bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlated measurement of platelet release and aggregation in whole blood
Authors: Ault Kenneth A. , Rinder H. M. , Mitchell J. G. , Rinder C. S. , Lambrew C. T. , Hillman R. S.
Published: August 2005

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two‐color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP‐140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP‐140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 μm. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)‐induced aggregates. After disaggregation the proportion of GMP‐140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)‐induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation. The ability to measure such aggregates both in vitro and in vivo may be very useful.
A rapid and sensitive flow cytometric method for the detection of multidrug‐resistant cells
Authors: Herweijer Hans , Engh Ger Van Den , Nooter Kees
Published: August 2005

Multidrug‐resistant (MDR) cells are characterized by a defect in drug accumulation caused by activity of an energy‐dependent rapid drug efflux pump. The action of this drug pump can be inhibited by specific agents, referred to as membrane transport modulating agents (MTMAs), resulting in a restoration of the intracellular drug accumulation. This paper presents a flow cytometric assay for the detection of MDR cells, which is based on the ability of these cells to respond to MTMAs. Daunorubicin net‐uptake kinetics were measured of anthracycline‐sensitive (A2780/S) and ‐resistant (A2780/R) human ovarian carcinoma cells in vitro. A2780/R cells accumulated significantly less (about a factor of 5) daunorubicin as compared to A2780/S cells. Addition of verapamil or cyclosporin A to A2780/R cells at steady‐state daunorubicin uptake led to a dose‐dependent increase in cellular daunorubicin accumulation. The sensitivity of the assay was determined by testing mixtures of A2780/S and A2780/R cells. Analysis of A2780/S cells contaminated with A2780/R cells showed that as few as 2.5% MDR cells could readily be detected in the mixture. In conclusion, this functional assay enables the detection of MDR cells in a heterogeneous cell suspension and is ideally suited for the study of the occurrence of typical MDR in human cancer.
Quantitative immunofluorescence in single Saccharomyces cerevisiae cells
Authors: Eitzman P. D. , Hendrick J. L. , Srienc F.
Published: August 2005

We have developed a staining procedure that allows the simultaneous determination of intracellular amounts of DNA and an antigen in Saccharomyces cerevisiae with a single laser flow cytometer. The antigen, β‐galactosidase from a cloned lacZ gene, is inducible and is detected with an indirect immunofluorescent stain. Cell preparation procedures, specifically cell fixation and cell wall removal, have significant effects on measured levels of immunofluorescence and have been optimized to prevent cell loss and maximize immunofluorescence. Average immunofluorescent levels of cell populations expressing different levels of β‐galactosidase show excellent correlation with measurements of average β‐galactosidase activity per cell based on cleavage of o‐nitrophenyl‐β‐D‐galactopy‐ranoside. Experiments with yeast populations containing various numbers of copies of the cloned gene indicate that the relationship between immunofluorescence and antigen content also holds at the single‐cell level. Correlated measurements of DNA and β‐galactosidase content on a single‐cell level permit the investigation of cellular enzyme content as a function of cell cycle position under various conditions. The procedure can be easily modified to detect other antigens by changing the primary antibody used.
Flow cytofluorimetric determination of protein distribution throughout the cell cycle
Authors: Khochbin Saadi , Chabanas Agnès , Albert Philippe , Lawrence Jean‐Jacques
Published: August 2005

Quantitative treatment of multiparametric determination on cells using flow cytofluorometry was made possible by the development of a computer program that allows the relative quantification of a specific protein as a function of the position of cells during the cell cycle. This type of analysis provides interesting information about the distribution of a given protein throughout the cell cycle. Four examples showing the distribution of specific proteins illustrate such a quantification during the cell cycle in two different cell lines. The program also allows for the handling of a series of histograms obtained by the analysis of protein distribution as a function of DNA content in relation to a third parameter. To illustrate possible applications for this program, the evolution of the distribution of two proteins, the oncoprotein p53 and the histone H1°, during the induced differentiation of murine erythroleukemia cells has been studied.
Flow cytometry and phytoplankton
Authors: Phinney D. A. , Cucci T. L.
Published: August 2005

Flow cytometry and sorting are now an important technology in aquatic research. Simultaneous measurements of individual particle cell size, fluorescence, and light scatter properties are directly applicable to current topics in aquatic research. Flow sorting may be employed to obtain subsets of cells for analysis by conventional methods. The manner in which rapid, precise measurements of single cells are made is complex, and the application of this technology to aquatic samples is subject to many analytical constraints. Flow cytometric measurements of algal cell size and pigment autofluorescence are relative and are therefore dependent on the optical configuration and variability of the instrument. Specific types of reference materials are used to establish the validity of analyses: (1) instrument standards, (2) fluorescence controls, and (3) internal stain standards. The selection and application of standards and controls are discussed in the context of allometric (cell size versus pigment fluorescence) and ataxonomic (pigment color groups) methods. The widespread acceptance of particular reference materials among research groups will result in comparable data sets describing aquatic particle distributions.
Separation and concentration of phytoplankton populations using centrifugal elutriation
Authors: Pomponi Shirley A. , Cucci Terry L.
Published: August 2005

Centrifugal elutriation is a technique for separating particles on the basis of their sedimentation velocity, an expression of size, shape, and specific gravity. Unialgal cultures, mixtures of two phytoplankton cultures, and natural seawater samples were elutriated to determine the feasibility of this technique for collecting fractions of different cell cycle phases, separating two phytoplankton species, and concentrating cells from dilute samples. Elutriation resulted in the separation of a culture of Dunaliella tertiolecta and Phaeodactylum tricornutum into homogeneous fractions of each species. Cells in the natural seawater sample were concentrated by nearly 2 orders of magnitude. Centrifugal elutriation provides an alternative cell separation and concentration technique when large numbers of cells are required.
Photobiology of natural populations of zooxanthellae from the sea anemone Aiptasia pallida: Assessment of the host's role in protection against ultraviolet radiation
Authors: Lesser Michael P.
Published: August 2005

Natural populations of the sea anemone Aiptasia pallida containing endosymbiotic dinoflagellates were acclimated to different irradiance regimes, with and without ultraviolet radiation (UV). They showed a compensatory response in the amount of chlorophyll and the activities of enzymes responsible for detoxifying active species of oxygen that are produced by the interaction between visible or ultraviolet radiation and photosynthetically produced oxygen. Protection from active species of oxygen is essential to prevent the photooxidation of chlorophyll and the concomitant loss of productivity. Bulk analyses of chlorophyll showed differences between the populations exposed to varying irradiance regimes, but revealed no significant independent effect of UV. However, analysis by flow cytometry of the individual cells from treated populations did detect statistically significant differences in cell size and the amount of chlorophyll fluorescence per cell, which could be attributed to treatment with ultraviolet radiation. With flow cytometry we are able to detect the population variability that is undetectable by bulk measurements which is important in assessing the effects of environmental parameters in both symbiotic and free‐living microalgae. Research using simultaneous measurements by flow cytometry could add considerable insight into the population dynamics of both zooxanthellae and host cells.
A simple and rapid method for determining the linearity of a flow cytometer amplification system
Authors: Bruce Bagwell C. , Baker David , Whetstone Sherry , Munson Mark , Hitchcox Shelly , Ault Kenneth A. , Lovett Edmund J.
Published: August 2005

We describe a simple and rapid method for determining the linearity of a flow cytometer amplification system. The method is based on a fundamental characteristic of linear amplifiers: The difference between two amplified signals increases linearly with increasing amplifier gain. Two populations of beads or cells, differing slightly in fluorescence intensity, are analyzed by the flow cytometer at increasing photomultiplier tube high‐voltage settings. The distribution of the populations' mean difference versus mean position is a straight line intersecting the origin for linear amplifiers. Although some types of nonlinearities cannot be detected with this technique, deviations from linearity indicate nonlinear components in the flow cytometer amplification system. The correlation coefficient is used to quantify degree of nonlinearity. We also describe a method for amplifier nonlinearity compensation.
Statistical evaluation of cell kinetic data from DNA flow cytometry (FCM) by the EM algorithm
Authors: Baldetorp Bo , Dalberg Mats , Holst Ulla , Lindgren Georg
Published: August 2005

Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A DNA histogram normally consists of a mixture of one or more constellations of G0/G1‐, S‐, G2/M‐phase cells, together with internal standards, debris, background noise, and one or more populations of clumped cells. We have modelled typical DNA histograms as a mixed distribution with Gaussian densities for the G0/G1 and G2/M phases, an S‐phase density, assumed to be uniform between the G0/G1 and G2/M peaks, observed with a Gaussian error, and with Gaussian densities for standards of chicken and trout red blood cells. The debris is modelled as a truncated exponential distribution, and we also have included a uniform background noise distribution over the whole observation interval. We have explored a new approach for maximum‐likelihood analyses of complex DNA histograms by the application of the EM algorithm. This algorithm was used for four observed DNA histograms of varying complexity. Our results show that the algorithm works very well, and it converges to reasonable values for all parameters. In simulations from the estimated models, we have investigated bias, variance, and correlations of the estimates.
Detection of bladder carcinoma in females by flow cytometry and cytology
Authors: Hermansen Dane K. , Badalament Robert A. , Fair William R. , Kimmel Marek , Whitmore Willet F. , Melamed Myron R.
Published: August 2005

The sensitivity of bladder wash flow cytometry (BWFCM), voided urinary cytology (VUC), and cytology of cathetrized urine obtained at the time of cystoscopy (CUC) were reviewed on all women evaluated for bladder cancer at Memorial Sloan‐Kettering Cancer Center between June 1985 and December 1986. This comprised sixty‐four episodes of pathologically proven bladder cancer in 48 women. Considering positive and suspicious results jointly the sensitivities of BWFCM, CUC and 3 VUC were 75%, 64% and 56%, respectively. If only positive results were considered (i.e., suspicious results considered as negative), the sensitivities of BWFCM, CUC and 3 VUC were 64%, 31% and 32%, respectively. The sensitivities of these tests are less than for a predominantly male population, presumably related to the presence of squamous epithelium and greater frequency of pyuria. However, bladder wash flow cytometry and conventional cytology are still a very valuable addition to cystoscopic examination, and the combination of BWFCM with conventional cytology is more sensitive than either procedure alone.
Separation of pigmented and albino melanocytes and the concomitant evaluation of endogenous peroxide content using flow cytometry
Authors: Boissy Raymond E. , Trinkle Linda S. , Nordlund James J.
Published: August 2005

Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non‐pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non‐pigmented melanocytes was compared by flow cytometry.

Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non‐pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non‐pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide‐related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay.

These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.
Sample station modification providing on‐line reagent addition and reduced sample transit time for flow cytometers
Authors: Kelley Keith A.
Published: August 2005

A flow cytometer was equipped with a modified sample station to facilitate online addition of mediators to the sample and reduce the time of delivery of the sample to the interrogation point. The ready availability of materials and straightforward nature of this design make modifications simple and facilitate measurements of cellular activation. Parameters such as pH, membrane fluidity, and calcium mobilization are easily measured in this system, because detection can be made less than 4 s after addition of mediator with no interruption of sample flow. The sample station modification is described in detail along with methods for mixing and temperature control.
Fluorescent staphylococci as microbeads
Authors: Szabó Gábor , Damjanovich Sándor
Published: August 2005

Fixed bacteria of the protein A‐rich Cowan I Staphylococcus strain were labelled with fluorescein isothiocyanate and used as the second‐step reagent in an indirect immunofluorescent assay of specific cell‐surface antigen expression. The results are documented with fluorescence microscopy and flow cytometry.
The athena semi‐automated karyotyping system
Authors: van Vliet Lucas J. , Young Ian T. , Mayall Brian H.
Published: August 2005

In this article we describe Athena, a system that provides for semi‐automated karyotyping of metaphase spreads. The system is based upon the Macintosh II computer. It uses software that is written entirely in C and consists of approximately 200 Kbytes of executable code. Athena provides automated segmentation of metaphase images into individual chromosomes, automated measurements on each banded chromosome, and automated classification into the standard Paris‐convention karyotype. Furthermore, the system provides the ability to construct one or more chromosome data bases to represent the types of metaphase spreads and staining techniques that may be used in a given laboratory. Because we believe that it is impossible to construct a system that can achieve perfect segmentation, perfect separation of touching and overlapping chromosomes, perfect localization of the centromeres, and perfect classification, the system offers the possibility for interaction at each of the above stages using the well‐accepted Macintosh user interface.
Multivariate chromosome analysis and complete karyotyping using dual labeling and fluorescence digital imaging microscopy
Authors: Arndt‐Jovin Donna J. , Jovin Thomas M.
Published: August 2005

The combination of multiple dye‐DNA interactions, a fluorescence digital imaging system with a scientific CCD camera, and multivariate image analysis allows the rapid karyotyping of fluorescent human metaphase chromosome spreads. Chromosomes are stained with the bisbenzimidazole dye Hoechst 33342 and chromomycin A3, a dye pair used frequently in bivariate flow analysis and sorting of metaphase chromosomes in suspension. The use of ratio functions involving the total and peak intensities of the two dyes provides increased resolution of the karyotype in the microscope, and it can be anticipated that the same approach could lead to improved performance with flow systems as well. High pass filtering with a Laplace operator yields characteristic banded images of the individual chromosomes, even with total fields that are less than 200 pixels on a side.
Discrepancies between flow cytometric and cytogenetic studies in the detection of aneuploidy in human solid tumors
Authors: Shackney Stanley E. , Burholt Dennis R. , Pollice Agnese A. , Smith Charles A. , Pugh Reginald P. , Hartsock Robert J.
Published: August 2005

Parallel flow cytometric (FCM) cell DNA studies and cytogenetic studies were performed on clinical samples from twenty human solid tumors of various types and on cell lines established in tissue culture from three of these tumors. Six of twenty clinical samples (30%) showed concordance between flow cytometry and cytogenetics with respect to the presence or absence of aneuploidy. Among the fourteen cases with discrepancies between the two methods, 8 (40% of all cases) showed hypodiploidy by cytogenetics and had diploid DNA histograms. Three cases (15%) had prominent discrete peaks in the triploid to tetraploid region by cytogeneties but had only barely discernible corresponding peaks in the DNA histogram. In two cases (10%)cytogenetic studies revealed diffuse aneuploidy. Cytogenetic studies demonstrated near‐tetraploidy in three samples, but only one of these was detected by FCM; all three cases exhibited other numerical chromosomal abnormalities. In one case aneuploidy was demonstrated by FCM and not by cytogenetics. Among the tumor cell lines established in culture, the DNA Index was often higher than the cytogenetic index. Overall, 13/20 or 65% of patients with solid tumors in this, study had numerical chromosomal abnormalities that were not detected by flow cytometry. Eleven of these patients had distant metastases at the time of tumor sampling, and nine of these died of their disease within 1–11 months of the time of study.
Rapid metaphase and interphase detection of radiation‐induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization
Authors: Cremer T. , Popp S. , Emmerich P. , Lichter P. , Cremer C.
Published: August 2005

Chromosomal in situ suppression (CISS)‐hybridization of biotinylated phage DNA‐library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the Iq12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co‐γ‐rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA‐library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS‐hybridization.
Localization of a mouse centromeric DNA repeat in interphase nuclei
Authors: Disteche Christine M. , Adler David A.
Published: August 2005

The position of a mouse DNA repeat located near the centromere of mouse chromosomes X, 11, 13, and 17 was examined in interphase nuclei of bone marrow and fibroblast cells by in situ hybridization of 3H‐ or biotin‐labeled DNA probe 70‐38. In most laboratory mouse strains this probe recognizes a single repeat cluster (DXWas70) close to the centromere of the mouse X chromosome. In a few mouse strains, a second locus (D11Was70, D13Was70, or D17Was70, depending on the mouse strain) is located near the centromere of an autosome. In interphase nuclei from mouse strains with the X‐linked locus only, two distinct sites of hybridization were found in female mice and one in male mice. These two sites remained separated during thedifferent phases of the cell cycle (G1, early S, late S, and G2) as demonstrated by in situ hybridization of the probe to flow‐sorted nuclei. In interphase nuclei from mouse strains with both the X‐linked locus and an autosomal locus, four distinct sites of hybridization were found in female mice and three in male mice. Further analysis of loci DXWas70 and D17Was70 showed that these loci were often located in the outer region of nuclei from bone marrow and fibroblast cells.
Multiple fluorescence in situ hybridization
Authors: Nederlof P. M. , van der Flier S. , Wiegant J. , Raap A. K. , Tanke H. J. , Ploem J. S. , van der Ploeg M.
Published: August 2005

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue.

This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.
System for flow sorting chromosomes on the basis of pulse shape
Authors: Bartholdi Marty F. , Parson Jimmie D. , Albright Kevin A. , Cram L. Scott
Published: August 2005

Sorting on the basis of the complex features resolved by chromosome slit‐scan analysis requires rapid and flexible pulse shape acquisition and processing for determining sort decisions before droplet breakoff. Fluorescence scans of chromosome morphology contain centromeric index and banding information suitable for chromosome classification, but these scans are often characterized by variability in length and height and require sophisticated data processing procedures for identification. Setting sort criteria on such complex morphological data requires digitization and subsequent computation by an algorithm tolerant of variations in overall pulse shape.

We demonstrate here the capability to sort individual chromosomes based on their morphological features measured by slit‐scan flow cytometry. To do this we have constructed a sort controller capable of acquiring an 128 byte chromosome waveform and executing a series of numerical computations resulting in an area‐based centromeric index sort decision in less than 2 ms. The system is configured in a NOVIX microprocessor, programmed in FORTH, and interfaced to a slit‐scan flow cytometer data acquisition system. An advantage of this configuration is direct control over the machine state during program execution for minimal processing time. Examples of flow sorted chromosomes are shown with their corresponding fluorescence pulse shapes.
Study of X chromosome abnormality in XX males using bivariate flow karyotype analysis and flow sorted dot blots
Authors: Carter N. P. , Ferguson‐Smith M. E. , Affara N. A. , Briggs H. , Ferguson‐Smith M. A.
Published: August 2005

We have used bivariate flow karyotype analysis to quantify aberrant X chromosome size in 11 XX males. With one exception, the patients could be grouped into those with an X homologue difference greater than normal (Group A, n = 3) and into those whose X homologue difference could not be distinguished from female controls (Group B, n = 7). The range of sizes of the aberrant X chromosome in Y‐sequence positive patients agrees with the variable nature of the X‐Y interchange in these individuals as determined by the use of Y‐specific DNA probes and Southern blotting analysis. In one patient it was possible to sort separately the normal and the X‐Y interchanged homologues for dot blot analysis. The presence of Y sequences and an increased dose of the zinc finger gene, ZFY, were detected in the X‐Y interchanged homologue. In preliminary studies of 5 male and 6 female controls, it was noted that a consistent difference between the two X homologues in females was found which could not be totally explained by errors of the fitting procedure. We suggest that this difference could be due to X inactivation and that the two X homologues in females might be distinguishable.
Light scattering measurement in an arc lamp‐based flow cytometer
Authors: Steen Harald B.
Published: August 2005

The epi‐illumination optics employed in most arc lamp‐based flow cytometers may be modified so as to produce a dark‐field configuration which facilitates highly sensitive detection of both forward and large angle light scattering in an instrument with a “jet on open surface” flow chamber. Forward scattering is detected at angles upwards from about 2°, while large angle scattering includes angles above 18°. Theoretical considerations suggest that large angle scattering measured around 20° may be as efficient as that measured at 90° for the purpose of distinguishing cells on the basis of intracellular structure. This was supported by the finding that dual parameter light scattering histograms of leukocyte suspensions obtained with the arc lampbased instrument were closely similar to such histograms recorded with a laserbased instrument with the large angle detector at 90°. Different species of bacteria could be distinguished by means of the dual parameter light scattering device, as could different species of sea algae. The sensitivity of the device is sufficient to measure 0.2 μm polystyrene particles in both forward and large angle scattering.
Vitality measurement using spectrum shift in Hoechst 33342 stained cells
Authors: Ellwart Joachim W. , Dörmer Peter
Published: August 2005

A bivariate flow cytometric technique has been developed in which Hoechst 33342 stained vital cells are discriminated from early damaged cells by differences in their fluorescence emission spectra. A discrete population of cells with intact cell membranes passing from vital to dead could be identified by using propidium iodide exclusion in combination with a concentration‐independent spectrum shift of Hoechst fluorescence to longer wavelengths. The appearance after injury of two subsets of cells with intact membranes representing vital and early damaged cells, respectively, indicates two types of binding of Hoechst 33342 to these cells, presumably resulting from differences in chromatin structure. The method was tested in murine and human hemopoietic cell lines by cytotoxic treatment with cytosine arabinoside and interleukin 3 deprivation in factor‐dependent lines. The power of the method was demonstrated by a cell cycle analysis of the early damaged cells after both cytotoxic treatment and growth factor deprivation.
Flow cytometric analysis of recombinant murine GM‐CSF (rmuGM‐CSF) induced changes in the distribution of specific cell populations in vivo
Authors: Pennline Kenneth J. , Pellerito Frances , DaFonseca Manuel , Monahan Peggy , Siegel Marvin I. , Smith Sidney R.
Published: August 2005

The changes in the distribution of granulocytes, monocytes, and lymphocytes in various tissue compartments following subcutaneous (SC) administration of recombinant murine GM‐CSF (rmuGM‐CSF) in vivo was determined by flow cytometry in time course studies. Balb/c mice were given single, daily SC injections of 1 or 4 μg of rmuGM‐CSF for 10 days. Flow cytometric analysis was performed on bone marrow (BMC), peritoneal exudate (PEC), and peripheral blood (PBC) cell preparations from mice treated for 1, 3, and 10 days. Dual fluorescence was employed to gate on leukocytes (T200+) and analyze for Ig+, Thy 1.2+, MAC+, and 8C5+ (granulocytes) cells. The analyses indicated that SC‐rmuGM‐CSF increased the percentage of 8C5+ cells in PBC after 1 day of treatment. However, significant changes in the cell composition of PEC and BMC were not observed until day 10 of treatment and included increases in 8C5+ cells and the myeloid cell population, respectively. Side scatter analysis (cell density) of PBC and PEC indicated that the percentage of the granulocytic cell population increased significantly in rmuGM‐CSF treated mice. The changes observed in PEC and BMC appeared to be doserelated whereas those observed in PBC were not. These data clearly demonstrate the utility of flow cytometric analyses for detecting selective effects of cytokines on cell populations that are involved in host defense mechanisms.
Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma
Authors: van Dam Peter A. , van Bockstaele Dirk R. , Keersmaeckers Guy H. , Uyttenbroeck Frans L.
Published: August 2005

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.
Introduction to flow cytometry data file standard
Authors: Dean Phillip N. , Bagwell C. Bruce , Lindmo Tore , Murphy Robert F. , Salzman Gary C.
Published: August 2005

The Data File Standards Committee of the Society for Analytical Cytology presents a Standard to be used for the storage of data associated with flow cytometric measurements. The Standard specifies a format that provides for the inclusion of all information necessary to fully describe: (1) the instrument used for the measurement; (2) the sample measured; (3) the data obtained; and (4) the results of analysis of the data. The Committee and the Society for Analytical Cytology point out that the use of this Standard by all those individuals and companies that generate or use data taken with flow cytometers or generate methods of analysis for the data will encourage the sharing of such data and methods of analysis.
Quantitative imaging of immunocytochemical (pap) estrogen receptor staining patterns in breast cancer sections
Authors: Sklarew R. J. , Bodmer S. C. , Pertschuk L. P.
Published: August 2005

“Receptogram Analysis” has been developed as a pattern‐oriented approach for predicting endocrine response in breast cancer based upon quantification of the estrogen receptor immunocytochemical assay (ERICA), using a Quantimet Imaging System. Response prediction was evaluated in 58 stage III and IV patients receiving endocrine therapy (primarily Tamoxifen). The Receptogram is a composite of the univariate distributions of nuclear receptor content, IOD(S), and concentration (MOD), and their bivariate contour plot; where (S) is the calculated nuclear radius in section. MOD distributions were classified into four types based upon peak modality and kurtosis (I–IV), and contour plots were classified into four subtypes (A–D) based upon contour slope. Patients failing therapy were ERICA ‐ or their receptogram revealed co‐existent ER+ and ER− tumor cells (type II), highly skewed MOD distributions lacking defined peaks (type IV), or contours with nearly horizontal slope (type C). Response was realized in 9/16 type I patients, with a single positive MOD peak, and in 9/15 type III patients, with discrete, multimodal MOD peaks. In contrast, 0/8 type II, 0/12 type IV, and 0/10 type C patients were responders. Receptogram analysis was superior to cytosol assay (DCC) as a response discriminant: positive predictive value, 53% vs. 33%; negative predictive value, 100% vs. 75%; sensitivity, 100% vs. 83%; specificity, 68% vs. 23%; and accuracy, 78% vs. 41%, respectively. Alternately, patients were assigned to potentially responsive or nonresponsive groups based upon thresholded mean receptor parameters: field MOD, mean nuclear MOD (NMOD), and mean NMOD(PF) where PF is the ER+ nuclear fraction. While these parameters correlated with DCC (r = .72, 0.69, and 0.69), they were only marginally better in predictive value.
System for acquisition and real‐time processing of multidimensional slit‐scan flow cytometric data
Authors: Robinson R. D. , Wheeless D. M. , Hespelt S. J. , Wheeless L. L.
Published: August 2005

The high‐speed sampling requirements of multidimensional slit‐scan signals (cell contours) have typically required custom hardware. This specialized hardware has often lacked the flexibility to adapt to varying instrument setups and experimental requirements. A hardware and software system capable of sampling multiple slit‐scan cell contours at rates of up to 40 MHz with 10‐bit resolution is described. It utilizes commercially available CAMAC transient recorders, a Digital Equipment Corp. PDP‐11/83 computer, and custom hardware for signal conditioning and trigger generation. The modular design of the software system allows various hardware options with minimal additional coding. Real‐time digital processing checks each cell contour for multiple peaks; extracts morphological features such as width, height, and area; accumulates gated histograms of these data; and optionally saves the derived data, selected contours, or both into list mode files on disk.
Flow cytometric evaluation of the cytotoxicity of novel antiviral compounds
Authors: Nassiri M. Reza , Hudson Jerry L. , Pudlo Jeffrey S. , Birch Gary M. , Townsend Leroy B. , Drach John C.
Published: August 2005

Two acyclic analogs of bromotubericidin were tested for cytotoxic effects on uninfected cells by monitoring cell growth and measuring cell cycle perturbations using flow cytometry. As reported elsewhere, 5‐bromotubercidin analogs in which ribose was replaced by 2‐hydroxyethoxymethyl (compound 102) or by 1,3‐dihydroxypropoxymethyl (compound 183) were potent inhibitors of human cytomegalovirus (HCMV) replication in vitro (Pudlo et al.: Journal of Medicinal Chemistry 31:2086–2092, 1988). Because these compounds also inhibited the growth of uninfected cells, we performed kinetic studies with an established neoplastic line of human cells (KB) using flow cytometry. Growth of KB cells treated with either compound 102 or 183 were inhibited in a dose‐dependent manner. Growth inhibition by compound 183, however, was not fully expressed for at least 24 h. DNA analysis by flow cytometry showed that a 4‐h incubation with 10 μM compound 102 caused a decrease of cells in G2/M phase. Cells began to accumulate in early S phase by 12 h of incubation, leading to mid S phase accumulation at 21 h. Compound 183 at 10 μM slightly decreased the number of cells in G2/M phase after a 4‐h incubation, and led to accumulation of DNA in S phase after a 12‐h incubation. By 24 and 30 h, DNA histograms appeared similar to those of control cells but with a slight accumulation of the population in early S phase. In separate experiments, drugs were removed following a 24‐h incubation. After removal of compound 102, KB cell growth resumed with a normal population doubling time. In contrast, the effects of compound 183 were not reversible, suggesting the two compounds acted by different biochemical mechanisms.
Evidence for false aneuploid peaks in flow cytometric analysis of paraffin‐embedded tissue
Authors: Joensuu Heikki , Alanen Kalle A. , Klemi Pekka J. , Aine Risto
Published: August 2005

It has recently been shown that bimodal histograms with false aneuploid peaks may be obtained by DNA flow cytometry from histologically normal tissue allowed to autolyze. To investigate if such peaks can be generated from surgically excised archival tissue, 198 paraffin blocks from 179 patients containing histologically normal spleen (n = 65), liver (n = 26), thyroid (n = 32), pancreas (n = 19), salivary gland (n = 49), or lymph node tissue (n = 7), obtained from the archives of two university pathology departments, were analyzed for nuclear DNA content. The great majority (n = 160, 83.8%) of the 191 interpretable histograms had a single symmetrical G1 peak; and 8 histograms, all produced from liver tissue had a tetraploid pattern. A slight or a prominent repeatable deviation in the G1 peak outline was present in 14 (7.3%) cases. A peak resembling an aneuploid G1 peak with a DNA index (DI) ranging from 1.14 to 1.38 was repeatedly produced from 9 (4.7%) blocks containing histologically normal or inflamed splenic (n = 3), pancreatc (n = 3), liver (n = 1), thyroid (n = 1), or lymph node (n = 1) tissue. The three abnormal peaks produced from pancreatic tissue were rounded in shape and resembled closely the ones that can be obtained from autolytic pancreatic tissue, and the six remaining extra peaks were all fused with the “diploid” peak. In conclusion, false peaks, probably caused by degradation of the nuclear contents during formalin fixation or before it, may rarely be obtained from surgical paraffin‐embedded samples.
Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry
Authors: Loken Michael R. , Brosnan Jeanne M. , Bach Bruce A. , Ault Kenneth A.
Published: August 2005

It is possible accurately to distinguish lymphocytes from other leukocyte populations in peripheral blood using the combination of fluorescence associated with CD45/CD14 and forward and orthogonal light scatter. By identifying the cell population of interest based on immunofluorescence, a light scattering window can then be drawn to include all (≥98%) of the lymphocytes. In this manner, maximal recovery of the lymphocytes within a sample can be consistently obtained. The combination of light scattering and immunofluorescence can also be used to define the purity of the gate. The identification of nonlymphocytes within the light scattering gate can then be used to establish an accurate denominator for the percent lymphocytes stained. Once the optimal data acquisition gate has been established and characterized, it is possible to correct subsequent analyses with that particular sample since the reactivity of monoclonal antibodies on monocytes and granulocytes can be accounted for once the nonlymphocytes have been identified as being within the acquisition gate.
Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry
Authors: Marder Philip , Maciak Ronald S. , Fouts Rebecca L. , Baker Robert S. , Starling James J.
Published: August 2005

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescence‐activated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein‐conjugated anti‐mouse‐IgG. The techniques were used to estimate successfully the frequency of immunoglobulin‐secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti‐IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric‐assisted single cell deposition provides positive attributes of several traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.
Increased light scattering resolution facilitates multidimensional flow cytometric analysis
Authors: Terstappen Leon W. M. M. , Mickaels Ronald A. , Dost Richard , Loken Michael R
Published: August 2005

Multidimensional flow cytometry identifies cell populations as clusters in a space created by the analysis of multiple parameters simultaneously. Optimal use of this multidimensional space requires each of the individual parameters to provide additional information for cell population discrimination as well as maximum utilization of the dynamic range available for each parameter. In this study we improve the visualization of the information present in light scattering signals from leukocytes to facilitate multidimensional flow cytometric analysis. Optimization of cell preparation techniques are essential to obtain high resolution light scattering signals that give complete separation of the granulocytes, monocytes, and granular and nongranular lymphocytes. The angle at which the forward scattered light was collected was modified to enhance the separation between leukocyte populations. Although orthogonal light scattering signals separate granular and nongranular lymphocytes, the resolution and dynamic range could not be displayed using linear or logarithmic functions. By applying a polynomial function to the orthogonal light scattering signals, all leukocyte populations could be displayed while maintaining high resolution. The combination of high resolution light scattering with a nonlinear display resulted in an equally spaced distribution of the cell populations distinguished by correlating forward and orthogonal light scattering signals. Using this approach, peripheral blood neutrophils, eosinophils, basophils, monocytes, and granular and nongranular lymphocytes were shown to occupy distinct locations in the correlation of orthogonal and forward light scattering. Surprisingly,the basophilic granulocytes were located close to granular lymphocytes and monocytes rather than near neutrophils and eosinophils.
Analysis of c‐erbB‐2 protein expression in conjunction with DNA content using multiparameter flow cytometry
Authors: Kelsten Martin L. , Berger Mark S. , Maguire Henry C. , Chianese David A. , Hellman Maria E. , Weiner David B. , Greene Mark I.
Published: August 2005

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c‐erbB‐2 protooncogene is amplified in 10–40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c‐erbB‐2 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c‐erbB‐2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c‐erbB‐2 expression in populations of ethanol‐fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c‐erbB‐2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.
Measurement of DNA and antigen expression of live cells using three fluorochromes and two detectors
Authors: de L. Davies Catharina , Kovacs Mary
Published: August 2005

In a multiparameter flow cytometric study, the fluorescence from the two different dyes fluorescein and dansyl‐lysine were detected in the same spectral interval on the same photomultiplier tube, using two lasers to excite the fluorochromes at two separate laser foci. The two signals were split by a T‐cable at the output of the photomultiplier tube and synchronized by delaying the first signal. Dansyl‐lysine binds to the membrane of heat‐inactivated cells and was used to distinguish between live and dead cells after a hyperthermic treatment. By gating on the dansyl‐lysine signal, the DNA distribution and surface antigen expression of live and dead cells were obtained separately, using Hoechst 33342 and a monoclonal antibody conjugated to fluorescein.