bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of the Papanicolaou and Feulgen staining methods for DNA quantification by image analysis
Authors: Gurley A. M. , Hidvegi D. F. , Bacus J. W. , Bacus S. S. 
Published: August 2005

AbstractThe possibility of using archival cytology material to study the evolution of neoplastic disease with regard to DNA content abnormalities was investigated. The accuracy of measuring the integrity optical density (OD) of nuclei that correlates to DNA amounts of those nuclei, on slides stained by the Papanicolaou method, was assessed and compared with a standard Feulgen method. Our data on rat liver nuclei peritoneal washings from patients with ovarian cystadenofibromas and ovarian cystadenocarcinomas suggested that analysis of cytological material using the Papanicolaou method is not reliable and that destaining the slides followed by Feulgen staining provides an optimal and reliable method of DNA quantification.
Variations in feulgen stainability of epithelial parenchymal cells extracted from paraffin‐embedded salivary gland specimens
Authors: Schimmelpenning Hendrik , Falkmer Ursula G. , Hamper Klaus , Seifert Gerhard , Auer Gert U. 
Published: August 2005

AbstractThe variations of Feulgen stainability of cells extracted from paraffin‐embedded archival specimens for DNA assessment by means of image cytometry (ICM) were investigated in normal salivary gland parenchyma. The Feulgen stainability of the deparaffinized, rehydrated, and disaggregated preparations was found to exhibit variations of up to 300%, expressed by the mean of integrated optical density (IOD), when a routine procedure was applied to a first series of Cytospin preparations of disaggregated specimens. When measured in nondisaggregated tissue sections, only negligible variations were observed. After minimization of the mechanical strains to the cellular material in the Cytospin preparations in a second series, the variations in Feulgen stainability were found to be considerably lower. The findings indicate that the main reason for variations in the Feulgen stainability of extracted cells is, most likely, the disaggregation procedure itself. Factors such as initial treatment of the specimens, duration and kind of formalin fixation, and length of storage time periods seem to be of minor importance. Retrospective studies on paraffin‐embedded specimens require a carefully controlled tissue type‐adapted disaggregation procedure. In addition, we concluded that the interpretation of histograms, obtained by means of ICM DNA assessments in Cytospin preparations of archival material, requires a well‐defined internal specific standard.
An improved flow cytometric assay for somatic mutations at the glycophorin A locus in humans
Authors: Langlois Richard G. , Nisbet Barbara A. , Bigbee William L. , Ridinger David N. , Jensen Ronald H. 
Published: August 2005

AbstractAn improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. The new assay, named the “BR6” assay, can be performed on a commercially available, single‐beam flow cytometer, in contrast to the previously described 1W1 assay that required a dual‐beam flow sorter. A modified cell labeling method developed for the BR6 assay results in improved separation of normal and mutant phenotype cells, as well as eliminating some cellular artifacts that affected the 1W1 assay. Parallel measurements on samples from 17 normal donors showed that the BR6 assay yields comparable variant cell frequencies and improved measurement precision compared with the 1W1 assay. A detailed analysis of three individuals who showed large differences in background variant frequency with the 1W1 assay confirmed that these differences could also be detected with the BR6 assay. A dramatically elevated variant cell frequency was seen with the BR6 assay of an individual exposed to a high level of ionizing radiation in an accident at Goiania, Brazil.
Isolation of giant DNA fragments from flow‐sorted human chromosomes
Authors: Minoshima Shinsei , Kawasaki Kazuhiko , Fukuyama Ryuichi , Maekawa Masahiko , Kudoh Jun , Shimizu Nobuyoshi 
Published: August 2005

AbstractWe have established a method using a conventional cell sorter equipped with a single argon laser to sort intact human chromosomes that can be used as a source for the production of giant DNA fragments. Various improvements were made to both the equipment and sorting method to enhance the sorting resolution and avoid destruction of chromosomal DNA. Using this improved method chromosomes 21 and 22 were sorted from the B‐lymphoblastoid line GM00130B, digested with the rare cutting restriction endonuclease NotI, and analyzed by pulsed field gel electrophoresis followed by Southern hybridization using the Alu repetitive sequence as a probe. More than 25 discrete NotI giant DNA fragments ranging from 50 kb to longer than 2.5 Mb were separated and the size distribution pattern was unique for each chromosome, indicating successful sorting of intact chromosomes. The cumulative size of these Alu‐positive NotI DNA fragments were 22.7 Mb and 25.5 Mb for chromosomes 21 and 22, respectively. These values are 47% and 49% of the estimated size of chromosomes 21 (48 Mb) and 22 (52 Mb).
Clinical applications of cytometry: 4th annual meeting
Authors: Giorgi J. V. , Hurtubise P. E. , Parker J. W. , Cram L. S. , La Via M. F. 
Published: August 2005

AbstractQuantitative cytology is undergoing rapid evolution because of dramatic advances in optics, lasers, photochemistry, computers and digital imaging. Novel clinical applications utilizing the latest flow and imaging technology as well as exciting new findings using more conventional methods are the focus of an annual meeting in Charleston, South Carolina. The 4th Annual Meeting, which featured sessions on image cytometry, fluorescence intensity, quantitation, leukemia/lymphoma analysis and transplantation, is summarized here.
Instrument for real‐time pulse‐shape analysis of slit‐scan flow cytometry signals
Authors: van Oven C. , Aten J. A. 
Published: August 2005

AbstractAn instrument is described which analyses shapes of fluorescence profiles generated by particles passing through the focussed laser beam of a flow cytometer. The output signal of this pulse‐shape analyzer is used as input for the signal processing electronics of a commercial flow cytometer system. The instrument detects dips in pulse‐profiles; a shape parameter named Pulse Dip Index (PDI) is defined as the ratio of the integrated signal from the beginning of the pulse until the first dip, relative to the integrated signal of the complete profile. This PDI is similar to the Centromeric Index of chromosomes. The composition of aggregates in mixtures of fluorescent particles of different sizes was evaluated by PDI analysis. In our experiments the PDI was determined within 30 μs from the onset of the pulse‐profile and particles with a specified morphology of interest were selected for on‐line registration of their profiles as digitized pulse‐shapes. In a cell sorter system, the PDI can be used as a parameter for sorting.
Effects of cellular fixatives on human immunodeficiency virus production
Authors: Cory Joan M. , Rapp Fred , Ohlsson‐Wilhelm Betsy M. 
Published: August 2005

AbstractEffects of cell fixation procedures appropriate for flow cytometric analysis on the infectivity of human T lymphoblastoid H9 cells infected with human immunodeficiency virus‐1 (HIV‐1) were evaluated to provide guidelines for choosing cell treatments for potentially infectious samples. H9 cells experimentally infected with HIV‐1 were treated by the test fixation procedure, washed, and cocultured with equal numbers of live, uninfected H9 cells. To estimate the reduction in infectivity due to the fixation procedure, dilution series of live infected H9 cells in uninfected H9 cells were simultaneously established in culture. Cell cultures were incubated 8–10 d, harvested, and evaluated for evidence of HIV‐1 infection by the presence of cell‐associated HIV‐1 antigens and/or by the presence of particle‐associated reverse transcriptase activity in cell culture supernatants. Thirty‐minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehyde (0.5%) reduced the infectivity of HIV‐1‐infected H9 cells by greater than 99.99%. To the same degree, a multi‐step fixation procedure utilizing formaldehyde and ethanol was effective in reducing HIV‐1 infectivity. Conversely, the erythrocyte fixative dimethylsuberimidate at 3 μg/ml was ineffective in reducing HIV‐1 infectivity.
Flow‐cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets
Authors: Tschoepe D. , Spangenberg P. , Esser J. , Schwippert B. , Kehrel B. , Roesen P. , Gries F. A. 
Published: August 2005

AbstractOcclusive vascular diseases are promoted by a “prethrombotic state” with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow‐cytometric bio‐assay and the related change in the actin status by using the DNase‐I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F‐actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein “actin” and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow‐cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.
A new principle of cell sorting by using selective electroporation in a modified flow cytometer
Authors: Schut Tom C. Bakker , de Grooth Bart G. , Greve Jan 
Published: August 2005

AbstractWhen a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill.We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 μs and resulting field strengths of 2.0 and 3.2 X 106 V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.).The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.
Flow cytometric identification of microorganisms by dual staining with FITC and PI
Authors: Miller Jane S. , Quarles John M. 
Published: August 2005

AbstractThe identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.
Fixation‐Associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs
Authors: Holtfreter Hiroko B. , Cohen Nicholas 
Published: August 2005

AbstractWe have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.
Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse
Authors: de Launoit Yvan , Kiss Robert , Danguy André 
Published: August 2005

AbstractWe compared cytomorphonuclear parameters–related to DNA histogram and chromatin distribution–on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen‐stained nuclei. The spectrophotometric results–assessed by integrated optical density values–indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol‐formalin‐acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well‐defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.
Histomorphometric study of the lower urogenital tract in pre‐ and post‐menopausal women
Authors: Semmelink H. J. F. , de Wilde P. C. M. , van Houwelingen J. C. , Vooijs G. P. 
Published: August 2005

AbstractIn this study 22 histomorphometric parameters were used to quantify the histologic changes in the vagina, trigone of the bladder, and the proximal and distal urethra of 37 women aged between 18 and 82 years.The results of this quantitative study revealed a hormonally induced, age‐independent atrophy of smooth muscle in the lamina propria of the mucosa and the longitudinal part of the muscular layer in the aforementioned anatomical sites in post‐menopausal women. In the lamina propria of the mucosa of the lower urogenital tract in post‐menopausal women hormonally induced quantitative changes of blood vessels were not observed. Senile atrophy of smooth muscles was only observed in the deeper part of the muscular layer of the vagina and the trigone of the urinary bladder and was less prominent than the hormonally induced smooth muscle atrophy.
Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes
Authors: Dietzel Andreas H. , Hain Jens , Virsik‐Peuckert R. Patricia , Harder Dietrich 
Published: August 2005

AbstractCHO‐K1 cells were irradiated in G0/G1 phase with 150 k V X‐rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose‐dependent changes in the flow karyotypes, they were split into peak‐ and background‐portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose‐dependent reduction in peak contents. The number of radiation‐induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non‐chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose‐effect relations and split‐dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.
Flow cytometric method to monitor the destruction of CD4+ cells following their fusion with HIV‐infected cells
Authors: Schols Dominique , Pauwels Rudi , Desmyter Jan , De Clercq Erik 
Published: August 2005

AbstractSyncytium formation between HUT‐78 cells persistently infected with human immunodeficiency virus type 1 (HIV‐1) and uninfected CD4‐bearing MOLT‐4 or CEM cells results in a rapid destruction of the MOLT‐4 or CEM cells. This syncytium formation is due to the interaction between the gp120 glycoprotein expressed by the persistently HIV‐1‐in‐fected HUT‐78 cells and the CD4 receptor present on MOLT‐4 or CEM cells. A flow cytometric method has been applied to separate the infected (HUT‐78) from the uninfected (MOLT‐4, CEM) cell populations. This method is based on a modified DNA staining protocol which clearly shows the differences in DNA content between HUT‐78 cells, on the one hand, and MOLT‐4 or CEM cells, on the other hand. Using this flow cytometric method we have demonstrated that those compounds (i.e., sulfated polysaccharides, aurintricarboxylic acid) that interact with gp120 (of the HIV‐infected cells) or CD4 (of the uninfected cells) suppress syncytium formation and concomitant destruction of the CD4+ cells.
A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis
Authors: Vindeløv Lars L. , Christensen Ib Jarle 
Published: August 2005

AbstractEstablishing flow cytometric DNA analysis as a clinical routine procedure requires adequate and proven guidelines, by which the data can be obtained and interpreted to directly influence management of the individual patient with a specific neoplasm. The present paper is intended as a contribution to such guidelines, of which only fragments are available today.We have previously described a system of methods, designed for routine flow cytometric DNA analysis. In the present status report our experience, based on approximately 18,000 samples (clinical and experimental) is summarised. Sample acquisition with fine‐needle aspiration, storage at −80°C, internal standardization by chicken (CRBC) and trout red blood cells (TRBC), staining with propidium iodide (PI), and analysis in the flow cytometer is recapitulated, with emphasis on previously unpublished aspects. The method of statistical analysis which has an integrating role is described in some detail. A lack of linearity between channel number and DNA content was determined experimentally, and the coefficient of variation (CV) was found to decrease with increasing channel number. The corrections in the algorithm of deconvolution made necessary by these findings are fundamental for estimating the end results. The zero point adjustment and procedures for changing from one batch of standards to another are described. A systematic approach to interpretation of DNA histograms is attempted and illustrated by data from clinical specimens of malignant lymphoma, breast cancer, small cell lung cancer, cancer of the oral cavity, and bladder cancer. Some problems are still unsolved and visual inspection is required to determine if the quality of the individual histogram is satisfactory. Inspection of the fluorescence/light scatter dot‐plot provides additional information for the recognition of artifacts. The results stress that good quality DNA histograms with as small CVs as possible are important for interpretation of the data. It is essential that statistical methods are employed to extract the key end‐point results. These are the number of subpopulations and their relative representation, and for each subpopulation the DNA index (DI) and the fractions of cells in the cell cycle phases.For the DNA data to have any rationally based impact on clinical decision making, it must be demonstrated that they have an independent prognostic value. Strategies for final evaluation are discussed. Multicenter trials on fresh material, to accrue quickly the number of patients necessary for firm conclusions, are suggested.