on a larger data set is required. Creatinine adjustment of urinary inorganic arsenic concentrations may not be required in population studies investigating environmental exposure.

Arsenic↗
PubMed1998

Lymph node sampling and survival in endometrial cancer.

OBJECTIVE: To determine the impact of pelvic lymph node sampling on survival in women with FIGO stage I and II endometrial adenocarcinoma. METHODS: We reviewed data from the National Cancer Institute's Surveillance, Epidemiology, and End Results program on 9185 women with stage I endometrial cancer and 881 women with stage II endometrial cancer. Life table analysis was used to compare survival rates. RESULTS: Overall, lymph node sampling did not appear to convey survival benefit. The 5-year relative survival for 6363 women with stage I endometrial cancer who did not undergo lymph node sampling was 0.98, compared to 0.96 for 2831 women who did undergo lymph node sampling at the time of hysterectomy, a nonsignificant difference. Lymph node sampling (LNS) was associated with increased survival among those with stage I, grade 3 disease, but not grade 1 or grade 2. Women with stage I, grade 3 disease who underwent LNS had a relative 5-year survival of 0.89, compared to 0.81 for those who did not undergo LNS (P = 0.0110). Nonsignificantly improved survival associated with LNS for women with grade 3 disease was observed in International Federation of Gynecology and Obstetrics stages Ib, Ic, and II. CONCLUSIONS: The observed survival benefit associated with lymph node sampling may be due to identification of women with more advanced endometrial cancer. Accurate determination of grade and extent of tumor is necessary to delineate which patients may benefit from lymph node sampling at hysterectomy. Effective cooperation between surgical pathology and gynecology services may be required to ensure adequate examination of the hysterectomy specimen. A surgeon with expertise in performing lymph node sampling should be available if operative findings render lymph node sampling appropriate.

Adenocarcinoma↗
PubMed1999

Differences in K-ras codon 12 mutation frequency between "high-risk" and "low-risk" HPV-infected samples.

OBJECTIVES: The current study was designed to determine the association between K-ras point mutations and the presence of high- and low-risk HPV types in noncancerous samples. METHODS: One hundred fifty-five noncancerous cytological samples from genital specimens positive for HPV-6, -16, or -18 were analyzed for codon 12 ras point mutations. DNA samples were subjected to nested PCR for the detection of HPV genome. Mutations at the first and second bases of K-ras codon 12 were detected using specific enriched PCR. RESULTS: Eleven percent of the HPV-positive samples analyzed showed mutation in K-ras codon 12 (17 of 155). Mutations were detected in 8 of 53 HPV-16- (15%) and 8 of 38 HPV-18-positive samples (21%). Nevertheless, when HPV-6-positive samples were screened for K-ras mutations only 1 of 64 samples was found mutated (1.56%). Statistical analysis using the chi(2) test showed a highly significant difference (chi(2) = 9.865, P < 0.01) when the rate of mutation for samples positive for high-risk HPV types (16 and 18) was compared with the frequency found in low-risk-infected samples (HPV-6). CONCLUSION: These results could be considered an indicator of the association between K-ras codon 12 mutations and the presence of high-risk HPV types.

DNA, Viral↗
PubMed1996

An Attempt to Interpret the Spread of Element Concentration in Antarctic Surface Snow: The Same Element in a Given Test Field, Several Elements in Different Sampling Fields

Sampling surface snow on a large test field always leads to a spread of analyte concentration data which partly follows a Gaussian distribution and partly a rectangular one as can be observed from the analysis of literature data. The spread depends on the nonuniformity of the air-snow interface in the field and on the extent of reproducibility of all the procedures used from sampling to analysis. Consequently a sample relevant to a restricted surface might be poorly representative of the surrounding area. Contamination of the sample during the gathering and storing steps is assumed to be the main source of nonrandom results (outliers). Using various statistical tools we were able to evaluate which part of the spread was due to the snow surface nonuniformity in the case of many samples collected in the same test field. In the case of samples gathered in different geographical areas, the possibility of finding correlations among points is greatly enhanced when three or more analytes are considered for each sample. When the same correlation is found for some analytes and a variable tentatively tested, information can be gained about the source of chemical content of snow samples. The use of UV pretreatment of snow samples has been proven to cut down the interference of organics on the electrochemical process in DPASV, allowing one to obtain accurate and reproducible data.

Journal Article↗
PubMed2002

Sampling wheat for deoxynivalenol.

The variability associated with testing wheat for deoxynivalenol (DON) was measured using a 0.454 kg sample, a Romer mill, 25 g of comminuted subsample and the Romer Fluoroquant analytical method. The total variability was partitioned into sampling, sample preparation, and analytical variability components. Each variance component was found to be a function of the DON concentration and equations were developed to predict each variance component using regression techniques. The effects of sample size, subsample size, and number of aliquots on reducing the variability of the DON test procedure were also determined. Using the test procedure described above, the coefficient of variation (CV) associated with testing wheat at 5 ppm DON was found to be 13.4%. The CVs associated with sampling, sample preparation, and analysis were 6.3, 10.0, and 6.3%, respectively. The sample variations associated with testing wheat are relatively small when compared to CVs associated with testing other commodities for other mycotoxins such as aflatoxin in peanuts. Even with the use of a small sample size (0.454 kg), the sampling variation was not the largest source of error as found in other mycotoxin test procedures.

Algorithms↗
PubMed1995

Sampling conformational hyperspace: techniques for improving completeness.

Three new strategies for sampling the conformation space accessible to a series of structurally diverse, flexible molecules are defined and compared to samples obtained using a fixed-grid torsion angle sampling strategy. A set of 28 potent inhibitors of angiotensin converting enzyme selected by Mayer et al. [J. Comput.-Aided Mol. Design, 1 (1987) 3] and the unrestricted active-site model proposed by Waller et al. [to be published] are used to produce a realistic experimental setting. We modified our Constrained Search algorithm [Dammkoehler et al., J. Comput.-Aided Mol. Design, 3 (1989) 3] to support these new sampling strategies, performing a series of 64 simulations (search experiments) and generating a large set of sterically allowed conformations. In each experiment, we systematically vary the internal torsion angles in each molecule using one of the sampling strategies. The common orientations of preselected functional groups thought to represent those dominating the interaction with the enzyme and presented by the set of molecules are classified and recorded for each experiment. Pairwise distances between groups are used to characterize the geometry of the common orientations. The results of each experiment, represented by a set of distance values, are compared and combined to evaluate the completeness of the conformational sampling. While no pure strategy or single search experiment was found to be adequate to fully explore the set of common sterically allowed conformations, a new sampling technique, called adaptive radial sampling, is shown to be significantly more complete than the commonly used fixed grid sampling.

Algorithms↗
PubMed1994

Improved resolution in triple-resonance spectra by nonlinear sampling in the constant-time domain.

Nonlinear sampling along the constant-time dimension is applied to the constant-time HNCO spectrum of the dimerization domain of Gal4. Nonlinear sampling was used for the nitrogen dimension, while the carbon and proton dimensions were sampled linearly. A conventional ct-HNCO spectrum is compared with a nonlinearly sampled spectrum, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension. Nonlinearly sampled data are processed by maximum entropy reconstruction. It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time. The constant intensity of the signal in the constant-time dimension allows for a variety of sampling schedules. A schedule of randomly distributed sampling points yields the best results. This general method can be used to significantly increase the quality of heteronuclear constant-time spectra.

Algorithms↗
PubMed1995

Amplifying dinucleotide microsatellite loci from bone and tooth samples of up to 5000 years of age: more inconsistency than usefulness.

We have studied the feasibility of using dinucleotide-repeat microsatellites in the analysis of DNA from ancient bones and teeth. We have used three microsatellites (IVS8CA, IVS17BTA, and IVS17BCA) within the cystic fibrosis transmembrane conductance regulator gene in 28 DNA samples from bones and teeth of up to 5000 years of age. PCR amplification was successful in 71.4% of cases. The repeated analysis of each marker produced different genotypes in 97% of samples, and the same individual genotype was reproduced at least once in 45.5% of cases. Alleles differing from the originals consisted of additions or deletions of 1-39 dinucleotides. The mechanism by which alleles differing from the originals were amplified can be related to the marked degradation of the DNA, with repeat sequences of different length interacting with the partially degraded repeats of the amplified loci. The repeated analysis of each sample allowed us to produce data with some anthropological interest. Among the haptotypes detected in samples from Easter Island, two (16-32-13 and 23-32-13) were found in more than one sample. Similarly, three haplotypes (16-7-17, 16-7-13, and 16-24-13) were detected more than once in samples from the Basque Country. Although haplotypes in the Basque Country are amongst the commonest in European chromosomes, most of those detected in the Easter Island samples are not frequent in Europeans. Thus, the repeated typing of microsatellites allowed us to postulate the genotypes that might be present in the samples but dinucleotide markers do not seem to be reliable enough for genotyping ancient bone and teeth samples.

Base Sequence↗
PubMed1994

Characterization of cytochrome P4501A induction in medaka (Oryzias latipes) by samples generated from the extraction and processing of coal.

The objective of this study was to characterize cytochrome P4501A induction in medaka liver as a biomarker for detecting polyaromatic hydrocarbon (PAH)-type compounds in samples of processed coal or petroleum. Ethoxyresorufin-O-deethylase (EROD) activity in individual medaka livers was used to asses induction of P4501A following the addition of various samples to aquaria water. Samples included a known P4501A inducer, beta-naphthoflavone, and various processed coal samples, as well as a petroleum-pitch. The sensitivity of detecting significant EROD induction by adding samples to aquaria water was approximately 0.1 mg/L for most samples; however, a coal-tar pitch significantly increased EROD activity at 0.01 mg/L. Different samples induced EROD activity to different extents. All samples elicited a concentration-dependent increase in EROD activity, with maximum EROD induction 2 days after a single administration of xenobiotics to aquaria water. Western blot studies established that induction of EROD activity by all xenobiotics tested was associated with corresponding increased amounts of immunoreactive P4501A. EROD induction was not influenced by gender, by single or multiple xenobiotic exposures, nor by feeding or fasting animals during the course of xenobiotic exposure. The ability of xenobiotics to induce EROD activity in medaka liver did not always correlate with their genotoxic potential determined by bacterial mutagenesis assays. Induction of P4501A in medaka liver appears to provide a convenient, economical, reliable and sensitive indicator for the presence of PAH-type compounds in coal- or petroleum-derived samples.

Animals↗
PubMed1994

Biological monitoring of cadmium exposure: reliability of spot urine samples.

Concentration-dilution of spot urine samples is a shortcoming of the biological monitoring of industrial xenobiotics. To ascertain whether the adjustment of urinary cadmium measured in spot samples is appropriate, urine samples were taken three times, once a week for 3 successive weeks, from 25 welders employed in the manufacture of jewelry (total 75 samples). Cadmium, creatinine, specific gravity, total urinary solutes, urinary volume and urinary flow rate were measured in 12-h collections and in spot samples taken immediately afterwards. Creatinine and total urinary solutes showed high inverse correlation with urinary flow rate (r = -0.858 and r = -0.768 respectively). Urinary cadmium displayed a similar trend but the correlation was not significant (r = -0.145). Creatinine adjustment of urinary cadmium values in spot samples increased the correlation with the same index in timed samples adjusted for urinary volume (r = -0.808) or urinary flow rate (r = 0.821) compared with non-adjustment (r = 0.732 and r = 0.738, respectively). Creatinine adjustment of spot sample values is also suitable for a wide range of urinary concentrations; discarding excessively diluted or concentrated urines, correlation of urine samples improved for non-adjusted or specific gravity-adjusted values, whereas no changes were observed for creatinine-adjusted values.

Adolescent↗
PubMed1993

DNA typing of urine samples following several years of storage.

The possibility of typing DNA polymorphisms on urine samples was investigated in a controlled storage experiment and for samples that were 1-7 years old. Female urine samples showed a higher amount of epithelial cells and therefore a higher DNA yield. Employing the polymerase chain reaction, specific amplification results were achieved for all samples over a 6 month storage period. The microscopical examination of the samples revealed not only differing degrees of contamination with bacteria, yeasts and fungi, but also the presence of still intact epithelial cells. Only 20% of the male samples and 32% of the female samples yielded specific amplification results. By separating the human cells from the contaminating organisms prior to DNA extraction, the number of successfully typed samples could be improved to 35% of the male and 77% of the female samples. This result confirms that excess amounts of co-extracted non-human DNA can inhibit the specific amplification of human target sequences.

Cell Count↗
PubMed1996

The characterization of VP7 (G type) and VP4 (P type) genes of bovine group A rotaviruses from field samples using RT-PCR and RFLP analysis.

Characterization of the VP7 (G type) and VP4 (P type) genes of bovine group A rotaviruses (BRV) from field samples was performed using RT-PCR and RFLP analysis. After RT-PCR amplification of the full length VP7 genes and partial length VP4 genes (nucleotides 1 to 1096), four enzymes, EcoRV, NlaIV, BamHI and HpaII were used for digestion analysis. For VP7, four RFLP profiles were observed after analysis of the digests: they were designated as G6, G6s (subtype, showed about 86% nucleotide and 90% amino acid identity to reference G6 strains), G8 and G10. For VP4, three RFLP profiles were observed: designated as P[1], P[5] and P[11]. The G typing analysis of 86 BRV fecal samples from 5 states, representing at least 11 different herds revealed that 60.5% (52/86) were G6, which included G6s (9/52); 19.8% (17/86) were G10; 7% (6/86) were G8; 10.4% (9/86) were G6 and G10 mixtures including two G6s samples; and 2.3% (2/86) were G6 and G6s mixtures. The P typing analysis of the same 86 fecal samples revealed that 64% (55/86) were P[5]; 28% (24/86) were P[11]; 1.2% (1/86) were P[1] and 6 samples (7%) were mixtures of either P[11] or P[5]. When the same samples were analyzed according to G and P type specificity, all possible combinations of G and P types existed in the field. The G6P[5] type was most prevalent and accounted for 46.7% (41/86) of the samples; 12.8% (11/86) were G10P[11]; 7% (6/86) were G10P[5] and an equal number were G6sP[11]. The G6P[11] (n = 2), G8P[1] (n = 1), G8P[5] (n = 1) and G8P[11] (n = 3) combinations were also observed. The following mixed BRV infections were observed in the field samples; G6sP[5 + 11] (n = 1), G8P[5 + 11] (n = 1), G6 + G10P[5] (n = 1) G6 + G10P[5 + 11] (n = 2), G6 + G6sP[11] (n = 1), G6 + G6sP[1 + 11] (n = 1), G6s + G10P[11] (n = 1) and G6s + G10P[5 + 11] (n = 1). Information on the G and P types and G/P combinations in the field samples should be useful for understanding the epidemiology of BRV and designing vaccination strategies to control BRV in the field.

Animals↗
PubMed2002

Small-scale variability of metals in soil and composite sampling.

Soil pollution data is also strongly scattering at small scale. Sampling of composite samples, therefore, is recommended for pollution assessment. Different statistical methods are available to provide information about the accuracy of the sampling process. Autocorrelation and variogram analysis can be applied to investigate spatial relationships. Analysis of variance is a useful method for homogeneity testing. The main source of the total measurement uncertainty is the uncertainty arising from sampling. The sample mass required for analysis can also be estimated using an analysis of variance. The number of increments to be taken for a composite sample can be estimated by means of simple statistical formulae. Analytical results of composite samples obtained from different fusion procedures of increments can be compared by means of multiple mean comparison. The applicability of statistical methods and their advantages are demonstrated for a case study investigating metals in soil at a very small spatial scale. The paper describes important statistical tools for the quantitative assessment of the sampling process. Detailed results clearly depend on the purpose of sampling, the spatial scale of the object under investigation and the specific case study, and have to be determined for each particular case.

Environmental Monitoring↗