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Detection of cervical cancer and high grade neoplastic lesions by a combination of liquid-based sampling preparation and DNA measurements using automated image cytometry.

OBJECTIVE: To establish if measurements of DNA ploidy could be used to assist cytopathologists and cytotechnologists in population based cervical cancer screening programs in countries where manually reading the slides is impossible due to the lack of sufficient skilled cytotechnologists. The goal of such program is to identify only clinically significant lesions, i.e. those where a clinical intervention to remove the lesion is required immediately. STUDY DESIGN: A total of 9905 women were enrolled in the study. Cervical samples were taken with a cervix brush that was then placed into a fixative solution. The cells were separated from mucus by mechanical and chemical treatment and then deposited onto microscope slides by a cytocentrifuge. Two slides were prepared from each case; one slide was stained by Papanicolaou stain for manual cytology examination, while the other slide was stained by a DNA specific stain. The latter slide was used to determine the relative amount of DNA in the cell nuclei. RESULTS: A total of 876 women were followed by colposcopy examination where biopsies were taken from the visible lesions or from suspicious areas and histopathology diagnosed 459 as normal or benign cases, 325 as CIN1, 36 as CIN2, 25 as CIN3/CIS, and 31 as invasive cancer. Of these 876 cases, manual cytology called 655 normal or ASCUS, 197 as LSIL, 16 cases as HSIL, and 8 as cancer. DNA measurements found 704 cases having no cells with DNA greater than 5c, 98 cases where there were 1 or 2 cells having DNA amount greater than 5c, and 74 cases where there were 3 or more cells having DNA amount greater than 5c. If manual cytology were to be used to refer all cases of HSIL and cancer to colposcopy and biopsy, 23 lesions that had to be removed would have been discovered (2 CIN2, 11 CIN3/CIS, and 10 cancers), for a sensitivity of 25.0+/-5.2% at specificity of 99.9+/-0.1%. If DNA assisted cytology were to be used instead, and all cases having 3 or more cells with DNA amount greater than 5c were to be referred to colposcopy and biopsy, then 50 lesions that had to be removed would have been discovered (10 CIN2, 15 CIN3/CIS and 25 cancers) for the sensitivity of 54.3+/-6.2% at specificity of 96.9+/-0.6%. CONCLUSIONS: The study suggests that screening for high grade cervical neoplastic lesions and cervical cancer by DNA assisted cytology could be implemented with minimal use of skilled cytotechnologists, at least in those countries where it would be difficult to introduce population based screening for cervical cancer due to the lack of availability of such skilled cytotechnologists.

Biopsy↗

Sample preparation and high-resolution separation of mycotoxins possessing carboxyl groups.

The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B1, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid-liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography-mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future.

Chromatography, Gas↗

A rapid method of sample preparation for detection of DNA viruses in human serum by polymerase chain reaction.

A rapid method of serum treatment is described that can be used for the detection of viral DNA by polymerase chain reaction (PCR). The key feature of the assay is inactivation of inhibitory serum factors by controlled heating of serum. This method avoids DNA extraction. It is very fast and limited sample handling decreases the chances of contamination by exogenous DNA. It has been successfully used for demonstration of parvovirus B19, hepatitis B virus, and human cytomegalovirus DNA in patient sera. Since pathological components in sera from patients with various diseases do not interfere in the assay, it can be used as a sensitive and safe screening assay for DNA viruses in a routine clinical setting.

Base Sequence↗

An evaluation of two sample preparation methods for measuring 3H and 14C in incinerator ash and spent lime.

This paper compares the performance of two methodologies, oxidation and gel suspension, for measuring the 3H and 14C specific activity in incinerator ash and lime. The two methods were judged on the basis of the count time needed to achieve a specified minimum detectable concentration. Both methods involve pretreatment of the sample matrix followed by liquid scintillation counting. Both methods perform adequately for measuring 3H in spent lime, but only oxidation delivers adequate sensitivity for the more demanding 14C case. In addition, the gel suspension method fails to detect over 95% of the 3H seen by the oxidation method in incinerator ash.

Carbon Radioisotopes↗

Automated solid-phase extraction for sample preparation followed by high-performance liquid chromatography with diode array and mass spectrometric detection for the analysis of resveratrol derivatives in wine.

A method has been developed for the simultaneous determination of resveratrol in all its forms (free isomers and glycosylates) in wines by high-performance liquid chromatography with diode array and mass spectrometric detection. Prior to injection into the column, preconcentration of the sample by automated solid-phase extraction is carried out. In the detection by UV absorption, quantitation was carried out at 280 and 305 nm, and in detection by mass spectrometry, quantitation was performed in the selected ion monitoring mode at m/z 228 and at m/z 238. A comparative study between both detection systems was carried out.

Automation↗

Rapid solid-phase extraction/derivatization system for sample preparation and gas chromatographic/mass spectrometric determination of drugs in human urine.

A novel automatic system has been developed for the rapid solid-extraction and derivatization of different drugs and their metabolites from urine, prior to further analysis. Manual injection of underivatized/derivatized drugs in a GC/MS system, in which the quadrupole mass filter was operated conventionally, provided high sensitivity and selectivity at the selected m/z value. The precision (2.1-6.8%) is acceptable and the overall recovery from urine was determined to be ca. 95%. The effectiveness of this approach is demonstrated with data obtained from different urine samples which had tested positively.

Calibration↗

Automated determination of polyamines by high-performance liquid chromatography with simple sample preparation.

Recently, a new fully endcapped reversed-phase packing material, Inertsil, was introduced, especially suitable for the determination of basic compounds. We used this packing material to separate ophthaldialdehyde (OPA) derivatives of amino acid derivatives completely from the OPA derivatives of spermine (SPM), spermidine (SPD), putrescine (PUT) and cadaverine (CAD). The obtained separation made the commonly used off-line extraction procedure redundant and thus an on-line sample clean-up was introduced. This enabled automation of the procedure resulting in a better reproducibility and a more efficient use of equipment. Furthermore, no studies are required to determine the extraction recovery. The present method has a cycle time of 30 min. A linear response for each polyamine was found up to 250 pmol, with an R2 ranging from 0.9981 (SPM) to 0.9998 (CAD). The limit of detection, calculated at a signal-to-noise ratio of 3, was 0.1 pmol, corresponding to a plasma concentration of 0.1 mumol/l. The coefficient of variation (C.V.) for the peak area was below 3% and for retention times below 0.5% (n = 15). In order to evaluate the applicability of the method, three different types of sample were chromatographed, e.g. urine (obtained from healthy human volunteers), pig plasma and sulfosalicylic acid homogenates of pig intestine biopsies. Tissue homogenates and urine-specimen could easily be quantitated, while plasma concentrations were just above the limit of detection, resulting in a plasma C.V. ranging from 4.8% (SPM) to 13.6% (SPD) and a tissue C.V. ranging from 2.1% (SPM) to 8.5% (CAD), The urinary C.V.s were not determined. In conclusion, the present method provides an easy way to measure polyamine concentrations for most applications.

Amino Acids↗

Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing.

An integrated and multiplexed on-line instrument starting from DNA templates to their primary sequences has been demonstrated based on multiplexed microfluidics and capillary array electrophoresis. The instrument automatically processes eight templates through reaction, purification, denaturation, preconcentration, injection, separation, and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were utilized to manage flow and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an eight-capillary array in a hot-air thermal cycler. Subsequently, the sequencing ladders are directly loaded into separate size exclusion chromatographic columns operated at approximately 60 degrees C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at approximately 70 degrees C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. The raw data allow base calling up to 460 bp with an accuracy of 98%. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also genetic typing.

Animals↗

Sample preparation for GC analysis of selected pesticides in surface water.

A new isolation procedure for the determination of nitrogen/phosphorous containing pesticides and organochlorine pesticides in water was tested, and statistical evaluation of the recoveries was performed. The procedure, designed specifically for the analysis of semi-volatile compounds in water containing Suspended Particulate Matter (SPM), utilizes a specially designed filtration vessel coupled directly to an SPE cartridge. The studies were based on surface water samples (from the Vistula River) spiked with pesticides. SPM separation and analyte isolation/concentration were carried out in a special filtration vessel. Pesticides were sorbed both on the SPE cartridge and on the suspended matter. The cartridge and the filter with the suspended matter were extracted separately with a solvent, which enabled the determination of analyte distribution between the two. For organochlorine pesticides, between 3 and 60% of the initial amount was found on the filter, while the recoveries in the filtrate ranged from 30 to 98%. Total recoveries of organochlorine pesticides from surface water samples spiked with pesticides using the method described were high, ranging from 90 to 101%. The amounts of nitrogen/phosphorus containing pesticides recovered from the filter were lower than 2%, while those from the filtrate ranged from 69 to 92%. Total recoveries of nitrogen/phosphorus containing pesticides from surface water samples were high, ranging from 71 to 92%.

Adsorption↗

Sample preparation and liquid chromatography mass spectrometry analysis of alkylphenolic compounds and steroid sex hormones in sediments.

A new methodology, based on the use of accelerated solvent extraction (ASE) and highly selective cleanup using restricted access material (RAM) on-line coupled with liquid chromatography-mass spectrometry (LC-MS), is presented for the simultaneous and unequivocal determination of alkylphenol ethoxylates (APEOs), their degradation products and halogenated derivatives, and steroid sex hormones in sediment samples. Using the integrated RAM-LC-MS system, the simultaneous determination of alkylphenolic compounds and sex hormones was achieved, yielding recoveries higher than 60% and producing low MS background noise.

Alkenes↗

Automated sample preparation on-line with thermospray high-performance liquid chromatography-mass spectrometry for the determination of drugs in plasma.

The combination of a solid-phase extraction module, the AASP, on-line with thermospray high-performance liquid chromatography-mass spectrometry for the automated determination of drugs in plasma is described. The technique was evaluated successfully using, as an example, the determination of labetalol in human plasma. [2H7]Labetalol was used as an internal standard to compensate for changes in ionization efficiencies between analyses. The chromatographic and mass spectrometric conditions were optimized for labetalol. The combined technique was demonstrated as being robust and reliable for the analysis of plasma samples from a clinical study.

Autoanalysis↗

Sample preparation by supercritical fluid extraction for quantification. A model based on the diffusion-layer theory for determination of extraction time.

A mathematical model based on the diffusion-layer theory was elaborated in order to calculate the extraction time in dynamic supercritical fluid extraction required to reach a predefined level of extraction recovery. The goodness of the model is demonstrated by application to the extraction of the main neutral cannabinoids from marihuana and hashish samples. For monitoring of the cannabinoid content of extracts normal-phase HPLC was applied. To obtain reliable quantitative results, the extraction time ensuring a predefined level of recovery should be calculated for each individual sample according to the model because the extraction recovery depends on the sample matrix. The systematic error caused by the unextracted compounds can be eliminated by correction of the experimental data. For semi-quantitative determinations, where a knowledge of the correct value of the extraction recovery is not important, as a rule of thumb the extraction of marihuana with carbon dioxide of density 0.9 g/ml at 40 degrees C for 34 min and of hashish for 18 min can be suggested. The application of the proposed extraction times ensured at least a 95% recovery for the main neutral cannabinoids.

Cannabinoids↗

A sample preparation for automated cervical cancer screening.

A procedure for cluster disruption, cell dispersal and the production of a monolayer of cells from fluid suspensions for automated cell-scanning machines in an interactive system has been developed. The combination of 0.1 per cent dithiothreitol and 40 per cent alcohol in a balanced salt solution provides a self-limiting form of mucolysis which is completed by a controlled syringing regime to produce an optimally dispersed cell sample without the production of cell debris from the more effete cells. The induction of a positive charge on a microscope slide to attract the negatively charged cells has been achieved by coating the slides with the cationic polymer, polylysine. The cells appear to be sufficiently well attached to permit wet fixation and subsequent stain processing without loss. Quantitative data is given to demonstrate the superior cell presentation that is achieved by this new technique for automated scanning.

Diagnosis, Differential↗

A rapid sample-preparation technique for thin-layer chromatographic analysis for 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in human urine.

In this rapid TLC method of analysis for the urinary cannabinoid metabolite, 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), hydrolyzed urine specimens are aspirated through a porous, alkyl-silica extraction layer located along the lower edge of a biphasic thin-layer chromatogram; THC-COOH is simultaneously extracted from the sample and concentrated on the TLC plate for subsequent migration and detection. The method, evaluated in a blind study, identified all samples containing THC-COOH greater than or equal to 20 micrograms/L. The technique increases samples throughput, sensitivity, and specificity as compared with conventional TLC methods and is applicable to a wide variety of compounds.

Chromatography, Thin Layer↗

[Solid phase-sample preparation of ifosfamide and chloroethyl metabolites in biological material. 1. Determination in plasma and urine].

The cytostatic drug ifosfamide (IFF) and its main metabolites 2-dechloroethyl ifosfamide (2-D-IFF) and 3-dechloroethyl ifosfamide (3-D-IFF) were isolated from the plasma matrix with high recovery by solid phase extraction using Bakerbond C18-cartridges. A further cleaning of the extracts is not necessary. An effective separation of IFF and metabolites from interfering compounds present in urine samples prior gc was performed with Extrelut-1 and dichloromethane/isopropanol (95:5, v/v). The described assays may be used for pharmacokinetic studies as well as drug monitoring in the clinical laboratory.

Humans↗

Liquid-phase microextraction combined with hollow fiber as a sample preparation technique prior to gas chromatography/mass spectrometry.

Two modes of liquid-phase microextraction (LPME) combined with hollow fiber (HF) were developed for gas chromatography/mass spectrometry (GC/MS). Both methodologies, that is, static LPME with HF and dynamic LPME with HF, involved the use of a small volume of organic solvent impregnated in the hollow fiber, which was held by the needle of a conventional GC syringe. In static LPME/HF, the hollow fiber impregnated with solvent was immersed in the aqueous sample, and the extraction processed under stirring; in dynamic LPME/HF, the solvent was repeatedly withdrawn into and discharged from the hollow fiber by a syringe pump. This is believed to be the first reported instance of a semiautomated liquid microextraction procedure. The performance of the two techniques was demonstrated in the analysis of two PAH compounds in an aqueous sample. Static LPME/HF provided approximately 35-fold enrichment in 10 min and good reproducibility (approximately 4%). Dynamic LPME/HF could provide higher enrichment (approximately 75-fold) in 10 min and even better reproducibility (approximately 3%). Both methods allow the direct transfer of extracted analytes to a GC/MS system for analysis.

Journal Article↗

Preparation of samples of plant material for chromatographic analysis.

The selection, collection, and preliminary treatment of plant material samples are discussed. Stages of sample preparation and extraction techniques presently used are described, and the most recent examples of sample preparation for chromatographic analysis are reviewed. Recent applications of gas chromatography for the assessment of emission of volatile organic compounds by plants are also described.

Chromatography, Gas↗

Sample preparation and in situ hybridization techniques for automated molecular cytogenetic analysis of white blood cells.

With the advent of in situ hybridization techniques for the analysis of chromosome copy number or structure in interphase cells, the diagnostic and prognostic potential of cytogenetics has been augmented considerably. In theory, the strategies for detection of cytogenetically aberrant cells by in situ hybridization are simple and straightforward. In practice, however, they are fallible, because false classification of hybridization spot number or patterns occurs. When a decision has to be made on molecular cytogenetic normalcy or abnormalcy of a cell sample, the problem of false classification becomes particularly prominent if the fraction of aberrant cells is relatively small. In such mosaic situations, often > 200 cells have to be evaluated to reach a statistical sound figure. The manual enumeration of in situ hybridization spots in many cells in many patient samples is tedious. Assistance in the evaluation process by automation of microscope functions and image analysis techniques is, therefore, strongly indicated. Next to research and development of microscope hardware, camera technology, and image analysis, the optimization of the specimen for the (semi)automated microscopic analysis is essential, since factors such as cell density, thickness, and overlap have dramatic influences on the speed and complexity of the analysis process. Here we describe experiments that have led to a protocol for blood cell specimen that results in microscope preparations that are well suited for automated molecular cytogenetic analysis.

Acetic Acid↗