bluuebunny/crossref_metadata_2025 huggingface dataset:
Boundary element technique for geotechnical problems of incompressible soils
Authors: Bu S. , Davies T. G.
Published: August 2005

Novel fundamental solutions for elastodynamics of fully incompressible media are derived, by reason of their many applications to engineering and physical problems. It is demonstrated that, when Poisson's ratio equals 1/2, the frequency domain fundamental solutions (Stokes's solutions) are independent of the dilatational wave velocity, which tends to infinity. Consequently, dynamic problems involving incompressible materials can be rigorously analysed by the boundary element method without special difficulty. Numerical solutions to geotechnical problems of incompressible half‐spaces are presented to demonstrate the applicability and accuracy of the formulations.
Remarks on the stability of enhanced strain elements in finite elasticity and elastoplasticity
Authors: de Souza Neto E. A. , Perić Djordje , Huang G. C. , Owen D. R. J.
Published: August 2005

The stability of a class of geometrically non‐linear assumed enhanced strain elements is addressed. By means of an eigenvalue analysis of element tangent stiffnesses at finitely deformed configurations, circumstances under which enhanced elements become unstable, with possible development of non‐physical hourglass patterns, are identified. Insight into the influence of spurious eigenvalues on the stability of solution of finite elasticity and elastoplasticity problems is provided by simple numerical tests with a patch of elements. Results of simulations of some practical engineering problems are presented and limitations on the applicability of enhanced strain elements in the finite strain regime are discussed.
A mass conservative least‐squares finite element method for the stokes problem
Authors: Nelson John J. , Chang C. L.
Published: August 2005

In the paper the simulation of incompressible flow in 2D by the least‐squares finite element method (LSFEM) in the velocity‐vorticity‐pressure version is studied. A problem with this method is that it does not conserve mass exactly, i.e. div uh ≠ 0 exactly. In the paper a modified LSFEM is developed which enforces a near zero residual of mass conservation, i.e. div u is nearly zero at every point of the discretization. This is accomplished by adding an extra restriction in the divergence free equation through the Lagrange multiplier strategy.
Treatment of distributed sources in the stochastic boundary element method
Authors: Kaljević I.
Published: August 2005

The contributions of distributed sources to the mathematical expectations and variances of the response variables are analysed in the paper using the stochastic boundary element method. The validity and the accuracy of the particular integral approach in representing statistical properties of the domain are investigated. To this end domain integration techniques to calculate the contributions of the distributed sources and their derivatives with respect to random variables that may represent both random configuration of the domain and random material parameter are developed. Several models for the random interior are presented and their influence on the response statistics is investigated. The accuracy of the particular integral approach is demonstrated through numerical comparisons with the direct integration formulation.
An accurate numerical algorithm for advective transport
Authors: Manson J. R. , Wallis S. G.
Published: August 2005

The paper describes a new numerical algorithm for pure advection at large Courant numbers. The DISCUS scheme uses an accurate and proven control volume algorithm (QUICKEST) within a Lagrangian treatment of advection. By being in sympathy with the information flow dictated by characteristic lines, the scheme performs exceedingly well at Courant numbers greater than one. In contrast to existing Lagrangian schemes which use the characteristics to determine the grid points from which concentrations are interpolated, in DISCUS the characteristics are used to determine the appropriate location for the control volume. The scheme is of comparable accuracy to Eulerian schemes at Courant numbers less than one, but it increasingly outperforms them as the Courant number increases. Indeed, the results reported illustrate the futility of using an Eulerian scheme at high Courant numbers.
Growth behaviour of bulk GaN single crystals grown with various flux ratios using solvent‐thermal method
Authors: Shin T. I. , Yoon D. H.
Published: August 2005

Bulk GaN single crystals were grown using a solvent‐thermal method. They were grown for 200 h at 600 °C and 800 °C using 8 MPa of N2 gas and 1–3 mm sized pyramid GaN single crystals. Pure Na, NaN3 and Ca were used as the flux. The mole fraction of the [flux]/([flux] + [Ga]) was 0.30–0.67. The growth behavior differed according to the flux ratio. The quality of the bulk GaN single crystals was improved by increasing the flux ratio. The bulk GaN single crystals formed by spontaneous nucleation were deposited on the BN crucible wall and bottom during the first step of synthesis. The wurtzite structure of the GaN grown single crystal was confirmed by x‐ray diffration. The chemical composition was analyzed by electron probe microanalysis. The quality and optical properties of the GaN single crystal were examined by Raman spectroscopy and photoluminesence analysis. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Morphological lattice complexes
Authors: Hauck J. , Mika K. , Sager W.
Published: August 2005

Binary and pseudobinary compounds AB like NaCl and CaCO3, with the center of charge at carbon position, are characterized by self‐coordination numbers of nearest, second and third neighborsT1T2T3of A and B positions. The maximum Madelung factor of compounds with identicalTivalues of A and B atoms is obtained for a maximum of B positions at the border of the A–A Dirichlet domain. The A and B positions form a packing with differentTivalues, if all B positions are on the border of the Dirichlet domain. This packing like the primitive cubic packing of Na and Cl atoms withTivalues 6 12 8 or the rhombohedrally distorted packing 6 8 6 of CaCO3as morphological lattice complexes can be related to cubic NaCl crystals or the cleaved rhombohedron of calcite. A different habit of crystals is expected for sphere, rod or layered packings. Many well‐defined nanoparticles can be obtained from microemulsions at variation of temperature, water content or water partial pressure. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Interactions between pairs of DNA‐binding dyes: Results and implications for chromosome analysis
Authors: Latt Samuel A. , Sahar Elhanan , Eisenhard Michael E. , Juergens Lois A.
Published: August 2005

A number of DNA‐binding dyes, with spectral properties making them suitable as components of energy donor‐acceptor pairs, are described. If such pairs are used to stain metaphase chromosomes, and if the energy acceptor (e.g., actinomycin D or methyl green) has a binding specificity opposite to the binding or fluorescence specificity of the donor (e.g., 33258 Hoechst, quinacrine or chromomycin A3), contrast in donor fluorescence can be enhanced, leading to patterns selectively highlighting standard or reverse chromosome bands or particular polymorphic regions. Such results presumably reflect chromosomal regions enriched in 10–20 base pair clusters to which the donor binds and fluoresces but to which the acceptor cannot bind. For other pairs, involving counterstains such as netropsin or echinomycin, which are not suitable as energy acceptors, specific changes observed in polymorphic region fluorescence are most likely due to binding competition between dyes. Dye pairs producing contrast by either method can be used to differentiate between homologous chromosomes or to facilitate detection of specific chromosomal rearrangements. Preliminary data indicate that contrast enhancement generated in fixed metaphase chromosomes spread on microscopic slides can also be observed in suspensions of unfixed metaphase chromosomes, reinforcing the expectation that the methodology described will be of use in flow cytometry.
Proteins from metaphase chromosomes treated with fluorochromes
Authors: Wray Wayne , Wray Virginia P.
Published: August 2005

The biochemical composition of any isolated organelle is affected by the agent(s) to which it has been exposed during the isolation and purification procedures. Many agents are known to extract molecular components either selectively or nonselectively. The protein composition of isolated chromosomes exposed to fluorochromes must be examined before proceeding to the biochemical analysis of the proteins from individual chromosomes purified by flow cytometry techniques. We have examined the poly‐acrylamide gel electrophoresis protein profiles from metaphase chromosomes exposed to the fluorochromes and staining conditions commonly used for flow cytometry. We find that the electropherograms of the protein of metaphase chromosomes which have been exposed to various fluorochromes are indistinguishable from controls. The analysis of proteins from individual chromosomes sorted by flow cytometry can now be undertaken.
Enhanced light collection in a flow cytometer
Authors: Fox Michael H. , Coulter James R.
Published: August 2005

A modified flow cell in a conventional flow cytometer is described. The refraction of light in the flow cell‐to‐air interface is analyzed. The analysis shows that much of the light that can be collected is refracted out of the collection optics. The efficiency of light collection is doubled by the addition of a plano‐convex lens to the flow cell. Also, more stable operation of the flow cell is achieved with the addition of a secondary sheath.
Further developments of a microscope‐based flow cytometer: Light scatter detection and excitation intensity compensation
Authors: Steen Harald B.
Published: August 2005

A microscope‐based flow cytometer, which is based on a new flow system, has been extended to include light scatter detection, dual parameter fluorescence analysis, and a device which compensates for variations in excitation light intensity. A dichroic mirror splits the fluorescence into two spectral components that are further purified by additional filters situated in front of the photomultiplier tube detectors (PMT). Light scatter detection is obtained by a dark field configuration. An objective focuses the scattered light emitted within the dark field onto an adjustable slit in front of a PMT. Resolution of light scatter histograms of 1.5‐m̈m latex spheres is about CV = 2.5%. Dual parameter analysis revealed a linear correlation between the light scatter signal and fluorescence of cells stained with fluorescein isothiocyanate, indicating that the light scatter signal is closely proportional to cell dry mass. The instrumental light scatter, which is proportional to the excitation light intensity, gives rise to a DC signal. This signal regulates the fluorescence signal amplifiers so that these signals are compensated for fluctuations in the excitation light intensity.
The kinetics of the formation of a G2 block from tritiated thymidine in phytohemagglutinin‐stimulated human lymphocytes
Authors: Pollack A. , Bagwell C. B. , Irvin G. L. , Jensen J. A.
Published: August 2005

Flow cytometry (FCM) was used to monitor the radiation effects promoted by incorporated tritiated thymidine (3H‐TdR) on phytohemagglutinin (PHA)‐stimulated human peripheral blood lymphocytes stained with propidium iodide (PI). Lymphocyte microcultures were continuously labeled or pulse‐labeled for various periods of time with different 3H‐TdR concentrations. Two types of DNA histogram analyses wer performed on unperturbed and 3H‐TdR perturbed lymphocytes. The data analyses consisted of statistical analyses between averaged groups of histograms (nonparametric analysis) and cell cycle analyses (parametric analysis) to determine the percentages of cells in G0 + G1, S and G2 + M. The results showed that (a) 3H‐TdR when added to proliferating lymphocytes under certain conditions (both short‐term continous and pulse‐labeling) caused a highly significant increase in the proportion of tetraploid (4C) cells by FCM, (b) the increase in the proportion of 4C cells represented a block in G2 and (c) the relative increase in the percentage of 4C cells was proportional to 3H‐TdR incorporation which was proportional to labeling time and concentration. Therefore, it was concluded that short labeling times be used to minimize adverse radiation effects when 3H‐TdR is used to assay substances affecting lymphocyte proliferation or in the estimation of cell cycle time.
The tradeoff of cell classifier error rates
Authors: Castleman Kenneth R. , White Benjamin S.
Published: August 2005

In gynecologic cytodiagnosis it is generally agreed that specimen false negative errors are more serious than false positives. When classifying individual cells, however, it is not obvious how one should adjust the parameters that control the two cell error rates. In the case where a specimen classifier follows a cell classifier, one can calculate the sample size required to achieve specified overall performance. This analysis shows that for the small abnormal cell proportions encountered in cervical cytology, cell false positives should be kept so low that a substantial portion of the abnormal cells are missed.
An optical model for fluorescence of mammalian sperm in flow cytometry
Authors: Kerker M. , Wang D.‐S. , Chew H. W.
Published: August 2005

When flat sperm heads that have been stained to fluoresce are examined in a flow cytometer, unexpectedly, skewed pulse height distributions are obtained despite the apparent homogeneity of the samples. This anomaly has been ascribed to an optical artifact that arises when the cells are oriented in flow. We have extended our model for fluorescent scattering to spheroids and here explore some aspects for oblate spheroids which serve to model sperm heads. Although computational limitations have restricted these studies to oblate spheroids about 1.5 μm in diameter and an eccentricity of 0.1, the results clearly show effects of particle size, shape, optical properties and particularly of orientation on the differential scattering cross‐sections. This plethora of information contained in the fluorescent signals may suggest further experiments.
Estimation of the performance of an Array‐Processor oriented system for automatic pap smear analysis
Authors: Abmayr Wolfgang , Gais Peter , Rodenacker Karsten , Burger Georg
Published: August 2005

The rapid progress in computer technology makes possible the automatic analysis of thousands of cells on a slide in the field of automatic uterine cancer cytology. Our approach starts with the high‐resolution scanning of visually selected and classified single cells determining the training set for discriminant analysis. On the basis of more than 15 morphologic and textural features measured, correct classification results of 95% are reached. Our aim is to speed up our image processing system by means of an Array‐Processor, the model AP 120 B from Floating Point Systems. In this study a realistic time estimation of the scanning, segmentation, feature extraction and classification of about 40,000 cells on a slide is performed.
Field test results using the BioPEPR cervical smear prescreening system
Authors: Zahniser D. J. , Oud P. S. , Raaijmakers M. C. T. , Vooys G. P. , Van de Walle R. T.
Published: August 2005

BioPEPR is a cathode ray tube scanning device that has been developed for the prescreening of cervical smears. Cervical samples are collected in suspension, syringed, counted and spread across a microscope slide. Feulgen‐Thionine with Congo Red is used as the staining procedure. Fields of 6 × 8 mm are scanned at 1 μ resolution, using an intermediate photographic step. Morphologic parameters of the cells are measured, as called for by a hierarchical decision strategy. Analysis speed is currently about 4 min per smear; improvements of at least a factor of four are expected. A field test of BioPEPR is being undertaken in cooperation with a local population screening program. In the study two smears are made from each woman; the first is prepared according to the Papanicolaou technique while the second is prepared and stained according to the BioPEPR methods. In addition to smears from the population screening program a number of possibly abnormal smears are obtained from several gynecological clinics. To date approximately 3500 “second” smears have been analyzed by the BioPEPR system. The results show a missed positive rate on the order of a few percent (no missed positives were more severe than a slight dysplasia), and a false alarm rate of about 24%. A further analysis of the age distribution of the false alarm rate shows that a large proportion came from women above the age of 50; the false alarm rate is about 20% for women under the age of 50.
A design for a real‐time fluorescence polarization computer
Authors: Stewart S. S. , Miller R. G. , Price G. B.
Published: August 2005

We have developed hardware components for our flow cytometer cell sorter which can perform real‐time calculations of either fluorescence polarization (FP) or anisotropy on a cell‐by‐cell basis and which can provide gating signals for the sorting of these cells into two populations. Artificial FP measurements, using electronic pulses varied over a 40dB range, were used to evaluate the computer's useful dynamic range. Real FP measurements, using human peripheral blood lymphocytes stimulated with phytohemagglutinin and labeled with fluorescein diacetate in saline, are presented to allow comparison with earlier work.
Flow cytometric analysis of the effect of 5‐bromodeoxyuridine on mouse teratocarcinoma cells
Authors: Swartzendruber Douglas E. , Travis Gayle L. , Martin John C.
Published: August 2005

Several flow cytometric techniques were used to study the effects of 5‐bromodeoxyuridine (BrdUrd) on the growth and differentiation of mouse teratocarcinoma cells. The teratocarcinoma system mimics embryogenesis, and the stem cells of this tumor, termed embryonal carcinoma (EC) cells, spontaneously differentiate in vitro to progeny that are morphologically, biochemically and biologically distinct from the EC cells. When cultures of EC cells were treated with 30 μM BrdUrd, a pleiotropic response was observed; i.e., certain parameters mimicked spontaneous differentiation but occurred 2–3 times more rapidly while others were inconsistent with, or diametric to, spontaneous differentiation. In one line of diploid EC cells in which tetraploidy accompanies spontaneous differentiation, the BrdUrd accelerated the appearance of the tetraploid population. Analysis of cell cycle distributions during the course of the BrdUrd treatment showed that virtually all of the cells had cycled 2 or more times and incorporated BrdUrd into both strands of DNA. Multiangle light‐scattering and electrical impedance analyses indicated that with respect to these two parameters, cultures treated with BrdUrd for 3 days resembled spontaneously differentiating cultures of 6–8 days. However, BrdUrd treatment dit not induce increases in the kinetics of esterase (EC3.1.1.) activities in teratocarcinoma cells that normally accompany spontaneous differentiation. Analysis of alkaline phosphatase (APase) EC (3.1.3.1.) activities showed that, unlike the decrease in APase activities which accompany spontaneous differentiation, BrdUrd Treatment causes an increase in APase activities.
Intracellular binding of fluorescein in lymphocytes
Authors: Meisingset Knut K. , Steen Harald B.
Published: August 2005

The fluorescence characteristics of intracellular fluorescein, formed by hydrolysis of fluorescein diacetate in peripheral human lymphocytes, were studied by fluorometry on cell suspensions and compared to those of albumin bound and free fluorescein in solution. The absorption and fluorescence spectra of both intracellular fluorescein and fluorescein in aqueous solutions of albumin and glycerol were red shifted by 2–10 nm as compared to the spectra of fluorescein in phosphate buffered saline. The fluorescence polarization (P) of both intracellular fluorescein and a mixture of albumin‐bound and free fluorescein showed a decrease towards low emission wavelengths and an increase toward high excitation wavelengths. The results were found to be consistent with a simple model assuming that part of the intracellular fluorescein is dissolved in the aqueous phase of the cytoplasm, giving P < 0.1, while the rest is bound to macromolecules, giving P = 0.33. The fraction of bound intracellular fluorescein was estimated to be about 70%. Fluorescein was found to bind with higher affinity and more rigidly (P = 0.43) to albumin than to intracellular macromolecules in general.
Correlation between cytometric features and mitotic frequency in human breast carcinoma
Authors: Stenkvist Björn , Bengtsson Ewert , Eriksson Olle , Jarkrans Torsten , Nordin Bo , Westman‐Naeser Sighild
Published: August 2005

An evaluation of cytometric features of 142 human breast cancers was performed. Twenty‐two cytometric features describing density, structure and shape were correlated with mitotic frequency and labeling index of some of the tumors and with the observed recurrence rate of the tumors within 3 years after removal of the breast. A new cytometric measure was also created by combining the four features most strongly correlated with mitotic frequency of the tumors. It was found that the variance of the nuclear area was the feature that was most strongly correlated with mitotic frequency and recurrence rate. No new cytometric measure created by combining the four features most strongly correlated with mitotic frequency, correlated as well as variance of nuclear area even though the four features were optimized by multiple regression analysis for their correlation with mitotic frequency.
Flow cytometric probes of early events in cell activation
Authors: Shapiro Howard M.
Published: August 2005

By now, it's well established that the surface of a cell

Contains transmembrane proteins which, on binding ligands, tell

The biochemical computer inside what to do

Without a need for any of the ligands coming through.

Receptor‐triggered cytoplasmic “interrupt routines”

May lead the nucleus to activate new sets of genes,

Or otherwise induce a cell, which never reasons why,

To twitch, secrete, endocytose, or reproduce, or die.

By merely watching labeled ligands bind, we cannot know

Down which of many paths the cells thus tagged are apt to go,

But other changes, shortly after binding, may predict

Which of its several options any given cell has picked.

Flow cytometric methods are described here which allow

Membrane potential to be estimated, to show how

Potential changes may occur in seconds after binding

Of ligand to a functional receptor, or for finding

No change when cells don't have the right receptor, or can't see

The ligand for antagonists of like affinity.

With chlorotetracycline fluorescence, one can find

Release of membrane calcium soon after ligands bind;

Both this and the potential measurement can help support

Decisions in an instrument about which cells to sort,

Though narrowing the distributions which these methods yield

Would greatly help to broaden applications in this field.
Flow cytometry of human reticulocytes based on RNA fluorescence
Authors: Tanke H. J. , Nieuwenhuis I. A. B. , Koper G. J. M. , Slats J. C. M. , Ploem J. S.
Published: August 2005

A fluorescent staining procedure based on pyronin Y is described. The technique has been used to stain RNA in human reticulocytes for subsequent flow analysis and sorting. Histograms of fluorescence values of mature red blood cells and reticulocytes obtained by flow cytometry of stained blood samples, contain more information than offered by conventional methods for counting reticulocytes. Analysis of the fluorescence histograms provides new descriptive parameters of the reticulocyte population. The potential importance of this method in experimental and routine hematology is illustrated for a number of clinical cases.
Solid KHT tumor dispersal for flow cytometric cell kinetic analysis
Authors: Pallavicini M. G. , Folstad L. J. , Dunbar C.
Published: August 2005

We used a bacterial neutral protease to disperse KHT solid tumors into single cell suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral protease disaggregation under conditions which would be suitable for routine tumor dispersal was compared with a trypsin/DNase procedure. Cell yield, clonogenic cell survival, DNA distributions of untreated and drug‐perturbed tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase‐specific cell loss were investigated. Tumors dispersed with neutral protease yielded approximately four times more cells than those dispersed with trypsin/DNase and approximately a 1.5‐fold higher plating efficiency in a semisolid agar system. Quantitative analysis of DNA distributions obtained from untreated and cytosine‐arabinoside‐perturbed tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated thymidine during the cell cycle were also similar with neutral protease and trypsin/DNase dispersal. Preferential phase‐specific cell loss was not observed with either technique. We find that neutral protease provides good single cell suspensions of the KHT tumor for cell survival measurements and for cell kinetic analysis of drug‐induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are often required.
Monocyte‐induced cell cycle synchronization of a leukemic cell line in vitro
Authors: Eggen Bjørn Magne
Published: August 2005

Human monocytes cultured in vitro exert cytostatic activity against neoplastic cell lines. The cytostatic effect was examinated on the nonadherent human leukemic cell line K‐562. After 24 hours of co‐culture between monocytes and K‐562 cells (in a ratio of 10: 1), the K‐562 cells were removed and their DNA‐synthesis and DNA‐content were examinated by methyl‐3H‐thymidine incorporation and flow cytofluorometry. Cell proliferation curves were obtained.

A partial and reversible cell cycle block in the G0G1‐phase was observed. After removal of the target cells from the monoeyte monolayers, the target cells regained their normal proliferation rate during the following 1 to 3 days, with a maximal number of cells in S‐phase at 7 to 9 hours after separation of target cells from the monocytes. The most marked effect was induced by monocytes cultured in vitro for 12 days, and with monocytes stimulated with lymphokine, i.e. supernatants from BCG‐exposed lymphocyte cultures.
Flow cytometric measurement of nuclear DNA content variations as a potential in vivo mutagenicity test
Authors: Otto F. J. , Oldiges H. , Göhde W. , Jain V. K.
Published: August 2005

Mutagen‐induced variations of the cellular DNA content have been studied in mouse bone marrow cells in vivo using high resolution flow cytometric techniques. During the first days after a single injection of the chemical mutagen cyclophosphamide an increased coefficient of variation in the G1 peak of the flow histograms was observed. The magnitude and duration of this effect were dose‐dependent. The reproducibility of the measurements was high, indicating that individual variability between animals and instrumental dispersion is small. The results demonstrate that on the basis of the flow cytometric measurement of cellular DNA content, a short‐term in vivo test for mutagenicity can be established which is much faster than conventional cytogenetic methods.
Calibration of flow cytometry detector systems
Authors: Durand Ralph E.
Published: August 2005

A method of determining the relative intensity of the fluorescence signal measured by a fluorescence activated cell sorter is described. The calibration process is a multistep procedure, in which the signal gain due to the amplifier module is first evaluated, followed by determination of the photomultiplier response as a function of tube voltage for each fluorochrome and filter combination of interest. Using these values, a simple mathematical calculation then determines the relative intensity of a fluorescent signal, even for samples differing in intensity by several orders of magnitude.
The look‐up table: A logarithmic converter for cell sorters
Authors: Koper Ger JM , Blanken Rieks
Published: August 2005

The employment of logarithmic amplifiers for flow cytometers is discussed and a logarithmic conversion algorithm is introduced. Two applications of the algorithm are given: the rescaling of a histogram and the use of a cheap programmable read‐only memory circuit. The performance of the algorithm is compared with a logarithmic amplifier.
Ploidy, proliferative activity and estrogen receptor content in human breast cancer
Authors: Raber Martin N. , Barlogie Barthel , Latreille Jean , Bedrossian Carlos , Fritsche Herbert , Blumenschein George
Published: August 2005

Tumor samples from 80 patients with breast cancer (43 primary, 37 metastatic) were analyzed for ploidy and proliferative activity using DNA flow cytometry. Sixty‐one tumors (40 primary, 21 metastatic) were also analyzed for estrogen receptor content. Eighty‐five percent of all tumors had an abnormal DNA content. The majority of tumors were hyperdiploid (65%). Seventy‐three tumors had a unimodal ploidy distribution, while in seven cases two distinct aneuploid subpopulations were identified. The degree of ploidy abnormality was not related to extent of disease or menopausal status, but was higher (DNA index > 1.5) in poorly differentiated tumors (p = 0.05). ER‐positive tumors were more often diploid (7 of 31) than ER‐negative tumors (3 of 30, p = 0.16). DNA content was constant in biopsies from multiple sites in 5 patients with metastatic disease and in serial biopsies over the course of the disease. High proliferative activity (percent cells in S‐phase) was more often associated with ER‐negative tumors than ER‐positive tumors (p = 0.03). However, in all subgroups analyzed a wide range of values was noted. We conclude that flow cytometric analysis of cellular DNA content provides a rapid means of quantitating ploidy and proliferative activity in human breast cancer. Ploidy abnormalities were common, stable, and usually unimodal. Proliferative activity was inversely related to estrogen receptor content.
Light scatter analysis and sorting of cells activated in mixed leukocyte culture
Authors: MacDonald H. Robson , Zaech Pierre
Published: August 2005

Cells activated in unidirectional mixed leukocyte cultures (MLC) have been analyzed on the basis of their light scatter characteristics. C57BL/6 spleen cells were cultured with irradiated (2000 rads) DBA/2 spleen cells for 5 days and the resulting suspension of activated cells was passed on a FACS II flow cytometer. Correlated parameter analysis of forward light scatter (FLS) and perpendicular light scatter (PLS) indicated that the MLC consisted of a heterogenous mixture of viable cells, dead cells, and subcellular debris. However, by appropriate gating of the FLS/PLS distribution, viable cells could be identified as a biphasic FLS histogram. Sorting and morphological analyses of these two FLS peaks demonstrated that they corresponded to almost pure populations of small lymphocytes (lower peak) and lymphoblasts (upper peak), respectively. Furthermore, when sorted cells were tested for their ability to lyse antigenically relevant (DBA/2) tumor target cells in a 51Crrelease assay, lymphoblasts were found to exhibit 40‐fold greater cytolytic activity (on a per‐cell basis) than small lymphocytes.
Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry
Authors: Szabó Gábor , Kiss Attila , Trón Lajos
Published: August 2005

The toxicity of polyethylene glycol 1000 (PEG) used similarly as in cell hybridization experiments, has been studied by flow cytometry, measuring the light scattering and fluorescence distributions of PEG‐treated human lymphocytes stained with propidium iodide, fluorescein diacetate and acridine orange (AO). The sensitivity of these tests to detect permeabilized, or potentially dead cells, was equal. In addition, PEG proved to interfere with AO staining most likely through the inhibition of its binding to nucleic acids. The decrease of AO fluorescence in cells killed by PEG was unexpected since intercalation of propidium iodide was the same as in alcohol fixed cells. Permeabilization of cells by PEG appears to be an all‐or‐none phenomenon, accompanied by entrance of PEG into the cells. The findings are described in the context of a review of the currently used flow cytometric techniques to discriminate viable and lethally affected cells; also, the problems of interpretation are discussed.
Changes in DNA content of human blood mononuclear cells with senescence
Authors: Staiano‐Coico Lisa , Darzynkiewicz Zbigniew , Melamed Myron R. , Weksler Marc
Published: August 2005

The mean DNA content per cell and heterogeneity of cell populations with respect to DNA content were measured in samples of peripheral blood mononuclear cells from donors of different ages. Nearly all of the cells in these populations are found in the G1Q phase of the cell cycle. While the mean DNA content was the same for all age groups, a significant increase in intercellular variability, reflected by an increase in coefficient of variation of the mean, was observed in the group of subjects between 67 and 83 years of age in comparison with the group of donors between 20 and 30 years of age. The difference was observed regardless of whether cells were stained with acridine orange or propidium iodide, and under conditions where the intercellular variability cannot be attributed to differences in chromatin structure or quantity of mitochondrial DNA. Increasing cell heterogeneity may reflect the age associated deterioration of the fidelity of DNA replication and/or repair and provide a quantitative marker of the accumulated defects in the DNA of a cell population. The observed changes may be responsible for the decline in immune function with age.
Cell‐Cycle position and nuclear protein content
Authors: Roti J. L. Roti , Higashikubo R. , Blair O. C. , Uygur N.
Published: August 2005

To determine the change in nuclear protein content as a function of cell cycle position, isolated heLa nuclei were stained for protein with fluorescein isothiocyanate (FITC) and for DNA with propidium iodide (PI) and analyzed by flow cytometry (FCM). The resulting FITC versus PI histogram consisted of four definable regions, a G1 region characterized by increasing FITC and relatively constant PI (2C DNA content), an S region characterized by increasing PI with relatively constant FITC, a G2 region characterized by increasing FITC and constant PI (4C DNA content), and a region of G1 FITC staining with near G2 PI staining. The relationship between cell cycle position and these regions of the histogram was confirmed by the two following studies: (1) The distribution of labeled nuclei throughout the histogram was observed after [14C]TdR pulse labeling. (2) Exit of cells from G1 was observed in the histogram after the addition of Colcemid to the HeLa cell cultures. Nuclear protein content did not appear to increase uniformly across the cell cycle (defined by DNA content). Rather, nuclear protein content showed the largest increase during G1. Thus, dual parameter FCM analysis based on nuclear DNA and protein content provides a more complete definition of cell cycle position than DNA content alone.
Sequential vincristine, arabinosylcytosine and adriamycin in acute leukemia: Cytologic and cytokinetic studies
Authors: Riccardi Alberto , Mazzini Giuliano , Montecucco Carlomaurizio , Cresci Roberto , Traversi Egidio , Berzuini Carlo , Ascari Edoardo
Published: August 2005

Cytokinetic and cytocidal effects exerted on peripheral blood blasts by sequential administration of vincristine (VCR) 2 mg on day 1, arabinosylcytosine (Ara‐c) 50–60 mg/m2/12 hr from day (2–4), and Adriamycin (ADM, Farmitalia, Milan, Italy, 40–60 mg/m2 on day 5) have been examined in 22 courses of treatment performed on 12 patients with nonlymphoblastic and in 4 with lymphoblastic acute leukemia. In 4 patients, the bone marrow blasts were examined before and also after VCR‐Ara‐c administration. In vitro tritiated thymidine autoradiography and propidium iodide‐DNA flow cytometry were employed for kinetic studies. Blasts disappeared from blood with a median half time of 35 hr. After VCR‐Ara‐c administration, a significant increase in labeling index (LI) and in the aliquot of cells with DNA content between the diploid (2n) and the tetraploid (4n) values was observed in 80% of the courses in peripheral blood blasts and in all courses in bone marrow blasts. The median grain count over the labeled nuclei was decreased, and the 4n cell percentage and the bone marrow blast mitotic index did not increase. These findings suggest that the increase in the S phase fraction of blast population is due to cell synchronization. Increase in the S phase appears to heighten the cytocidal effect of ADM. The aliquot of the blasts cleared from blood after ADM were in fact related directly to the degree of labeling index increase observed during the previous administration of VCR and Ara‐c.
Rapid detection of Legionella pneumophila by flow cytometry
Authors: Ingram Marylou , Cleary Timothy J. , Price Brandon J. , Price Relf L. , Castro Albert
Published: August 2005

Legionella pneumophila labeled with specific fluorescent antibody was detected in a suspension of organisms by means of flow cytometry. The presence of other microorganisms in the suspension did not interfere with the recognition of Legionella when present at concentrations of 104 to 105 cells per ml. These data suggest that flow cytometry may be useful for the detection of Legionella in environmental samples.
Chromosome sorting and DNA sequence localization
Authors: Lebo Roger V.
Published: August 2005

This article focuses on current techniques and possible future developments in chromosome isolation and sorting, and DNA analysis of sorted chromosomes. The strategy of subchromosomal gene mapping by chromosome sorting is outlined and a list of cell lines containing translocated chromosomes is provided which may be used to map genes to a single chromosome with a standard fluorescence activated cell sorter. The usefulness of this and other gene mapping methods for localizing unique DNA sequences and characterizing recombinant DNA libraries constructed from sorted chromosomal DNA is also discussed.
Analysis of ligand‐receptor interactions with the fluorescence activated cell sorter
Authors: Sklar Larry A. , Finney David A.
Published: August 2005

The binding of a fluorescent peptide to human neutrophils is analyzed with a fluorescence activated cell sorter. We examine steady‐state and kinetic features of the ligand‐receptor interaction (in the presence of unbound ligand) and we show that the number of receptors may be estimated without resorting to any external references for calibration. These methods are applicable to other fluorescent ligands with affinities > 108 M−1.
Flow cytofluorometric study of lipoprotein interactions with cultured endothelial cells
Authors: Preobrazhensky Sergey N. , Antonov Alexander S. , Perova Natalya V. , Sherbakova Inna A. , Samokhin Gennady P. , Gerasimova Elena N. , Repin Vadim S. , Smirnov Vladimir N.
Published: August 2005

Accumulation of rhodamine B isothyocyanate‐conjugated low density lipoproteins (R‐LDL) in cultured endothelial cells from human umbilical cord was studied with a fluorescence activated cell sorter. R‐LDL uptake was blocked at 0°C, inhibited by addition of excess of nonlabeled low density lipoprotein, high density lipoprotein‐2 or high density lipoprotein‐3. High density lipoprotein‐2 was about twice as effective in inhibition of R‐LDL uptake as high density lipoprotein‐3. Endothelial cells that formed a contact‐inhibited monolayer lost the ability to incorporate R‐LDL via a receptor‐mediated pathway. Using R‐LDL, it was possible to distinguish cells with different levels of R‐LDL incorporation.
Two color light scattering indentifies physical differences between lymphocyte subpopulations
Authors: Otten Gillis R. , Loken Michael R.
Published: August 2005

Forward angle light scattering of two different wavelengths by cells in a flow cytometer was used to investigate physical differences between lymphocytes of different lineage, functional subclass and developmental stage. Correlation of the ultraviolet (UV: 351 nm and 364 nm) and 488 nm light scattering signals produced by lymphoid cells demonstrated that the two signals were not equivalent and that they placed different emphasis on the physical parameters characterizing lymphocytes. Both small T and B lymphocytes from peripheral lymphoid tissues and mitogenically activated large T and B lymphocyte blasts were discriminated by both wavelengths. Differences between the Lyt‐2 negative and Lyt‐2 positive T lymphocyte subsets were also apparent. Two color light scattering could also discriminate between immature thymocytes and mature peripheral T cells and between small bone marrow cells and mature peripheral B cells. In bone marrow an increase in UV light scattering coincided with the appearance of cell surface immunoglobulin on small cells. These data establish that two color light scattering is a sensitive probe for distinguishing cells of apparently similar morphology and that it can be used to study the physical changes that occur during lymphoid cell differentiation.
A new flow cytometric transducer for fast sample throughput and time resolved kinetic studies of biological cells and other particles
Authors: Kachel V. , Glossner E. , Schneider H.
Published: August 2005

In state of the art flow cytometric transducers, the cells are supplied through tubes. Passage through the tube and washing between different samples is time consuming and limits the number of samples that can be processed in a given time. This is a drawback particularly with automatic routine instruments. For kinetic studies in the time range of seconds, it is necessary to perform the cell reactions directly in the transducer in order to have a short delay between the suspension vessel where the cell reaction is in progress and the point of measurement. A new one parameter electrical sizing transducer without a particle supplying tube is described and compared with a conventional Metricell transducer. The cells are directly supplied from an exchangeable vessel to the measuring point in the transducer. The vessel which is an inexpensive mass produced product, serves as the injection tip for passing the cell suspension into the focusing flow path. There is no interconnected tube to delay or intermix the cells in the stage between the reaction in progress in the vessel and flow analysis. Delay times of only 1 second are achieved with the new transducer, and by supplying each sample with its own vessel subsequent samples are analyzed without the necessity of cleaning a cell supplying tube. In this way a high throughput of samples per time unit is achieved and time kinetic experiments in the time range of seconds can be performed. The design of a tubeless multiparameter Fluvo Metricell transducer is explained.
Slit‐scan flow cytometry: Separability properties of cell features
Authors: Mann R. C.
Published: August 2005

A model is presented to compare the separability of cell populations described by features measured in low resolution slitscanning flow systems with their separability when the features are extracted from high resolution digitized cell images. The results show that although the accuracy of the feature measurements deteriorates for increasing slit width, this is not necessarily true for the discriminatory power of the features. Depending on their original position in the high resolution feature space, the cell populations may be located even farther apart in the space of low resolution slit‐scan features for reasonably small widths of the slit. The results presented with high resolution images of cells from gynecological specimens and simulated slit‐scan measurements can be explained by the model. For the features nuclear DNA content and diameter the abnormal populations are shifted closer to the normal populations in the slit‐scan simulations as compared to the high resolution measurements. The cell classifier errors rates are unacceptably high.
Quantification of the degree of cell spreading of human fibroblasts by semi‐automated analysis of the cell perimeter
Authors: Brugmans Mia , Cassiman Jean‐Jacques , Vanderheydt Luc , Oosterlinck André J. J. , Vlietinck Robert , Van Den Berghe Herman
Published: August 2005

Cell flattening and spreading on a substratum is of major importance in cellular and developmental biology. To study the mechanisms of cell spreading, quantitative and reproducible measures of the degree of cell spreading must be available.

Normal human fibroblasts, spreading on a substratum, were fixed with glutaraldehyde, stained with acridine orange and photographed (×40) under a fluorescence microscope. The photonegatives (containing 10–30 cells) were scanned with a drum scanner and a complete picture containing 128 gray levels was constructed. Each cell contour was calculated with the use of a local threshold. The image and the superimposed cell contours were displayed on a television screen (16 gray levels) and errors were corrected interactively. With this system the spreading of normal human skin fibroblasts as a function of time could be quantified reproducibly. Compared to surface area or shape, the cell perimeter proved to be a very sensitive parameter of the degree of spreading. By using cell perimeter measurements, differences in the degree of spreading on various substrata could be quantified.
Flow cytometric analysis of factors which influence the BrdUrd‐Hoechst quenching effect in cultivated human fibroblasts and lymphocytes
Authors: Kubbies M. , Rabinovitch P. S.
Published: August 2005

Human fibroblasts and lymphocytes were grown in bromodeoxyuridine (BrdUrd) containing medium, harvested after 27–71 hours and stained with the fluorochrome Hoechst 33258. Parameters that influence the BrdUrd quenching effect of Hoechst fluorescence at a given pH and ionic strength of the staining solution were determined. The parameters were determined in order to obtain high resolution histograms with differentiation of each cell cycle compartment for noncycling cells and for cycling cells in the first and second cycle. The BrdUrd concentration was found to be the most significant determinant, but cell density, cell type, dye concentration, type of Hoechst stain and incubation temperature during staining also contribute to the modulation of fluorescence of BrdUrd substituted nuclei. Optimization of these factors is required in order to achieve complete and well resolved cell cycle histograms by the BrdUrd‐Hoechst flow cytometric technique.