bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA slit‐scan flow cytometry of bladder irrigation specimens and the importance of recognizing urothelial cells
Authors: Wheeless Leon L. , Reeder Jay E. , O'Connell Mary J. , Robinson Roy D. , Cosgriff Janice M. , Fradet Yves , Frank Irwin N. , Cockett Abraham T. K. 
Published: August 2005

AbstractDNA slit‐scan flow cytometry was used to analyze 150 bladder irrigation specimens from 83 patients. Specimens were categorized into groups based on cystoscopy, histology, and cytopathology. Cells were stained for DNA with propidium iodide using a whole cell protocol. Nonspecific fluorescence in the cytoplasm of some urothelial cells together with differential DNA staining of cell types in certain specimens was noted. DNA frequency distributions were analyzed using a semi‐automated technique. Data were gated using slit‐scan morphological features to remove cellular debris, multiple nuclei, and cells exhibiting nonspecific cytoplasmic fluorescence. Specimens were classified abnormal if they were aneuploid or had a hyperdiploid fraction (HDF) greater than 8%. The sensitivity to abnormality was 89% for grade 3 transitional cell carcinoma (TCC), 70% for grade 2 TCC, and 67% for grade 1 TCC. Specificity was 61%. Specimen data were then reprocessed using slit‐scan morphological features to enrich for urothelial cells. The urothelial cells were identified by the ratio of nuclear diameter to cell diameter. This method was found to be in good agreement with immunofluorescent labeling of urothelial cells using the urothelium‐seleetive T16 monoclonal antibody. The sensitivity to abnormality remained 89% for grade 3 TCC and 70% for grade 2 TCC, but fell to 52% for grade 1 TCC. Specificity for the urothelial cell enriched data increased to 77%. Reprocessing of data to enrich for urothelial elements resulted in 16 fewer specimens with an aneuploid DNA distribution and 2 fewer specimens with increased HDF. These data illustrate the necessity for recognition of urothelial cells in the flow analysis of unfixed bladder irrigation specimens stained with propidium iodide using a whole cell technique. Differential staining of blood and urothelial cells can mimic aneuploidy or contribute to an increased hyperdiploid fraction. These may result in the specimen being classified as abnormal if they are not recognized as such.
Detection and analysis by dual‐laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli
Authors: Sanders Cynthia A. , Yajko David M. , Nassos Patricia S. , Hyun William C. , Fulwyler Mack J. , Hadley W. Keith 
Published: August 2005

AbstractBacteriophage T4 DNA was detected and analyzed inside E. coli by dual‐laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A‐T‐ and G‐C‐rich regions of DNA, respectively. An exponentially‐growing culture of E. coli ATCC 11303 was infected with T4 bacteriophage at a 1:1 multiplicity of infection. Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication. The samples were then centrifuged, ethanol‐fixed, stained with H33258 and CA3, and analyzed by flow cytometry. Twenty‐five minutes post‐infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data. By 35 min, T4 DNA‐containing cells could be distinguished from E. coli cells containing little or no T4 DNA. The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4 DNA inside E. coli. A value of 35.6 ± 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that dual‐laser flow cytometry can be used to study viral DNA inside the bacterial host.
Comparison of microscopic imaging strategies for evaluating immunocytochemical (PAP) steroid receptor heterogeneity
Authors: Sklarew R. J. , Bodmer S. C. , Pertschuk L. P. 
Published: August 2005

AbstractReceptogram analysis was compared with three other imaging strategies for immunocytochemical assay of estrogen receptors. These included nuclear‐specific methods for analysis of nuclear integrated optical density (IOD) or mean optical denstiy (MOD) histograms, and field‐specific methods, where the pixel optical density (POD) histogram was evaluated for the composite nuclear phase. Measurements in culture and in breast cancer cryosections were treated separately to isolate geometric considerations. In culture receptograms the modality of IOD and MOD histograms and their bivariate contour maps revealed one, two, or more subpopulations with discrete receptor content and concentration. However, when the field of nuclei was imaged as a whole, regardless of the number of subpopulations, POD histograms showed two minima, defining three intranuclear phases. This was due to mottling and variegation of intranuclear chromatin and nucleolar immunostaining and not to differences between nuclei. These limitations were also revealed in breast cancer sections. In POD histograms, % unstained pixels did not provide a reliable estimate of % receptor negative nuclei, as determined by their enumeration. In sections, correction of IOD for nuclear volume variability was essential to suppress artifactual peaks not representing differences in receptor content. This was achieved by multiplying nuclear IOD by the spherical nuclear radius (S) of individual slab sections. Peaks of IOD(S) then reflected receptor content on a true ratio scale. Only receptogram analysis, which incorporates these strategies, permitted objective evaluation of receptor heterogeneity at the level of tumor subpopulations.
Evaluation of flow cytometric methods for determining population potential doubling times using cultured cells
Authors: Terry Nicholas H. A. , White R. Allen , Meistrich Marvin L. , Calkins Dennise P. 
Published: August 2005

AbstractVarious methods have been proposed for determining the potential doubling times (Tpot) of mammalian cell populations by using flow cytometric techniques after labeling the cells with bromodeoxyuridine (BrdUrd). We show here that, in a well‐defined in vitro system where multiple time measurements are possible, all the methods give similar results that are close to the true population doubling time. Of ultimate interest, however, is the accuracy of determination of Tpot from a single time point. In this paper we compare the accuracy and precision of the methods in making such determinations at different times after labeling.The relative movement (RM) of BrdUrd‐labeled cells that have not divided at the time of assay allows for computation of the length of S phase (Ts). The precision of estimation of Ts was enhanced when a quantity, ν (a function of the fraction of BrdUrd‐labeled divided and the fraction of BrdUrd‐labeled undivided cells), was used to estimate the initial intercept of RM. Furthermore, calculation of Tpot from the formula, Tpot = ln(2) Ts/v, gave values closest to the observed population doubling time. It is suggested that the use of RM with v be the analytical method of choice for the calculation of Tpot from single time‐point observations, preferably made at times between the length of the G2 and M phases (TG2M) and Ts.
Mimosine reversibly arrests cell cycle progression at the G1–S phase border
Authors: Watson Paul A. , Hanauske‐Abel Hartmut H. , Flint Alan , Lalande Marc 
Published: August 2005

AbstractIt has previously been demonstrated that the compound mimosine inhibits cell cycle traverse in late G1 phase prior to the onset of DNA synthesis (Hoffman BD, Hanauske‐Abel HM, Flint A, Lalande M: Cytometry 12:26–32, 1991; Lalande M:Exp Cell Res 186:332–339, 1990). These results were obtained by using flow cytopmetric analysis of DNA content to compare the effects of mimosine on cell cycle traverse with those of aphidicolin, an inhibitor of DNA polymerase α activity. We have now measured the incorporation of bromodeoxyuridine into lymphoblastoid cells by flow cytometry to determine precisely where the two inhibitors act relative to the initiation of DNA synthesis. It is demonstrated here that mimosine arrests cell cycle progression at the G1–S phase border. The onset of DNA replication occurs within 15 min of releasing the cells from the mimosine block. In contrast, treatment with aphidicolin results in the accumulation of cells in early S phase. These results indicate that mimosine is a suitable compound for affecting the synchronous release of cells from G1 into S phase and for analyzing the biochemical events associated with this cell cycle phase transition.
Genetic stock assessment of spawning Arctic cisco (Coregonus autumnalis) populations by flow cytometric determination of DNA content
Authors: Lockwood Samuel F. , Bickham John W. 
Published: August 2005

AbstractIntraspecific variation in cellular DNA content was measured in five Coregonus autumnalis spawning populations from the Mackenzie River drainage, Canada, using flow cytometry. The rivers assayed were the Peel, Arctic Red, Mountain, Carcajou, and Liard rivers. DNA content was determined from whole blood preparations of fish from all rivers except the Carcajou, for which kidney tissue was used. DNA content measurements of kidney and blood preparations of the same fish from the Mountain River revealed statistically indistinguishable results. Mosaicism was found in blood preparations from the Peel, Arctic Red, Mountain, and Liard rivers, but was not observed in kidney tissue preparations from the Mountain or Carcajou rivers. The Liard River sample had significantly elevated mean DNA content relative to the other four samples; all other samples were statistically indistinguishable. Significant differences in mean DNA content among spawning stocks of a single species reinforces the need for adequate sample sizes of both individuals and populations when reporting “C” values for a particular species.
Sorting minor subpopulations of cells: Use of fluorescence as the triggering signal
Authors: McCoy J. Philip , Chambers William H. , Lakomy Robert , Campbell Joseph A. , Stewart Carleton C. 
Published: August 2005

AbstractFlow cytometric cell sorting is commonly used to obtain purified subpopulations of cells for use in in vitro and in vivo assays. This can be time‐consuming if the subpopulations of interest represent very low percentages of the cell suspension under study. Often the desired subpopulations are identified by two‐color immunofluorescence staining. Generally, cell sorting is performed with a flow cytometer configured to trigger on light scatter signals, then sort windows are set based upon the signals from both fluorescent markers. We demonstrate that triggering the cytometer with the fluorescence signal from antibody staining common to both of the desired subpopulations, then sorting the subpopulations based upon staining of a second marker, substantially increases the speed of cell sorting vis‐à‐vis traditional methods. This is because undesired events are not analysed, allowing an increase in the throughput rate. While desired subpopulations of cells can be obtained by this method, undesired (i.e., nonstaining) cell “contaminants” increase and may require a second sort. The combined time for the initial enrichment sort and a second sort can be less than sorting once using standard methodology. Alternatively, the degree of contamination may be controlled by adjusting the concentration of the cell suspension and by the sample flow rate.
Use of diploid and triploid trout erythrocytes as internal standards in flow cytometry
Authors: Pierrez José , Ronot Xavier 
Published: August 2005

AbstractDNA content determination requires the use of standards. Vindelov has shown the need to use two standards. Chicken and trout erythrocytes are commonly used, but they are not ideal standards. On the one hand, their DNA contents rarely frame the studied sample DNA content, and, on the other hand, as their base compositions are different in terms of A+T/G+C, their relative indices change according to the stains used. Use of triploid trout erythrocytes instead of chicken erythrocytes allows elimination of these two drawbacks; however, diploid trout must be differentiated from triploid trout. The present paper shows that an anatomic malformation is found with the triploid trout and so justifies the use of paired diploid and triploid trout as standards to measure nuclear DNA content.
A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification
Authors: Schmid Ingrid , Uittenbogaart Christel H. , Giorgi Janis V. 
Published: August 2005

AbstractA method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4°C with 0.25% buffered paraformaldehyde then for 15 min at 37°C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1‐peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90° angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual‐color surface antigen staining combined with DNA staining with 7‐amino‐actinomycin D (7‐AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF‐CEM for expression of CD3ϵ and on NALM‐6 for expression of μ. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.
3‐D image analysis system and megakaryocyte quantitation
Authors: Zhang Y. J. 
Published: August 2005

AbstractA quasi‐automatic computer image analysis system has been developed for 3‐D reconstruction of stained serial sections and implemented on an IBAS system. Some new automatic image analysis techniques have been designed and incorporated into the system. For image segmentation, a transition region determination based thresholding method is introduced. Neither histogram calculation nor empirical parameters are needed in the automatic threshold selection. A two step 3‐D reconstruction procedure—symbolic and pictorial reconstructions—is designed to improve the flexibility and the computational capability of the system. The global level registration and local level registration are separated. The former consists of establishing the relationship among a large numbers of profile pairs dispersed in adjacent sections. A pattern matching method based on pattern recognition principles is devised to exploit the information about the statistical character of mismatch caused by deformation of sections and about the relationship of nearby objects. For the latter, an equivalent elliptical approximation method based on the physical theory of the rotation of rigid bodies is proposed. The system has been used for 3‐D reconstruction and quantitation of megakaryocytes in human bone marrow tissue. Features about individual 3‐D megakaryocyte cell and the spatial distribution of megakaryocytes are determined. The latter is a new to megakaryocyte quantitation and is not possible by using conventional stereologic techniques. These experimental results have demonstrated the ability of the system to perform quantitative analysis.
Dicentric chromosome frequency analysis using slit‐scan flow cytometry
Authors: Lucas Joe N. , Mullikin James C. , Gray Joe W. 
Published: August 2005

AbstractSlit‐scan flow cytometry (SSFCM) was used to quantify the frequency of dicentric chromosomes in human lymphoblastoid cells following γ irradiation. In this study, cultured human cells were irradiated with 0, 0.25, 0.5, 1.0, and 2.0 Gy of 0.66 MeV γ‐rays, cultured for an additional 11 h, and treated for 5 h with colcemid. Chromosomes were then isolated, stained with propidium iodide, and analyzed using SSFCM for total fluorescence and slit‐scan profile. The frequency of chromosomes having DNA contents greater than once and less than twice the DNA content of the number 1 chromosome and producing trimodal profiles was determined at each dose. This frequency was used as an estimate of the relative dicentric chromosome frequency at that dose. The estimated dicentric chromosome frequency per cell, f(D), increased with dose, D, in a linear‐quadratic manner according to the relation
Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide
Authors: Prosperi Ennio , Giangarè Maria Chiara , Bottiroli Giovanni 
Published: August 2005

AbstractA flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA‐specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single‐strandspecific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15—30% above the value of undigested control samples. No significant modifications were observed with the restriction enymzes Eco RI, Alu I, and Not I. The DNAse I‐induced increase in fluorescence intensity was also observed with the non‐intercalating dye Hoechst 33258, but not with mithramycin. Nuclease‐induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA‐P ratios > 0.11. Below this value (2 μg/ml PI), the fluorescence intensity of digested samples was 15—30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation‐ but not sequence‐specific nucleases induce a relaxation of DNA supercoils.
Flow cytometric monitoring of glutathione content and anthracycline retention in tumor cells
Authors: Nair Swagata , Singh Shivendra V. , Krishan Awtar 
Published: August 2005

AbstractWe have used an enzymatic (spectrophotometric) and a flow cytometric (GSH‐MBCL) method to compare the glutathione (GSH) content of doxorubicin sensitive (P388) and resistant (P388/R‐84) murine leukemic and human lung cancer cells. The flow cytometric analysis revealed that GSH‐MBCL conjugate formation was dependent on glutathione‐S‐transferase (GST) activity. The human solid tumor cell lines exhibited extensive heterogeneity, high GSH content, and GST activity. In contrast to the enzymatic method, the flow cytometric method did not accurately reflect the 95% reduction in GSH content of cells treated for 24 h with 100 μM BSO. Possible reaction of MBCL with other sulfhydryl groups (other than GSH) in BSO‐treated cells may be responsible for this discordance. We have also shown the feasibility of using dual parameter flow cytometry to monitor cellular anthracycline (daunorubicin) retention and GSH‐MBCL conjugate fluorescence in human tumor cells. These two parameters, which measure drug retention and cellular detoxification, are believed to be the important determinants of chemoresistance in tumor cells.
Concomitant delineation of surface Ig, B‐cell differentiation antigens, and HLADR on lymphoid proliferations using three‐color immunocytometry
Authors: Segal Glenn H. , Edinger Mark G. , Owen Matthew , McNealis Michael , Lopez Phillip , Perkins Alvin , Linden Michael D. , Fishleder Andrew J. , Stoler Mark H. , Tubbs Raymond R. 
Published: August 2005

AbstractAccurate and consistent enumeration of B‐cell subpopulations in lymphoid tissue was achieved through multiparameter three‐color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)‐anti‐CD19 (Leu12) and biotinylated anti‐HLADr/streptavidin‐Duochrome® (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti‐surface immunoglobulin (SIg) antibodies, effectively separated non‐specific binding and background fluorescence from true B‐cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion. Autofluorescence was measured to establish a fluorescence threshold. A second control measured non‐specific binding of isotypic control mouse Ig and non‐immune rabbit IgG. Cell suspensions from 128 samples of various lymphoid proliferations were studied. In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis. Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated. SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively. Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas.
The effect of improvements in cytometer sensitivity on the detection of CD5‐positive B cells with dim fluorescence
Authors: Givan Alice L. , Shenton B. K. , Culvert Jane E. 
Published: August 2005

AbstractWhen antigen density on the surface of a cell population is low and variable, the percentage of that population determined to express the antigen (i.e., to be positively stained) depends directly on the sensitivity of the flow cytometer for resolving particles which are dimly fluorescent from those which are unstained. In this study, the sensitivity of a commercial flow cytometer has been improved by changes in the photomultiplier tube, the fluorescence filter, and the amount of stray light entering the fluorescence channel. In a model system with human lymphocytes, modifications to these factors increased the percent of the B‐lymphocyte population found to express the CD5 antigen.