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PubMed1999

[The standardized techniques for preparing samples used in pathologic diagnostic transmission electron microscopy].

Transmission electron microscopy (TEM) has been used widespreadly in the field of diagnostic pathology. Based on the practical domestic condition and aimed at fully satisfying the needs of the pathologic diagnostic TEM, a set of standardized techniques were established for preparing different kinds of samples. The techniques included the preparation and storage of fixatives and embedding media, the requirement of sampling, the 5 h and 24 h embedding procedures for fresh specimens, the embedding procedure for suspension specimens and specimens collected from fine needle aspiration biopsy, as well as the procedures for preparing samples taken from paraffin embedded blocks or paraffin sections. In practical use, we found that the advantage of these techniques was that the ultrastructures of the tissues and cells were well preserved and they perfectly met the needs for diagnosis of tumors, kidney diseases, et al. The main points of the standardized techniques have been discussed in detail in this paper.

Breast Neoplasms↗
PubMed2000

An automated method of sample preparation of biofluids using pierceable caps to eliminate the uncapping of the sample tubes during sample transfer.

Biological samples are normally collected and stored frozen in capped tubes until analysis. To obtain aliquots of biological samples for analysis, the sample tubes have to be thawed, uncapped, samples removed and then recapped for further storage. In this paper, we report an automated method of sample transfer devised to eliminate the uncapping and recapping process. This sampling method was incorporated into an automated liquid-liquid extraction procedure of plasma samples. Using a robotic system, the plasma samples were transferred directly from pierceable capped tubes into microtubes contained in a 96-position block. The aliquoted samples were extracted with methyl-tert-butyl ether in the same microtubes. The supernatant organic layers were transferred to a 96-well collection plate and evaporated to dryness. The dried extracts were reconstituted and injected from the same plate for analysis by liquid chromatography with tandem mass spectrometry.

Blood Chemical Analysis↗
PubMed1980

Sampling and preparation of samples of peanut butter for aflatoxin analysis.

Procedures are discussed for sampling peanut butter and preparing those samples for alatoxin analysis. Special emphasis is placed on sampling the product from shipping pallets and comminuting chunk stype peanut butter in order to reduce the variability in the analysis associated with the nonuniform distribution of aflatoxin in the product. The slurry method of preparation is a convenient means of obtaining a sample which is representative of a nonhomogeneous product.

Aflatoxins↗
PubMed1980

Effect of sample preparation on cerebrospinal fluid protein patterns in polyacrylamide gels.

The method of preparing CSF and dilute serum samples for polyacrylamide gel electrophoresis has a marked effect on the pattern of the resolved proteins. The incorporation of sucrose or glycerol into the sample seriously impairs the quality of the resolution and affects the actual number of bands resolved and the relative mobility of certain proteins. The use of sucrose as an anti-convection medium in dense samples such as serum and CSF is not recommended.

Blood Protein Electrophoresis↗
PubMed1982

Interaction between sample preparation techniques and three methods of nitrite determination.

Meat samples containing nitrite and varying concentrations of ascorbate, cysteine, and sodium chloride were prepared for nitrite analysis. The methods used were the AOAC method of dilution and heating; the addition of mercuric chloride, charcoal, and Carrez reagents at 2 different pH values; and direct analysis of sample supernatants with no treatment (control). The effect of these initial conditions and preparation methods on measured nitrite was determined by using 3 different Griess reagent combinations and chemiluminescent and differential pulse polarographic techniques. Systematic variations were observed in samples treated with mercuric chloride, while the addition of Carrez reagents had little or no effect. Best results were obtained by the AOAC dilution/heating method under alkaline conditions, or by charcoal addition followed by chemiluminescent or colorimetric nitrite determination. Statistical analysis of the nitrite concentrations determined in the several samples showed that these 3 procedures were precise to about 5-6% CV, which was not significantly different from the CV value of 4% determined from replicate analyses.

Indicators and Reagents↗
PubMed2002

Surface topography modulates the osteogenesis in human bone marrow cell cultures grown on titanium samples prepared by a combination of mechanical and acid treatments.

Titanium samples of different roughness R(a) and morphology were prepared using a combination of mechanical (grinding with a SiC paper or blasting with aluminum oxide particles with 65 or 250 microm) and chemical (attack with a sulphuric acid based solution or a hydrofluoric acid based solution) treatments. The biological performance of the prepared surfaces was evaluated using human bone marrow osteoblastic cell cultures. Mechanically treated samples presented different R(a) values and surface morphology. The hydrofluoric acid solution was more effective than the sulphuric acid solution in smoothing titanium surface and also in eliminating aluminum contamination resulting from the blasting process. Bone marrow cells seeded on the different titanium samples showed a similar pattern of behavior during cell attachment and spreading. Cells proliferated very well on all the titanium surfaces and cell growth was observed during approximately two to three weeks. The samples treated with the hydrofluoric acid solution presented higher alkaline phosphatase activity. Only the blasted samples treated with the acid solutions allowed seeded bone marrow cells to form a mineralized extracellular matrix. The best biological performance was found in the blasted samples treated with the hydrofluoric acid solution, which could be related to the characteristic microtopography of these samples that presented a homogeneous and smooth roughness.

Journal Article↗
PubMed2000

Plasma sample preparation by ultrafiltration for FTIR analysis.

Total body water (TBW) may be significantly altered with disease. Isotope dilution techniques, considered to be the "gold standard" methods for measuring TBW, are expensive, time consuming and require considerable expertise, especially during the sample preparatory phase. In this study, a new method, ultrafiltration (UF), was hypothesised to be an efficient alternative to vacuum sublimation (VS) in the preparation of plasma samples for Fourier Transform Infra Red (FTIR) determination of TBW. Deuterium Oxide (D2O) concentrations were prepared in human plasma and subjected to both techniques. FTIR analysis was carried out on the resulting VS and UF solutions and on D2O concentrations in distilled water. The resulting absorbance values were then statistically compared. Urea concentrations prepared in D2O-containing plasma were also compared to "blank" plasma to investigate the effect of high plasma urea concentration on the resulting H2O/D2O mixture obtained during UF Paired t-tests showed that the VS plasma samples (p=0.003), but not the UF samples (p=0.9), were significantly different to D2O standards prepared in distilled water. While there was no evidence of an effect of urea on UF at low (0.4 g/L) D2O concentration, a marginal (p=0.04) effect occurred at a higher (1.6 g/L) D2O level. Throughput of samples was much more efficient with the UF technique. These findings indicate that the new UF method is an accurate, more efficient method of plasma sample preparation than the VS method in the FTIR determination of TBW.

Blood Chemical Analysis↗
PubMed1995

The effect of sample preparation and storage on maternal triple-marker screening.

OBJECTIVE: To evaluate the effect of different sample collection, storage, and preparation techniques on serum alpha-fetoprotein (AFP), beta-hCG, and unconjugated estriol (E3) concentrations. METHODS: A solution containing known concentrations of AFP, hCG, and unconjugated E3 was diluted in blood samples obtained from seven healthy male volunteers. Serum from each blood sample was removed immediately, and either assayed or frozen at -70C. Portions of the remaining blood were handled as follows: centrifuged and refrigerated, centrifuged and left at room temperature, not centrifuged and refrigerated, or not centrifuged and left at room temperature. Serum was removed from these samples for triple-marker analysis at 24, 48, 96, and 168 hours after the initial sample collection. RESULTS: Immediate freezing of serum and subsequent thawing resulted in a significant increase in beta-hCG and unconjugated E3 levels, but no change in AFP levels. There was a significant effect over time on AFP, hCG, and unconjugated E3 concentrations. The change in AFP levels was influenced by centrifugation status, whereas all three analytes were influenced by refrigeration status. CONCLUSION: Different sample collection, storage, and preparation techniques may affect maternal triple-marker screening.

Chorionic Gonadotropin↗
PubMed1987

Plasma amino-acids analysis: effects of delayed samples preparation and of storage.

This paper describes changes occurring in plasma amino-acid concentrations of: samples which are not prepared for analysis immediately after blood collection and samples stored for 5 to 6 months in the form of either plasma, or of deproteinized plasma, or of deproteinized and at pH 2.2 buffered plasma. Results showed that in order to avoid these changes, plasma should be deproteinized and buffered as soon as possible after blood collection. When analysis could not be performed immediately, storage of samples in a freezer at -18 degrees C in the form of deproteinized and at pH 2.2 buffered plasma showed after 5 to 6 months the best recovery of the initial concentrations, but did not exclude all changes.

Amino Acids↗
PubMed1980

A comparison of methods of blood withdrawal and sample preparation for potassium measurements.

The effect on measured potassium values of a number of factors involved in sample acquisition and preparation were investigated. Measured potassium values in either plasma or serum were not influenced by the site of sample withdrawal, the presence of a tourniquet, or the time elapsed between blood sampling and analysis. However, increasing heparin concentrations and/or volumes decreased potassium values. The highest values were obtained in samples obtained in a syringe that had been washed with minimum volumes of heparin. It is concluded that this common clinical practice is based on a sound scientific rationale.

Arteries↗
PubMed2002

New sample preparation technique for the determination of avoparcin in pressurized hot water extracts from kidney samples.

A new approach is presented for the determination of avoparcin in tissue. Complete recovery from spiked swine kidney was achieved with hot water modified with 30% ethanol (v/v). The samples were extracted at 75 degrees C and 50 atm by accelerated solvent extraction. In situ sample clean-up was achieved by using matrix solid-phase dispersion utilizing the acrylic polymer XAD-7 HP, and by adding triethylammonium phosphate (TEAP) to the extraction solvent. The aqueous extracts were concentrated by solid-phase extraction (SPE) on the hydrophilic interaction chromatography (HILIC) material polyhydroxyethyl aspartamide. Complete analyte retention was possible during SPE when the kidney extracts were modified with 70% ethanol. A 200 A, 5 microm HILIC column with UV225 detection was used for the separation of avoparcin. The retention time was less than 15 min with 47% aqueous component in acetonitrile and 15 mM TEAP as eluent. The average recovery of avoparcin from kidney samples was 108%.

Animals↗
PubMed1979

Elemental composition of platelets. Part I. Sampling and sample preparation of platelets for trace-element analysis.

Sampling of platelets for trace-element analysis poses special problems: obtaining adequate sample material, achieving a sufficient cell purity, preserving viability (integrity), correcting for trapped plasma, and controlling contamination. We used a blood-cell separator for the primary isolation of platelets from blood, and differential centrifugation in natural plasma to further isolate them. The pyrimidopyrimidine RA233 was used as a stabilizer to maintain viability. 131I-labeled human serum albumin was used to estimate trapped plasma. Contamination was controlled by using five-times-distilled water to simulate donor's blood in the system and by comparing three fractions: the serum, the first portion of the platelet-rich plasma, and the supernatant plasma after the final centrifugation. Neutron activation analysis was used for the elemental analysis. A single differential centrifugation of the platelet-rich plasma from the blood-cell separator at 400 x g for 8 min was optimum (mean mass fractions:erythrocytes/platelets less than 5 mg/g and leukocytes/platelets less than 20 mg/g). The trapped plasma in the wet platelet samples amounted to about 0.40 g/g. No appreciable contamination from the sampling system was found for the elements Ag, Cd, Co, Cr, Cs, Cu, Fe, Mo, Rb, Sb, Se, and Zn.

Blood Platelets↗
PubMed1997

Cross-section TEM sample preparation of multilayer and poorly adhering films.

The preparation of TEM cross-section samples from multilayer films or poorly adhering films is discussed in detail in a step-by-step approach designed to enable a competent experimentalist to reproduce the technique. The samples are mounted on an aperture grid and mechanically polished to 2-3 microns in thickness. After ion beam milling for a short period of time (less than 1 hour), a large electron transparent area is obtained. Examples from several thin film systems are discussed.

Microscopy, Electron↗
PubMed1992

InP sample preparation for the TEM by photochemical etching, ion milling, and chemical thinning.

Photochemical etching (PCE) as a method for preparation of InP semiconductor plan view samples for the transmission electron microscope is demonstrated and compared to the methods of ion milling and chemical thinning. PCE can produce small area samples for TEM analysis quickly and accurately. Also, the resulting thin regions are surrounded by a built-in stabilizing structure that improves handleability and reduces the occurrence of handling induced fracture.

Microscopy, Electron↗
PubMed2003

An efficient fusion expression system for protein and peptide overexpression in Escherichia coli and NMR sample preparation.

An efficient fusion expression system with a small fusion partner, His6-tagged N-terminal fragment of staphylococcal nuclease R, has been constructed and tested with two genes. The results show that the system is not only suitable for overexpression of small proteins and peptides but simplifies purification of target proteins and peptides. The study also provides a practical method for preparation of isotope-labeled protein sample for NMR analysis.

Crotalid Venoms↗
PubMed1993

A rapid method for microbial sample preparation for the scanning electron microscope.

A synthetic aromatic polymer has been used for preparing replicas of different microorganisms. This method of preparing highly concentrated (9.6 k) microbiological samples for scanning electron microscopy was compared with a standard method. The micrographs of the replicated samples are satisfactory. This method is rapid, cost effective and produces good results, especially in the case of spore-forming mycelial microorganisms.

Aspergillus fumigatus↗