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PubMed2004

Use of the quantitative fluorescent-PCR assay in the study of fetal DNA from micromanipulated transcervical samples.

AIM: The purpose of this study was to evaluate the validity of the combined use of micromanipulation and quantitative fluorescent (QF)-PCR assay for the identification of fetal elements in transcervical cell (TCC) samples collected in early pregnancy. METHODS: TCC samples were obtained by intrauterine lavage (IUL) in 113 pregnant women who were between 7 and 12 weeks pregnant before termination of pregnancy. All IUL samples were screened under an inverted microscope, at which time the isolation of fetal cells by micromanipulation was attempted. QF-PCR assay, using 9 small tandem repeat (STR) markers for chromosomes 13, 18, 21, X, and Y, was performed in all specimens to identify fetal cells in TCC samples and the corresponding placental tissue and blood specimens. TCC samples from male fetuses in which either the micromanipulation or QF-PCR analysis were unsuccessful, were studied with fluorescent in situ hybridization (FISH), using probes for X and Y chromosomes. RESULTS: Isolation of supposed fetal material from IUL samples was carried out by means of micromanipulation in 93 cases (82.3%), where discernible chorionic villous filaments or cell clumps of probable trophoblastic origin were present. The QF-PCR analysis was performed in all 93 IUL samples and paternal peaks could be documented in 88 cases (94.6%) thus confirming the presence of fetal cells. Thirteen cases negative to micromanipulation and derived from male fetuses and four male cases not informative with QF-PCR analysis, after micromanipulation, were then tested with FISH assay using probes for sexual chromosomes. In six samples, rare (2-3%) male fetal cells were detected. Considering the combined results obtained from QF-PCR and FISH assays, the overall fetal sexing was correct in 83.2% of cases (94 of 113). CONCLUSION: This study provides evidence that fetal cells are present in a high proportion of IUL samples. Micromanipulation appears to be an extremely efficient method for the isolation of trophoblastic elements. This study also confirms the potential of IUL as a possible alternative to the traditional prenatal diagnostic procedures for the recovery of fetal cells in precocious stage of gestation, and validates the combination of the isolation of such fetal elements by means of micromanipulation and analysis with the QF-PCR assay for the identification of the most frequent prenatal chromosomal aneuploidies.

Cervix Uteri↗
PubMed1999

Measurement of blood serum cyclosporine levels using capillary "fingerstick" sampling: a validation study.

Capillary blood sampling as a means of monitoring blood cyclosporine levels has replaced venipuncture in some medical centers. As the validity of capillary venipuncture for analysis of cyclosporine has not been documented, we sought to validate the capillary blood collection technique by comparing it with serum samples collected simultaneously by venous phlebotomy. Forty paired capillary- and venous samples were collected from 36 cardiac transplantation patients and analyzed, using a polyclonal immunoassay. The values obtained were compared using regression correlation. The correlation coefficient for all 40 samples was 0.859. However, we discovered that the first 7 capillary specimens were processed incorrectly. The correlation coefficient for the other 33 samples was 0.995 (99% confidence interval 0.987-0.998). The excellent correlation between serum samples obtained from capillary sampling and from venous sampling, together with the ease of obtaining capillary blood specimens, make "fingerstick" sampling the method of choice for monitoring cyclosporine levels in infants and children.

Adult↗
PubMed2002

Determination of gallium in soil by slurry-sampling graphite-furnace atomic-absorption spectrometry.

A graphite-furnace atomic-absorption spectrometric method, utilizing ultrasonic slurry-sampling has been developed for the determination of Ga in soils. Calibration with aqueous standards and with slurries prepared from a certified soil reference material were both employed. When calibration with soil slurries was used no modifier was needed. Because lower and more variable sensitivity was obtained for Ga in aqueous standards than for Ga in slurry soil samples, external calibration with aqueous Ga standards required a suitable chemical modifier to level out the sensitivity difference. Of the many potential modifiers tested, i.e. Al, As, Co, Mg, Mo, Ni, Pd, Pd+Mg, Se, and Te, Ni was found to be best. When Ni (1.0 mg mL(-1), 10 micro L) was injected to the graphite tube with the aqueous standards or slurry samples (10 micro L) accurate results were obtained. Both methods of calibration gave acceptable accuracy and precision. The repeatability was <or=4.6% (RSD) for slurry sampling for both methods of calibration. The intermediate precision for analyses performed on three different days was 7.3% and 4.8% when using aqueous standards and slurry standards, respectively. The limit of detection for Ga was 3.0 micro g L(-1)in soil slurry solution, equivalent to 0.08 micro g g(-1) in the soil samples. Results from slurry sampling were compared with results from microwave-digested samples. Chemical interference was observed when the acid-mixture used to dissolve the samples contained HF. This interference was eliminated when Ni was added as chemical modifier. The repeatability was <or=7.2% (RSD) for analysis of decomposed samples.

Journal Article↗
PubMed2003

Determination of methylmercury in environmental samples using static headspace gas chromatography and atomic fluorescence detection after aqueous phase ethylation.

A rapid and automated method for the determination of monomethylmercury (MMHg) in environmental samples was developed using headspace gas chromatography with atomic fluorescence detection in combination with aqueous phase ethylation. Sample preparation steps were optimized for sediments, biological samples, and water samples using certified reference materials and real samples with a broad range of MMHg concentrations. Different extraction procedures were compared for both sediments and biological samples. The methods were applied in the intercomparison exercises for the certification of MMHg in sediments (IAEA 405) and in Oyster tissue (BCR 710) and the results were accepted for certification. The detection limits for MMHg are 0.002 ng Hg/g for sediments and biological samples and 0.01 ng Hg/L for water samples. The method was tested for methylation artifacts; no artifact was observed in the sediment samples and CRMs tested.

Journal Article↗
PubMed2004

Determination of trace amounts of gold in acid-attacked environmental samples by atomic absorption spectrometry with electrothermal atomization after preconcentration.

A method for determination of trace amounts of gold in environmental samples (rocks, soils, sediments, and waters) by atomic absorption spectrometry with electrothermal atomization (ETAAS) after preconcentration using a chelating sorbent Spheron Thiol 1000 is described. The method accurately determines gold between 0.001 and several tens of grams per ton in samples having complex variations in mineralogy. Pulverized samples are roasted at 650 degrees C to oxidize any sulfide and/or carbonaceous material. Samples are then subjected to a series of acid treatments to eliminate any silica matrix and to dissolve the sample. The Spheron Thiol 1000 is added to the sample solution, and then with sorbed gold is filtered out, washed, and ignited at 550 degrees C. The residue is dissolved in aqua regia, evaporated, dissolved in distilled water, transferred to a volumetric flask, and analyzed by ETAAS. The limits of detection of gold, based on the 3 sigma definition, were 0.5 ng g(-1) for 10-g samples (rocks, sediments, soils) and 0.05 ng mL(-1) for 1-L water samples. Precision of determination expressed by the relative standard deviation varied from 2.9% to 16.4%. The accuracy of the method is verified by analysis of certified reference materials. The obtained analytical results are in good agreement with attested values. The developed method was applied for gold determination in environmental samples affected by the acidification (acid mine drainage which is mainly a product of pyrite oxidation) from an open quartzite mine in the Sobov region situated NE of the city of Banská Stiavnica (Slovakia).

Journal Article↗
PubMed2005

Simultaneous multi-species determination of trimethyllead, monomethylmercury and three butyltin compounds by species-specific isotope dilution GC-ICP-MS in biological samples.

An accurate and sensitive multi-species species-specific isotope dilution GC-ICP-MS method was developed for the simultaneous determination of trimethyllead (Me3Pb+), monomethylmercury (MeHg+) and the three butyltin species Bu3Sn+, Bu2Sn2+, and BuSn3+ in biological samples. The method was validated by three biological reference materials (CRM 477, mussel tissue certified for butyltins; CRM 463, tuna fish certified for MeHg+; DORM 2, dogfish muscle certified for MeHg+). Under certain conditions, and with minor modifications of the sample pretreatment procedure, this method could also be transferred to environmental samples such as sediments, as demonstrated by analyzing sediment reference material BCR 646 (freshwater sediment, certified for butyltins). The detection limits of the multi-species GC-ICP-IDMS method for biological samples were 1.4 ng g(-1) for MeHg+, 0.06 ng g(-1) for Me3Pb+, 0.3 ng g(-1) for BuSn3+ and Bu3Sn+, and 1.2 ng g(-1) for Bu2Sn2+. Because of the high relevance of these heavy metal alkyl species to the quality assurance of seafood, the method was also applied to corresponding samples purchased from a supermarket. The methylated lead fraction in these samples, correlated to total lead, varied over a broad range (from 0.01% to 7.6%). On the other hand, the MeHg+ fraction was much higher, normally in the range of 80-100%. Considering that we may expect tighter legislative limitations on MeHg+ levels in seafood in the future, we found the highest methylmercury contents (up to 10.6 microg g(-1)) in two shark samples, an animal which is at the end of the marine food chain, whereas MeHg+ contents of less than 0.2 microg g(-1) were found in most other seafood samples; these results correlate with the idea that MeHg+ is usually of biological origin in the marine environment. The concentration of butyltins and the fraction of the total tin content that is from butyltins strongly depend on possible contamination, due to the exclusively anthropogenic character of these compounds. A broad variation in the butylated tin fraction (in the range of <0.3-49%) was therefore observed in different seafood samples. Corresponding isotope-labeled spike compounds (except for trimethyllead) are commercially available for all of these compounds, and since these can be used in the multi-species species-specific GC-ICP-IDMS method developed here, this technique shows great potential for routine analysis in the future.

Animals↗
PubMed2000

Determination of analytical limits in solid sampling ETAAS: a new approach towards the characterization of analytical quality in rapid methods.

In the present study the lower analytical limits of solid sampling electrothermal atomization atomic absorption spectrometry (SS-ETAAS) were characterized by means of blank measurements and--for the first time--by means of the calibration curve method, where a calibration near the range of these limits (limit of decision, detection and quantification) was performed. The limit of decision as derived from blank measurements was calculated according to the 3sigma-criterion to be 0.003 and 0.019 ng for Cd and Pb, respectively. For Pb and Cd a roughly three-fold increase of these limits was observed when the calibration method according to DIN 32 645 was applied. When solid reference material was used, only a slight increase could be observed. The analytical limits were 2 to 20 times lower than reported for sample decomposition methods. The blank measurement and conventional calibration curve method, however, do not account for factors relating to solid sampling such as sample mass and matrix. Therefore, the calibration curve model was applied to data derived from comparisons between direct solid sampling ETAAS and a compound reference method (ETAAS following sample homogenization and digestion). The observed analytical limits were not found to be substantially increased if enough samples with low element contents were available for calibration. Coupling of the calibration curve model with the comparison of methods included real test samples and thus the relevant maximum sample mass and analyte content in the range of the lower analytical limits. As validation procedures frequently include comparisons of methods, the present approach might prove to be of some general interest for the characterization of analytical quality in rapid methods.

Animals↗
PubMed1999

Development of a general method of limited sampling for the determination of AUC for a drug that displays two-compartment pharmacokinetics.

OBJECTIVES: To develop a method of limited sampling that would enable accurate estimation of the area under the concentration time curve (AUC) when using the log trapezoidal method. METHODS: A series of datasets were simulated. Each dataset comprised 1000 subjects. Each subject was "administered" an intravenous bolus dose of a drug that displays two compartment pharmacokinetics. In the first series of simulations, a variety of combinations of the number of sampling times (K) and number of replicate measurements (R) at each of these times were tested, where K x R = 12 (i.e. N = 12). The times that each of the K samples were taken were chosen to be those that divided the AUC into K - 1 trapezoids of equal area. The concentration-time curves were estimated based on a priori estimates of the population parameters. The best combination of K and R was tested under various conditions of parameter variability and assay variability. The combinations were compared with a conventional sampling strategy, where N = 12, K = 12 (R = 1). RESULTS: The combination K = 4 and R = 3 proved to be the "best". It had similar accuracy to the conventional method. The best limited sampling combination was superior to the conventional method when assay variability was high (CV= 30%), was similar when assay variability was 15%, but the conventional method became statistically superior when assay variability was 7.5% or less. The accuracy of the best limited sampling combination was inversely related to the parameter variability. If K was set to 4 and R allowed to increase to 6 (i.e. N is not equal to 12), there was no further gain in accuracy. CONCLUSION: The proposed method of limited sampling is at least as accurate as the conventional intensive sampling technique, but more efficient in terms of sampling.

Area Under Curve↗
PubMed1998

Design, construction and six years' experience of an integrated system for automated handling of discrete blood samples.

The present paper describes the design of an integrated system to aid in the taking and measurement of manual blood samples during nuclear medical examinations requiring blood sampling. In contrast to previously published systems, the present system is not used in the actual sampling of the blood, but aims to aid in all other aspects of handling and measurement. It consists of two main parts. One part is a distributed software system running on the scanner host computer used to register sample times, to display information pertaining to the ongoing examination and to collect data from a number of well crystals. The other main part consists of an industrial robot used to perform the actual weighing, centrifugation, pipetting and measurement of the samples. The system has been operational for 6 years, during which time it has had an "up-time" in excess of 95% and has handled and measured the blood samples from more than 5000 examinations, each comprising an average of 15 blood samples. The throughput of the system is 50 whole blood samples or 21 plasma samples per hour. In addition it has to a large extent removed the "human factor" from the process, thereby increasing the reliability of the data.

Automation↗
PubMed1999

Time-related bisphenol-A content and estrogenic activity in saliva samples collected in relation to placement of fissure sealants.

It was recently reported that estrogenic activity was detected in saliva samples collected during 1 h after placement of one fissure sealant (Delton) and this related to Bisphenol-A (BPA) content. The aim of the present study was to determine the time-related BPA content and estrogenic activity in saliva samples collected before and after placement of two fissure sealants each with a different monomer composition. Eight healthy male volunteers with no history of prior placement of fissure sealants or composite resin fillings had four molars sealed with either Delton LC (four people) or Visio-Seal (four people). Base-line saliva samples were collected preexperimentally, in the morning when fasting. Fissure sealants were placed and saliva samples collected immediately, 1 h and 24 hs after placement of the fissure sealant. BPA was found in saliva samples collected immediately after placement of Delton LC (range 0.3-2.8 ppm). No detectable amounts of BPA were determined 1 h and 24 h after Delton treatment (detection limit < or = 0.1 ppm). In base-line samples and in all samples collected from Visio-Seal treated individuals, no BPA was detected. In a recombinant yeast cell assay, significantly increased estrogenic activity was found in saliva samples collected immediately after placement of Delton LC sealant (P < 0.05; ANOVA) whereas no statistically significant estrogenic activity was observed in the remaining groups. In conclusion, minute amounts of BPA, however considerably lower than previously reported, were detected in saliva samples collected immediately after but not 1 and 24 h(s) after placement of Delton LC fissure sealant. BPA was not detected after placement of Visio-Seal fissure sealant.

Adult↗
PubMed2006

Estimation of heavy metals in different berberis species and its market samples.

Popularity of herbal drugs is increasing all over the world because of lesser side effects as compared to synthetic drugs. Besides, it costs less and easily available to poor people particularly in developing countries. But quality assurance of herbal drugs is very necessary prior to its use. Because in today's polluted environment, even herbal drugs are not safe. Berberis spp. is very important medicinal plant, having various medicinal properties. It is also included in Indian and British pharmacopoeias. Its demand is quite high in herbal drug market. So, to check the quality of market samples of this drug, ten different samples were procured from different drug markets of India for heavy metal estimation. Besides, genuine samples of four species of Berberis viz. B. aristata, B. chitria, B. lycium, B. asiatica were also collected from natural habitats to compare heavy metal concentration in both market and genuine samples. It was found that market samples were much more contaminated than genuine samples. Lead (Pb) concentration is far beyond from WHO permissible limit (10 ppm) for herbal drugs, reaching to maximum 49.75 ppm in Amritsar market sample. Likewise, concentration of all other metals like Cd, Cr and Ni were also very high in market samples as compared to genuine samples.

Berberis↗
PubMed2006

Evaluation of storage and filtration protocols for alpine/subalpine lake water quality samples.

Many government agencies and other organizations sample natural alpine and subalpine surface waters using varying protocols for sample storage and filtration. Simplification of protocols would be beneficial if it could be shown that sample quality is unaffected. In this study, samples collected from low ionic strength waters in alpine and subalpine lake inlets and outlets in the western United States were used to evaluate (1) effects of refrigerated storage time on the chemistry of unfiltered samples, and (2) differences in sample filtration protocols. No analytes exhibited significant changes when stored less than 48 h. Six analytes (pH, sodium, ammonium, potassium, chloride, sulfate) exhibited statistically significant (but small) changes when storage time exceeded 48 h. Two analytes (calcium, nitrate) were significantly higher when samples were field filtered than when filtered in the laboratory, but the differences were also small. For waters similar to those in this test, unfiltered refrigerated samples may be stored up to 48 h without compromising sample quality. The small differences between field and lab filtration do not justify the expense, training, and contamination risk of field filtration.

Colorado↗
PubMed2005

Optimal blood sampling time windows for parameter estimation using a population approach: design of a phase II clinical trial.

The objective of this paper is to determine optimal blood sampling time windows for the estimation of pharmacokinetic (PK) parameters by a population approach within the clinical constraints. A population PK model was developed to describe a reference phase II PK dataset. Using this model and the parameter estimates, D-optimal sampling times were determined by optimising the determinant of the population Fisher information matrix (PFIM) using PFIM_ _M 1.2 and the modified Fedorov exchange algorithm. Optimal sampling time windows were then determined by allowing the D-optimal windows design to result in a specified level of efficiency when compared to the fixed-times D-optimal design. The best results were obtained when K(a) and IIV on K(a) were fixed. Windows were determined using this approach assuming 90% level of efficiency and uniform sample distribution. Four optimal sampling time windows were determined as follow: at trough between 22 h and new drug administration; between 2 and 4 h after dose for all patients; and for 1/3 of the patients only 2 sampling time windows between 4 and 10 h after dose, equal to [4 h-5 h 05] and [9 h 10-10 h]. This work permitted the determination of an optimal design, with suitable sampling time windows which was then evaluated by simulations. The sampling time windows will be used to define the sampling schedule in a prospective phase II study.

Administration, Oral↗
PubMed2006

Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins.

The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.

Carbon Isotopes↗
PubMed1982

A comparative study of some physico-chemical properties of human serum albumin samples from different sources--I. Some physico-chemical properties of isoionic human serum albumin solutions.

Human serum albumin samples from different sources were investigated. The fatty acid content of the albumin before and after deionization on a mixed bed ion-exchange column varied from sample to sample. When an albumin sample from one source was deionized under standard conditions the amount of fatty acid bound by the albumin was reduced to a reproducible amount. In samples from different sources, however, the amount bound varied considerably. Also the isoionic pH of the albumin varied from sample to sample. This variation could be attributed to the difference in the fatty acid content and the different number of titratable histidines and acid amino-acid residues in the albumin from different sources. It can be concluded from the specific conductance of these isoionic solutions that ions other than H-, OH and protein are effectively removed by a mixed bed ion-exchange column. The specific conductance of the albumin samples is directly related to the isoionic pH. Therefore, the isoionic pH and the specific conductance of the albumins reflect the heterogeneity of the albumin samples with respect to their primary structure and fatty acid content.

Chemical Phenomena↗
PubMed1985

Evolution of efficient methods to sample lead sources, such as house dust and hand dust, in the homes of children.

Efficient sampling methods to recover lead-containing house dust and hand dust have been evolved so that sufficient lead is collected for analysis, and to ensure that correlational analyses linking these two parameters to blood lead are not dependent on the efficiency of sampling. Precise collection of loose house dust from a 1-unit area (484 cm2) with a Tygon or stainless steel sampling tube connected to a portable sampling pump (1.2 to 2.5 liters/min) required repetitive sampling (three times). The Tygon tube sampling technique for loose house dust less than 177 microns in diameter was around 72% efficient with respect to dust weight and lead collection. A representative house dust contained 81% of its total weight in this fraction. A single handwipe for applied loose hand dust was not acceptably efficient or precise, and at least three wipes were necessary to achieve recoveries of greater than 80% of the lead applied. House dusts of different particle sizes less than 246 microns adhered equally well to hands. Analysis of lead-containing material usually required at least three digestions/decantations using hot plate or microwave techniques to allow at least 90% of the lead to be recovered. It was recommended that other investigators validate their handwiping, house dust sampling, and digestion techniques to facilitate comparison of results across studies. The final methodology for the Cincinnati longitudinal study was three sampling passes for surface dust using a stainless steel sampling tube; three microwave digestions/decantations for analysis of dust and paint; and three wipes with handwipes with one digestion/decantation for the analysis of six handwipes together.

Adult↗
PubMed1988

Adaptive sampling of cardiac waveforms.

In recent years cardiac electrocardiograms and electrograms often have been measured using automated sampling systems that record samples periodically, with a fixed sample-to-sample interval. In contrast, adaptive sampling systems envision variable sample-to-sample intervals, and new information about such systems has emerged in the last few years. The author summarizes these reports. On the whole, they show that the fan algorithm is an effective basis for adaptive sampling of cardiac waveforms, that it works well on electrocardiograms, electrograms, and action potentials, and that prototype sampling systems based on the fan can be constructed to run in real time.

Algorithms↗
PubMed1996

Rapid sampling, cell inactivation and evaluation of low extracellular glucose concentrations during fed-batch cultivation.

A method for rapid extracellular sampling, cell inactivation and handling of a large number samples has been developed and evaluated. This method might be used during experiments, where concentrations of extracellular components in the range of some milligrams per litre has to be evaluated. The analysis should be performed by enzymatic or colorimetric analysis and is shown to be suitable for measuring low nutrient concentrations present in fed-batch cultivation. The test organisms were Escherichia coli and Saccharomyces cerevisiae and the model substance was glucose. Using this technique, the sample can be taken in less than 0.15 s, a time during which negligible glucose is shown to be consumed from the sample. The glucose consumption is stopped by a rapid pH decrease using perchloric acid in a defined concentration that depended on the organism studied. This concentration was chosen in order to avoid cell lysis that could affect the glucose concentration by the intracellular release of glucose containing compounds and thereby expose them to the acid. It was seen that the sample mixed rapidly with the acid and that no hydrolysis of glucose related compounds interfered with the analysis. The samples were neutralised and the precipitation centrifuged to minimise the effect on the chosen enzymatic analysis. This analysis was modified and the accuracy determined in order to analyse concentrations in the milligram range. A method to increase the handling of a large number of samples was also devised which is based on measurements on microtiter plates and allows samples to be evaluated with a high statistical accuracy with minimum sample waste in a short time. The ability of the method is shown by a study of the metabolic response of a shift in glucose feed, as followed by glucose analysis.

Cell Division↗