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PubMed1992

Determination of substituted purines in body fluids by micellar electrokinetic capillary chromatography with direct sample injection.

Many substituted purines (theobromine, caffeine, paraxanthine, theophylline and uric acid, as well as other methylated xanthines and uric acids) can easily be separated and analysed in one run using micellar electrokinetic capillary chromatography with a boratephosphate buffer containing 75 mM sodium dodecyl sulphate (pH approximately 9). Serum, saliva and urine samples collected after the self-administration of caffeine and serum samples from patients receiving theophylline or caffeine pharmacotherapy were screened for substituted purines. The data presented show the ease of using on-column multi-wavelength detection for investigating the feasibility of direct sample application, the characterization of sample pretreatment procedures and peak confirmation by comparing absorption spectra. It is shown that the determination of purines in serum and saliva samples, including therapeutic concentrations of caffeine and theophylline, can be accomplished without any sample pretreatment, whereas sample extraction is required for the determination of purines in urine. Quantitative data for the determination of micromolar amounts of theophylline (samples from adult patients) and caffeine (samples from infants born prematurely) in serum samples compared well with data obtained by non-isotopic immunoassays. Micellar electrokinetic capillary chromatography with the direct injection of serum or saliva samples requires only microlitre volumes of sample and several different compounds can be determined within a few minutes.

Adult↗
PubMed1984

A comparison between different high volume sampling systems for collecting ambient airborne particles for mutagenicity testing and for analysis of organic compounds.

Samples of urban air were collected simultaneously using different sampling systems, including electrostatic precipitation (ESP) and high volume filtration (HVF) on various filters for particle sampling and absorption on activated carbon and organic polymers for sampling of volatiles. Acetone extracts of the samples were analyzed for polycyclic aromatic hydrocarbons (PAH) and tested for mutagenicity with the Ames Salmonella/microsome assay. The results show that the concentrations of PAH found in the various particle-samples were in good agreement, whereas the mutagenic activity of these samples showed large variations. The highest mutagenic activity was found in the samples collected by ESP and on the teflon-coated glassfibre filters, whereas samples collected by high volume filtration with size-fractionation showed the lowest mutagenic activity. We do not know whether the higher activity in samples from the teflon-coated filters compared to those from ordinary glassfibre filters represent filter artifacts or if it represents a more pronounced degradation of mutagenic compounds on the non-coated glassfibre filters. Extracts from filter blanks seemed to interfere with the expression of the mutagenic activity of the positive controls, benzo[a]pyrene and nitropyrene. When sampling volatile compounds, two organic polymers, polyurethane (PUR) and XAD-2, were found suitable for collecting PAH, whereas no PAH could be detected in extracts from the activated carbon. The XAD-2 adsorbent was the most effective for sampling bicyclic PAH. None of the adsorbents yielded extracts well suited for mutagenicity testing, since blank extracts were toxic to the test bacteria. Some extracts of the PUR blanks were weakly mutagenic as well. More emphasis should be placed upon developing more efficient and unreactive adsorbents and on the adaptation of such adsorbents in samplers suited for routine use.

Air Pollutants↗
PubMed2006

Monitoring of polychlorinated dibenzo-p-dioxins and dibenzofurans, dioxin-like PCBs and polycyclic aromatic hydrocarbons in food and feed samples from Ismailia city, Egypt.

Concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyl (PCBs) and polycyclic aromatic hydrocarbons (PAHs) have been determined using GC/HRMS in food (butter, seafood and meat) and feed samples (chicken, cattle and fish) purchased from Ismailia city, Egypt. PCDD/F concentrations in food samples ranged between 0.12 and 3.35 pg WHO TEQ/g wet w, while those in feed samples were between 0.08 and 0.2 pg WHO TEQ/g dry w. Levels of PCB TEQ ranged from 0.14 to 3.2 pg/g wet w in the food samples. The feed samples have an average of 0.35 pg PCB TEQ /g dry w. In this study, butter samples showed the highest contamination levels of PCDD/Fs and PCBs. The PCBs contribution to the total TEQ was on average 63% in seafood and on average 49% for meat and butter. The highest contamination levels of PCDD/Fs and PCBs were found in butter samples. The butter TEQ content is several times higher than that reported in all EU countries and exceeded the EU limits, while the PCDD/F levels in seafood and the feed samples is far below the current EU limit. Generally, congener profiles in the food samples reflect the non-industrialized nature of the city and suggest solid waste burning as a significant source of emission. Nevertheless, the profiles for butter suggest an impact from various sources. In the case of the sum of 16 PAH contamination levels in food samples were in the range of 11.7-154.3 ng/g wet w and feed samples had a range of 116-393 ng/g dry w. Benzo(a)pyrene (BaP) has been detected in the range of 0.05-3.29 ng/g wet w in the food samples; butter showed the highest contamination which exceeded the EU standard set for fats and oil. Fingerprints of PAHs suggested both petrogenic and pyrolytic sources of contamination.

Animal Feed↗
PubMed2005

Optimized method for dissolved hydrogen sampling in groundwater.

Dissolved hydrogen concentrations are used to characterize redox conditions of contaminated aquifers. The currently accepted and recommended bubble strip method for hydrogen sampling (Wiedemeier et al., 1998) requires relatively long sampling times and immediate field analysis. In this study we present methods for optimized sampling and for sample storage. The bubble strip sampling method was examined for various flow rates, bubble sizes (headspace volume in the sampling bulb) and two different H2 concentrations. The results were compared to a theoretical equilibration model. Turbulent flow in the sampling bulb was optimized for gas transfer by reducing the inlet diameter. Extraction with a 5 mL headspace volume and flow rates higher than 100 mL/min resulted in 95-100% equilibrium within 10-15 min. In order to investigate the storage of samples from the gas sampling bulb gas samples were kept in headspace vials for varying periods. Hydrogen samples (4.5 ppmv, corresponding to 3.5 nM in liquid phase) could be stored up to 48 h and 72 h with a recovery rate of 100.1+/-2.6% and 94.6+/-3.2%, respectively. These results are promising and prove the possibility of storage for 2-3 days before laboratory analysis. The optimized method was tested at a field site contaminated with chlorinated solvents. Duplicate gas samples were stored in headspace vials and analyzed after 24 h. Concentrations were measured in the range of 2.5-8.0 nM corresponding to known concentrations in reduced aquifers.

Environmental Monitoring↗
PubMed2006

Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry.

Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 microg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5 microg/ml PerCP-conjugated anti-CD45, 5 microg/ml FITC-conjugated anti-CD11b, or 80 microM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior to antibody staining caused a decrease in overall fluorescence; it can be used to successfully identify extra-cellular markers.

Animals↗
PubMed2005

Electronic monitoring of salivary cortisol sampling compliance in daily life.

Naturalistic research methods have been developed to collect data in the daily environment, providing ecological valid measures. Recent reports suggest, however, that compliance with fixed time sampling protocols may be problematic and can bias results. This study investigated compliance with an intensive, random time sampling protocol for salivary cortisol and effects of non-compliance on cortisol results. Twenty female twin pairs and nineteen of their sisters were instructed to take saliva samples when signaled at ten unpredictable moments on each of five consecutive days. Subjects recorded collection times, unaware that compliance with the sampling protocol was being investigated by means of electronic monitoring devices. Samples taken < or = 15 min after the signal, according to self-report, were defined as adherent to the protocol. Samples taken < or = 10 min after the self-reported collection time, according to the monitor, were defined as accurate. Self-reported adherence to the sampling protocol was 96.4%. Verified compliance was somewhat lower, with 81% of all saliva samples accurately timed. Contrary to previous reports, inclusion of non-compliant samples in the analysis did not distort the cortisol diurnal profile. Intensive, random time sampling appears to have advantages over fixed time sampling for obtaining valid cortisol profiles when researchers do not have devices to monitor compliance. Results indirectly support the validity of momentary self-report data about daily experiences obtained with the same sampling methods.

Adult↗
PubMed2006

Direct identification of bacteria in clinical respiratory samples using fluorescent amplicon length analysis of 16S-23S rRNA spacer-region.

We describe the development and application of a rapid and universal molecular technique for direct identification of multiple bacteria in clinical samples. Amplification of the 16S-23S rRNA spacer-region using universal primers led to fragment patterns distinct for different bacterial species and that were analyzed with fluorescent amplicon length analysis (FALA). 136 pure cultures of clinical isolates and 20 culture collection strains belonging to 22 different medically important species were used to create a primary database of fragments with sizes between 100 and 1000 bp. Subsequently, 127 respiratory samples were analyzed with culture-based techniques and via FALA of the 16S-23S rRNA spacer-region. Two DNA extraction methods were evaluated: Instagene (FALA-I) and Fastprept (FALA-P). Of the 127 samples, 26 culture-negative samples were also negative with FALA-P. Of 18 samples with growth of commensal oral flora, 10 gave a mixed oral flora pattern with FALA-P and 8 gave a negative result. For 54 samples with growth of a single bacterial species, FALA-P gave an identical result for 46. For 29 samples with growth of more than one bacterial species, identical results were obtained in 19 samples. False-negative results with FALA-P were mostly due to paucity (less than 10(3) CFU/ml) of bacteria (12 out of 18 false-negatives) or difficulties with homogenization of viscous samples (6 out of 18 false-negatives).With regard to identification of all significant pathogens of clinical samples tested, the sensitivity of FALA-P was 77% and its specificity was 100%. With FALA-I, the number of false-negative results was higher than with FALA-P due to less efficient extraction of DNA, particularly with Staphylococcal species. FALA-P allows rapid and direct identification of multiple species directly from clinical samples; pauci-cellular samples may give false-negative results.

Bacteria↗
PubMed2000

SnifProbe: new method and device for vapor and gas sampling.

SnifProbe is based on the use of 15 mm short pieces of standard 0.53 mm I.D. capillary or porous layer open tubular columns for sampling airborne, headspace, aroma or air pollution samples. A miniaturized frit-bottomed packed vial named MicroSPE was also prepared which served for the sampling of solvent vapors and gases as well as liquid water. The short (15 mm) trapping column is inserted into the SnifProbe easy-insertion-port and the SnifProbe is located or aimed at the sample environment. A miniature pump is operated for pumping 10-60 ml/min of the air sample through the short piece of column to collect the sample. After a few seconds up to a few minutes of pumping, the short column is removed from the SnifProbe with tweezers (or gloved hands) and placed inside a glass vial of a direct sample introduction device (ChromatoProbe) having a 0.5 mm hole at its bottom. The ChromatoProbe sample holder with its glass vial and sample in the short column are introduced into the GC injector as usual. The sample is then quickly and efficiently desorbed from the short sample column and is transferred into the analytical column for conventional GC and/or GC-MS analysis. We have explored the various characteristics of SnifProbe and demonstrated its applicability and effectiveness in many applications. These applications include: the analysis of benzene, toluene and o-xylene in air, SO2 in air, perfume aroma on hand, beer headspace, wine aroma, coffee aroma, cigarette smoke, trace chemical warfare agent simulants, explosives vapors, ethanol in human breath and odorants in domestic cooking gas. SnifProbe can be operated in the field or at a chemical process. The sample columns can be plugged and stored in a small union storage device, placed in a small plastic bag, marked and brought to the laboratory for analysis with the full power of GC and/or GC-MS. Accordingly, we feel that the major and most significant feature of SnifProbe is that it brings the field and process to the laboratory. Thus, SnifProbe can extend the "arm" of the GC and GC-MS laboratory and enable high-quality field and process analysis.

Chromatography, Gas↗
PubMed2006

Risk of prostate cancer on first re-biopsy within 1 year following a diagnosis of high grade prostatic intraepithelial neoplasia is related to the number of cores sampled.

PURPOSE: We determined the influence of the extent of needle biopsy sampling on the detection rate of cancer on first biopsy within 1 year following a diagnosis of HGPIN. MATERIALS AND METHODS: We identified 791 patients with HGPIN on the initial biopsy who had a followup biopsy within 1 year of their diagnosis. The mean interval from diagnosis of HGPIN to re-biopsy was 4.6 months. In the initial biopsy with HGPIN, 323 men had 8 or more cores (median 10, range 8 to 26) and 332 men had 6 core biopsies. RESULTS: In the 6 core initial sampling group, the risk of cancer on re-biopsy was 20.8% compared to only 13.3% following an initial 8 core or more sampling (p = 0.011). With 6 core biopsies for both the initial and re-biopsy the risk of cancer was 14.1% (group 1). With an initial 6 core biopsy and 8 core or more biopsy on followup, the risk of cancer was 31.9% (group 2). With 8 core or more biopsy sampling for both initial and repeat biopsies, the risk for cancer was 14.6% (group 3). The differences between groups 1 and 3 as compared to group 2 were statistically significant (p = 0.001 and p <0.0001, respectively). CONCLUSIONS: With relatively poor sampling (6 cores) on the initial biopsy, associated cancers are missed resulting in only HGPIN on the initial biopsy, and with relatively poor sampling on re-biopsy there is also a relatively low risk of finding cancer on re-biopsy (group 1). With poor sampling on the initial biopsy and better sampling on re-biopsy, some of these initially missed cancers are detected on re-biopsy yielding a higher detection of cancer (group 2). Sampling more extensively on the initial biopsy detects many associated cancers, such that when only HGPIN is found they often represent isolated HGPIN. Therefore, re-biopsy even with good sampling does not detect many additional cancers (group 3). Our study demonstrates that the risk of cancer on biopsy within 1 year following a diagnosis of HGPIN (13.3%) is not that predictive of cancer on re-biopsy if good sampling (8 or more cores) is initially performed. For patients diagnosed with HGPIN on extended initial core sampling, a repeat biopsy within the first year is unnecessary in the absence of other clinical indicators of cancer.

Adult↗
PubMed2001

Evaluation of a second derivative UV/visible spectroscopy technique for nitrate and total nitrogen analysis of wastewater samples.

A method for nitrate analysis based on second derivative UV/Visible spectroscopy was developed by Simal et al. (1985: Simal J., Lage M. A., and Iglesias I. (1985) Second derivative ultraviolet spectroscopy and sulfamic acid method for determination of nitrates in water. J. Assoc. Analyt. Chem. 68, 962-964) and Suzuki and Kuroda (1987: Suzuki, N. and Kuroda R. (1987) Direct simultaneous determination of nitrate and nitrite by ultraviolet second-derivative spectrophotometry. Analyst 112, 1077-1079), and later modified for the analysis of total nitrogen in aqueous samples of varying nitrate:organic nitrogen ratios (Crumpton et al., 1992: Crumption W. G., Isenhart T. M. and Mitchell P. D. (1992) Nitrate and organic N analyses with second-derivative spectroscopy. Limnol. Oceanogr. 37, 907-913). The procedure uses the second derivative of the absorption spectrum for nitrate (NO3-), which has a peak at approximately 224 nm that is proportional to the NO3- concentration. Samples for total N analysis are first oxidized to NO3- by persulfate digestion. The objectives of this study were to: (1) test the accuracy and precision of the second derivative method through the use of NIST-traceable wastewater check samples; (2) determine whether the second derivative method for nitrate analysis can be used for wastewater samples and whether the method compares favorably with other currently used nitrate analysis methods; and (3) use the method to analyze wastewater samples containing a range of nitrate and total nitrogen concentrations. Our results indicated that the method needed to be modified to include a longer digestion time (60 min) and dilution of samples prior to digestion (if needed). With the modified method, nitrogen recoveries were not significantly different (P > or = 0.05) from samples with known N concentrations. In addition, nitrate concentrations in constructed wetland and wastewater samples analyzed by both second derivative spectroscopy and ion chromatography were not significantly different. Total nitrogen concentrations in wastewater samples also compared favorably to the same samples analyzed by Kjeldahl digestion. The method is faster, simpler, requires smaller sample volumes, and generates less waste than many EPA-approved methods of N analysis, and may offer a suitable alternative to current methods for analysis of nitrate and total N in wastewater samples.

Chromatography, Ion Exchange↗
PubMed1999

Analysis of the viability of freezer stored serum samples for hepatitis C virus RNA analysis by the SUPERQUANT method: results of a 16 year retrospective study.

Prior to the discovery of the hepatitis C virus (HCV), virological analysis of serum from patients with non-A non-B hepatitis was not possible. Since the finding that HCV is the causative agent of most non-A non-B hepatitis, several reliable methodologies have been developed that allow for quantification of HCV RNA. To determine the viability of stored serum samples for HCV RNA analysis. 256 samples were examined for HCV RNA using a multi-cycle RT-PCR assay. All samples were stored unopened in a -70 degree C freezer until the time of testing. Collection years ranged from 1981 to 1995. To examine the integrity of stored serum samples, the distribution of quantitative HCV RNA values for each year was compared: year-to-year; and, to the distribution of HCV RNA concentrations from 1510 chronic HCV patients determined by the same assay in 1996 and 1997. Pairwise year-to-year analysis revealed that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). Likewise, comparison of the stored samples to the 1510 fresh samples demonstrated that samples collected prior-to-and-including 1991 had significantly lower HCV RNA concentrations as compared to samples collected after 1991 (P < 0.001). The results demonstrate a method for determination of the integrity of stored serum samples from chronic HCV patients. The mechanism of RNA degradation is unknown but it is most likely to be due to poor sample collection procedures in place prior to 1991.

Adult↗
PubMed1996

Diagnosis of subclinical cryptosporidiosis in captive snakes based on stomach lavage and cloacal sampling.

The applicability of stomach lavage and cloacal swab techniques for diagnosis of subclinical cryptosporidiosis were tested in eight captive snakes subclinically infected with Cryptosporidium serpentis. Two feeding regimes were employed. The snakes were first fed 7 days prior to stomach and cloaca sampling, and then 3 days prior to sampling, and the oocysts were detected by fluorescein labeled monoclonal antibody (mAb) and by acid-fast stained (AFS) direct wet smear (DWS). The overall sensitivity of AFS DWS was 95% for stomach samples and 57% for cloacal samples, with false-negativity of 5% and 43%, respectively. A significant relationship (P < 0.01) was found between stomach and cloacal samples when mAb were used for oocyst detection. Stomach sampling was diagnostically superior to cloacal sampling for identifying snake subclinical cryptosporidiosis. Based on gastric aspirates, cryptosporidial infection was diagnosed in all eight animals, and only in two or four snakes when cloacal swab material was processed by AFS or by mAb, respectively. Feeding snakes 3 days prior to sampling facilitated diagnosis based on stomach samples; however, it did not improve diagnosis when cloacal samples were used. The fraction of oocyst-positive stomach samples was significantly higher (P < 0.05) for snakes fed 3 days prior to gastric lavage when compared with the fraction of positive samples of snakes fed 7 days prior to lavage. If subclinical cryptosporidiosis is suspected in a non-eating snake patient, force-feeding and stomach lavage, 3 days after the meal, is recommended.

Animals↗
PubMed2004

Salivary cortisol sampling compliance: comparison of patients and healthy volunteers.

OBJECTIVE: Problems of compliance with in vivo data collection and treatment protocols have been identified. This study investigated compliance with salivary cortisol sampling in a 7-day protocol. Impact of non-compliance on cortisol data was evaluated. METHODS: Female fibromyalgia patients were matched with healthy female volunteers and randomized to Aware or Unaware conditions regarding objective monitoring of their sampling compliance. The protocol entailed collecting five saliva samples at prescribed times on each of 7 consecutive days. Participants self-reported time of each sample, and electronic monitor caps provided an objective date and time stamp of each sample. RESULTS: Objective compliance among participants unaware of monitoring was 71%, though their self-reported compliance was 93%. Aware participants' objective compliance was 90% which was consistent with self-reported compliance of 93%. Within-subject comparison of early morning rise and day slope of cortisol for compliant and non-compliant samples found significant differences with non-compliant samples resulting in flatter slopes. Patients were somewhat more compliant than healthy volunteers. Slight decrements in compliance were found for the afternoon sample (1600 h) and for the last 3 days of sampling. Compliance did not differ on weekdays versus weekends. CONCLUSIONS: Self-report of compliance in a salivary cortisol sampling protocol substantially overestimates actual compliance in the absence of objective monitoring. Non-compliance with the sampling protocol results in cortisol data that significantly differs from compliant data. Awareness of electronic monitoring of sampling results in satisfactory compliance.

Adult↗
PubMed1997

Sperm head morphometry analysis of ejaculate and dismount stallion semen samples.

The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility. Conventional semen evaluation includes subjective determination of sperm concentration, motility, and gross morphology. Due to the subjectivity and variability of the manual morphology assessment, computer automated sperm morphology analyses has been developed. Computer automated sperm morphology analysis was applied in the current study to determine if the morphometric measurements of sperm heads from collected and dismount samples of the same ejaculate were similar. If the post-ejaculate dismount sample is representative of the entire ejaculate, this sample may be utilised in determining the fertility of the ejaculate. Ejaculate samples were collected from ten stallions using an artificial vagina. Post-ejaculate dismount samples of the same ejaculate were taken from the head of the penis. A thin smear of the collected and dismount samples were prepared onto microscope slides and spermatozoa were stained for 40 min in haematoxylin. At least 200 properly digitised sperm heads from each slide were analysed using computer automated sperm morphometry analysis. The mean values for length, width, width/length, area, and perimeter were recorded from each analysis of collected and dismount samples and compared by paired t-test. The coefficients of variation of each analysis was also recorded and compared between collected and dismount samples by paired t-test. No significant differences (P > 0.10) in any measurements were found between collected and dismount samples. The mean values for all stallions for collected and dismount samples were length = 5.96 microM and 6.06 microM, width = 2.95 microM and 2.98 microM, width/length = 0.49 and 0.49, area = 13.31 microM2 and 13.65 microM2 and perimeter = 15.54 microM and 15.74 microM respectively. No significant differences were detected in the coefficients of variation of sperm head measurements from collected and dismount samples. These results indicate sperm head measurements from dismount semen are representative of those of the ejaculate. Hence, sperm head measurements of dismount samples may be viably applied to studies of fertility or in case of clinical fertility assessment. This finding will further assist in the development of normal sperm head morphometry criteria in the stallion. Clinically, a slide can be prepared in the field after natural services matings and analysed accurately and objectively by ASMA.

Animals↗
PubMedDate not supplied

A comparison of sample preparation methods for PCR detection of pathogenic Yersinia enterocolitica from ground pork using swabbing and slurry homogenate techniques.

Two sample preparation methods for multiplex polymerase chain reaction (PCR) for detection of plasmid-bearing virulent Yersinia enterocolitica (YEP(+)) from ground pork were compared. Two sets of ground pork samples were inoculated with 10, 1, and 0.5 CFU/cm(2) of a YEP(+) strain, one set was swabbed and the second set was dispersed into a slurry homogenate. Both swab and slurry homogenate samples were enriched in sterile Whirl Pak bags containing modified trypticase soy broth for 48 h at 12 degrees C. From the enriched swab samples, the bacterial cells were pelleted, washed, boiled in sterile distilled water, and treated with proteinase K to prepare cell lysates to use as a DNA template. Since slurry homogenate samples contained food material, DNA extraction was performed using a commercial kit. The DNA from cell lysates and from extracted slurry homogenate samples were evaluated as templates for multiplex PCR employing primers for the chromosomal ail and plasmid virF genes. The enrichment of the YEP(+) strain was more efficient using the sponge-swabbed samples than the slurry homogenate samples at all three inoculum levels tested. It was necessary to dilute the DNA extracted from slurry homogenate to determine the optimal concentration of each sample for PCR amplification. No amplification signal was detected using undiluted DNA, possibly due to DNA inhibitors present in the slurry homogenate that were not removed in the process of extraction. However, DNA could be detected in undiluted cell lysates from swab samples. Thus, the cell lysates from swab samples are more advantageous than DNA extracted from ground pork slurry homogenate samples for the PCR assay.

Animals↗
PubMed2003

Establishment of stably EBV-transformed cell lines from residual clinical blood samples for use in performance evaluation and quality assurance in molecular genetic testing.

Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P = 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P = 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (10 of 16). Implementation of this process could help alleviate the shortage of positive control materials for clinical molecular genetic testing.

Adult↗
PubMed1998

The prevalence of Vibrio spp. in drinking water and environmental samples in Vellore South India.

The prevalence of Vibrio cholerae in drinking water, lakes and sewage outfalls during July and August 1996 in Vellore, India was determined. Drinking water samples were collected on single occasions from 12 sites in different geographic areas of the town where cholera had been reported. Samples of water, plankton and sediment were collected from fixed sites at three lakes on three occasions separated by at least 3 days during the course of the study. Samples from open sewers were taken from two representative sites in four areas of the town. Bacteria isolated from samples were identified by standard biochemical tests and isolated strains of V. cholerae tested for their ability to agglutinate O1 and O139 antisera. Water samples from lakes were also tested for the presence of V. cholerae O1 and O139 by fluorescent antibody staining. Non-O1, non-O139 strains of V. cholerae were detected in 41% of drinking water samples and 100% of water, sediment and plankton samples from the test lakes. Eighty-seven per cent of open sewers sampled contained viable non-O1, non-O139 V. cholerae. Fluorescent antibody staining gave positive results for V. cholerae O1 and O139 for all water samples from the three lake sites. Strains of Aeromonas spp. were isolated from 58% of drinking water samples and from 66% of sediment, 77% of plankton and 55% of water samples from lakes. All open sewers sampled contained Aeromonas spp. PCR amplification employing specific primers demonstrated that none of the non-agglutinating V. cholerae isolates contained the ctx operon. The non-O1, non-O139 V. cholerae isolates showed different patterns of antibiotic resistance to ampicillin, ciprofloxacin, chloramphenicol, tetracycline and trimethoprim.

Cholera↗
PubMed2003

A device for automated direct sampling and quantitation from solid-phase sorbent extraction cards by electrospray tandem mass spectrometry.

A new solid-phase extraction (SPE) device in the 96-well format (SPE Card) has been employed for automated off-line sample preparation of low-volume urine samples. On-line automated analyte elution via SPE and direct quantitation by micro ion spray mass spectrometry is reported. This sample preparation device has the format of a microtiter plate and is molded in a plastic frame which houses 96 separate sandwiched 3M Empore sorbents (0.5-mm-thickness, 8-microm particles) covered on both sides by a microfiber support material. Ninety-six discrete SPE zones, each 7 mm in diameter, are imbedded into the sheet in the conventional 9-mm pitch (spacing) of a 96-well microtiter plate. In this study one-quarter of an SPE Card (24 individual zones) was used merely as a convenience. After automated off-line interference elution of applied human urine from 24 samples, a section of SPE Card is mounted vertically on a computer-controlled X, Y, Z positioner in front of a micro ion spray direct sampling tube equipped with a beveled tip. The beveled tip of this needle robotically penetrates each SPE elution zone (sorbent disk) or stationary phase in a serial fashion. The eluted analytes are sequentially transferred directly to a microelectrosprayer to obtain tandem mass spectrometric (MS/MS) analysis. This strategy precludes any HPLC separation and the associated method development. The quantitative determination of Ritalin (methylphenidate) from fortified human urine samples is demonstrated. A trideuterated internal standard of methylphenidate was used to obtain ion current response ratios between the parent drug and the internal standard. Human control urine samples fortified from 6.6 to 3300 ng/mL (normal therapeutic levels have been determined in other studies to be between 50 and 100 ng/mL urine) were analyzed and a linear calibration curve was obtained with a correlation coefficient of 0.9999, where the precision of the quality control (QC) samples ranged from 9.6% at the 24 ng/mL QC level to 1.2% at the 3000 ng/mL QC level, and the accuracy for the four levels of QC samples ranged from 98.1% to 100.3%. The QC samples were prepared at four concentrations which included 24, 240, 1200, and 3000 ng/mL, respectively. The run time per sample in this work was 1.5 min not including the sample preparation time.

Automation↗