bluuebunny/crossref_metadata_2025 huggingface dataset:
Applicability of a noncooled video‐rated CCD camera for detection of fluorescence in situ hybridization signals
Authors: Vrolijk J. , Sloos W. C. R. , Verwoerd N. P. , Tanke H. J.
Published: June 2005

Cooled CCD cameras provide good sensitivity and linearity with a high dynamic range and are therefore well suited for quantification of fluorescence in situ hybridization signals. However, for a fraction of the cost, conventional noncooled, video‐rated CCD cameras can also be applied for most applications in the field of fluorescence in situ hybridization, provided that they allow for longer integration times. This paper describes the use of the Sony camera, model XC‐77RR‐CE, for this purpose. Tests were carried out to compare the dark current, linearity, and signal‐to‐noise ratio of this camera with a Photometrics cooled CCD camera model KAF 1400, and the suitability for quantitative measurements was investigated on a model system of fluorescent beads. It is shown that if the dark current of the video‐rated camera is internally corrected, integration times of up to 30 s can be used; under such conditions good linearity is maintained. The camera was found suitable for the detection of in situ hybridization spots in interphase nuclei using centromere‐specific probes. The fast readout rate of the camera offers interesting facilities for quickly locating objects with relatively strong fluorescence, such as counterstained metaphases. The less intense probe signals may then be analyzed at higher magnification. © 1994 Wiley‐Liss, Inc.
Evaluation of sensitivity in DNA aneuploidy detection using a mathematical model
Authors: Benson Neal A. , Braylan Raul C.
Published: June 2005

Detection of DNA aneuploid populations in samples analyzed by flow cytometry generally requires recognition of more than a single G0/G1 peak on the resulting DNA histogram. This assessment is critical in clinical samples if diagnosis or subsequent patient treatment are influenced. In this paper we evaluate three major distribution parameters that contribute to recognition or obscuration of closely spaced peaks on a DNA histogram. These parameters are (1) coefficient of variation (CV), (2) proportion of cells within each peak, and (3) separation of peaks (DNA, index, DI). Through the analysis of computer‐synthesized DNA histograms, we produced a single chart illustrating the minimum CV required for two peaks (representing DNA diploid and DNA aneuploid cells) in mixtures of various proportions (1:99 to 99:1) and DI values (1.01 to 1.20) to be resolved. This information can be utilized to provide insight into the ability of flow cytometry to resolve a DNA aneuploid population in a histogram. © 1994 Wiley‐Liss, Inc.
Light scatter based lymphocyte gate–helpful tool or source of error?
Authors: Krömer Elisabeth , Grossmüller Friedrich
Published: June 2005

During flow cytometric analysis of 29 samples of human peripheral blood mononuclear cells, we observed that a light scatter‐based lymphocyte gate would fail to cover all TCR (CD3)‐expressing cells. A mean of 7.9 ± 5.3% of all CD3+ cells was found outside the gate. In addition, this shifted cell subset showed an altered CD4/CD8 ratio (8.1 ± 5.0% of all CD4+ and 13.7 ± 5.8% of all CD8+ cells). The use of standard light scatter‐based lymphocyte gates in acquisition and/or analysis of flow cytometry (FCM) data may thus lead to erroneous results, particularly if T cells are to be studied. © 1994 Wiley‐Liss, Inc.
Simultaneous measurement of two cellular antigens and DNA using fluorescein‐isothiocyanate, R‐phycoerythrin, and propidium iodide on a standard FACScan
Authors: Corver Willem E. , Cornelisse Cees J. , Fleuren Gert Jan
Published: June 2005

Multiparameter flow cytometry is a powerful tool for analyzing the phenotypic, cell kinetic, and ploidy heterogeneity of tumor cell populations. Because of the substantial spectral overlap of propidium iodide (PI) and R‐phycoerythrin (PE) fluorescence emission, this combined use of these fluorochromes has been thought not to be feasible on a standard flow cytometer for these kind of studies. Instead of PI, 7‐amino‐actinomycin D (7‐AAD) is used as DNA stain. In this paper however, we show that PI can be used as a DNA stain in combination with fluorescein isothiocyanate (FITC) and R‐phycoerythrin (PE) on a standard FACScan. Three established ovarian cancer cell lines (IGROV1, NIH: OVCAR‐3, and COV362. c14) were used for these experiments. Cells were fixed with 1.0% paraformaldehyde and permeabilized, with various concentrations of lysolecithin for the simultaneous detection of surface antigens by monoclonal antibodies MOv18, BMA180 or OV632, intermediate filament antigens (keratin 18 or vimentin), and DNA. A final concentration of 80 μg/ml lysolecithin was found to give optimal results. The emission spectrum overlap from PI into the orange fluorescence channel (FL2) used for PE fluorescence detection could be sufficiently compensated up to a photomultiplier tube potential of about 440 Volts (V) required at the FL2 channel. Using the same instrument settings, 5.10×104 PE equivalents were detectable. Under these conditions, CVs obtained for the DNA histograms ranged from 3.0–4.1. Application of the method on a mixture of activated peripheral blood lymphocytes and ovarian tumor cells resulted in a clear separation of the two populations both by surface and cytoplasmic antigen expression and DNA content. © 1994 Wiley‐Liss, Inc.
Cell membrane changes induced by the cytolytic peptide, melittin, are detectable by 90° laser scatter
Authors: Weston Kathryn M. , Alsalami Mohammed , Raison Robert L.
Published: June 2005

The 90° light scatter parameter of the flow cytometer was used to observe changes in the membrane of human lymphoblastoid cells (HMy2) as a result of the action of the cytolytic peptide, melittin. There was a rapid and concentrationdependent Increase in 90° light scatter after incubation of the cells with melittin, with the level of 90° light scatter reaching a maximum after 2 min. Even after all the cells were killed, as determined by ethidium bromide incorporation, the 90° light scatter continued to increase. Further, the 90° light scatter changes were temperature dependent. The data are consistent with the formation of lipid vesicles, either attached to the cell membrane or intracellular, as confirmed by electron microscopy of cells treated with melittin. The results demonstrate the use of the flow cytometer to detect changes in the integrity of the cell membrane. © 1994 Wiley‐Liss, Inc.
Hydrochloric acid denaturation of colorectal tumour tissue infiltrated with bromodeoxyuridine
Authors: Williamson Kate , Gilliland Robert , Weir Helen , Grimes John , Hamilton Peter , Anderson Neil , Crockard Alistair , Rowlands Brian
Published: June 2005

The thymidine analogue, bromodeoxyuridine, is substituted into DNA during DNA synthesis and can be used to identify those cells which have passed through the S‐phase of the cell cycle. Incorporated bromodeoxyuridine can be detected using anti‐bromodeoxyuridine monoclonal antibodies which bind to the exposed bromodeoxyuridine in singlestranded DNA after a suitable denaturation step. Hydrochloric acid is the most commonly employed denaturation agent in bromodeoxyundine monoclonal antibody methodologies. This preliminary study was to validate the hydrochloric acid denaturation step for colorectal tumour tissue infiltrated in vivo with bromodeoxyuridine. Standard immunohistochemistry and flow cytometric techniques using Bu20a were employed across a range of hydrochloric acid concentrations. Although high labelling indices were achieved using acid concentrations of 0. 10 M HCl, the optimal hydrochloric acid concentration was not the same in all tumours. Sensitivity of DNA to hydrochloric acid denaturation should be carefully considered in bromodeoxyuridine methodologies using the monoclonal antibody Bu20a. © 1994 Wiley‐Liss, Inc.
Detection of apoptosis‐associated DNA strand breaks in fine‐needle aspiration biopsies by in situ end labeling of fragmented DNA
Authors: Gorczyca Wojciech , Tuziak Tomasz , Kram Andrzej , Melamed Myron R. , Darzynkiewicz Zbigniew
Published: June 2005

The predominant mode of either spontaneous or drug‐induced death of cells in tumors is apoptosis. A flow cytometric method was developed in our laboratory to identify apoptotic cells, based on labeling DNA strand breaks, which appear as a result of extensive DNA cleavage by the apoptosis‐associated endonuclease, with biotinylated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The aim of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine‐needle biopsy. Twentytwo tumors, consisting of 11 breast carcinomas; three metastatic anaplastic carcinomas; three adenocarcinomas of colon, endometrium, and lung; two metastatic lymphnode squamous cell carcinomas of the larynx; and three malignant lymphomas were examined. It was possible to identify cells with DNA strand breaks in all these tumors Extremely high variability in the proportion of cells with DNA strand breaks Was observed between the individual tumors. In diploid tumors (n=12) the percentage of cells with DNA strand breaks varied from 1% to 43%, and the mean value, was 19%. In aneuploid tumors this percentage varied from 15% to 51% and the mean value was 37%. In the latter tumors the presence of cells with DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, showed the presence of DNA strand breaks. No correlation was observed between the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of tumor cells than among normal cells of the same organs. The higher percent of cells with DNA strand breaks compared to the percent of cells with morphology typical of apoptosis suggests that the early phase of apoptosis, characterized by the appearance of DNA strand breaks, which precedes morphological changes (chromatin condensation, nuclear fragmentation), may be very long in individual cells of the tumors studied. The present method may by applicable to estimate DNA strand breaks in solid tumors, either during spontaneous apoptosis or during apoptosis induced by chemo or radiotherapy, to evaluate tumor cell response early during treatment. © 1994 Wiley‐Liss, Inc.
High resolution mapping of overlapping cosmids by fluorescence in situ hybridization
Authors: Houseal Timothy W. , Dackowski William R. , Landes Gregory M. , Klinger Katherine W.
Published: June 2005

The constituents of two cosmid contigs were analyzed by high resolution mapping using two‐color fluorescence in situ hybridization (FISH) to extended DNA molecules. Samples were prepared by lysing the nuclei in situ followed by his tone depletion. This treatment results in elongate DNA filaments appropriate for high resolution mapping. The hybridization signals appeared as a strong of fluorescent spots separated by non‐fluorescing gaps. Probe‐specific features of the hybridization patterns were detected and some of the non‐fluorescing gaps were found to represent regions of repetitive DNA suppressed during hybridization. © 1994 Wiley‐Liss, Inc.
Characterization of exocytosis in electropermeabilized neutrophils by flow cytometric analysis: Difference in sensitivity to calcium and guanosine‐5′‐[γ‐thio]triphosphate
Authors: Boonen Ger J. J. C. , de Koster Ben M. , van der Keur Maarten , Vansteveninck John , Tanke Hans J. , Elferink Jan G. R.
Published: June 2005

When rabbit neutrophils were subjected to two electrical discharges of 4.75 kV/cm, the cells became permeable to propidium iodide. Measurement of propidium iodide fluorescence using flow cytometry showed that all cells in the suspension were permeabilized. The cells remained permeable for ≥ 20 min when the cells were stored at 0°C. When exocytosis was induced by Ca2+alone, the orthogonal light scatter (a sensitive parameter for cell granularity) of the complete population changed depending on the concentration. All the cells were equally sensitive to Ca2+and showed a similar degree of exocytosis at the same time. In the presence of a fixed concentration of Ca2+and a variable concentration of guanosine‐5′‐[v‐thio]triphosphate (GTPγS), a division of the cell population was observed in the orthogonal light scatter histogram. At low GTP‐γS concentrations, a part of the population showed complete exocytosis and a part of the population showed almost no exocytosis. With increasing GTP‐γS concentrations, the light scatter pattern of the population changed indicating that the cells were gradually sensitive to GTP‐γS. Electropermeabilized neutrophils showed an equal sensitivity to Ca2+and a graded sensitivity to GTP‐γS. Flow cytometry is considered as an ideal tool to study such an effect on a cell‐to‐cell basis. © 1994 Wiley‐Liss, Inc.
Neural network analysis of flow cytometric data for 40 marine phytoplankton species
Authors: Boddy Lynne , Morris C. W. , Wilkins M. F. , Tarran G. A. , Burkill P. H.
Published: June 2005

Flow cytometry data (time of flight, horizontal and, vertical forward light scatter, 90° light scatter, and “red” and “orange” integral fluorescence) were collected for laboratory cultures of 40 species of marine phytoplankton, from the following taxonomic classes, the Dinophyceae, Bacillariophyceae, Prymnesiophyceae, Cryptophyceae, and other flagellates. Single‐hidden‐layer “back‐propagation” neural networks were trained to discriminate between species by recognising patterns in their flow cytometric signatures, and network performance Was assessed using an independent test data set. Two approaches were adopted employing: (1) a hierarchy of small networks, the first identifying to which major taxonomic group a cell belonged, and then a network for that tax‐onomic group identified to species, and (2) a single large network. Discriminating some of the major taxonomic groups was successful but others less so. With networks for specific groups, cryptophyte species were all identified reliably (probability of correct classification always being ≥ 0.75); in the other groups half of the species were identified reliably. With the large network, dinoflagellates, cryptomonads, and flagellates were identified almost as well as by networks specific for these groups. The application of neural computing techniques to identification of such a large number of species represents a significant advance from earlier studies, although further development is required. © 1994 Wiley‐Liss, Inc.
Evaluation of the number of positive cells from flow cytometric immunoassays by mathematical modeling of cellular autofluorescence
Authors: Lampariello Francesco
Published: June 2005

A method, for automated evaluation of the percentage of positive cells from flow cytometric, immunofluorescence histograms is presented. The method is based on a suitable mathematical representation of cellular autofluorescence distribution which is used to identify the negative cell distribution in the test histogram. An algorithm is developed and implemented in a computer program that quantifies the positive cells from the routinely available control and test histograms. Its accuracy is compared to that of the other currently used automated methods of analysis on the basis of a set of histograms with overlapping distributions and with known percentages of positive cells, generated by artificial mixing of real data. © 1994 Wiley‐Liss, Inc.
Determination of the viability of trichomonas vaginalis using flow cytometry
Authors: Humphreys Michelle J. , Allman Richard , Lloyd David
Published: June 2005

In clinical laboratories, viability of Trichomonas vaginalis is determined by using light microscopy (differential count of motile to nonmotileorganisms). Alternative methods are proposed that utilise flow cytometry. Under an epifluorescence microscope, live organisms fluoresce intensely green with fluorescein diacetate (FDA), whereas dead cells fluoresce orange with propidium iodide (PI). Flow cytometric histograms of green versus red fluorescence reveal distinct populations for live and dead cells. The anionic oxonal probe DiBAC4(3) is a membrane potential sensitive dye that distributes between the inside of the cell and the medium. Live organisms are less fluorescent than dead organisms when stained with the oxonol probe. Valinomy: cin, dicyclohexylcarbodiimide, and vana‐date all give significant changes in the fluorescence intensities of cultures stained with the oxonol probe compared with control cultures, indicating that this probe is detecting changes in plasma membrane potential. Both FDA/PI and oxonol staining protocols allow good discrimination between populations and permit counts that are more statistically significant than those obtained by light microscopy. These methods remove the subjectiveness of microscopic counts and would increase the accuracy of susceptibility assays. © 1994 Wiley‐Liss, Inc.
Measurement of ceroid accumulation in macrophages by flow cytometry
Authors: Hunt J. V. , Bottoms M. A. , Skamarauskas J. , Carter N. P. , Mitchinson M. J.
Published: June 2005

The accumulation in macrophages of ceroid, an sutofluorescent polymer composed of oxidised protein and lipid, can be monitored seiniquantitatively by staining techniques. However, such methods are crude and give little infor mation about the amount of ceroid within cells. Flow cytometry, however, can give a quantitative assessment of cellular ceroid accumulation in vitro. Recently, flow cytometry was explored as a method for measurement of the accumulation in macrophages of ceroid. The accumulation appeared to be diminished in the presence of the antioxidant, α‐tocopherol. This is consistent with the role of lipoprotein oxidation in ceroid accumulation. Here the optimum wavelengths of emission and excitation, using both conventional fluorescence spectroscopy of cellular ceroid and w cytometric measurements with a number of optical filters, are determined. The use of optimal wavelengths determined in these studies enhances overall sensitivity. The findings are discussed in the context of future investigation of cell‐mediated lipid oxidation and its potential antagonists. © 1994 Wiley‐Liss, Inc.
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

Interphase 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

A 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

Determination 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

A 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

We 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

We 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

Automatic 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

Cell 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

Chinese 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

The 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

Scanning 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

The 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

Expression 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

The 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.
Factors underlying the variability of lipid droplet fluorescence in MA‐10 leydig tumor cells
Authors: Gocze Peter M. , Freeman Dale A.
Published: June 2005

Neutral lipids accumulate in cellular lipid droplets. These droplets vary remarkably in number and amount between cells. In the present studies, the variability in lipid content was quantified by comparing the coefficient of variation of fluorescence histograms of nile red lipid‐stained cells to the variability of cell size or cell protein distributions. This measure of lipid droplet variability persisted through a wide range of cell lipid content and averaged 4.4‐fold more variability than cell size and 2.6‐fold more variability than cell protein content. While looking for possible explanations for this variability, it was determined that cell to cell variability could not be explained by multiple clonal populations of cells or the confluence of the cell monolayer. Analysis of lipid variability using a more droplet‐specific fluorescent dye, bodipy, reduced variability by about 44%. Cell cycle analysis revealed that G2 + M cells contained more lipid than S‐phase cells, which in turn contained more lipid than G0 + G1 cells, but that variability was equally large throughout the cell cycle. The cholesteryl ester hydrolase inhibitor, diethylumbelliferyl phosphate, inhibited hydrolysis of both cholesteryl esters and triglycerides. Lipid content of diethylumbelliferyl phosphate‐treated cells increased while the variability in lipid staining decreased by an average of 72%. Thus, the excess lipid fluorescence variability compared to cell size or protein fluorescence could in part be explained by variability in cellular hydrolysis of triglyceride and cholesteryl ester. Excess lipid fluorescence variability could be reduced by an average of 44% when a more lipid droplet‐specific stain was used instead of nile red. © 1994 Wiley‐Liss, Inc.
Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature
Authors: Wheeless Leon L. , Robinson Roy D. , Lapets Oleg P. , Cox Christopher , Rubio Ana , Weintraub Michael , Benjamin Lennette J.
Published: June 2005

Sickle cell anemia is a disease for which there is currently no effective treatment. One method of evaluating clinical status is the counting of cell types based on morphology. There is a need for a rapid, reproducible method, superior to human inspection, for classification of these cells. Quantitative digital‐image analysis is being applied to this need. Blood from 24 patients with sickle cell anemia (SS) and SC disease and ten hematologically normal volunteers (AA) was stressed by bubbling with nitrogen. One hundred fifty cells were analyzed from each sickle specimen, and 100 were analyzed from each nonsickle specimen. Expert observers classified each cell as normal (N), sickle (S), or other abnormal (A). Cells were analyzed with a custom, high‐resolution image‐analysis instrument. A total of 42 features including metric, optical density‐derived, and textural features were extracted. The metric feature Form Factor (4πArea/Perimeter2) was selected by recursive partitioning analysis as the sole feature needed for segregating cells into the classes of N, A, and S. The agreement of automated classification (using cutpoints determined by recursive partitioning analysis) with a human expert for specimens from individuals with sickle cell anemia was 89% for N‐, 73% for A‐, and 92% for S‐classified cells. For specimens from individuals with sickle cell anemia, rates of agreement between two human experts were compared and found to be 86% for N, 84% for A, and 80% for S. For specimens from AA individuals, the agreement was 90% for N and 87% for A. © 1994 Wiley‐Liss, Inc.
Flow cytometry distinction between adherent and phagocytized yeast particles
Authors: Giaimis Jean , Lombard Yves , Poindron Philippe , Muller Christian D.
Published: June 2005

Our laboratory recently developed a light microscopy staining technique that provides a mean to distinguish between yeast that are simply bound to the surface of macrophages and yeast that have actually been phagocytized by macrophages (7). We adapted this technique by using fluorescent probes in order to test phagocytic activity by flow cytometry. Thus we are able to distinguish unambiguously extracellular from intracellular yeast during phagocytosis with the fast rate of flow cytometry (∼200 cells/s). The fluorescence quenching induced by a 1% tannic acid solution (w/v) can be applied to any FITC‐labeled, heat‐killed yeast cell or bacteria. The yeast cells already engulfed in the macrophage remain with their native fluorescence (internal and external pH equilibrated by 50 μ monensin 30 min/4°C) protected from the action of tannic acid, a nonmembrane permeable molecule. The results presented here validate this new technique. An application is presented showing the inhibition of endocytosis by cytochalasin‐B. © 1994 Wiley‐Liss, Inc.
Detection of immunocytochemically stained rare events using image analysis
Authors: Mesker Wilma E. , v.d. Burg Marja J. M. , Oud Peter S. , Knepfle Cecile F. H. M. , Ouwerkerk‐v. Velzen Maria C. M. , Schipper Nellie W. , Tanke Hans J.
Published: June 2005

The detection of rare‐event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation.

SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS‐red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counterstaining based on ethyl green. Slides were automatically screened for cytokeratin‐positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 × 106 negative cells. A comparison between manual screening and automated screening for cytokeratin‐positive cells showed a high level of correlation (0.9998).

For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study.

It is concluded that optimization of preparation and staining procedures for the detection of rare‐event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events. © 1994 Wiley‐Liss, Inc.