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PubMed2006

Views of female breast cancer patients who donated biologic samples regarding storage and use of samples for genetic research.

Although social and ethical issues related to the storage and use of biologic specimens for genetic research have been discussed extensively in the medical literature, few empiric data exist describing patients' views. This qualitative study explored the views of 26 female breast cancer patients who had consented to donate blood or tissue samples for breast cancer research. Participants generally did not expect personal benefits from research and had few unprompted concerns. Few participants had concerns about use of samples for studies not planned at the time of consent. Some participants did express concerns about insurance or employment discrimination, while others believed that current privacy protections might actually slow breast cancer research. Participants were generally more interested in receiving individual genetic test results from research studies than aggregate results. Most participants did not want individual results of uncertain clinical significance, although others believed that they should be able to receive such information. These data examined the range of participants' views regarding the storage and use of biologic samples. Further research with different and diverse patient populations is critical to establishing an appropriate balance between protecting the rights of human subjects in genetic research and allowing research to progress.

Breast Neoplasms↗
PubMed1992

Advantages of achiral h.p.l.c. as a preparative step for chiral analysis in biological samples and its use in toxicokinetic studies.

1. Achiral reverse-phase h.p.l.c. with semi-automated post-column fraction collection and solid-phase sample reconcentration, has been applied as the purification procedure during the enantiomeric quantification of two widely differing experimental drugs; an HMG-CoA reductase inhibitor (I) and an alpha 2-adrenoceptor antagonist (II). 2. The robust and specific achiral methodologies were available prior to the need for chiral analyses and recovery of drug from the fractions provided clean samples from a variety of biological matrices, without the need to develop compatible achiral/chiral mobile phases. 3. Compared with direct chiral chromatography of plasma extracts, this approach decreased the potential for metabolites and endogenous components to interfere or impair the performance of the chiral stationary phase. 4. The availability of quantitative data from achiral analysis of samples negated the need for internal standardization of the chiral analyses, helped confirm assay specificity and provided potential to determine enantiomeric ratios where only one isomer could be accurately measured. 5. Routine enantiomeric analyses were successfully carried out on samples taken from animals dosed orally with the racemic drugs, providing important data on the possible levels of exposure to individual enantiomers during toxicity testing.

Animals↗
PubMed1998

A round-robin determination of boron in botanical and biological samples.

The accurate determination of boron (B) at trace and ultratrace concentrations is an important step toward establishing the role of B in biological functions. However, low-level B concentrations are difficult to determine accurately, especially for many botanical and biological matrices. A round-robin study was conducted to assess analytical agreement for low-level B determinations. Ten experienced research groups from analytical laboratories extending across Europe, Asia, and the US participated in this study. These groups represent a cross-section of academic, commercial, and government facilities. The researchers employed both ion-coupled plasma and neutron techniques in the study. Results from this round-robin study indicate good agreement between participating laboratories at the mg/kg level, but at the lowest levels, microg/kg, only three laboratories participated, and agreement was poor. By encouraging discussion among scientists over these data, the secondary goal of this round-robin study is to stimulate continued improvement in analytical procedures and techniques for accurate low-level B determinations. Furthermore, it is intended to encourage the development of a variety of low-level (low mg/kg and microg/kg) B certified reference samples in biological and botanical matrices. The results from the round-robin analyses were compiled and are summarized in this article.

Animals↗
PubMed1993

Column-switching techniques for high-performance liquid chromatography of drugs in biological samples.

In recent years, an increasing number of publications have demonstrated the potential of column-switching techniques for the chromatographic separation, determination and preparative isolation of analytes from biological matrices. Column-switching systems greatly facilitate drug analysis, by on-line sample clean-up and trace enrichment, or by improving the analytical separative process. In this paper, the main applications of column-switching techniques to drug analysis in biological samples, are reviewed.

Animals↗
PubMed2004

Electron tomography of biological samples.

Electron tomography allows computing three-dimensional (3D) reconstructions of objects from their projections recorded at several angles. Combined with transmission electron microscopy, electron tomography has contributed greatly to the understanding of subcellular structures and organelles. Performed on frozen-hydrated samples, electron tomography has yielded useful information about complex biological structures. Combined with energy filtered transmission electron microscopy (EFTEM) it can be used to analyze the spatial distribution of chemical elements in biological or material sciences samples. In the present review, we present an overview of the requirements, applications, and perspectives of electron tomography in structural biology.

Animals↗
PubMedDate not supplied

[A foldable cardboard box for drying and storage of by cotton swab collected biological samples].

The ability to perform successful DNA analysis on biological evidence obtained at a crime scene or during a sexual assault examination depends very much on the first step--how specimens are collected and preserved. Body fluids and their wet or dry stains, are often recovered using dry cotton swabs or cotton swabs moistened with sterile water or saline. In order to prevent decomposition and deterioration of a specimen, resulting in degradation or loss of DNA, it is recommended to either air dry or freeze these swabs as soon as possible after collection. We designed a simple, foldable cardboard box, which is suitable for the drying and storage of biological evidence collected on cotton swabs. Immediately after collection swabs are placed into the drying racks within the cardboard box, which is subsequently folded, labeled, sealed and initialed. At room temperature swabs completely air dry within the sealed box within 6-9 hours. In this box the evidence is properly packed, labelled and sealed, thus preventing cross contamination, degradation and sample switch. It is a valuable device for the collection of biological evidence at a crime scene, during sexual assault examinations, and for the collection of buccal swabs for PCR-based databasing and paternity testing.

DNA↗
PubMedDate not supplied

A simple method for the determination of ethylene-thiourea (ETU) in biological samples.

A direct, simple, and rapid high-performance liquid chromatographic method has been developed for the determination of ethylene-thiourea (ETU) in biological fluids. Samples were chromatographed on a Lichrosorb RP8 (5 pm) column after extraction with dichloromethane. The mobile phase was a mixture of hexane/isopropyl alcohol/ethyl alcohol (93:6:1 v/v) added with 0.6 mL/L butylamine. Detection was done with a UV detector set at 243 nm. This method was validated to standard criteria. Calibration curves for ETU in 100 microL of 0.9% saline, 500 microL plasma, and 10 mL urine were linear (r2 > 0.99) from 0.05 to 30 microg/mL, 0.025 to 30 microg/mL, and 1 to 100 ng/mL, respectively. The lower limit of detection was 20 ng/mL in plasma, 25 ng/mL in 0.9% saline, and 0.5 ng/mL in urine.

Animals↗
PubMed1998

Low-voltage backscattered electron imaging of non-coated biological samples in a low-vacuum environment using a variable-pressure scanning electron microscope with a YAG-detector.

A variable-pressure scanning electron microscope (VP-SEM) equipped with a high-sensitive backscattered electron (BSE) detector of the YAG type was applied to studies of biological tissue samples. The rat kidney and trachea were fixed, dehydrated in ethanol, critical point-dried and examined in the VP-SEM under a specimen chamber pressure of 1 to 150 Pa. The high-resolution surface images of the non-coated specimens were obtained in a low-vacuum (1-20 Pa) environment at an accelerating voltage of 5 kV, while the images at 10-20 kV contained information beneath the surface of the specimens. The observation in the VP-SEM equipped with YAG detector in a low-vacuum (1-20 Pa) environment at low (3-5 kV) accelerating voltages is useful for the three-dimensional analysis of the surface morphology of biological non-coated samples.

Animals↗
PubMed2003

Low-temperature electrothermal vaporization of thenoyltrifluoroacetone complex of Sc(III) and Y(III) for sample introduction in an inductively coupled plasma atomic emission spectrometry, and their determination in biological samples.

A new method for inductively coupled plasma atomic emission spectrometry (ICP-AES) determination of trace Sc and Y, based on gaseous compound introduction into the plasma as their thenoyltrifluoroacetone (TTA) complexes by electrothermal vaporization was developed. Using the reagent TTA as chemical modifier can not only enhance the analytical signals, but also reduce the vaporization temperature. At a temperature of 1,000 degrees C the trace Sc and Y can be vaporized completely into ICP. The factors affecting the formation of the chelate and its vaporization behavior, such as drying time, vaporization temperature/time, reaction medium and the amount of TTA, were investigated in detail. Under the optimized conditions (drying temperature/time 100 degrees C/10 s, vaporization temperature/time 1,000 degrees C/4 s), the limits of detection for Sc and Y were 19 pg and 34 pg (3sigma), respectively, and the relative standard deviations for Sc and Y were 4.2% (cSc=0.2 microg mL(-1); n=7) and 2.6% (cY=0.5 microg mL(-1); n=7). The linear ranges of the calibration graphs cover three orders of magnitude. The method was applied to the analysis of the biological reference materials (GBW 07602, bush branches and leaves; GBW 07604, poplar leaves), and the results obtained were in good agreement with the certified values.

Electrochemistry↗
PubMed2003

Reconstructing the temporal ordering of biological samples using microarray data.

MOTIVATION: Accurate time series for biological processes are difficult to estimate due to problems of synchronization, temporal sampling and rate heterogeneity. Methods are needed that can utilize multi-dimensional data, such as those resulting from DNA microarray experiments, in order to reconstruct time series from unordered or poorly ordered sets of observations. RESULTS: We present a set of algorithms for estimating temporal orderings from unordered sets of sample elements. The techniques we describe are based on modifications of a minimum-spanning tree calculated from a weighted, undirected graph. We demonstrate the efficacy of our approach by applying these techniques to an artificial data set as well as several gene expression data sets derived from DNA microarray experiments. In addition to estimating orderings, the techniques we describe also provide useful heuristics for assessing relevant properties of sample datasets such as noise and sampling intensity, and we show how a data structure called a PQ-tree can be used to represent uncertainty in a reconstructed ordering. AVAILABILITY: Academic implementations of the ordering algorithms are available as source code (in the programming language Python) on our web site, along with documentation on their use. The artificial 'jelly roll' data set upon which the algorithm was tested is also available from this web site. The publicly available gene expression data may be found at http://genome-www.stanford.edu/cellcycle/ and http://caulobacter.stanford.edu/CellCycle/.

Algorithms↗
PubMed2003

Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry.

BACKGROUND: Cardiac troponins are modified during ischemic injury and are found as a heterogeneous mixture in blood of patients with cardiovascular diseases. We present a strategy to isolate cardiac troponins from human biological material, by use of affinity chromatography, and to provide samples ready for direct analysis by mass spectrometry. METHODS: Cardiac troponins were isolated from human left ventricular tissue by affinity chromatography. Isolated troponins were either eluted and analyzed by Western blot or enzymatically digested while bound to affinity beads. The resulting peptide mixture was subjected to mass spectrometry for protein identification and characterization. The same method was used to analyze serum from patients with acute myocardial infarction (AMI). RESULTS: Affinity chromatography with antibodies specific for one cardiac troponin subunit facilitated the isolation of the entire cardiac troponin complex from myocardial tissue. The three different proteases used for enzymatic digestion increased the total protein amino acid sequence coverage by mass spectrometry for the three cardiac troponin subunits. Combined amino acid sequence coverage for cardiac troponin I, T, and C (cTnI, cTnT, cTnC) was 54%, 48%, and 40%, respectively. To simulate matrix effects on the affinity chromatography-mass spectrometry approach, we diluted tissue homogenate in cardiac troponin-free serum. Sequence coverage in this case was 44%, 41%, and 19%, respectively. Finally, affinity chromatography-mass spectrometry analysis of AMI serum revealed the presence of cardiac troponins in a wide variety of its free and/or complexed subunits, including the binary cTnI-cTnC and cTnI-cTnC-cTnT complexes. CONCLUSIONS: Affinity chromatography-mass spectrometry allows the extraction and analysis of cardiac troponins from biological samples in their natural forms. We were, for the first time, able to directly confirm the presence of cardiac troponin complexes in human serum after AMI. This approach could assist in more personalized risk stratification as well as the search for reference materials for cardiac troponin diagnostics.

Amino Acid Sequence↗
PubMed1987

Influence of preanalytical factors on the atomic absorption spectrometry determination of trace elements in biological samples.

In the analysis of the concentration of trace elements in biological material there are many possible preanalytical sources of error. Precision and accuracy of trace element analyses are not so much determined by the analytical procedure--atomic absorption spectrometry--as by individual biological factors, contamination and loss of elements during sampling and sample preparation, influences resulting from the calibration procedure, or from the analytical equipment itself. Genetical factors like sex or race, ecological factors like age or pregnancy and short term factors like food intake, parenteral feeding or drug therapy can influence the final results. Different blood sampling methods with different duration and strength of compression may lead to hemolysis resulting in falsely elevated values of trace elements. Needles, syringes and tubes contribute to contamination by leaching trace elements from their walls or lead to loss of material by adsorption to the surface of the walls. As there is still no primary standard in order to guarantee precision and accuracy the calibration procedures have to be compared individually depending on the different matrices of the sample.

Calibration↗
PubMed2004

Application of in-gel protease assay in a biological sample: characterization and identification of urokinase-type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-3.

A considerable problem in proteomics is to separate and identify functional proteins that participate in specific biological processes. To expedite the analysis of active proteases, we have developed a substrate-specific, sensitive in-gel trypsin activity assay after two-dimensional (2-D) separation in a sodium dodecyl sulphate (SDS)-polyacrylamide gel [22]. Using this method, we detected and characterized Arg-specific protease activity in the secreted protein sample of a prostate cancer cell line, PC-3, in 1-D and 2-D gels. Mass spectrometry (MS) identified the protease as urokinase-type plasminogen activator (uPA). Western blotting using anti-uPA antibody and protease inhibition tests confirmed the identification. Since no antibody was involved in the procedure, the result clearly demonstrates the feasibility of this method for identifying novel proteases in biological samples.

Blotting, Western↗
PubMed1980

Combined liquid chromatograph/mass spectrometer for involatile biological samples.

A new liquid chromatograph/mass spectrometer has been developed in our laboratory for application to analysis of biological molecules of extremely low volatility. Oxyhydrogen flames rapidly vaporize the total liquid-chromatographic effluent, and molecular and particle beam techniques are used to efficiently transfer the sample to the ionization source of the mass spectrometer. This new instrument is comparable in cost and complexity to a combined gas chromatograph/mass spectrometer, but extends the capabilities of combined chromatography/mass spectrometry to a broad range of compounds not previously accessible. We are currently testing biologically significant samples with this instrument, using reversed-phase liquid-chromatographic separation and both positive and negative ion chemical-ionization mass spectrometry. Results have been obtained from mixtures of nucleic acid components--bases, nucleosides, and nucleotides--and from amino acids, peptides, saccharides, fatty acids, vitamins, and antibiotics. In all cases investigated to date, ions indicative of molecular mass are obtained in at least one of the operating modes available. Detection limits are typically in the 1-10 ng range for full mass scans (about 80-600 amu); sub-nanogram quantities are usually detectable with single-ion monitoring.

Amino Acids↗
PubMed1998

Attomole detection of 3H in biological samples using accelerator mass spectrometry: application in low-dose, dual-isotope tracer studies in conjunction with 14C accelerator mass spectrometry.

This is the first demonstration of the use of accelerator mass spectrometry (AMS) as a tool for the measurement of 3H with attomole (10(-18) mol) sensitivity in a biological study. AMS is an analytical technique for quantifying rare isotopes with high sensitivity and precision and has been most commonly used to measure 14C in both the geosciences and more recently in biomedical research. AMS measurement of serially diluted samples containing a 3H-labeled tracer showed a strong correlation with liquid scintillation counting. The mean coefficient of variation of 3H AMS based upon the analysis of separately prepared aliquots of these samples was 12%. The sensitivity for 3H detection in tissue, protein, and DNA was approximately 2-4 amol/mg of sample. This high sensitivity is comparable to detection limits for 14C-labeled carcinogens using 14C AMS and demonstrates the feasibility of 3H AMS for biomedical studies. One application of this technique is in low-dose, dual-isotope studies in conjunction with 14C AMS. We measured the levels of 3H-labeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 14C-labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rat liver tissue and bound to liver DNA and protein 4.5 h following acute administration of individual or coadministered doses in the range of 4-5100 pmol/kg of body weight. Levels of PhIP and MeIQx in whole tissue and bound to liver protein were dose-dependent. MeIQx-protein and -DNA adduct levels were higher than PhIP adduct levels, which is consistent with their respective carcinogenicity in this organ. Coadministration of PhIP and MeIQx did not demonstrate any measurable synergistic effects compared to administration of these compounds individually. These studies demonstrate the application of AMS for the low-level detection of 3H in small biological samples and for its use in conjunction with 14C AMS for dual-labeling studies.

Animals↗
PubMed1994

Differential-pulse polarographic determination of zinc and manganese in various pharmaceutical and biological samples after adsorption of their morpholine-4-carbodithioates on microcrystalline naphthalene.

A highly selective, sensitive, rapid and economical differential-pulse polarographic method was developed for the simultaneous determination of trace amounts of zinc and manganese in various standard alloys and pharmaceutical, biological and environmental samples after adsorption of their morpholine-4-carbodithioates on microcrystalline naphthalene in the pH range 4.1-9.0 for manganese and 6.5-10.2 for zinc. After filtration, the metal complexes are shaken with 10 ml of 1 mol l-1 HCl and determined simultaneously using a differential-pulse polarograph. There is no need to remove naphthalene from the solution. Dissolved oxygen is removed by adding a few millilitres of 4% NaBH4 solution. The detection limits are 0.06 ppm for Mn and 0.1 ppm for Zn (signal-to-noise ratio = 2). Linearity is maintained in the concentration ranges 0.06-10 ppm for Mn and 0.1-12 ppm for Zn with a correlation factor of 0.9997 and relative standard deviations of 0.95 and 0.81%, respectively. The characterization of the electroactive process included an examination of the degree of reversibility. Various parameters such as the effect of pH, volume of aqueous phase and interference of a number of metal ions on the determination of manganese and zinc were studied in detail to optimize the conditions for their simultaneous determination (based on the large difference in their Ep values) in various biological, pharmaceutical and environmental samples.

Humans↗
PubMed2003

[Legal aspects of biological sample banks].

In order to frame the research on the human biological products, the French legislator has chosen an option consisting in several laws. This situation represents serious difficulties for the researcher. In the first way he will have to apply various general principles corresponding to the protection of persons since the sampling. To these general principles it has to be add more specific rules related to sampling organisation. The complexicity of this system being the references to be taken by the researcher following the organisation level of the samplings. Consequently legislation has to be adapted at this particular subject: human research.

Bioethical Issues↗