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PubMed2003

A rapid and sensitive cytotoxicity screening assay for trichothecenes in cereal samples.

The application of a bioassay for toxicity screening of cereal samples has been studied. The sensitive Swiss mouse 3T3 fibroblasts (3T3 cells) were used to determine the cytotoxicity of Fusarium trichothecenes T-2 toxin (T-2), HT-2 toxin (HT-2), deoxynivalenol (DON) and nivalenol (NIV) in cereal samples. Clean-up of the sample extracts was performed using the MycoSep #225 column and the cytotoxic effects of the samples were determined using the 5-bromo-2'-deoxyuridine (BrdU) bioassay assessing DNA-synthesis. Trichothecene-free samples of wheat, wheat bran and barley had no toxic effect on the 3T3 cells at sample concentrations up to 400 mg/ml cell culture medium while a few oat samples had an inhibiting effect on the DNA-synthesis. IC(50) values (50% response compared with untreated cells) of trichothecene-free wheat and oats spiked with T-2, HT-2, DON and NIV were similar to IC(50) values of pure toxins. A cytotoxicity screening of 33 different cereal samples showed that all samples contaminated with a trichothecene concentration higher than the IC(50) values of T-2, HT-2, DON and NIV had a clear inhibiting effect on DNA-synthesis of the 3T3 cells. The culture of 3T3 cells on microtiter plates in combination with the simple and rapid sample clean-up using the MycoSep #225 column provides a sensitive and easy method for the screening of trichothecenes in cereal samples.

3T3 Cells↗
PubMed1997

Mutagenic heterocyclic aromatic amines (HAAs) in 'processed food flavour' samples.

Eight samples of 'processed food flavours' (PFFs), chosen from five different categories, were analysed for their mutagenic activity using the Ames Salmonella assay, and also for the presence of eight heterocyclic aromatic amines (HAAs), namely 2-amino-3-(trideuteromethyl)imidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (4,7,8-TriMeIQx), 2-amino-I-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) using liquid chromatography and mass spectrometry (LC/MS). The isolation of HAAs was based on sequential liquid-liquid extraction procedures of samples at both acidic and basic pH values. The recoveries and the clean-up were monitored by introduction of quality control samples and by spiking with three tri-deuterated standards of HAAs. Although the results for the mutagenicity assay were comparable by testing less-purified and highly-purified extracts, the analysis for identification and quantification of HAAs by LC/MS required highly purified concentrates. Four samples had little or no mutagenic activity and these results were in agreement with their LC/MS results: they had no detectable levels (detection limits 1-3 ppb) of any of the HAAs monitored. The mutagenic activity of one sample was in complete agreement with the quantification of HAAs by LC/MS. Two samples produced strong mutagenic responses (3115 and 2664 revertants/g). In one sample, LC/MS analysis revealed the presence of 9.6 ppb IQ, whereas LC/MS of the other could not confirm the presence of any of the eight HAAs monitored. Two samples produced mild mutagenic activity (204 and 160 revertants/g), but relatively elevated concentrations of IQ (6.7 and 6.8 ng/g) by LC/MS. The extracts from all samples were tested for their modifying effects on mutagenicity of four HAAs. The discrepancy between the Ames test and the LC/MS analysis of some samples indicates several possibilities, such as the presence of some other HAAs, of their isomers or of other mutagens. In addition, the presence of mutagen modifiers (inhibitors or synergists) was observed in most samples. The results indicate that although chemical tests (e.g. LC/MS) can provide quantitative data for the HAAs monitored, the Ames mutagenicity test should also be conducted to determine the mutagenic activities of PFFs, in order to assess their health risk potential.

Amines↗
PubMed1999

Charged cyclodextrin-mediated sample stacking in micellar capillary electrophoresis. A simple method for enhancing the detection sensitivity of hydrophobic compounds.

The development of on-line sample stacking techniques for enhancing limits of detection of neutral analytes in micellar capillary electrophoresis (MCE) has recently gained much attention. Utilizing high-conductivity sample matrices to invoke sample stacking is promising, but requires the limited use of sample solubilizing agents such as alcohols in the sample matrix. In this study, we show how simple replacement of the sample solvent (methanol) with a solution of sulfated beta-cyclodextrin (s beta-CD) allows a significant increase in the sensitivity of detection of model hydrophobic analytes. This increase in sensitivity is accompanied by significant peak sharpening. Sulfated CDs in the sample matrix allow for effective solubilization of hydrophobic analytes without the use of organic solvents such as methanol. The testing of various sample matrix s beta-CD concentrations for their effect on peak sharpening identified 3 to 5% as optimal for the estrogen standards. The use of a s beta-CD sample matrix allowed for hydrostatic injections (3.5 kPa) of 297 s, compared with 4 s when the analytes were dissolved in methanol. A mechanism explaining the s beta-CD-induced effect involves an analyte transfer mechanism where the s beta-CDs, despite providing anodic mobility to analytes in the sample zone, are able to transfer analytes to trailing separation buffer micelles for "recycling" back into the sample zone without compromising the stacking process. The overall improvement in sensitivity allows detection of estrogens in the parts-per-billion range and stands to improve the utility of MCE as a bioanalytical technique.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2003

Contamination of perilymph sampled from the basal cochlear turn with cerebrospinal fluid.

Our understanding of the perilymph kinetics of drugs depends largely on data obtained by the analysis of perilymph samples. Although a number of studies have demonstrated qualitatively that perilymph samples may be contaminated by cerebrospinal fluid (CSF), and some investigations adopt specific methods to minimize CSF contamination of their samples, many other studies fail to consider the influence of this potential artifact on their measurements. In the present study we have attempted to quantify the degree of CSF contamination of perilymph samples taken from the basal turn of the guinea pig cochlea using the ionic marker trimethylphenylammonium (TMPA). TMPA solution was irrigated across the round window membrane while a TMPA-selective electrode sealed into the perilymphatic space continuously monitored perilymph TMPA concentration. After a period of TMPA loading, a perilymph sample was aspirated and its TMPA content determined. Differences between the sample concentration and the measured TMPA time course during perilymph loading and sampling were analyzed using a finite element computer model for simulation of solute movements in the inner ear fluids. The experimental results were consistent with the aspirated fluid sample from the cochlea being replaced by CSF drawn into the perilymphatic space through the cochlear aqueduct. The dependence of perilymph sample purity on the location of sampling and on the volume withdrawn was quantified. These relationships are of value in the design and interpretation of experiments that utilize perilymph sampling.

Animals↗
PubMed2000

Integrated sample collection and handling for drug discovery bioanalysis.

An integrated sample handling process for drug discovery bioanalysis is described. The streamlining of study design, sample collection and automatic bioanalytical sample processing is demonstrated. Specific details for the entire procedure regarding the time saved, ease of automation and integration are defined. Details of sample handling involved a sample collection map, sample collection formatting and volume, dilution schemes for high concentration samples, choice of biological fluid and evaluating the capabilities of two liquid-handling workstations. Numerous comparisons were conducted between the new approaches and the conventional sample handling approaches. The precision and accuracy obtained from the new integrated sample handling process were comparable to those obtained from a conventional approach, as were pharmacokinetic profiles and parameters. This new sampling process greatly improved the efficiency of drug discovery bioanalysis. The integration of pre-clinical protocol design, sample collection and bioanalysis processes was also achieved.

Animals↗
PubMed1999

Direct determination of selenium and other trace elements in serum samples by ICP-MS.

Selenium belongs to a group of trace elements of special interest in biological samples for clinical diagnosis. Selenium has antioxidizing functions and is essential for providing the organism with triiodothyronine produced from thyroxine. Among several analytical techniques used to determine the Se concentration in serum, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has been used in the past because of its high sensitivity. Interference problems originating from different ions on the major Se isotopes have been described to be a limiting factor for the direct determination of Se in these matrices. Standard addition calibration or isotope dilution is often required to overcome carbon-enhanced ionisation effects in biological sample matrices. In most cases, the typical serum sample volume which is available for the analysis is limited to 0.5 ml or less, making multiple sample preparation for standard addition calibration impractical. Isotope dilution requires enriched isotopes and substantial sample preparation. Furthermore, the approximate Se concentration in every sample has to be known to adjust the appropriate amount of spike to each sample. Matrix matching with methanol has been described to overcome ionisation effects but we found limiting factors of this application when other trace elements are also determined within one sample run. This paper describes an effective sample preparation method which allows the direct determination of Se in serum without limiting the analytical capabilities for the additional determination of Al, Cu, Ni, Co, Cd, Mn and Zn in a single sample run by ICP-MS. Optimization procedures are presented and results of the analysis of reference samples are discussed, with a comparison of more than 150 serum data with those obtained by the GF-AAS method.

Animals↗
PubMedDate not supplied

Enhancing local health department disaster response capacity with rapid community needs assessments: validation of a computerized program for binary attribute cluster sampling.

INTRODUCTION: Local health departments are among the first agencies to respond to disasters or other mass emergencies. However, they often lack the ability to handle large-scale events. Plans including locally developed and deployed tools may enhance local response. Simplified cluster sampling methods can be useful in assessing community needs after a sudden-onset, short duration event. METHODS: Using an adaptation of the methodology used by the World Health Organization Expanded Programme on Immunization (EPI), a Microsoft Access-based application for two-stage cluster sampling of residential addresses in Louisville/Jefferson County Metro, Kentucky was developed. The sampling frame was derived from geographically referenced data on residential addresses and political districts available through the Louisville/Jefferson County Information Consortium (LOJIC). The program randomly selected 30 clusters, defined as election precincts, from within the area of interest, and then, randomly selected 10 residential addresses from each cluster. The program, called the Rapid Assessment Tools Package (RATP), was tested in terms of accuracy and precision using data on a dichotomous characteristic of residential addresses available from the local tax assessor database. A series of 30 samples were produced and analyzed with respect to their precision and accuracy in estimating the prevalence of the study attribute. Point estimates with 95% confidence intervals were calculated by determining the proportion of the study attribute values in each of the samples and compared with the population proportion. To estimate the design effect, corresponding simple random samples of 300 addresses were taken after each of the 30 cluster samples. RESULTS: The sample proportion fell within +/-10 absolute percentage points of the true proportion in 80% of the samples. In 93.3% of the samples, the point estimate fell within +/-12.5%, and 96.7% fell within +/-15%. All of the point estimates fell within +/-20% of the true proportion. Estimates of the design effect ranged from 0.926 to 1.436 (mean = 1.157, median = 1.170) for the 30 samples. CONCLUSIONS: Although prospective evaluation of its performance in field trials or a real emergency is required to confirm its utility, this study suggests that the RATP, a locally designed and deployed tool, may provide population-based estimates of community needs or the extent of event-related consequences that are precise enough to serve as the basis for the initial post-event decisions regarding relief efforts.

Cluster Analysis↗
PubMed1992

Determination of selenium by inductively coupled plasma mass spectrometry utilizing a new hydride generation sample introduction system.

An inductively coupled plasma mass spectrometer with a newly designed continuous flow hydride generator was used for the determination of Se in biological materials. The design of the hydride generator was important in minimizing interference from HCl and in maximizing analytical sensitivity. Two sample preparation procedures incorporating either 3.8 or 7.2 M HCl in the final sample solutions were compared. Interference from Cu was eliminated by the addition of 0.2 M NaI to the sodium borohydride solution (3.8 M method) or by maintaining a high concentration of HCl in the sample solution (7.2 M method). The 3.8 M method had the advantage of minimizing exposure of expensive equipment to corrosive HCl fumes, whereas the 7.2 M method did not contaminate equipment with I and had no measurable sample-to-sample cross-contamination. In practice, cross-contamination from sample to sample in both methods was negligible during analysis. An important factor in minimizing cross-contamination from sample to sample was the elimination of the air bubble normally entrained between samples. Determination of isotopic tracer enrichment was linear from 0 to 320% enrichment, which provided a broad range for isotope dilution analysis. A detection limit of 6.4 pg of Se was observed under optimum conditions, whereas a detection limit of 1.3 ng of Se was found for routine analysis of 1-g samples of plant material. Selenium was accurately determined by isotope dilution analysis in a variety of biological reference materials.

Animals↗
PubMed2001

Purge-and-membrane mass spectrometry, a screening method for analysis of VOCs from soil samples.

Purge-and-membrane mass spectrometry (PAM-MS) is a combination of dynamic headspace sampling and membrane extraction. A new and simple purge-and-membrane sampler is introduced and its basic testing results for the analysis of VOCs in soil samples are reported. Soil moisture had no effect on desorption times in the case of sand, but the desorption times increased when the content of organic matter in the soil sample (garden soil) increased. The longest desorption times were measured with dry garden soil samples. For both types of samples, minor differences in desorption peak areas were observed between 10 and 20% moisture. Detection limits of the VOCs varied in the range 2-150 microg/kg, depending on the soil type. Good linearity (correlation coefficient > 0.990) was observed in the range 0.5-50 mg/kg. Aging of the spiked soil samples had only a slight effect on desorption peak areas for samples stored at 5 degrees C up to two weeks, but after six months of storing, differences were observed between dry sand and moistened garden soil. In both cases, peak areas were diminished. On average, 46% of compounds could be desorbed from the aged sand and 86% from the aged garden soil. The modified vapor fortification method was used in preparing standard soil samples, which were analyzed by static headspace gas chromatography (HSGC) and PAM-MS. Some authentic soil samples were also analyzed using both of these techniques. Many of the vapor fortification samples and the authentic samples were also analyzed in another laboratory by HSGC. The agreement between the methods and the laboratories was generally good.

Environmental Monitoring↗
PubMed2002

Sample preparation for quantitation of tritium by accelerator mass spectrometry.

The capability to prepare samples accurately and reproducibly for analysis of tritium (3H) content by accelerator mass spectrometry (AMS) greatly facilitates isotopic tracer studies in which attomole levels of 3H can be measured in milligram-sized samples. A method has been developed to convert the hydrogen of organic samples to a solid, titanium hydride, which can be analyzed by AMS. Using a two-step process, the sample is first oxidized to carbon dioxide and water. In the second step, the water is transferred within a heated manifold into a quartz tube, reduced to hydrogen gas using zinc, and reacted with titanium powder. The 3H/1H ratio of the titanium hydride is measured by AMS and normalized to standards whose ratios were determined by decay counting to calculate the amount of 3H in the original sample. Water, organic compounds, and biological samples with 3H activities measured by liquid scintillation counting were utilized to develop and validate the method. The 3H/1H ratios were quantified in samples that spanned 5 orders of magnitude, from 10(-10) to 10(-15), with a detection limit of 3.0 x 10(-15), which is equivalent to 0.02 dpm tritium/mg of material. Samples smaller than 2 mg were analyzed following addition of 2 mg of a tritium-free-hydrogen carrier. Preparation of organic standards containing both 14C and 3H in 2-mg organic samples demonstrated that this sample preparation methodology can also be applied to quantify both of these isotopes from a single sample.

DNA↗
PubMed2003

Functional microfabricated sample targets for matrix-assisted laser desorption/ionization mass spectrometry analysis of ribonucleic acids.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful analytical tool for the structural characterization of proteins and nucleic acids. However, many proteomics or genomics methodologies that employ MALDI-MS require external sample manipulation, which limits the overall throughput of analysis. We have focused on fabricating functional MALDI sample plates that would permit the on-probe characterization of nucleic acids. Here, we present results arising from the fabrication of functional sample plates composed of poly(methyl methacrylate) (PMMA). The PMMA sample plates were fabricated by a CNC milling technique. The key structural feature of our microfabricated samples plates is the presence of individual cylindrical posts (360 microm x 360 microm), which serve as individual sample targets within the overall PMMA-based MALDI sample plate. Functionality is added to these microposts via the covalent attachment of enzymes. As an example of the applicability of these microfabricated sample plates, enzymatic digestion of ribonucleic acids was performed on probe (i.e., on the micropost) with subsequent analysis by MALDI-MS. Advantages to such an approach include a reduction in sample handling (and concomitant sample losses) and a reduction in the amount of sample required for analysis due to the small surface area of the microposts.

Microscopy, Confocal↗
PubMedDate not supplied

Integrated sampling procedure for metabolome analysis.

Metabolome analysis, the analysis of large sets of intracellular metabolites, has become an important systems analysis method in biotechnological and pharmaceutical research. In metabolic engineering, the integration of metabolome data with fluxome and proteome data into large-scale mathematical models promises to foster rational strategies for strain and cell line improvement. However, the development of reproducible sampling procedures for quantitative analysis of intracellular metabolite concentrations represents a major challenge, accomplishing (i) fast transfer of sample, (ii) efficient quenching of metabolism, (iii) quantitative metabolite extraction, and (iv) optimum sample conditioning for subsequent quantitative analysis. In addressing these requirements, we propose an integrated sampling procedure. Simultaneous quenching and quantitative extraction of intracellular metabolites were realized by short-time exposure of cells to temperatures < or =95 degrees C, where intracellular metabolites are released quantitatively. Based on these findings, we combined principles of heat transfer with knowledge on physiology, for example, turnover rates of energy metabolites, to develop an optimized sampling procedure based on a coiled single tube heat exchanger. As a result, this sampling procedure enables reliable and reproducible measurements through (i) the integration of three unit operations into a one unit operation, (ii) the avoidance of any alteration of the sample due to chemical reagents in quenching and extraction, and (iii) automation. A sampling frequency of 5 s(-)(1) and an overall individual sample processing time faster than 30 s allow observing responses of intracellular metabolite concentrations to extracellular stimuli on a subsecond time scale. Recovery and reliability of the unit operations were analyzed. Impact of sample conditioning on subsequent IC-MS analysis of metabolites was examined as well. The integrated sampling procedure was validated through consistent results from steady-state metabolite analysis of Escherichia coli cultivated in a chemostat at D = 0.1 h(-)(1).

Adenosine Triphosphate↗
PubMed2002

Preserving the distribution of inorganic arsenic species in groundwater and acid mine drainage samples.

The distribution of inorganic arsenic species must be preserved in the field to eliminate changes caused by metal oxyhydroxide precipitation, photochemical oxidation, and redox reactions. Arsenic species sorb to iron and manganese oxyhydroxide precipitates, and arsenite can be oxidized to arsenate by photolytically produced free radicals in many sample matrices. Several preservatives were evaluated to minimize metal oxyhydroxide precipitation, such as inorganic acids and ethylenediaminetetraacetic acid (EDTA). EDTA was found to work best for all sample matrices tested. Storing samples in opaque polyethylene bottles eliminated the effects of photochemical reactions. The preservation technique was tested on 71 groundwater and six acid mine drainage samples. Concentrations in groundwater samples reached 720 microg-As/L for arsenite and 1080 microg-As/L for arsenate, and acid mine drainage samples reached 13 000 microg-As/L for arsenite and 3700 microg-As/L for arsenate. The arsenic species distribution in the samples ranged from 0 to 90% arsenite. The stability of the preservation technique was established by comparing laboratory arsenic speciation results for samples preserved in the field to results for subsamples speciated onsite. Statistical analyses indicated that the difference between arsenite and arsenate concentrations for samples preserved with EDTA in opaque bottles and field speciation results were analytically insignificant. The percentage change in arsenite:arsenate ratios for a preserved acid mine drainage sample and groundwater sample during a 3-month period was -5 and +3%, respectively.

Adsorption↗
PubMed1996

Serial versus sparse sampling in toxicokinetic studies.

PURPOSE: Sparse sampling in rodent toxicokinetics usually involves the collection of a single blood sample on a given study day from each animal in a treatment group. The samples are allocated to different time points, often allowing some replicates, and statistical inferences are then made about the concentration-time behavior of the test compound. The present study compared the results of one such analysis with those obtained from serial sampling as might be applied using satellite animals. METHODS: Ten rats each received a single oral dose of tritium-labeled compound X. Blood concentrations in each rat at 10 time points post-dose were determined by liquid scintillation counting. Individual peak concentrations of blood radioactivity (Cmax) and peak times (tmax) were recorded, and area-under-curve (AUC) values were calculated by trapezoidal rule. The mean AUC and Cmax of all 10 animals over all 10 time points, referred to as AUCtrue and Cmax,true, were used as points of reference. These values were then estimated using subsets of the data that simulated satellite-animal or sparse-sampling designs. First, several different sampling schedules of 5 bleeding times were stimulated by taking subsets of the full data set. For analysis using satellite animals, serial blood concentrations from subsets of 3 or 4 rats were used to calculate point and confidence-interval estimates of AUCtrue and Cmax,true by standard methods; all possible subsets of 3 or 4 of the 10 rats were considered. For sparse data analysis, a single concentration from each of the 10 rats was used to calculate both point and confidence-interval estimates of AUCtrue by the Bailer-Satterthwaite method, and point estimates of Cmax,true, according to several different designs of replication. Animals were randomly assigned to time points, and 1000 of over 50000 possible combinations were evaluated for each bleeding schedule. The average percent absolute errors of the point estimates were computed and, for the 95% confidence intervals, average widths were determined. RESULTS: For point estimates of AUCtrue, sparse sampling yielded average percent absolute errors of 7-13%. Percent errors for 3 and 4 satellite animals were 6-12% and 5-10%, respectively. For 95% confidence intervals, sparse sampling yielded widths of 24-90% of AUCtrue, whereas for 3 and 4 satellite animals widths were 37-50% and 24-34%, respectively. For Cmax,true point estimates from sparse-sampling and satellite-animal approaches had average percent absolute errors of 5-12% and 3-8%, respectively. The confidence-interval widths for Cmax,true from the satellite-animal approach were 15-24% of Cmax,true, but coverage did not achieve the nominal 95% for some choices of sampling times. CONCLUSIONS: By using proper study designs, one can limit the number of samples and the amount of blood drawn so as not to affect the animals' health status, yet still achieve the customary pharmacokinetic objectives in a toxicity study.

Animals↗
PubMed2000

Ventilator associated pneumonia: quality of nonbronchoscopic bronchoalveolar lavage sample affects diagnostic yield.

The importance of predefined criteria for acceptable samples of respiratory therapists obtained lower respiratory samples were studied, using a nonbronchoscopic bronchoalveolar lavage (BAL) protocol for ventilated patients in the intensive care unit. Therapists were instructed and asked to follow guidelines for obtaining samples. Over one year, 219 samples were obtained by respiratory therapists. Of these, 115 were considered to be adequate samples using the following criteria: 60 mL of instilled volume, at least 5 mL of fluid aspirated, specimens sent for semiquantitative culture, a differential cell count of <5% bronchial epithelial cells. Overall, 52 samples grew one or more pathogen at >10,000 colony forming units (cfu).mL(-1) of BAL. The most common pathogen was Staphylococcus aureus (S. aureus) (11 samples), although Gram-negative bacilli were the single pathogen in 21 specimens. Of the 115 acceptable samples, 40 (35%) grew > or =1 pathogen at >10,000 cfu.mL(-1). For the 80 not acceptable samples which were sent for appropriate culture, 12 (15%) grew >10,000 cfu.mL(-1) BAL. This difference was significant (Chi-squared=9.44, p<0.01). Nonbronchoscopic bronchoalveolar lavage can be safely performed by respiratory therapists'. The authors recommend that a protocol be used to evaluate the quality of a bronchoalveolar lavage sample in the same manner sputum samples are screened prior to interpretation.

Bronchoalveolar Lavage Fluid↗
PubMed1999

Vapor pressure osmometry: minimum sample microvolumes.

PURPOSE: Vapor pressure osmometers are currently designed to handle sample volumes as small as 2.0 microliters (microl) but smaller sample volumes are desirable in tear fluid studies. We determined the minimum sample size required for adequate validity and repeatability of osmolality measurements. METHODS: A standard saline solution (290 mmol/kg) was sampled by a variable, calibrated pipette. Forty samples were processed with a Wescor 5520 vapor pressure osmometer at each of the following volumes: 2.0, 1.6, 1.2, 1.0, 0.8, 0.7, 0.6 and 0.5 microl. Prior to each test series, the instrument was calibrated with the identical volume of 290 mmol/kg saline. Relevant descriptive statistics were computed and an analysis of variance (ANOVA) was performed on the resulting data. RESULTS: The mean osmolalities of the eight 40-sample groups ranged from 288.42 to 290.68 mmol/kg and were not significantly different from 290 mmol/ kg or each other (p>0.05). The standard deviations were inversely correlated with the sample volumes, gradually increased to approximately 1% as sample volume was reduced to 0.8 microl, then more rapidly increased as the sample volume was lowered still further. CONCLUSION: Sample microvolumes as small as 0.8 microl can be collected for accurate and repeatable results with the Wescor 5520 vapor pressure osmometer when a standard deviation of approximately 1% is acceptable. Microvolumes from 0.7 to 0.5 microl may also be used if the expanded spread of data can be offset by multiple repeated readings. Using a 2.0 microl sample volume, the ultimate accuracy and repeatability of the Wescor vapor pressure osmometer was +/-2% at 290 mmol/kg: 99%, of all readings (+/-3 standard deviations) should fall within +/-6 mmol/kg of the true value.

Equipment Design↗
PubMed2005

Learning processes in matching and oddity: the oddity preference effect and sample reinforcement.

Eight pigeons learned either matching (to sample) or oddity (from sample) with or without reward for sample responding. The training stimuli were coarse-white, fine-black, or smooth-mauve gravels in pots with buried grain as the reinforcer. Oddity without sample reward was learned most rapidly, followed by matching with sample reward, oddity with sample reward, and matching without sample reward. Transfer was related to acquisition rate: The oddity group without sample reward showed full (equal to baseline) color and texture transfer; the matching group with sample reward showed partial texture transfer; other groups showed no transfer. Sample reward was shown to determine rate of acquisition of matching and oddity and the oddity preference effect. The results are discussed in terms of item-specific associations operating early in learning prior to any relational learning between sample and comparison stimuli.

Analysis of Variance↗
PubMed1994

Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay.

The methyl-thiazol-tetrazolium (MTT) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > or = 80% leukaemic cells at the start of culture give similar results in the MTT assay and the differential staining cytotoxicity assay, in which a discrimination between malignant and non-malignant cells can be made. However, the percentage of leukaemic cells may change during culture, which might affect the results of the MTT assay. We studied 106 untreated childhood acute lymphoblastic leukemia (ALL) samples with > or = 80% leukaemic cells at the start of culture. This percentage decreased below 80% in 28%, and below 70% in 13%, of the samples after 4 days of culture. A decrease below 70% occurred more often in case of 80-89% leukaemic cells (9/29) than in case of > or = 90% leukaemic cells at the start of culture (5/77, P = 0.0009). Samples with < 70% leukaemic cells after culture were significantly more resistant to 6 out of 13 drugs, and showed a trend towards being more resistant to two more drugs, than samples with > or = 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > or = 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunoamagnetic beads. Using a beads to target cell ratio of 10:1, the percentage of leukaemic cells increased from mean 72% (s.d. 9.3%) to mean 87% (s.d. 6.7%), with an absolute increase of 2-35%. The recovery of leukaemic cells was mean 82.1% (range 56-100%, s.d. 14.0%). The procedure itself did not influence the results of the MTT assay in three samples containing only leukaemic cells. We conclude that it is important to determine the percentage of leukaemic cells at the start and at the end of the MTT assay and similar drug resistance assays. Contaminating mononuclear cells can be successfully removed from ALL samples using immunomagnetic beads. This approach may increase the number of leukaemic samples which can be evaluated for cellular drug resistance with the MTT assay or a similar cell culture drug resistance assay.

Cell Separation↗