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PubMed2004

Comparative study of methods for DNA preparation from olive oil samples to identify cultivar SSR alleles in commercial oil samples: possible forensic applications.

Virgin olive oil is made from diverse cultivars either mixed or single. Those ensure different tastes and typicity, and these may be also enhanced by the region of production of cultivars. The different olive oil labels correspond to their chemical composition and acidity. Labels also may correspond to a protected origin indication, and thus, such oils contain a given composition in cultivars. To verify the main cultivars used at the source of an olive oil sample, our method is based on DNA technology. DNA is present in all olive oil samples and even in refined oil, but the quantity may depend on the oil processing technology and oil conservation conditions. Thus, several supports were used to retain DNA checking different techniques (silica extraction, hydroxyapatite, magnetic beads, and spun column) to prepare DNA from variable amounts of oil. At this stage, it was usable for amplification through PCR technology and especially with the magnetic beads, and further purification processes were checked. Finally, the final method used magnetic beads. DNA is released from beads in a buffer. Once purified, we showed that it did not contain compounds inhibiting PCR amplification using SSR primers. Aliquot dilution fractions of this solution were successfully routinely used through PCR with different SSR primer sets. This enables confident detection of eventual alien alleles in oil samples. First applied to virgin oil samples of known composition, either single cultivars or mixtures of them, the method was verified working on commercial virgin oil samples using bottles bought in supermarkets. Last, we defined a protocol starting from 2 x 40 mL virgin olive oil, and DNA was prepared routinely in about 5 h. It was convenient to genotype together several loci per sample to check whether alleles were in accordance with those of expected cultivars. Thus, forensic applications of our method are expected. However, the method needs further improvement to work on all oil samples.

Alleles↗
PubMed2002

Power and sample sizes for linkage with extreme sampling under an oligogenic model for quantitative traits.

Extreme sampling of sibling pairs has been shown to be efficient in terms of statistical power and sample sizes (in number of sibling pairs needed to genotype) to detect a quantitative trait locus (QTL) when the residual distribution is normal. In the present study, the efficiency of extreme sampling strategies to detect each locus under an oligogenic model is analytically explored with a test statistic based on identical-by-descent (IBD) statuses of independent sibling pairs. In the oligogenic model, the joint effect of oligogenes is the sum of the effects of each locus. Under this model, detecting each single locus will depend, in part, on the allele frequencies and magnitudes of effect of the other loci. Effects of two QTLs with different magnitudes of displacement and acting nonepistatically are considered. Three types of extreme sampling-that is, extreme concordant high (ECH), extreme concordant low (ECL), and extreme discordant (ED)-are primarily considered herein. Among these, ED sampling under the oligogenic model is shown to be most efficient in most situations considered here in terms of allele frequency and mode of inheritance. Differences in results between ECH and ECL sampling are purely arbitrary, brought up mostly by the directions of displacement effects. However, power to detect a locus with the lesser (in magnitude) displacement effect does not necessarily increase with extremity of sampling. Combinations of extreme discordant and extreme concordant sibling pairs are briefly discussed.

Alleles↗
PubMed2002

Within-sample and between-sample variation of antimicrobial resistance in fecal Escherichia coli isolates from pigs.

The present study was initiated to evaluate the effect of sampling time and within-sample variability on the diversity in antimicrobial resistance patterns in fecal Escherichia coli from healthy pigs. Isolates were tested against 11 antimicrobials. A total of 25 different profiles were observed, involving resistance to ampicillin, streptomycin, tetracycline, sulfonamides, trimethoprim, and/or a trimethoprim/sulfonamide combination. No isolates were resistant to enrofloxacin, gentamicin, or chloramfenicol, whereas resistance against neomycin and nalidixic acid was sporadically detected in isolates from grower pigs. A model that clusters pigs within-sampling time as a repeated factor and clusters isolates within individual pigs as a random factor was used. For sows, the variance component ratio of sampling time to residuals was 0.28-0.56 for the different antimicrobials (except ampicillin) and 0.85-1.79 for grower pigs. The variance components for within-sample variation were zero or close to zero, except in isolates from sows where resistance to ampicillin explained 14.8 times more of the variation compared to residuals. Thus, the effect of an animal's status at a given sampling time was more influential on the variability in antimicrobial resistance than within-animal diversity. We conclude that repeated sampling and analysis of one isolate per animal each time may be preferable for screening general tendencies, whereas several isolates have to be tested when individual animals are focused.

Animals↗
PubMed2005

Genetic research and donation of tissue samples to biobanks. What do potential sample donors in the Swedish general public think?

BACKGROUND: The aim of this study was to identify perceptions of the general public regarding research involving human tissues; to assess the public's willingness to donate samples to biobanks; and to identify factors associated with the willingness to donate samples. METHODS: Cross-sectional survey. Postal questionnaires to a random sample of the general public in Sweden, 18-80 years of age (n = 6000) in October 2002 (response rate 49.4%; n = 2928). RESULTS: A majority of the respondents had a positive attitude towards genetic research. Their trust in authorities' capability to evaluate the risks and benefits of genetic research varied. Individual university/hospital-based researchers received the greatest trust, while the county councils (health care providers), and the Swedish Parliament received the lowest trust. Most respondents (86.0%) would donate a linked blood sample for research purposes. Another 3.0% would provide an anonymous sample. In total, 78% of the respondents would agree to both donation and storage. The most common motive was benefit of future patients. The majority was indifferent to the funding source for the research and would delegate this judgment to the research ethics committee. After adjusting for covariates, those more likely to donate a sample were middle-aged, had children, had personal experience of genetic disease, were blood donors, had a positive attitude toward genetic research, and had trust in experts/institutions. CONCLUSIONS: The majority of the general public is willing to donate a sample to a biobank. The willingness is mainly driven by altruism, and depends on the public being well-informed and having trust in experts and institutions.

Adolescent↗
PubMedDate not supplied

Monitoring of occupational exposure to methylene chloride: sampling protocol and stability of urine samples.

A sampling protocol for biomonitoring of the volatile solvent methylene chloride (MeCl(2)) by analysis of urine from exposed workers was established. Storage temperature, sample volume in headspace vial (HSV), and time to sealing HSV on determination of MeCl(2) in urine were evaluated. MeCl(2) was analyzed by a solid-phase microextraction technique combined with gas chromatography. Volume of urine in HSV has no effect on MeCl(2) analysis. Delays of 30 and 60 min from collection of urine until sealing the HSV caused 14.47 +/- 6.98% and 26.17 +/- 9.57% decreases from baseline concentration, respectively. MeCl(2) concentration in spiked urine samples stored in sealed HSVs decreased on day 2 and then remained stable for 2 weeks. Refrigeration did not improve recovery although it seems to be associated with less variability. MeCl(2) in urine samples of seven exposed workers was in the range of 0.02-0.06 mg/L. Sampling of MeCl(2)-containing urine should include collection of urine in closed plastic bottles, transfer to HSV within 15 min, sealing and clamping of HSV within 15 s, and storage of HSV in refrigeration until analysis, but no longer than 2 weeks. Standard samples should be prepared on the day of test sample collection and handled under the same conditions.

Adult↗
PubMed1996

Gastric contamination of postmortem blood samples during blind-stick sample collection.

A blood sample drawn by a blind stick through the chest was collected for toxicologic examination in a suspected natural death. Drug screen results on the blood sample indicated a combined overdose of alcohol and amitriptyline. An autopsy was later performed and a peripheral blood sample was drawn. A drug screen of this sample showed the presence of therapeutic quantities of amitriptyline and a trace of alcohol. Cytologic examination of the blind-stick sample showed the presence of columnar cells and food debris, confirming that the sample was contaminated either by stomach contents or gastric aspirate during the collection procedure. We strongly encourage all agencies to refrain from the use of blind-stick sample collection for toxicologic specimens.

Autopsy↗
PubMed2006

Sample bias among women with retained DNA samples for future genetic studies.

OBJECTIVE: To evaluate whether women who agree to future use of their biologic specimens for genetic studies reflect the larger study population from which they are derived. METHODS: Women were questioned as to the future disposition of their maternal and fetal DNA samples upon enrollment in a multicenter, observational study originally designed to identify factor V Leiden mutation carriers and prospectively ascertain the estimated rate of pregnancy-related venous thromboembolism and adverse pregnancy outcome. Univariate and multivariate analyses was carried out on the 5,003 of 5,188 enrolled women who indicated their desire regarding future disposition of their DNA samples. RESULTS: Among these 5,003 women, 20.1% desired that their samples be discarded and not available for future genetic studies. Multivariate analysis demonstrated that women who agreed to subsequent use of samples were less likely African-American (odds ratio [OR] 0.6, 95% confidence interval [CI] 0.4-0.7) or Hispanic (OR 0.4, 95% CI 0.3-0.5), and more likely to use tobacco (OR 1.2, 95% CI 1.0-1.6) than those who desired that their samples be discarded. CONCLUSION: Genetic samples from women agreeing to their use in a sample repository may not be representative of the index study cohort. This should be considered in their subsequent interpretation and generalizability. LEVEL OF EVIDENCE: III.

Adult↗
PubMedDate not supplied

Shanxi Province cervical cancer screening study II: self-sampling for high-risk human papillomavirus compared to direct sampling for human papillomavirus and liquid based cervical cytology.

The objective of this study was to compare the sensitivity and specificity of a new method for self-sampling for high risk human papillomavirus (HPV) with direct sampling and liquid based cervical cytology. In Shanxi Province, China, 8,497 women (ages 27-56) underwent a self-sample for HPV using a conical-shaped brush placed into the upper vagina and rotated. Three to sixteen months later the women were screened with liquid-based cytology and direct HPV tests. Subjects with any abnormal test underwent colposcopy and multiple biopsies. Mean age was 40.9 years. 4.4 percent of subjects had >or=CIN II, 26% a positive self-sample and 24% a positive direct test for HPV. The sensitivity for detection of >or=CIN II was 87.5% for self-sampling, and 96.8% for the direct test (P < 0.001). The specificity was 77.2% for the self-sample and 79.7% for the direct test. With an abnormal Pap defined as ASCUS or greater the sensitivity of the Pap for the detection of >CIN II was 88.3% and the specificity was 81.2%. We conclude that self-sampling for HPV is less sensitive for >CIN II than the direct test, but similar to liquid based cytology.

Adult↗
PubMed2004

Rapid quantification of Yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR.

The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 +/- 0.021 and 1.007 +/- 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 +/- 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y. enterocolitica was possible down to a level of at least 4.2 x 10(3) CFU per ml of meat juice, even in the presence of 10(6) CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.

Animals↗
PubMed1994

Effects of sample storage time, temperature and syringe type on blood gas tensions in samples with high oxygen partial pressures.

BACKGROUND: Although plastic arterial sampling syringes are now commonly used, the effects of sample storage time and temperature on blood gas tensions are poorly described for samples with a high oxygen partial pressure (PaO2) taken with these high density polypropylene syringes. METHODS: Two ml samples of tonometered whole blood (PaO2 86.7 kPa, PaCO2 4.27 kPa) were placed in glass syringes and in three brands of plastic blood gas syringes. The syringes were placed either at room temperature or in iced water and blood gas analysis was performed at baseline and after 5, 10, 20, 40, 60, 90, and 120 minutes. RESULTS: In the first 10 minutes measured PaO2 in plastic syringes at room temperature fell by an average of 1.21 kPa/min; placing the sample on ice reduced the rate of PaO2 decline to 0.19 kPa/min. The rate of fall of PaO2 in glass at room temperature was 0.49 kPa/min. The changes in PaCO2 were less dramatic and at room temperature averaged increases of 0.47 kPa for plastic syringes and 0.71 kPa for glass syringes over the entire two hour period. These changes in gas tension for plastic syringes would lead to an overestimation of pulmonary shunt measured by the 100% oxygen technique of 0.6% for each minute left at room temperature before analysis. CONCLUSIONS: Glass syringes are superior to plastic syringes in preserving samples with a high PaO2, and prompt and adequate cooling of such samples is essential for accurate blood gas analysis.

Blood Gas Analysis↗
PubMed1999

Influence of sampling practices on the appearance of DNA image histograms of prostate cells in FNAB samples.

Twenty-one fine needle aspiration biopsies (FNAB) of the prostate, diagnostically classified as definitely malignant, were studied. The Papanicolaou or H&E stained samples were destained and then stained for DNA with the Feulgen reaction. DNA cytometry was applied after different sampling rules. The histograms varied according to the sampling rule applied. Because free cells between cell groups were easier to measure than cells in the cell groups, two sampling rules were tested in all samples: (i) cells in the cell groups were measured, and (ii) free cells between cell groups were measured. Abnormal histograms were more common after the sampling rule based on free cells, suggesting that abnormal patterns are best revealed through the free cells in these samples. The conclusions were independent of the applied histogram interpretation method.

Biopsy, Needle↗
PubMed2006

Reverse transcription-polymerase chain reaction on pooled samples to detect bovine viral diarrhea virus by using fresh ear-notch-sample supernatants.

Ear-notch samples from 3,599 yearling heifers were collected to detect persistently infected (PI) animals with suspect bovine viral diarrhea virus (BVDV). Individual immunohistochemistry (IHC), individual antigen-capture enzyme-linked immmunosorbent assay (AC-ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) tests with pooled ear-notch supernatants were compared with samples from 3,016 heifers, whereas RT-PCR ear-notch pools and individual AC-ELISA tests were compared with samples from all 3,599 heifers. Four heifers were identified positive by both IHC and AC-ELISA, whereas the remaining heifers were identified negative by both tests. When supernatant from ear notches from 100 animals was randomly pooled and RT-PCR was accomplished on each pool, RT-PCR identified 2 pools that contained 1 positive AC-ELISA sample and 1 pool that contained 2 positive AC-ELISA samples. Further evaluation of the pooled RT-PCR ear-notch supernatant detected 100% (n = 36) samples spiked with supernatant from a single randomly selected positive AC-ELISA ear notch. Although follow-up confirmatory tests were not completed, all 3 methods correlated 100% in detecting suspect PI animals, with a kappa value of 1. The use of RT-PCR on pooled ear-notch supernatant could provide an initial, rapid, cost-effective method of screening cattle herds for BVDV PI animals. Subsequent serial testing with an AC-ELISA to evaluate individual samples included in the positive pool could minimize the length of time other animals are exposed to the virus.

Animals↗
PubMed2005

Self-sampling versus physician-sampling for human papillomavirus testing.

We evaluated the detection of human papillomavirus (HPV) infection using two sampling methods of cervical exfoliated cells, consisting of self-sampling of vaginal cells and cervical sampling performed by the physician. Women included were 379 patients of the general population attending outpatient clinics in Northern Greece for routine cytological cervical dysplasia screening. HPV DNA detection was similar with both sampling techniques. The HPV prevalences in self-collected samples were 4.7% and 3.7% in the physician-collected samples (P>0.05). The Kappa statistic for HPV DNA agreement between the two methods was 0.54 (95% Confidence interval = 0.33-0.75). Self-sampling of cervico-vaginal exfoliated cells could be used as an alternative option to test for HPV infection.

Cervix Uteri↗
PubMed2003

Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns.

We have measured 29 pesticides in plasma samples collected at birth between 1998 and 2001 from 230 mother and newborn pairs enrolled in the Columbia Center for Children's Environmental Health prospective cohort study. Our prior research has shown widespread pesticide use during pregnancy among this urban minority cohort from New York City. We also measured eight pesticides in 48-hr personal air samples collected from the mothers during pregnancy. The following seven pesticides were detected in 48-83% of plasma samples (range, 1-270 pg/g): the organophosphates chlorpyrifos and diazinon, the carbamates bendiocarb and 2-isopropoxyphenol (metabolite of propoxur), and the fungicides dicloran, phthalimide (metabolite of folpet and captan), and tetrahydrophthalimide (metabolite of captan and captafol). Maternal and cord plasma levels were similar and, except for phthalimide, were highly correlated (p < 0.001). Chlorpyrifos, diazinon, and propoxur were detected in 100% of personal air samples (range, 0.7-6,010 ng/m(3)). Diazinon and propoxur levels were significantly higher in the personal air of women reporting use of an exterminator, can sprays, and/or pest bombs during pregnancy compared with women reporting no pesticide use or use of lower toxicity methods only. A significant correlation was seen between personal air level of chlorpyrifos, diazinon, and propoxur and levels of these insecticides or their metabolites in plasma samples (maternal and/or cord, p < 0.05). The fungicide ortho-phenylphenol was also detected in 100% of air samples but was not measured in plasma. The remaining 22 pesticides were detected in 0-45% of air or plasma samples. Chlorpyrifos, diazinon, propoxur, and bendiocarb levels in air and/or plasma decreased significantly between 1998 and 2001. Findings indicate that pesticide exposures are frequent but decreasing and that the pesticides are readily transferred to the developing fetus during pregnancy.

Adult↗
PubMed2000

A study of factors that may influence the determination of copper, iron, and zinc in human milk during sampling and in sample individuals.

The aim of this study was to establish the possible effects of the sampling protocol (between-breast, within-feed, and diurnal differences) and the mother's personal factors (age, parity, iron supplementation, smoking habits, and lactation period) on the copper, iron, and zinc contents in human milk. One hundred thirty-six human milk samples identified by their origin and sampling conditions were analyzed. The samples were obtained from the 2nd to 15th d postpartum from 62 women. The data on the individuals required for the study were available. Mineral determinations were analyzed by flame atomic absorption spectrometry following a standardized protocol. The results showed that iron contents were higher in hind-milk samples and at the nighttime feeding and depended on the breast from which the sample was taken. The copper and zinc concentrations showed no significant variations. There was no significant relationship among the mothers' age, parity, smoking habits, iron supplementation, and copper content. Milk from older women had lower zinc contents than that of younger women. Increased amounts of iron were found in multiparous women. Between colostrum and transitional milk, a sharp decrease in zinc content was observed, whereas copper and iron contents remained constant. All of these results make it clear that standardized sampling protocols are needed in order to obtain comparable values.

Adult↗
PubMed2004

Accounting for cluster sampling in constructing enumerative sequential sampling plans.

Green's sequential sampling plan is widely used in applied entomology. Green's equation can be used to construct sampling stop charts, and a crop can then be surveyed using a simple random sampling (SRS) approach. In practice, however, crops are rarely surveyed according to SRS. Rather, some type of hierarchical design is usually used, such as cluster sampling, where sampling units form distinct groups. This article explains how to make adjustments to sampling plans that intend to use cluster sampling, a commonly used hierarchical design, rather than SRS. The methodologies are illustrated using diamondback moth, Plutella xylostella (L.), a pest of Brassica crops, as an example.

Animals↗
PubMed2005

Nasal cytologies--impact of sampling method, repeated sampling and interobserver variability.

The influence of sampling technique, repeated sampling and the interobserver variability on quantitative analysis of nasal cytologies were explored. Nasal cytologies from 12 healthy and 4 allergic volunteers were repeatedly collected with a total of three different sampling techniques. Cells were demonstrated by May-Grünwald-Giemsa staining and by immunostaining for CD45 and eosinophil peroxidase (EPO). To determine the interobserver variability specimens were evaluated by three observers with various levels of nasal cytology experience. When adjusted for the variability due to sampling method and repeated sampling, the staining index for CD45+ cells was 53.4 +/- 11% (interindividual coefficient of variation: 0.21) with an average intraindividual coefficient of variation of 0.14. For CD45, staining indices differed significantly between the washing techniques and the nasal swabs (p<0.01). Repeated sampling did significantly influence the staining indices of specimens collected with nasal swabs (p<0.05). Subjects with nasal allergy had less CD45+ cells in their nasal cytologies than the non-allergic subjects (p=0.02). A high level of compliance for the quantitative analysis of nasal cytologies between three observers was found (kappa(w) range 0.95-0.98). For studies that require repeated sampling, the lavage will be recommended as best suitable to obtain nasal cytologies.

Adult↗
PubMed2006

Effects of syringe material, sample storage time, and temperature on blood gases and oxygen saturation in arterialized human blood samples.

BACKGROUND: The practice of on-ice storage of arterial-blood samples in plastic syringes for delayed analysis continues, and the effects of storage time and temperature on the measurement of blood-oxygen-saturation values (S(aO2)) have not been adequately described. OBJECTIVE: To determine the effects of syringe material, storage time, and storage temperature on normal arterialized blood gas and S(aO2) values. METHODS: We used a temperature-controlled extracorporeal circuit to "arterialize" 500 mL of fresh, whole human blood at 37 degrees C, and we used certified calibration gases of 12% O2 and 5% CO2 to produce normal blood-gas values. From that arterialized blood we took 90 samples and randomly assigned them to 6 groups, until there were 15 samples in each group. The groups were (1) plastic syringe, analyzed immediately, (2) plastic syringe, stored 30 min at 0-4 degrees C, (3) plastic syringe, stored 30 min at 22 degrees C, (4) glass syringe, analyzed immediately, (5) glass syringe, stored 30 min at 0-4 degrees C, and (6) glass syringe, stored 30 min at 22 degrees C. RESULTS: Compared to the samples that were analyzed immediately, the P(O2) of the samples stored in plastic syringes for 30 min at 22 degrees C and at 0-4 degrees C was significantly higher, with a clinically important magnitude of 11.9-13.7 mm Hg. The P(CO2) of blood stored in glass for 30 min at 0-4 degrees C was significantly lower, although the magnitude of the difference (1.5 mm Hg) was not clinically important. There were no statistically significant differences in pH or oxygen saturation among the 6 groups. CONCLUSION: For accurate arterial-blood-gas results, samples drawn in plastic syringes should be analyzed immediately. If the analysis is going to be delayed, the samples should be drawn and stored in glass.

Analysis of Variance↗