bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cytophotometric analysis of cervical intraepithelial neoplasia grade III, with and without synchronous invasive squamous cell carcinoma
Authors: Hanselaar A. G. J. M. , Vooijs G. P. , Van't Hof‐Grootenboer A. E. , Pahlplatz M. M. M. 
Published: August 2005

AbstractCytophotometric analysis was performed in nuclei retrieved from paraffinembedded cervical tissue from 57 cases of CIN III. CIN III lesions of patients without invasive squamous cell carcinoma (N=37) were regarded to represent a mixture of progressive and nonprogressive lesions. The CIN III lesions of patients with a synchronous invasive squamous cell carcinoma (N=20) were regarded as representing truly progressive precursor lesions (CIN.INV). Twenty‐one photometric features describing geometrical, density, and texture characteristics were extracted from the digitized nuclear images. Statistical analysis of cytophotometric data indicated significant differences between the group of CIN III lesions and CIN.INV lesions. A cluster analysis, using one co‐occurrency texture feature (S‐HOMOG), one density feature (S‐DI), and two geometrical features (S‐AREA and M‐CIRC), showed that two clusters (C1 and C2) were present in the total group of CIN III and CIN.INV lesions. The vast majority of CIN.INV lesions was member of one and the same cluster C1. The CIN III group appeared to consist of a mixture of two clusters, 54% C1 and 46% C2 lesions. Of patients 45 years or younger, the majority (62%) of CIN III lesions had feature values, corresponding with those of cluster C1, and as such possibly with a potentially progressive course. In patients older than 45 years the percentage of CIN III lesions with C1 feature values was 27%.
Cytophotometric analysis of corresponding cytological and histological cervical intraepithelial neoplasia grade III specimens
Authors: Hanselaar A. G. J. M. , Vooijs G. P. , Van't Hof‐Grootenboer A. E. , Gemmink J. H. , De Leeuw H. , Pahlplatz M. M. M. 
Published: August 2005

AbstractCytophotometric analysis of cervical intraepithelial neoplasia grade III (CIN III) was performed in 22 cytological smears (CS) and in 22 corresponding cytospin specimens retrieved from selected areas of paraffin‐embedded tissues (PEC). The average time interval between cytological and histological diagnosis was 6 weeks. CIN III nuclei in CS and PEC specimen were Thionin‐Feulgen stained and digitized. Beside the visual classification of DNA ploidy patterns, the 2.5c and 5c exceeding rates and the specimen mean and standard deviation values of 21 photometric features were also analyzed. It was shown that, although there was a significant correlation between DNA ploidy patterns in corresponding PEC and CS specimen, the DNA patterns were dissimilar in eight of 22 cases. The DNA index, as represented by 2.5c and 5c exceeding rates, was significantly higher in the CS specimen. High‐resolution cytophotometric analysis of cell nuclei in CS and PEC specimens showed significant differences for a large number of nuclear photometric features. These findings can possibly be explained by differences in selection of CIN III cells from CS and PEC specimens and by differences between fixation procedures as used for the two techniques. It was concluded that cytophotometric data of CS and PEC specimens representing CIN III lesions should not be regarded as interchangeable.
Contextual analysis and intermediate cell markers enhance high‐resolution cell image analysis for automated cervical smear diagnosis
Authors: Zahniser D. J. , Wong K. L. , Brenner J. F. , Ball H. G. , Garcia G. L. , Hutchinson M. L. 
Published: August 2005

AbstractUntil now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell‐by‐cell approach can provide.A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high‐resolution, single cell analysis and the analysis of intermediate cell markers. Thirty‐four samples prepared as mono‐layers and stained with the Feulgen‐Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.
Simultaneous quantification of c‐myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry
Authors: Engelhard Herbert H. , Krupka Jeri L. , Bauer Kenneth D. 
Published: August 2005

AbstractVariations in total cellular protein content can confound interpretation of the significance of modulations of specific cellular proteins. In an effort to overcome this problem, a techniaque is described for the simultaneous measurement of a specific cellular protein, total cellular protein, and DNA content. The method utilizes dual‐laser (uv and 488 nm) excitation and three fluorescent dyes: FITC, SR 101, and DAPI. FITC‐labelled antibody coupled with indirect immunofluorescence was used ot quantify the c‐myc oncoprotein, whereas SR 101 and DAPI were used ot measure total cellular protein and cellular DNA, respectively.Flow cytometric measurements of c‐myc oncoprotein were compared to densitometric readings of p64c‐myc SR 101 protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements corrlated well with those obtained by the biochemical methods.The usefulness of the technique was further examined following treatment of exponentially growing HL‐60 cells with 2.5 μg/ml cycloheximide for 0 to 12 h. Cycloheximide treatment was found to cause a significant decrease in c‐myc oncoprotein content within 2 h (P<0.05), a relative increase in the proportion of G0/G1 cells and a modest decrease in total cellular protein. This technique appears to provide a rapid, quantitative approach, useful for investigating alterations in cellular growth balance occurring with cell differentiation, neoplastic transformation, or cell treatment with radiation of cytostatic drugs.
DNA distributions in maldescended testes: Hyperdiploid aneuploidy without evidence of germ cell neoplasia
Authors: Clausen O. P. F. , Giwercman A. , Jøsrgensen N. , Bruun E. , Frimodt‐Møller C. , Skakkebæk N. E. 
Published: August 2005

AbstractFine‐needle aspiration biopsies and surgical biopsies were obtained from maldescended testes of 149 consecutive men. The aspirates were subjected to quantitative DNA flow cytometry and the surgical biopsy to histological evaluation. From more than 80% of the gonads, sufficient material was obtained for both examinations. A significant hyperdiploid cell population with a mean DNA index of 1.23 (range 1.17–1.31) was found in six gonads. Hyperdiploid aneuploidy was found in gonads without, as well as with, complete spermatogenesis. In none of the six cases did the surgical biopsy show evidence of early testicular neoplasia by morphology or by immunohistochemical methods with antibodies against carcinoma in situ. This indicates that aneuploidies in maldescended testes do not necessarily indicate malignancy. It may be speculated that hyperdiploid aneuploidy is related to the development of preneoplastic lesions.
Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines
Authors: Dooley William C. , Allison David C. , Robertson Joel 
Published: August 2005

AbstractHeteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non‐modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE‐5, MCa‐11, and HL‐60. The WCHE‐5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa‐11 and HL‐60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa‐11 and HL‐60 M‐ and T‐cell DNA peaks were similar to those of their chromosome number‐peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.
Method for kinetic analysis of drug‐induced cell cycle perturbations
Authors: Ubezio Paolo , Filippeschi Stefania , Spinelli Laura 
Published: August 2005

AbstractA method is described for quantitative study of the flux of cells through the cell cycle phases in in vitro systems perturbed by chemicals, such as chemotherapeutic agents. The method utilizes cell count and the flow cytometric technique of bromodeoxyuridine (BrdUrd) labeling, according to an optimized strategy. Cells are exposed to BrdUrd during the last minutes of drug treatment and fixed for analysis at 0,1/3Ts, 2/3Ts, Ts, and Tc + TG1 recovery times, where Ts, TG1, Tc are the mean durations of phases S and G1 and of the whole cycle of control cells. As an example of application of the proposed procedure, a kinetic study of the effect of l‐(2‐chloroethyl)‐l‐nitrosourea (CNU) on the L1210 cell cycle is described. Simple data analysis, requiring only a pocket calculator, showed that cells in phases G1 and G2M at the end of a 1 h treatment with 1 μg/ml CNU were fully able to leave these phases but were destined to remain blocked in the following G2M phase (G1 for a minority of them). We also found that cells initially in S phase were slightly delayed in completing their S phase and that 50% of them remained temporarily blocked in the subsequent G2M phase, irrespective of their position in the S phase.
Simultaneous use of Rhodamine 123, phycoerythrin, Texas red, and allophycocyanin for the isolation of human hematopoietic progenitor cells
Authors: Srour Edward F. , Leemhuis Tom , Brandt John E. , Vanbesien Koen , Hoffman Ronald 
Published: August 2005

AbstractThe supravital, mitochondrial specific dye Rhodamine 123 (R123) was used in conjunction with three monoclonal antibodies to isolate a population of human bone marrow (BM) cells enriched for hematopoietic progenitor cells. BM cells stained with phycoerythrin‐HLA‐DR, Texas red‐CD34, allophycocyanin‐CD15, and R123 were fractionated using four‐color immunofluorescence cell sorting. Cells expressing CD34 but not HLA‐DR and CD15 (CD34+ HLA‐DR− CD15−) were subdivided according to their reactivity with R123 into quiescent, R123 dull (R+) or cycling, R123 bright (R++) subpopulations. Morphological analysis and hematopoietic progenitor cell assays indicated that CD34+ HLA‐DR− CD15− R+ cells contained larger numbers of blast cells and colony forming units than CD34+ HLA‐DR− CD15− R++ cells. The flow cytometer settings used to accommodate the detection of the R123 fluorescence in combination with that of three other fluorochromes are described.
Flow cytometric analysis of cell cycle‐dependent changes in cell thiol level by combining a new laser dye with hoechst 33342
Authors: Poot Martin , Kavanagh Terrance J. , Kang Hee Chol , Haugland Richard P. , Rabinovitch Peter S. 
Published: August 2005

AbstractBy halogenation of methylfluoresceindiacetate (MFDA) or eosin‐diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)‐excitation system as required by bimane‐based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37°C, and fluorescence is sensitive to pre‐treatment with diethylmaleate but not to buthionine sulfoximine. In a dual‐laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position.
Retinoblastoma cell cycling
Authors: Kaleta‐Michaels Shawnya , Char Devron H. , Kroll Stewart M. , Waldman Frederic M. 
Published: August 2005

AbstractTechniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material.Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesisphase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48).DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.
Comparison of autofocus methods for automated microscopy
Authors: Firestone Lawrence , Cook Kitty , Culp Kevin , Talsania Neil , Preston Kendall 
Published: August 2005

AbstractTraditional autofocus methods were designed for microscopes driven by single processor computers. As computers are developed that exploit massive parallelism when acquiring and analyzing images, parallel cellular logic techniques became available to focus automatically. This paper introduces the reader to both cellular logic techniques for autofocus and a new spectral moment autofocus measure. It then compares these methods with more traditional autofocus methods. It is shown that traditional methods based on measurements of image power‐give the best results when tested on one set of real images and two sets of synthetic images. The next best methods are the cellular logic and spectral moment techniques, while the worst are those based on the image probability density function or histogram.
Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry
Authors: Collins James M. , McLean Grogan W. 
Published: August 2005

AbstractThe transverse location normal to the bilayer surface of a series of n‐(9‐anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cu+2, was 2 ≥ 3 > 6 ≥ 7 > 9 > 12 > 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells.
Pertussis toxin effects on chemoattractant‐induced response heterogeneity in human PMNs utilizing fluo‐3 and flow cytometry
Authors: Omann Geneva M. , Harter Josephine M. 
Published: August 2005

AbstractFlow cytometric methods were utilized to determine N‐formylpeptide‐induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo‐3. Fluo‐3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo‐3 extrusion was minimal (<10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N‐formyl‐met‐leu‐phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP‐induced cytosolic calcium levels were similar in cells loaded over a 20‐fold range of Fluo‐3‐acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A‐protomer of PT or a very sharp threshold of coupled G‐proteins required to transduce the responses.
Improved FACS‐Gal: Flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs
Authors: Fiering Steven N. , Roederer Mario , Nolan Garry P. , Micklem David R. , Parks David R. , Herzenberg Leonard A. 
Published: August 2005

AbstractThe previously reported FACS‐Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603–2607, 1988) measures E. coli lacZ‐encoded β‐galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, ussing a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence‐activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: (1) use of phenylethy‐lβ‐D‐thiogalactoside, a competitive inhibitor, to inhibit β‐galactosidase activity; (2) reduction of false positives by two‐color measurements; and (3) inhibition of interfering mammalian β‐galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by β‐galactosidase in this assay and the intracellular concentration of β‐galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS‐Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.
An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo‐ and chlorodeoxyuridine incorporated into DNA
Authors: Bakker P. J. M. , Stap J. , Tukker C. J. , van Oven C. H. , Veenhof C. H. N. , Aten J. 
Published: August 2005

AbstractIn this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat antiBrdUrd monoclonal antibody from Seralab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti‐BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and Texas‐Red conjugated goat antimouse.
Immunofluorescent labeling using covalently linked anti‐phycoerythrin antibodies and phycoerythrin polymers
Authors: Wilson M. R. , Crowley S. , Odgers G. A. , Shaw L. 
Published: August 2005

AbstractWe have developed methods for covalently attaching anti‐phycoerythrin (PE) MAbs to other antibodies, and for using PE or polymers of PE in conjunction with these conjugates to rapidly produce specific, high intensity fluorescent labeling of antigens. The performance of these systems was examined on the surface of microspheres and on the cell surface. The noncovalent means by which PE is incorporated into the label complex in this method makes it possible to use crude algal homogenate successfully as a source of PE in immunofluorescence assays. The intensity of labeling achieved using this method is comparable to, or in some cases better than, that obtained using a direct PE conjugate. With the aim of amplifying the intensity of fluorescence obtained, we produced two types of covalently linked complexes. These were an anti‐PE MAb, designated PE6, linked to itself (i.e., PE6—PE6), and PE linked to itself (i.e., PE—PE). When used in a twostage procedure in place of monomeric PE alone, these complexes increased the intensity of fluorescence obtained on the surface of microspheres by more than fourfold. On the cell surface, the performance of this system varied from one antigen to the next but in most cases was restricted to, at best, a 60% increase in the intensity of fluorescence and in many cases only about a 30% increase.
Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data
Authors: Kamentsky Louis A. , Kamentsky Lee D. 
Published: August 2005

AbstractWe describe a computer‐controlled 10 μm spot size laser scanning cytometer for making multiple wavelength fluorescence and scatter measurements of unconstrained cells on a surface such as a microscope slide. Designated areas of slides placed on a microscope stage are automatically scanned, and cells which generate above‐threshold scatter or fluorescence values are found and individually processed to determine a list of measurement parameters. For each fluorescence or scatter measurement parameter, this list contains the integrated and peak values and bit pattern images of a scan window centered on the cell. The measurement time, the position of the cell on the slide, and two segmentation indices are also included in the list. Measurement time, cell position, and properties derived from the bit patterns are used interchangeably with integrated or peak measurement values as coordinates of multiproperty displays. Cells may be selected for counting, data display in various forms, or visual observation based on their meeting complex criteria among a chain of two property screens. Cells with selected properties may be viewed during an experiment or retrospectively. A designated specimen field may be repeatedly remeasured to perform kinetic cell studies. An argon ion and a HeNe‐ based laser instrument have been constructed and software has been written and evaluated with the specific goal of increasing the precision of propidium iodide‐stained cellular DNA measurements. Some of the capabilities of the instrument and its current performance are described.