bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative analysis of chromosome in situ hybridization signal in paraffin‐embedded tissue sections
Authors: Dhingra K. , Sneige N. , Pandita T. K. , Johnston D. A. , Lee J. S. , Emami K. , Hortobagyi G. N. , Hittelman W. N. 
Published: June 2005

AbstractInterphase cytogenetic analysis using chromosome‐specific probes is increasingly being used to detect chromosomal aberrations on paraffin‐embedded tissue sections. However, quantitative analysis of the hybridization signal is confounded by the nuclear slicing that occurs during sectioning. To determine the sensitivity and accuracy of chromosome in situ hybridization for detecting numerical chromosomal aberrations on paraffin‐embedded sections, in situ hybridization was performed on sections derived from mixtures of cell populations with known frequencies of numerical chromosomal aberrations and the Chromosome Index (CI) was calculated (i.e., total number of signal spots/number of nuclei counted) as a quantitative measure of chromosome copy number. The presence of 25% or more monosomic or tetrasomic cells in a given population was easily detected as a change in CI (P 0.05). Lower degrees of polysomy could be detected as a small percentage of nuclear fragments with >2 signal spots. The CI was not significantly influenced by a change in section thickness from 4 to 8 μM, by an increase in cell size from 478 to 986 μM3, or by the choice of detection method (fluorescence vs. conventional bright‐field microscopy). Comparative analysis of touch preparations and tissue sections from the corresponding breast tumors showed that CI accurately reflects the average copy number of chromosomes in intact nuclei and may actually be superior to in situ hybridization on whole nuclei for the detection of numerical chromosomal changes in defined histologic areas. This method is thus a sensitive and accurate means of studying genetic changes in premalignant and malignant tissue, and of assessing the genetic changes associated with specific phenotypes. © 1994 Wiley‐Liss, Inc.
Use of ferro‐electric liquid crystal shutters for time‐resolved fluorescence microscopy
Authors: Verwoerd N. P. , Hennink E. J. , Bonnet J. , Van der Geest C. R. G. , Tanke H. J. 
Published: June 2005

AbstractA technique is described to modify a standard fluorescence microscope for time‐resolved visualization of delayed luminescing substances with decay times from 50 μs to several milliseconds. The modification consists of synchronized operation of a mechanical shutter, positioned in an aperture plane in the excitation pathway, simultaneously with a ferro‐electric liquid crystal (FLC) shutter on the emission side. Operation of the microscope is through a microprocessor interfaced keypad by which all timing parameters can be adjusted for optimal suppression of fast decaying luminescence. Accuracy of the timing was within 1 μs. Prompt fluorescence was suppressed up to 106 times, as determined for bright prompt fluorescing microspheres. The use of the FLC shutter resulted in a reduction in emission intensity by a factor of 8 (due to the use of polarizers, the lower transmission of the FLC devices, and IR blocking filters). No significant image degradation due to shutter operations was observed. The modified microscope was successfully used for the visualization of delayed luminescing immunolabels, such as inorganic phosphor particles and lanthanide chelates, as well as naturally phosphorescing materials. © 1994 Wiley‐Liss, Inc.
Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry
Authors: Pechhold Klaus , Pohl Thomas , Kabelitz Dieter 
Published: June 2005

AbstractDetermination of the number of viable cells or quantification of lymphocyte subsets in heterogeneous cell populations is critically important for cytotoxicity assays, apoptosis assays, or the analysis of differential activation of T‐cell subsets by distinct stimuli. In this report, we describe a rapid flow cytometry method termed Standard Cell Dilution Analysis (SCDA) specifically to quantify any subset of phenotypically definable, viable cells in heterogeneous populations using a FACScan flow cytometer. This method combines: (1) specific detection of lymphocyte subsets by phycoerythrin‐conjugated monoclonal antibodies, (2) electronic exclusion of dead cells or cell debris by propidium‐iodide staining and gating on forward vs. sidescatter, respectively, and (3) admixture of a known amount of fixed, fluorescein isothiocyanate stained cells immediately before analysis as a constant parameter to allow for calculation of cell quantity. We have used SCDA to analyze the in vitro growth characteristics of various human T‐lymphocyte subpopulations in response to different activation stimuli. © 1994 Wiley‐Liss, Inc.
Time‐resolved flow cytometry for the measurement of lanthanide chelate fluorescence: II. Instrument design and experimental results
Authors: Condrau Marc A. , Schwendener Reto A. , Zimmermann Marcel , Muser Markus H. , Graf Urs , Niederer Peter , Anliker Max 
Published: June 2005

AbstractA time‐resolved flow cytometer capable of measuring a luminescence with a decay time in the range of 10 μs to 2 ms, typical for some lanthanide chelates, is presented. The instrument permits acquisition of conventional light scatter and prompt fluorescence signals as well as detection of slowly decaying luminescence by a photon counting unit for a selectable time period of 1 μs to 1 ms. During photon counting, the laser beam is turned off by an acoustooptic deflector. The design of a flow chamber with an average geometrical light collection efficiency of 35% over a distance of 1.7 mm is presented and analyzed by ray tracing. A pulse processing system featuring digital integration of the conventional signals and a transputer system for the acquisition and the transfer of the measured parameter values to a host computer is described.Instrument function is verified with lyophilized human lymphocytes stained for the CD8 antigen with dye‐loaded liposomes. Quantitation of cell‐associated europium chelate fluorescence, displaying a decay time of 1.6 ms, is demonstrated. Elimination of fast decaying background emission generated by DNA‐associated ethidium bromide is shown. The background generated by instrument components in the time‐gated measurement channel is characterized, and measures for its complete elimination are discussed. © 1994 Wiley‐Liss, Inc.
Automated peak detection and cell cycle analysis of flow cytometric DNA histograms
Authors: Kallioniemi Olli‐P. , Visakorpi Tapio , Holli Kaija , Isola Jorma J. , Rabinovitch Peter S. 
Published: June 2005

AbstractWe describe an algorithm for fully automated flow cytometric DNA histogram classification and analysis that provides rapid, reproducible determination of DNA index and S‐phase fraction (SPF). Automated classification agreed with subjective assessment of DNA ploidy in 96–98% of DNA histograms. Automated and conventional analyses of DNA index (r = 0.95) and SPF (r = 0.89) were also highly correlated with one another. In a series of 86 node‐negative breast carcinomas, SPF calculated with the fully automated method was a significant predictor of 10 year survival (P = 0.009). Automation greatly increased the speed of DNA histogram analysis, allowing evaluation of the same set of histograms with different methods. In a preliminary study exploring the optimization of DNA histogram analysis, the best association between SPF and prognosis of breast cancer patients was achieved using sliced nuclei debris modeling, reporting only the aneuploid SPF (in aneuploid histograms), while excluding small aneuploid clones (<15% of total cell count) from evaluation. In conclusion, automated DNA histogram analysis does not replace the need for close human supervision but provides a useful guideline for less experienced users, facilitates interlaboratory comparisons, and makes possible extensive reanalyses of large data sets. © 1994 Wiley‐Liss, Inc.
Direct flow cytometry of anaerobic bacteria in human feces
Authors: van der Waaij L. A. , Mesander G. , Limburg P. C. , van der Waaij D. 
Published: June 2005

AbstractWe describe a flow cytometry method for analysis of noncultured anaerobic bacteria present in human fecal suspensions. Nonbacterial fecal compounds, bacterial fragments, and large aggregates could be discriminated from bacteria by staining with propidium iodide (PI) and setting a discriminator on PI fluorescence and by exclusion of events with large forward scatter. Since anaerobic bacteria, which account for over 99.9% of all fecal bacteria, die during sample preparation, a fixation step was not necessary.A second aim of this study was to investigate the technical possibility of measurement of in vivo IgA coating of fecal anaerobic bacteria as well as their bacterial size. Fecal samples of 22 healthy human volunteers were analyzed. The fluorescence distribution of IgA‐coated bacteria labeled with fluorescein isothiocyanate (FITC)‐anti‐Hu‐IgA had overlap with noncoated bacteria. However, with match region subtraction, detection of low levels of specific FITC fluorescence on IgA‐coated bacteria was achieved. The median bacterial two‐dimensional surface area was 1.0 μm2. To validate flow cytometry data, all samples were analyzed with an image analysis system as well. With this new method, a rapid evaluation of fecal flora with high sensitivity for specific FITC fluorescence is possible without culturing. © 1994 Wiley‐Liss, Inc.
Automatic detection of clustered, fluorescent‐stained nuclei by digital image‐based cytometry
Authors: Lockett Stephen J. , Herman Brian 
Published: June 2005

AbstractAutomatic image‐based cytometry (IC) can conveniently quantify the distributions of several specific, fluorescencelabeled molecules within individual, isolated cells of slide‐ or tissue‐based specimens. However, many specimens contain clusters of cells or nuclei that are not detected as individual entities by existing automatic methods. We have developed analysis algorithms which detect individul nuclei occurring in clusters or as isolated nuclei. Specimens were labeled with a fluorescent DNA stain, imaged and the images were segmented into regions of nuclei and background. Clusters of nuclei, identified by their size and shape, were divided into individual nuclei by searching for dividing paths between nuclei. The paths, which need not be straight, possessed the highest average gradient per pixel. In addition, both high‐ and low‐pass filtered images of the original image were analyzed. For each individual nucleus, one of the three segmented regions representing the nucleus (from either the original or one of two filtered images) was chosen as the final result, based on the closeness of the regions to average nuclear morphology. The algorithms correctly detected a high proportion of isolated (328/333) and clustered (254/271) nuclei when applied to images of 2 μm prostate and breast cancer sections. Thus, these algorithms should enable much more accurate detection and analyses of nuclei in intact specimens. © 1994 Wiley‐Liss, Inc.
Flow‐cytometric quantification in human gliomas of alpha satellite DNA sequences specific for chromosome 7 using fluorescence in situ hybridization
Authors: Kwak Taeshik , Nishizaki Takafumi , Ito Haruhide , Kimura Yuka , Murakami Tomoyuki , Sasaki Kohsuke 
Published: June 2005

AbstractCell suspensions prepared from freshly frozen tissue specimens were used to examine aberrations in the number of chromosome 7 signals in 10 human gliomas. Nuclear DNA was hybridized in vitro with an alpha satellite DNA probe specific for the centromeric regions of chromosome 7, using fluorescence in situ hybridization (FISH). The intensity of the fluorescence signal from the hybridized probe was measured, together with the nuclear DNA content, by flow cytometry. The mean probe fluorescence of all nuclei was compared to the mean copy number per nucleus found with microscopic scoring. Moreover, the mean probe fluorescence ratio of DNA aneuploid nuclei relative to DNA diploid nuclei (FISHa/FISHd) was calculated to determine how the numerical aberration of chromosome 7 signals contributes to the DNA ploidy of the sample.The results from the flow‐cytometric analysis and from microscopic evaluation were compatible. One of four tumors with DNA diploidy had a higher average intensity of FISH signal and a broader coefficient of variation in FISH signal than normal brain tissue; this was shown to be due to the gain of chromosome 7 signals. Although FISHa/FISHd correlated with DNA indices (P < 0.01), there were some disparities, probably due to other complex genotypic associations involving several gains or losses of chromosomes. Thus gain of chromosome 7 in gliomas is related to both DNA ploidy change and chromosome specific gain. It is concluded that flow‐cytometric quantification of FISH is useful in investigating numerical aberrations of chromosomes and nuclear DNA content simultaneously. © 1994 Wiley‐Liss, Inc.
Isolation and characterization of a Chinese hamster ovary cell mutant with improved staining for indo‐1
Authors: Wieder Eric D. , Fox Michael H. 
Published: June 2005

AbstractChinese hamster ovary (CHO) 10B2 cells do not stain well with indo‐1 and thus cannot be used for experiments to measure intracellular calcium using this dye. We have isolated a mutant CHO cell line (CHO IS1) that stains quite well with indo‐1 and that has virtually identical growth characteristics and heat sensitivity as the parent line. The mutant was isolated by sorting individual mutagenized cells with high indo‐1 fluorescence and cloning them. Since it has been reported that cells with multiple drug resistance (MDR+) can pump out various fluorescent dyes, the mutant and parent lines were characterized for Hoechst 33342 staining, Adriamycin toxicity, and P‐glycoprotein expression, which are markers of the MDR phenotype. P‐Glycoprotein was measured with the C219 antibody using flow cytometry. Multidrug‐resistant cells (CHRC5) were used as positive controls. The IS1 cells stained as well with Hoechst 33342 as fixed 10B2 cells, and much better than unfixed 10B2 cells. The IS1 cells were 10‐ to 30‐fold more sensitive to Adriamycin that the 10B2 cells, and both cell lines were much more sensitive than the CHRC5 cells. The amount of P‐glycoprotein was similar in both 10B2 and IS1 cell lines, but was about fivefold lower than the CHRC5 cells. Thus, the poor staining for indo‐1 in the 10B2 cells may not be caused by the P‐glycoprotein MDR pump, but by a different efflux pathway. Alternatively, the P‐glycoprotein may be altered and less efficient in the CHO IS1 cells. © 1994 Wiley‐Liss, Inc.
Effect of fixation on quantification of the expression of leucocyte function‐associated surface antigens
Authors: McCarthy D. A. , Macey Marion G. , Cahill Mary R. , Newland A. C. 
Published: June 2005

AbstractThe surface expression of adhesion molecules and other function‐associated antigens on peripheral blood leucocytes may be measured by flow cytometry. However, quantification of these antigens is difficult, because their expression can be rapidly and artefactually modulated if the cells are activated in vitro. Consequently, it is common, when analyzing these antigens either: (1) to label leucocytes in whole blood at 4°C, to lyse erythrocytes and then fix the leucocytes, or (2) to fix the leucocytes in whole blood, to lyse the erythrocytes, and then label the leucocytes. We have compared the mean fluorescence intensity (MFI) values for CD11b, CD18, and L‐selectin (Leu‐8 and TQ1 epitopes) on human peripheral blood leucocytes, using these two approaches. In addition, we have simultaneously evaluated how anticoagulants (acid citrate, K3EDTA, and heparin) and the presence or absence of divalent metal ions (Ca2+ and Mg2+) affect the expression levels of these antigens. The results for all four epitopes varied markedly depending on the preparation procedure used but were less affected by the choice of anticoagulant and whether divalent cations were or were not present in the media used for cell preparation and labelling. Comparison of the results obtained using these procedures, which involve fixation with formaldehyde, with those obtained by a recently developed procedure in which unfixed leucocytes were labelled with the vital nuclear dye LDS‐751 and antibodies together, then analysed in unlysed whole blood at 4°C, showed that formaldehyde‐based preparation techniques underestimated the expression (MFI) of CD18, Leu‐8, and TQ1. It is recommended that, whenever practicable, measurements are made on unfixed cells stained using the newer procedure. © 1994 Wiley‐Liss, Inc.
Scanning microspectrofluorometry of rhodamine 123 in multidrug‐resistant cells
Authors: Millot Jean‐Marc , Sharonov Sergueï , Manfait Michel 
Published: June 2005

AbstractScanning microspectrofluorometry has been developed to perform the mapping of fluorescence spectra from all locations in a living cell. This new method has been applied to study the molecular environment of rhodamine 123 (R123) in sensitive (K562, CEM) and multidrug‐resistant (K562‐R, CEM/VLB100) tumor cells.All cells exposed to R123 showed a similar distribution of fluorescence in the perinuclear region. A lower cytoplasmic fluorescence intensity corresponding to a reduced drug accumulation was observed in resistant cells, as expected in the multidrug resistance process.Fluorescence emission spectra of R123 are useful to probe the polarity of the R123 environment. Thus, fluorescence spectra of R123‐treated cells have been analyzed as a linear combination of model spectra: R123 in water and R123 in tensio‐active Triton X‐100. In sensitive cells, emission spectra of R123 underwent a red shift, equivalent to those observed in isolated coupled mitochondria. This suggests the formation of a complex in hydrophobic sites. In contrast, R123 spectra were less shifted in resistant cells, showing two types of both hydrophobic and hydrophilic binding sites. This could be related to an intracellular redistribution of R123 in resistant cells. © 1994 Wiley‐Liss, Inc.
Flow cytometric detection and quantitation of the epidermal growth factor receptor in comparison to Scatchard analysis in human bladder carcinoma cell lines
Authors: Brockhoff Gero , Hofstaedter Ferdinand , Knuechel Ruth 
Published: June 2005

AbstractThe epidermal growth factor receptor (EGFR) is considered a tumor‐related marker with potential diagnostic and prognostic value. In order to assess the sensitivity of flow cytometry to detect EGFR and to quantify receptors objectively, two human bladder carcinoma cell lines with different urothelial differentiation, RT4 and J82, were grown in vitro, and their membrane EGFR content was measured by flow cytometry. Exponential monolayers showed decrease of EGFR content after 20 min pulses with 10 ng/ml EGF in medium, as detected with the antibody EGFR1 in a double staining technique with propidium iodide for DNA evaluation. Further decrease of green fluorescence intensity was seen in cells constantly exposed to EGF.Absolute receptor numbers were determined by Scatchard analysis with radioactive EGF and resulted in relatively low receptor numbers for both cell lines (∼3–4 × 104 EGFR/cell), as well as one affinity class.These findings could be matched by absolute receptor quantification by flow cytometry, adding beads with defined antigenic sites (Quantum Simply Cellular, Microbead Corporation) to the cell suspension for staining.Our data suggest that flow cytometric EGFR detection and quantitation may be supplied to in vivo tumor samples and that measurements by multiparameter analysis may define subpoplations valuable for tumor diagnosis and judgment on tumor progression. © 1994 Wiley‐Liss, Inc.
Flow cytometric determination of the multidrug‐resistant phenotype in acute leukemia
Authors: Maslak Peter , Hegewisch‐Becker Susanna , Godfrey Loren , Andreeff Michael 
Published: June 2005

AbstractExpression of the multidrug‐resistant (MDR) phenotype was investigated in acute leukemia using a monoclonal antibody (HYB‐241) directed against a cell surface epitope of the 180 kd P‐glycoprotein (gp180) by flow cytometric analysis of clinical samples. Samples from sixtyfour patients were tested (37 with acute myelocytic leukemia, 20 with acute lymphocytic leukemia, and 7 with blastic chronic myelocytic leukemia). A D value (derived from Kolmogorov‐Smirnov test) greater than 0.15 was considered positive (+). Eight of 32 newly diagnosed patients were positive for gp180 compared with 22 of 32 relapsed/refractory (R/R) patients (P < 0.001). Of the new patients, vinca/anthracycline‐based induction therapy failed in 3/6 gp180(+) and 5/18 gp180(−) patients. In the R/R group, 15/16 gp180(+) and 3/6 gp180(−) patients failed to achieve complete remission (P < 0.05). In vitro drug accumulation studies performed with verapamil failed to show a correlation with clinical response. However, in a subset of patients, a striking correlation (r = .97, P = .001) was noted between the presence of gp180 as determined by the D value and the functional activity of the P‐glycoprotein as expressed by increased daunorubicin accumulation in the presence of verapamil. The results suggest that (1) newly diagnosed patients can express gp180, (2) P‐glycoprotein is expressed in 69% of R/R patients, (3) response in R/R patients is effected by the presence of gp180, and (4) expression of gp180 is highly correlated with its function as a drug‐efflux pump in a subset of the patients studied. The complexity of clinical drug resistance is underscored by the finding that the MDR model is not applicable to all cases. In such instances, other mechanisms may play a predominant role. © 1994 Wiley‐Liss, Inc.
Use of human leukocyte‐specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys
Authors: Reimann Keith A. , Waite Barry C. D. , Lee‐Parritz David E. , Lin Wenyu , Uchanska‐Ziegler Barbara , O'Connell Michael J. , Letvin Norman L. 
Published: June 2005

AbstractThe rhesus monkey (Macaca mulatta) is an important experimental animal frequently utilized for studies of infectious diseases, immunity, hematopoiesis, and transplantation. Since the structure of cell surface molecules is phylogenetically conserved, monoclonal antibodies raised against human leukocyte antigens can sometimes recognize the homologous determinant on monkey leukocytes. To facilitate better utilization of this animal model, we tested 89 commercially available monoclonal antibodies which define 27 human cell surface antigens for reactivity with rhesus monkey PBL. Certain antigens which delineate clinical useful lymphocyte subsets such as CD2, CD4, CD8, CD14, CD16, CD20, and MHC class II are apparently well conserved since most human cell‐specific antibodies identified the homologous cell subset in monkeys. However, other antigens such as CD3, CD19, CD45, and CD56 were identified infrequently by human cell‐specific antibodies. FITC‐modification of antibodies which had no effect on their binding to human cells occasionally inhibited antibody binding to monkey cells. Never‐theless, an adequate number of cross‐reactive monoclonal antibodies was identified to allow gating of lymphocytes for accurate flow cytometric analysis and quantitation of the major lymphocyte subsets of the rhesus monkey. The T lymphocyte subset distribution in blood and lymphoid tissue of rhesus monkeys was similar to man. However, the B subset was significantly larger in monkeys. The daily variation in absolute PBL subset size was marked and found to be due mainly to daily fluctuations in total lymphocyte number. © 1994 Wiley‐Liss, Inc.