bluuebunny/crossref_metadata_2025_split huggingface dataset:
Precision of DNA flow cytometry in inter‐institutional analyses
Authors: Wheeless Leon L. , Coon John S. , Cox Christopher , Deitch Arline D. , White Ralph W. Devere , Fradet Yves , Koss Leopold G. , Melamed Myron R. , O'Connell Mary J. , Reeder Jay E. , Weinstein Ronald S. , Wersto Robert P. 
Published: August 2005

AbstractA Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter‐ and intra‐laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter‐ and intra‐laboratory differences. Results were evaluated by a two‐way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra‐ and inter‐laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories. This study demonstrates the need for measurement and control of laboratory variability and for the routine use of laboratory standards and check samples if the results from one laboratory are to be compared with the results of another or if classification criteria are to be transferred between laboratories.
Calibration of fluorescence intensities to quantify antibody binding surface determinants of cell subpopulations by flow cytometry
Authors: Dux R. , Kindler‐Röhrborn A. , Lennartz K. , Rajewsky M. F. 
Published: August 2005

AbstractQuantitative indirect immunofluorescence analysis by flow cytometry was used to determine the mean number of antibody binding sites per cell in a small subpopulation of rat brain cells expressing low levels of a cell surface differentiation antigen recognized by monoclonal antibody (Mab) RB13‐6 (Kindler‐Röhrborn et al.: Differentiation 30:53–60, 1985). For these non‐disjunct distributions of fluorescence intensities, the cut‐off border between antigen‐positive and antigen‐negative cells was defined by a statistical test. To eliminate the influence of accidental disturbances leading to incorrect statistical decisions, the curves for antigen‐negative cells were fitted according to cell number and shape. The flow cytometer was calibrated with the use of a clonal cell line for which the average number of Mab RB13‐6 binding sites per cell had previously been determined by radioimmunoassay and Scatchard‐plot analysis. Using this analytical procedure, both the proportion of Mab binding brain cells and the mean number of Mab binding sites per Mab binding cell could be determined as a function of developmental stage.
Flow cytometric measurement of p53 protein expression and DNA content in paraffin‐embedded tissue from bronchial carcinomas
Authors: Mørkve Odd , Laerum Ole Didrik 
Published: August 2005

AbstractThe nuclear protein p53 has been measured in archival lung cancer biopsies. The monoclonal antibody PAb 1801, which recognizes human p53, was used. After immunostaining, the nuclei prepared from paraffin‐embedded tissue were stained with propidium iodide for simultaneous measurement of DNA content; 17 of 24 lung cancers were p53 positive. The S‐phase fraction in positive tumors was 22.9±6.4%, as compared to 13.6±6.1% in negative tumors (P < 0,02). In ten of the positive tumors (two small cell carcinomas and eight non‐small cell carcinomas), the p53 expression varied through cell cycle, whereas in seven tumors (five small cell carcinomas and two non‐small cell carcinomas), no such variation of p53 expression was observed. Freezing the nuclear suspensions did not substantially reduce the p53 signals. Control experiments with the SV40‐transformed human foreskin fibroblast cell line HSF4‐T12 showed that the enzymatic digestion utilized to dissociate paraffin‐embedded tissue did not significantly reduce p53 fluorescence. Immunohisto‐chemical staining of biopsy specimens indicated that only cancer cells were overexpressing p53. In conclusion, using the monoclonal antibody PAb 1801, p53 is detectable in cell nuclei prepared from paraffin‐embedded bronchial carcinoma biopsies. P53 positive tumors have increased proliferative activity compared to p53 negative tumors. Furthermore, the lack of cell cycle variation of p53 in small cell carcinomas indicates that this pattern may be related to high‐grade malignancy.
Cell kinetic analysis of mixed populations using three‐color fluorescence flow cytometry
Authors: Begg A. C. , Hofland I. 
Published: August 2005

AbstractThe development of antibodies to DNA‐incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters. More recently, these techniques have been applied to clinical tumor material. One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material. In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro. An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative). The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours.Selecting out cells from the mixture required the application of three‐color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd‐DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA). This allowed single laser excitation. The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC‐conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE‐conjugated second antibody) and lastly by the DNA stain containing RNase. Two analysis methods of the IdUrd/DNA cytograms were applied: a mid‐S window analysis and a relative movement (RM) analysis. Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately.A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms. By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis. Little difference was seen, with both the mid‐S and RM analyses, between cells gated from mixtures or stained separately. It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors.
Very early detection of changes associated with cellular activation using a modified flow cytometer
Authors: Kelley Keith A. 
Published: August 2005

AbstractA sample station modification previously described has been redesigned to provide greater flexibility and enhanced performance. The improved modification provides mixing and temperature regulation in a compact unit that mounts close to the nozzle holder for reduced transit times, allowing for addition of mediators to a sample in place on the flow cytometer, with observation of results in approximately 1 s. An electronic circuit activated at the time of injection generates full‐scale pulses in the forward scatter channel. This provides the data with a time stamp for direct correlation of injection and cellular response. A detailed description of the modification, performance verification data, and practical applications in the measurements associated with cellular activation are presented.
Experimental and theoretical investigations on the validity of the geometrical optics model for calculating the stability of optical traps
Authors: Schut Tom C. Bakker , Hesselink Gerlo , De Grooth Bart G. , Greve Jan 
Published: August 2005

AbstractWe have developed a computer program based on the geometrical optics approach proposed by Roosen to calculate the forces on dielectric spheres in focused laser beams. We have explicitly taken into account the polarization of the laser light and thd divergence of the laser beam. The model can be used to evaluate the stability of optical traps in a variety of different optical configurations.Our calculations explain the experimental observation by Ashkin that a stable single‐beam optical trap, without the help of the gravitation force, can be obtained with a strongly divergent laser beam. Our calculations also predict a different trap stability in the directions orthogonal and parallel to the polarization direction of the incident light.Different experimental methods were used to test the predictions of the model for the gravity trap. A new method for measuring the radiation force along the beam axis in both the stable and instable regions is presented. Measurements of the radiation force on polystyrene spheres with diameters of 7.5 and 32 μm in a TEM00‐mode laser beam showed a good qualitative correlation with the predictions and a slight quantitative difference.The validity of the geometrical approximations involved in the model will be discussed for spheres of different sizes and refractive indices.
Application of laser optical tweezers in immunology and molecular genetics
Authors: Seeger S. , Monajembashi S. , Hutter K.‐J. , Futterman G. , Wolfrum J. , Greulich K. O. 
Published: August 2005

AbstractOptical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis).In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the Chinese hamster karyotype (CHV 79) can be easily micro‐dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.
Laser induced cell fusion in combination with optical tweezers: The laser cell fusion trap
Authors: Steubing Rosemarie Wiegand , Cheng Steve , Wright William H. , Numajiri Yasuyuki , Berns Michael W. 
Published: August 2005

AbstractA single‐beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 μ, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen‐pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of ≈ 1 μJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.
Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks
Authors: Rigaut Jean Paul , Vassy Jany , Herlin Paulette , Duigou Françgoise , Masson Eric , Briane Dominique , Foucrier Jean , Carvajal‐Gonzalez Santos , Downs Angela M. , Mandard Anne‐Marie 
Published: August 2005

AbstractA method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three‐dimensional images, 60 μm deep, were obtained by stacking up confocal fluorescent images obtained with an MRC‐500 (Bio‐Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.
Quantitative flow cytometric analysis of ABO red cell antigens
Authors: Sharon Raphael , Fibach Eitan 
Published: August 2005

AbstractA flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluoroscein‐conjugated anti‐H lectin and the A and B antigens by indirect staining first with monoclonal anti‐A or anti‐B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti‐mouse immunoglobulin (Ig) antibodies. More than a ten‐fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of >200 and >30, respectively, while heterozygotes (AO or BO) had ratios of <5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti‐A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population. These results suggest that in addition to a specific gene dosage effect, other factors (e.g., aging of the RBC) may affect the expression of all these antigens in a coordinate way.
Membrane cell permeabilisation with saponin and multiparametric analysis by flow cytometry
Authors: Jacob Marie Christine , Favre Mireille , Bensa Jean‐Claude 
Published: August 2005

AbstractSaponin, a detergent like molecule, can permeabilise cell membranes without destroying them, and thus can be used for the detection of intracellular antigens on intact cells with a flow cytometer.First experiments were reported that demonstrated the detection of intracytoplasmic antigens such as intermediate filaments and CD3 in T acute lymphoblastic leukemia (ALL). Further experiments were also performed to prove that intranuclear structures were equally accessible: dyes such as propidium iodide (PI) and monoclonal antibodies (mAb) such as Ki67 could penetrate the nucleus and lead to the analysis of DNA content and to the discrimination between the different cell cycle phases (G0, G1, S, G2‐M).This rapid and sensitive method retained sufficient integrity of cells being treated to enable differentiation of cell types on the basis of morphology. Furthermore, it did not alter membrane expression of most antigens. Therefore, it was of particular interest for multiparametric analysis, especially for simultaneous study of membrane and intracellular structures.
Bivariate flow karyotyping of human chromosomes: Evaluation of variation in Hoechst 33258 fluorescence, chromomycin A3 fluorescence, and relative chromosomal DNA content
Authors: Boschman Gert A. , Rens Wim , van Oven Carel H. , Manders Erik M. M. , Aten Jacob A. 
Published: August 2005

AbstractThe total variation of chromosome peak positions, in bivariate distributions of Hoechst 33258 and chromomycin A3 fluorescence of 19 healthy individuals, was compared with the experimental variation, determined from 23 bivariate distributions of chromosomes prepared separately from a single cell lineage. The experimental variation in Hoechst and chromomycin fluorescence and the relative chromosomal DNA content were determined from experiments performed over several days. The additional variance contributed by time was the same as the daily variance. The accuracy by which the relative chromosomal DNA content can be calculated from bivariate peak positions was investigated. A least squares method was used to fit the distributions of relative DNA content, obtained, respectively, from mono‐ and bivariate flow analyses of chromosomes from the same cell lineage. In general the DNA contents match quite well, but for a few chromosomes a difference was found, statistically discernible at the 5% level. The average relative chromosomal DNA content of the chromosomes from the 19 normal individuals, calculated from bivariate peak positions, showed a linear relation with the estimates published by other investigators.
CUBIC: A three‐dimensional colored projection of consort 30 generated trivariate flow cytometric data
Authors: Greimers Roland , Rongy Anne Michel , Schaaf‐Lafontaine Nicole , Boniver Jacques 
Published: August 2005

AbstractThe CUBIC program displays three‐dimensional colored dot plots of flow cytometric trivariate data collected by unmodified commercial instruments (FACScan flow cytometer, FACS 440 cell sorter). Assuming a bimodal distribution of the fluorescence intensity of the cells, the eight theorical subpopulations involved in a three‐color fluorescence histogram are clearly localized in the 3‐D space by colored dots that are clustered near each corner of a cubic frame. Rotation, tilting, and zoom functions are available. Table look‐up is not needed. CUBIC was illustrated by two experiments: 1) three‐color immunofluorescence of antigens on human lymphocytes using monoclonal antibodies conjugated either to fluorescein (FITC), to R‐phycoerythrin (PE), or to biotin revealed by a streptavidin coupled to a PE‐Texas red tandem conjugate (TC); 2) two‐color immunofluorescence of CD4 and CD8 antigens on thymocytes of healthy or preleukemic mice correlated to the DNA content quantified by 7‐amino‐actinomycin D (7‐AAD). The three fluorescences were excited by a single argon‐ion laser emitting at 488 nm.
Towards a quantitative grading of bladder tumors
Authors: De Meester Uneco , Young Ian T. , Lindeman Jan , van Der Linden Hans C. 
Published: August 2005

AbstractThe histological inspection of tumor tissue for the purpose of reporting a tumor grade is a problem of significant clinical importance. The grading by a pathologist is only partly reproducible due to vaguely defined, subjective criteria. In this article we describe and evaluate a set of measurable features that quantitate the differences in tumor tissue. Different aspects of the reproducibility of the measurements under varying conditions of image selection, focus, and noise have been investigated.Three hundred thirty‐three images were digitized from 111 bladder tissue sections (4 μm thick, Feulgen stained), using the ICAS microscope‐camera platform. A segmentation routine was developed to segment the images into nuclei and background without any user interaction. Size, shape, optical density, and texture features were measured on and among the objects found by this segmentation routine using the image analysis package Acuity.The results of the measurements showed that there is a significant quantitative difference between grade 1 and grade 3 tumors. Grade 2 tumors can be described as “in between grade 1 and grade 3” and falling somewhere on an increasing scale between grades 1 and 3. Grade 2 tumors do not seem to represent a statistically distinct population.The procedure described here is shown to be quite reproducible in the presence of noise, reasonably reproducible in the event of a modest amount of defocusing (with grade 3 tumors exhibiting the most sensitivity), and less reproducible in the context of which fields‐of‐view are chosen for analysis.
Influence of tissue preparation techniques on p53 expression in bronchial and bladder carcinomas, assessed by immunofluorescence staining and flow cytometry
Authors: Mørkve Odd , Høstmark Jens 
Published: August 2005

AbstractIn a series of bronchial and bladder carcinomas, p53 protein expression was examined. Samples from formalin‐fixed, paraffin‐embedded tissue (routine‐treated) were compared with parallel samples of fresh tissue and tissue fixed in paraformaldehyde and ethanol. The expression of p53 was measured by immunofluorescence staining and dual parameter flow cytometry, with simultaneous monitoring of DNA content. For each tumor, p53 fluorescence with different fixatives was expressed relative to fresh tissue. The p53 fluorescence signals were on average brighter from routine‐treated tissue than from fresh tissue. The tissue fixed in paraformaldehyde showed no difference from fresh tissue. In the ethanol‐fixed tissue, however, fluorescence signals were reduced by nearly 70%, and the fraction of detectable p53 positive cells in tumor tissue was reduced by more than 50%. This loss of fluorescence was probably due to a leakage of the antigen from nucleus to cytoplasm. Pepsin treatment did not influence p53 fluorescence. Within the same tumor, the Sphase fraction in p53 positive cells was significantly higher than in p53 negative cells (13.1 ± 2.0% vs. 6.5 ± 0.8%). This pattern was not influenced by formalin fixation or pepsin treatment.Our study demonstrates that in measuring a nuclear antigen, tissue handling may influence the results, and care should be taken to optimize the preparation procedure. Using the antibody PAb 1801, p53 expression measured in archival material is not reduced as compared to fresh tissue.