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At least 1,081 records · Page 60Linked to original sources
PubMed2006

Differences in platelet growth factor release and leucocyte kinetics during autologous platelet gel formation.

Three commercial systems for whole blood separation were compared to obtain the buffy coat composed of platelet-rich plasma (BC-PRP) and leucocytes . These samples of the buffy coat were used to make a platelet gel (PG), which was used to measure platelet growth factor (PGF) release, to perform a white blood cell (WBC) count and to measure myeloperoxidase (MPO) release from WBCs. Aliquots of whole blood obtained from ten volunteers were distributed either to a blood cell separator (The Electa Cell-Separator, E-CS) or to a tabletop centrifuge (Gravitational Platelet Sequestration System, GPS) to prepare the BC-PRP. The third system combines the BC-PRP production by E-CS with a micro porous filter (Autologous Growth Factor filter, AGF) to enrich for the BC-PRP. Autologous thrombin was used to activate the BC-PRP and to prepare the PG and subsequently to degranulate the platelet concentrate. Platelet-derived growth factor-AB and transforming growth factor-beta1 were present in high levels after thrombin activation of the E-CS or GPS prepared samples. However, the AGF prepared samples released their growth factors before thrombin activation. The WBCs were significantly increased with each of the three systems. Contrary to the AGF, no leucocyte degranulation occurred with the E-CS or GPS prepared samples, based upon the low MPO concentrations in the BC-PRP. The three types of apparatus had different harvesting capacities for collecting the enriched platelets and the release of high concentrations of PGF. When the E-CS and GPS, but not the AGF, were used, low levels of MPO were maintained in the PG, which potentially contributes to antimicrobial properties of platelet gel at the site of application.

Adult↗
PubMed2006

Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS.

We have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein. Optimization of the reactor operational parameters for trypsin digestion using a temperature-controllable microreactor was carried out. The entire trypsin digestion procedure took about 11 min, and consisted of 1 min for the thermal denaturation of the sample protein (3 microl, 0.2 microM) at 85 degrees C, and 10 min for digestion of the protein at 37 degrees C. The resulting sequence coverage ranged from 24% to 57%, which was sufficient for practical protein identification.

Microchemistry↗
PubMed2004

Effects of long-term storage on Fusarium toxin concentrations in wheat--sources of error of the analytical results.

The concentrations of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) of a heavily contaminated wheat grain batch were followed over a period of 1 year by taking samples 15 times every 28 days. The air temperature and relative humidity at the top of the wheat batch ranged between 7 and 22 degrees C and 44 and 55%, respectively, and corresponded to a variation in the moisture content of the wheat grain between 11.5 and 12.3%. None of these fluctuations were related to ZON and DON concentrations, which varied between 0.46 and 0.66 and 15.0 and 19.5 mg/kg DM. Therefore, the data were used to analyse the error sources for the analytical results. It was found that the variance proportions due to sampling and sample preparation plus analysis were not similar for DON and ZON. The variance proportion due to sampling was found to be 0.62 for ZON, which corresponded to a variance proportion of 0.38 due to sample preparation plus analysis. In contrast, the latter variance proportion for DON was estimated to be 1.0 and consequently completely superimposed the sampling error. It is concluded that long-term storage of contaminated wheat grain does not affect the concentrations of DON and ZON considering the measured fluctuations in ambient temperature, relative humidity and moisture content of the grain. Therefore, no degradation of DON and ZON occurred during the storage of wheat for a period of one year under ambient conditions. The effects of sampling and sample preparation plus analysis on the final analytical results are different for DON and ZON and require further consideration.

Animal Feed↗
PubMed2004

Pre-PCR processing: strategies to generate PCR-compatible samples.

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

Animals↗
PubMed1999

A novel strategy for the preparation of liposomes: rapid solvent exchange.

During the preparation of multi-component model membranes, a primary consideration is that compositional homogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipid mixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membrane components. A new preparative method has been developed which is specifically designed to avoid this intermediary state. This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organic solvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapment volumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content, suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared by rapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional sample preparation are discussed.

Buffers↗
PubMed1996

Optimization of a continuous real-time computerized semen analysis system for ram sperm motility assessment, and evaluation of four methods of semen preparation.

Sampling conditions that affect the biological validity of computer-assisted analysis of ram sperm motion were examined using a continuous real-time computerized semen analysis system (Hobson Sperm Tracker). Search radius (SR, 10 settings) and minimum track point (MTP, 10 settings) were varied factorially to evaluate their effects on the inclusion of sperm subpopulations within derived datasets. Low SR (< 12 microns) or high MTP values (> 26 frames) precluded measurements of rapidly moving cells, whereas high SR (> 17 microns) and low MTP settings (< 22 frames) led to erroneous tracking and poor data quality. Suitable settings for these set-up parameters were derived and tested for their biological consistency with four methods of preparing ram semen for computerized analysis. The preparation techniques tested were: centrifugation through sucrose-based Ficoll and Percoll media, a swim-up technique and simple dilution in Tyrode's media. The "selective' Percoll and swim-up methods generated sperm populations with significantly higher linear velocities and a lower tendency to deviate from linear trajectories than from either the Ficoll method or dilution technique. Deleterious effects of centrifugation were evident, particularly on sperm survival in vitro over several hours. It is concluded that computer-assisted semen analysis provides useful information about the behaviour of ram spermatozoa in vitro, but the measurement conditions must be defined carefully.

Animals↗
PubMed2005

Mechanical compression affecting the thermal-induced conformational stability and denaturation temperature of human fibrinogen.

Thermal-induced conformational stability and changes in denaturation temperature of human fibrinogen (FBG) after different mechanical compressions were investigated by a simultaneous Fourier transform infrared microspectroscopy equipped with thermal analyzer (thermal FTIR microscopic system). The confocal Raman microspectroscopy was also applied to determine the thermal reversibility of solid FBG. FBG powder was pressed on one KBr pellet (1 KBr method) or sealed within two KBr pellets (2 KBr method) by different mechanical compressions. The result indicates that there was no marked difference in the thermal behavior for the solid FBG samples prepared by 1 KBr method in the heating process even under different mechanical compression pressures, in which the thermal-induced denaturation temperatures from native to denatured state were maintained constant at 66-67 degrees C. However, the denaturation temperature for the solid FBG samples prepared by 2 KBr method was shifted from 55 to 62 degrees C with the increase of mechanical compression pressure. A good linear correlation was also found between the denaturation temperature and mechanical compression pressure for FBG samples prepared by 2 KBr method. The solid FBG sample, whether prepared by 1 KBr or 2 KBr method, was also found to show the thermal-irreversible property.

Bromides↗
PubMed2005

Solvent-free MALDI-MS for the analysis of biological samples via a mini-ball mill approach.

An efficient, low sample load mini-ball mill (MBM) sample preparation procedure was developed for solvent-free MALDI analysis of peptides and proteins. Picomole sample amounts can be handled conveniently, with 30 s grinding times being sufficient. Matrix purity and molar analyte/matrix ratios are not as critical as with methods employing solvent. Ammonium salt is employed for protonation of the peptide and suppression of sodiation. This strategy allows for peptide mapping and other biochemical manipulations to be performed prior to MBM sample preparation and mass analysis. The analysis of bovine serum albumin (66 kDa) yielded good results, indicating that higher molecular weight proteins are accessible. A semi-solvent-free strategy by the MBM sample preparation method is also described.

Animals↗
PubMed2003

Detection of inorganic species by chemical reaction laser desorption ionization mass spectrometry.

The oxidation numbers of metals in inorganic compounds were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by using their acetylacetonates, which are soluble in acetone. For the MALDI analysis of inorganic species insoluble in common solvents used for matrices, such as acetone, methanol, water, etc., a suspension method of sample preparation was developed. Turbid suspensions of inorganic species in the solvent were spotted on the sample holder with chelating reagents, as in the conventional sample preparation for MALDI-MS. Chemical reaction between the inorganic species and the chelating reagents occurred in the plume after irradiation by laser light. Metal oxides were also analyzed by this method, and samples with different oxidation numbers gave different mass spectra. These results suggest that many other metal oxides with different oxidation numbers could be identified if suitable chelating reagents are chosen for sample preparation for MALDI-MS.

Journal Article↗
PubMed2002

A strategy for a post-method-validation use of incurred biological samples for establishing the acceptability of a liquid chromatography/tandem mass-spectrometric method for quantitation of drugs in biological samples.

Validated liquid chromatography/tandem mass spectrometric (LC/MS/MS) methods are now widely used for quantitation of drugs in post-dose (incurred) biological samples for the assessment of pharmacokinetic parameters, bioavailability and bioequivalence. In accordance with the practice currently accepted within the pharmaceutical industry and the regulatory bodies, validation of a bioanalytical LC/MS/MS method is performed using standards and quality control (QC) samples prepared by spiking the drug (the analyte) into the appropriate blank biological matrix (e.g. human plasma). The method is then declared to be adequately validated for analyzing incurred biological samples. However, unlike QC samples, incurred samples may contain an epimer or another type of isomer of the drug, such as a Z or E isomer. Such a metabolite will obviously interfere with the selected reaction monitoring (SRM) transition used for the quantitation of the drug. The incurred sample may also contain a non-isomeric metabolite having a molecular mass different from that of the drug (such an acylglucuronide metabolite) that can still contribute to (and hence interfere with) the SRM transition used for the quantitation of the drug. The potential for the SRM interference increases with the use of LC/MS/MS bioanalytical methods with very short run times (e.g. 0.5 min). In addition, a metabolite can potentially undergo degradation or conversion to revert back to the drug during the multiple steps of sample preparation that precede the introduction of the processed sample into the LC/MS/MS system. In this paper, we recommend a set of procedures to undertake with incurred samples, as soon as such samples are available, in order to establish the validity of an LC/MS/MS method for analyzing real-life samples. First, it is recommended that the stability of incurred samples be investigated 'as is' and after sample preparation. Second, it is recommended that potential SRM interference be investigated by analyzing the incurred samples using the same LC/MS/MS method but with the additional incorporation of the SRM transitions attributable to putative metabolites (multi-SRM method). The metabolites monitored will depend on the expected metabolic products of the drug, which are predictable based on the functional groups present in the chemical structure of the drug. Third, it is recommended that potential SRM interference be further investigated by analyzing the incurred samples using the multi-SRM LC/MS/MS method following the modification of chromatographic conditions to enhance chromatographic separation of the drug from any putative metabolites. We will demonstrate the application of the proposed strategy by using a carboxylic acid containing drug candidate and its acylglucuronide as a putative metabolite. Plasma samples from the first-in-man (FIM) study of the drug candidate were used as the incurred samples.

Animals↗
PubMed1999

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers.

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.

Cholesterol↗
PubMed2006

Cell handling using microstructured membranes.

Gentle and precise handling of cell suspensions is essential for scientific research and clinical diagnostic applications. Although different techniques for cell analysis at the micro-scale have been proposed, many still require that preliminary sample preparation steps be performed off the chip. Here we present a microstructured membrane as a new microfluidic design concept, enabling the implementation of common sample preparation procedures for suspensions of eukaryotic cells in lab-on-a-chip devices. We demonstrate the novel capabilities for sample preparation procedures by the implementation of metered sampling of nanoliter volumes of whole blood, concentration increase up to three orders of magnitude of sparse cell suspension, and circumferentially uniform, sequential exposure of cells to reagents. We implemented these functions by using microstructured membranes that are pneumatically actuated and allowed to reversibly decouple the flow of fluids and the displacement of eukaryotic cells in suspensions. Furthermore, by integrating multiple structures on the same membrane, complex sequential procedures are possible using a limited number of control steps.

Cell Line, Tumor↗
PubMed1998

Automated high-throughput mass spectrometric analysis of synthetic oligonucleotides.

The use of automated sample preparation and data collection coupled with delayed ion extraction matrix-assisted laser desorption ionization with time-of-flight detection mass spectrometry (DE MALDI-TOF MS) enables the rapid mass analysis of oligonucleotide samples. We have incorporated a matrix-diluting and liquid-spotting automated work station and a DE MALDI mass spectrometer to provide high-throughput analysis of synthetic oligonucleotides. Automated sample preparation greatly speeds processing while the spotting function allows for precise sample placement on a 100-well sample plate. The DE MALDI MS typically determines the mass of each sample to within 0.13 +/- 0.10% of the theoretically expected mass. This mass accuracy allows confirmation of identity and detection of low-level impurities present in synthetic oligonucleotide samples with an unprecedented degree of confidence. Using this automation suite, sample preparation, spotting, and analysis of 100 samples ranging from 12 to 70 bases in length can be routinely completed in less than 90 min.

Calibration↗
PubMed1989

Bioassay alpha spectrometry: energy resolution as a function of sample source preparation and counting geometry.

Alpha particle counting is based on the response of an electronic counting system to an incident alpha particle. Alpha spectrometry is used in our employee surveillance bioassay program to measure the concentration of isotopes of Am, Pu and U contained on sample source preparations. Nuclides of Am, Pu and U are separated from the sample matrix by anion exchange and are electroplated on a stainless steel disc (Gautier and Gladney 1986). The plated source diameter is 12.7 mm. A tracer is added to the sample before anion exchange as a quality control procedure to provide a measure of chemical yield. Tracer alpha-particle emissions are recorded in a preassigned calibrated area of the energy spectrum and chemical recovery is calculated by the ratio of tracer counts per second divided by the tracer activity in becquerels (Bq). Percent tracer recovery may also be calculated by introducing the average counting efficiency factor in the denominator. Tracer yield is then used to provide a reliable estimate of the sample's analyte counts that are recorded in other preassigned energy dependent areas of the spectrum. The tracer spectrum in the presence or absence of other nuclides also provides evidence of the performance characteristics of the alpha spectrometer, for example, chamber vacuum and electronics. Electroplated samples are counted in any one of 96 detectors. The backgrounds of these detectors are maintained at less than 3 counts per 70,000 s over a 190 keV energy window to provide a limit of detection of less than 0.37 mBq per sample at the 95% confidence level. In this paper, resolution of the photopeak is shown to be a function of the source to detector distance and a function of degraded alpha energies due to Fe or other extraneous materials on the plated surface. Since our program is concerned only with low activity samples (less than 0.2 Bq) we encounter no significant problem with tailing when the resolution is less than 50 keV FWHM.

Alpha Particles↗
PubMed2006

Pharmaceuticals analysis by capillary electrophoresis at nanolevel detection.

There is an urgent need to analyze drugs and pharmaceuticals at nanoscale detection, both now and in the future. Capillary electrophoresis is a growing technique that is continuously developing with improved detectors, sample preparation units, and other components. This article describes the analysis of drugs and pharmaceuticals by capillary electrophoresis at nanoscale detection in various standard solutions, dosage formulations, and biological samples. Nanoscale detection analysis is discussed in terms of the advancement in the detectors and sample preparation methods. The coupling of the detectors and sample preparation units to capillary electrophoresis is also discussed. Attempts have been made to evaluate the optimization strategies of capillary electrophoresis conditions to achieve detection at the nanoscale. Applications of capillary electrophoresis for the analyses of simple and chiral mixtures of several drugs and pharmaceuticals at nanoscale detection are included.

Electrophoresis, Capillary↗
PubMed1996

Organochlorine pesticide analysis in oil seeds and vegetable oil: simplification of silica gel clean-up and elimination of chlorinated solvents.

For the determination of organochlorine pesticides (OCPs) in vegetable oils and oil seed samples, a solid-phase extraction method is described, modified from that developed by Steinwandter, with silica gel columns activated with a 10% water content. In comparison to the method developed by Steinwandter, we reduced the amount of solvents and chemicals required for sample preparation and substituted dichloromethane by the much less toxic tert-butylmethyl ether in the eluent mixture. In this manner the sample preparation technique becomes more convenient, cheaper and ecologically more justifiable. Under the specified sample preparation and elution conditions 1 g silica gel retained about 16 mg triglycerides whereas the OCPs were quantitatively eluted. The recoveries for hexachlorobenze, (HCB) alpha-HCH, Lindane (gamma-HCH) gamma-HCH, Heptachlor, Heptachlorepoxid, o,p'-DDE, p,p'-DDE, Dieldrin, o,p'-DDT, p,p'-DDT were between 75 and 90%. The reproducibility of the total method was excellent as well as its ruggedness.

Chromatography, Gas↗
PubMed2006

EPR properties of intact and deproteinated dentin.

The effect of sample preparation on dentin electron paramagnetic resonance (EPR) spectra was investigated. The analysis was performed on dentin samples prepared by pure mechanical treatment or by an alkaline deproteination method. It was observed that (1) mechanical treatment induces both stable and transient signals, depending on the specific mechanical operation applied; and (2) sodium hydroxide deproteination removes the native signal, but at the same time introduces new confounding signals in the EPR spectrum. Also, it increases the radiation sensitivity of dentin. These findings suggest that attention must be paid to dentin sample preparation.

Dentin↗
PubMed2005

Elemental analysis of soil samples for forensic purposes by inductively coupled plasma spectrometry--precision considerations.

Major and trace elemental composition provides a powerful basis for forensic comparison of soils, sediments and rocks. However, it is important that the potential 'errors' associated with the procedures are fully understood and quantified, and that standard protocols are applied for sample preparation and analysis. This paper describes such a standard procedure and reports results both for instrumental measurement precision (repeatability) and overall 'method' precision (reproducibility). Results obtained both for certified reference materials and example soils show that the instrumental measurement precision (defined by the coefficient of variation, CV) for most elements is better than 2-3%. When different solutions were prepared from the same sample powder, and from different sub-sample powders prepared from the same parent sample, the CV increased to c. 5-6% for many elements. The largest variation was found in results for certified reference materials generated from 23 instrument runs over an 18 month period (mean CV=c. 11%). Some elements were more variable than others. W was found to be the most variable and the elements V, Cr, Co, Cu, Ni and Pb also showed higher than average variability. SiO2, CaO, Al2O3 and Fe2O3, Rb, Sr, La, Ce, Nd and Sm generally showed lower than average variability, and therefore provided the most reliable basis for inter-sample comparison. It is recommended that, whenever possible, samples relating to the same investigation should be analysed in the same sample run, or at least sequential runs.

Forensic Medicine↗