bluuebunny/crossref_metadata_2025_split huggingface dataset:
Validation of a mathematical procedure for computer analysis of flow cytometric DNA data in human tumors
Authors: Del Bino G. , Bruni C. , Koch G. , Mazzini G. , Costa A. , Silvestrini R. 
Published: August 2005

AbstractThe determination of tritiated thymidine labeling index and the percentage of cells with S phase DNA content was performed on cell suspensions obtained from 69 patients with non‐Hodgkin's lymphoma. The distributions of cells in the cell cycle by computer analysis of flow cytometric data were obtained by two mathematical procedures: the widely adopted Fried model and a new one proposed by Bruni et al. A significant agreement was observed by checking the Spearman index (rs) between the percentages of cells in the different cell cycle phases (G0/1, rs = 0.76; S, rs = 0.60; and G2+M, rs = 0.43; p < 0.001) determined by the two procedures. Similarly, a good correlation was observed between the labeling index (LI) and the S phase values obtained by the Fried (rs = 0.45, p < 0.001) and Bruni (rs = 0.69, p < 0.001) models, but with a higher agreement for the latter one. The S phase by the Bruni model was also superior in predicting LI: in fact, by employing the S cutoff value of 12%, a better agreement between low LI and low S phase or high LI and high S phase was observed with the Bruni procedure (90%) than with the Fried model (72%). Finally, the analysis of the prognostic significance of the different kinetic variables confirmed the prognostic relevance of LI at any time; the S phase percentage as determined by Bruni et al. was discriminant of survival only at shorter times, and no prognostic significance could be ascribed to S phase according to the Fried procedure.
Helpful hints in flow cytometry and sorting
Authors: Dean Phillip N. 
Published: August 2005

AbstractMost users of flow cytometers and sorters become proficient in their use through direct operation of the instruments. There are no text books or operation manuals that describe all of the problems that will be encountered and that may not be apparent. We have discovered many of these problems through finding an explanation for unexpected data and in resolving difficulties in achieving and maintaining high resolution and stable operation of the instruments. This paper presents a summary of problems encountered and solutions found at this laboratory and others.
Linearity and noise sources in flow cytometry
Authors: Ubezio Paolo , Andreoni Alessandra 
Published: August 2005

AbstractA model of pulse formation in flow cytometers is presented that demonstrates the proportionality between the area (or the peak height) of the fluorescence signal produced by the photomultiplier and the number of fluorochrome molecules present in the cell that cause the signal. The model clarifies the possible instrumental origins of inaccuracy in this linearity that results in a broadening of the histograms obtained. A comprehensive formula for the coefficient of variation of the unimodular histograms of an homogeneous population is presented that clearly discriminates among the different contributions of staining, possible inhomogeneity of the examined population, photon statistics, and instrumental instabilities.Finally, some experimental data are presented that show the agreement with the proposed formula.
Inexpensive upgrading of a FACS I and isolation of rare somatic variants by double‐fluorescence sorting
Authors: Weichel Walter , Liesegang Bernhard , Gehrke Klaus , Göttlinger Christoph , Holtkamp Bodo , Radbruch Andreas , Stackhouse Thomas K. , Rajewsky Klaus 
Published: August 2005

AbstractWe have modified a FACS I by addition of a tunable dye laser and an optical system for fluorescence detection that allows physically independent measurement of green and red immunofluorescence. These modifications are inexpensive and should be applicable to most single‐laser system. By using the modified machine and double‐fluorescence activated selection with H‐2Kk‐specific monoclonal antibodies labelled with FITC or Texas Red, we have isolated from a murine T‐cell lymphoma line variant subclones expressing structurally altered histocompatibility class I (H‐2Kk) molecules.
Dual‐parameter scatter‐flow immunofluorescence analysis of bacillus spores
Authors: Phillips A. P. , Martin K. L. 
Published: August 2005

AbstractUsing a commercial flow cytometer (Cytofluorograf), narrow‐forward‐angle (NFA) light‐scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein‐conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter‐FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell‐free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right‐hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.
Evaluation of colony‐forming ability experiments using normal and DNA repair‐deficient human fibroblast strains and an automatic colony counter
Authors: Thielmann Heinz Walter , Hagedorn Roland , Freber Werner 
Published: August 2005

AbstractAn automated procedure for evaluation of colony‐forming ability experiments has been developed using normal and xeroderma pigmentosum fibroblast strains. Technically this consists of an image analyzer equipped with a contrast‐intensifying video camera, monitor, and a desk‐top computer. Complex structures composed of overlapping colonies could be reduced to the consitituent single colonies by special correction programs. Eighteen experiments were evaluated, and the D0 values for colony‐forming ability were determined. A systematic but correctable deviation between visual and automated counting was found. The source of this deviation is described and a solution for correction is provided. The automatic procedure reduced the evaluating time by a factor of five to ten.
Quantitation of anthracyclines in human hematopoietic cell subpopulations by flow cytometry correlated with high pressure liquid chromatography
Authors: Speth Paul A. J. , Linssen Peter C. M. , Boezeman Jan B. M. , Wessels Hans M. C. , Haanen Clemens 
Published: August 2005

AbstractThe major white cell subpopulations present in bone marrow and peripheral blood can be discriminated by forward and perpendicular light scatter two‐parameter flow cytometry (FCM). Fluorescent properties of anthracycline antibiotics allow measurement of the concentration of these cytotoxic drugs in hematopoietic cells by FCM as a third parameter.Analysis of scatter‐gated fluorescence histograms provides quantitative information about the cellular concentration of at least four cell categories in human blood and bone marrow cells. A good correlation was found between the mean cellular fluorescence measured by FCM and the overall cellular concentration of adriamycin, daunomycin, and their main metabolites determined with high‐pressure liquid chromatography (HPLC).In incubation experiments with human hematopoietic tissues, the final concentration of various anthracyclines in subpopulations of white cells appeared to be dependent on cell density, incubation time, temperature, and type of compound and its concentration.FCM analysis is a rapid, sensitive, and quantitative method for measurement of cellular anthracycline concentrations in subpopulations and therefore provides an useful new tool in monitoring chemotherapy.
Single‐cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells
Authors: Rudolph Natalie S. , Ohlsson‐Wilhelm Betsy M. , Leary James F. , Rowley Peter T. 
Published: August 2005

AbstractMultiparameter single‐cell analysis by flow cytometry was used to distinguish between size‐related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light‐scatter pulse‐width time‐of‐flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell‐cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true “down‐regulation” and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand‐receptor interaction.
Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of friend erythroleukemia cells
Authors: Darzynkiewicz Zbigniew , Traganos Frank , Kapuscinski Jan , Melamed Myron R. 
Published: August 2005

AbstractDNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right‐angle direction.The AO‐induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau‐phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 μM with the midpoint of the transition (Cd) at 70 μM. DNA in nuclei of differentiated cells (dimethyl‐sulfoxide‐induced erythroid differentiation) was more resistant (Cd = 77–83 μM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 μM). Extraction of proteins with 0.1 M HCl at 0°CC abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 μM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 μM of AO, for rRNA, tRNA, and denatured and native‐calf thymus DNA, respectively.Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permealized or fixed cells provides a new approach to discriminate cell sub‐populations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.
Flow cytometric detection of a two‐step cell death induced by hyperthermia
Authors: Böhmer Ralph‐M. 
Published: August 2005

AbstractA human leukaemic cell line (REH) was subjected to various temperatures °C42 °C for various time intervals; the cells were stained with a mixture of ethidium bromide and acridine orange, and red and green fluorescence were analysed by flow cytometry. Nontreated cells appeared as one cluster (V) in the biparameter histograms, but with time of heat treatment, two further discrete clusters (D1,D2) of cells appeared successively. They were distinguished by both the degree of red and green fluorescence. The kinetics of transit from one cluster to the other was dependent on temperature, the time lag between both steps becoming shorter with higher temperatures.It was shown previously that the same effect occurred during incubation with various cytostatic agents, and that only the D2 stage correlated with the stage of cell death monitored by the usual trypan blue exclusion test. Therefore the ethidium bromide technique seems to monitor an earlier stage of cell death.The decrease in the number of dye‐excluding (V) cells during heat exposure occurred in two phases. After an initial decrease a plateau of number of dye‐excluding cells was reached; the duration and level of this plateau depended on the temperature. The plateau was followed by a second phase where the remaining cells ceased to exclude the dye.
The cytodisk: A cytometer based upon a new principle of cell alignment
Authors: le Grooth B. G. , Geerken T. H. , Greve J. 
Published: August 2005

AbstractA new method is described for one‐dimensional alignment of small particles such as biological cells. A drop of the particle suspension is spread out on a flat disk or plate equipped with V‐shaped grooves such as are present on a gramophone disk. After drying, the particles are located on the bottom of the grooves and are thus aligned in a one‐dimensional array. The new alignment procedure is demonstrated with a suspension of fluorescent polystyrene micropheres (diameter 3.8 μm) and a suspension of the unicellular algae chlorella vulgaris (diameter about 3 μm). It appears that the alignment of cells and spheres is very good.When using microspheres, more than 95% of the particles in the grooves are located within ±2 μm of the centre line of the groove. Based upon this cell‐alignment principle, a new cytometer, named the cytodisk, is proposed. The proposed system has a number of advantages over the flow cytometer, among which is the unique ability of relocating a previously measured cell for further measurement or visual examination.A prototype of a cytodisk, developed for initial test measurements, was built in our laboratory. The apparatus, constructed from a record player and ordinary long‐playing records, uses a simple mechanical tracking system and a single optical fiber for fluorescence excitation and detection. With this apparatus it is demonstrated that a cytodisk can indeed perform quite well: A histogram of fluorescing microspheres could be measured with a coefficient of variation of 4.1%. The performance of this prototype is limited by the quality of the mechanical tracking system and the optical system used. It is expected that considerable improvements may be obtained by using a more sophisticated optical detection system such as the tracking system in use in optical disk players.
Preparation of monolayer smears from paraffin‐embedded tissue for image cytometry
Authors: van Driel‐Kulker A. M. J. , Mesker W. E. , van Velzen I. , Tanke H. J. , Feichtinger J. , Ploem J. S. 
Published: August 2005

AbstractA method is described for the preparation of monolayer smears from paraffin‐embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin‐embedded tissue this method allows retrospective studies of, for instance, the significan of DNA content with regard to the behavior of a tumor.
Differential of light‐scattering detection in an arc‐lamp‐based epi‐illumination flow cytometer
Authors: Steen Harald B. , Lindmo Tore 
Published: August 2005

AbstractLight‐scattering histograms of blood cell suspensions were recorded for various ranges of scattering angles by means of an arc‐lamp‐based flow cytometer (AL‐FCM). The results were compared with those obtained with a conventional, laser‐based flow cytometer (L‐FCM) with forward scattering (2–20°) and scattering at right angles. Measuring with the AL‐FCM in the angle range upward from 13°, the relative light‐scattering intensities of lymphocytes, monocytes, and granulocytes were essentially independent of scattering angle and closely similar to the values measured as right‐angle scattering in the L‐FCM. With a range of scattering angles upward from 2° the AL‐FCM yielded histograms similar although not identical to that of the forward‐scattering detector in the L‐FCM. Differentiation between live and dead cells in this mode of operation was similar in the two instruments.
Automated identification of subpopulations in flow cytometric list mode data using cluster analysis
Authors: Murphy Robert F. 
Published: August 2005

AbstractThe application of K‐means (ISODATA) cluster analysis to flow cytometric data is described. The results of analyses of flow cytometric data for mixtures of fluorescent microspheres and samples of peripheral blood mononuclear cells are presented. A method for simultaneously displaying list mode data for any number of parameters, which had previously been applied to a continuous set of parameters such as multi‐angle light scattering data, is used to present the results of cluster analysis on physically unrelated parameters; this method allows rapid evaluation of the success of subpopulation identification. The factors that influence automated identification of subpopulations are examined, and methods for determining optimal values for these factors are described.
Flow cytometric determination of circulating immune complexes with the indirect granulocyte phagocytosis test
Authors: Terstappen L. W. M. M. , de Grooth B. G. , Nolten G. M. J. , ten Napel C. H. H. , van Berkel W. , Greve J. 
Published: August 2005

AbstractA method for the determination of circulating immune complexes (CIC) was adapted for flow cytometric analysis. Human granulocytes were used to phagocytose IgG‐bearing CIC of serum from systemic lupus erythematosus (SLE) patients. A method for labeling the phagocytosed CIC with FITC‐conjugated anti‐human IgG was developed where the granulocytes remain in suspension during fixation and labeling. The fluorescence per cell, measured with a flow cytometer, is a measure of the total amount of the phagocytosed IgG. The results indicate that a rapid and quantitative method for the detection and measurement of phagocytosed CIC is possible using the flow cytometer.
Application of DNA flow cytometry to paraffin‐embedded archival material for the study of aneuploidy and its clinical significance
Authors: Hedley David W. , Friedlander Michael L. , Taylor Ian W. 
Published: August 2005

AbstractBy using a recently developed flow cytometric method we have analyzed cellular DNA content of paraffin‐embedded histological material from cancer patients. This method allows the retrospective study of tumors from patients whose clinical outcome is already known, and we have applied it to ovarian cancers, stage II breast cancers, and to metastatic adenocarcinoma of unknown primary site. In addition to knowledge of patient survival, comprehensive information was available about other prognostic determinants and treatment received, and we have used multivariate analysis in an attempt to determine the prognostic significance of cellular DNA content. In ovarian cancer, it is a major prognostic variable except in stage IV disease, whereas in metastatic adenocarcinoma of unknown primary site cellular DNA content has no influence on survival. For stage II breast cancer the situation is more complex and requires larger numbers to be studied. However, aneuploid tumors tend to have more extensive involvement of axillary lymph nodes and a poorer overall disease‐free survival. This influence of DNA content on disease‐free survival appears to be confined to premenopausal patients, and has no effect on patient survival following disease recurrence. Although we need to study more patients and more turnor types, taken together the results so far show a generally more favorable prognosis for patients with diploid tumors, except in the presence of recurrent or metastatic disease. The better prognosis associated with diploid tumors could be due to the fact that they are more commonly found in earlier clinical stages rather than to their being inherently less aggressive than aneuploid tumors.
Estimation of nonproliferating cells in the neural retina of embryonic chicks by flow cytometry
Authors: Morris Valerie B. , Taylor I. W. 
Published: August 2005

AbstractNonproliferating cells (N cells) in the neural retina of embryonic chicks were estimated after isolating them in the 2C peak of a DNA distribution by exposure to the cell‐cycle inhibitor ICRF 159. ICRF 159 inhibits cell division but not DNA synthesis, so proliferating cells can leave the 2C peak but not reenter it. Cells left in the 2C peak after exposure to ICRF 159 were assumed to be N cells. The effectiveness of ICRF 159 in inhibiting cell division but not inhibiting DNA synthesis was demonstrated in neural retinae from stage 17 embryos which showed no evidence of a 2C peak after 6‐h exposure to ICRF 159 and which were thus shown to have no N cells. A test to detect escape from the cell division block in older embryos with an N cell population in the neural retina showed some escape after longer exposures to ICRF 159. The escape was suppressed by a second dose of ICRF 159, given some hours after the first.
Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry
Authors: Le Bouteiller Philippe P. , N'Guyen Catherine , Lemonnier François A. , Jordan Bertrand R. , Caillol DanièLe , Mishal Zohair 
Published: August 2005

AbstractBy using a calibrated cell sorter and monoclonal antibodies conjugated to fluorochromes, a quantitative analysis of the levels of expression of HLA class I molecules at the surface of cloned murine L cells transfected with purified A3, B7, or CW3 genes was performed and compared with radioimmunoassay data. We selected clones of heterogenous levels of HLA class I expression, which were shown to remain constant over a period of 4 mo in absence of HAT selection and not to be correlated to the DNA copy number of the corresponding integrated gene.