bluuebunny/crossref_metadata_2025 huggingface dataset:
Stylized chromosome images
Authors: Martin Alice O. , Isenstein Barry S. , Thomas Cecil W. , Rzeszotarski Mark S. , Johnson Walter E.
Published: August 2005

Stylized chromosome images 1) serve as a format to test effects of preprocessing algorithms used in automated karyo‐typing; 2) enhance the ability of humans to perform quantitative analysis of chromosomal aberrations; 3) provide an alternative format for karyotype hard copies produced by automated systems. Stylized chromosomes are two‐dimensional computer‐generated images based on information extracted from one‐dimensional width and density profiles. These profiles correspond to what cytogeneticists observe through the microscope as the shape and banding patterns of stained chromosomes. Stylized presentation sharpens chromosome band boundaries and perimeters, reduces “noise,” and enhances gray level variations, which are difficult to distinguish by humans on photographic or computer generated karyotypes.

Karyotyping accuracy using stylized images was used to detect difficult areas for automated chromosome identification. Landmark bands sufficient to classify chromosomes were identified; shapes of chromosomes reflected in width profiles were said to aid classification. A two‐step automated karyotyping strategy proposed is: 1) classify chromosomes by landmarks, minimum information needed for identification; 2) subsequently employ the full banding pattern with maximum resolution to detect aberrations. Stylized images of abnormal chromosomes have potential for testing hypothesis regarding breakpoints and quantitative analysis, but improvements are needed in homologue normalization and definition of termini of chromosomes.
Experience with the athena semi‐automated karyotyping system
Authors: Mayall Brian H. , Tucker James D. , Christensen Mari L. , van Vliet Lucas J. , Young Ian T.
Published: August 2005

The traditional analysis and assembly of metaphase chromosomes into, a karyogram is a slow and tedious process requiring intermediate photographic steps and manual manipulation of the chromosome images. Much of this task is highly repetitive and readily lends itself to partial automation. Semi‐automated karyotyping systems now are being used increasingly in both clinical and research cytogenetic laboratories. Digital image processing techniques are used to capture, manipulate, and make an initial classification of chromosome images. The Athena system uses commercially available components based on a Macintosh II personal computer. Digital image processing procedures automatically isolate chromosome images from the metaphase and arrange them into a karyogram, using information about relative chromosome length, centromeric index, and banding pattern. The operator uses the intuitive graphics interface of the Macintosh computer to monitor each phase of the analysis, to resolve any problems in isolating chromosome images, and to rearrange the individual chromosome images while assembling the final karyogram. Athena is designed as a semi‐automated karyotyping system that is easy to learn and has sufficient power and versatility for routine use in the analysis of human metaphase chromosomes.
Automatic detection of fragile X chromosomes using an X centromere probe
Authors: Piper Jim , Fantes Judith , Gosden John , Ji Liang
Published: August 2005

In order to score for the fragile X syndrome, blood samples are prepared with absorption stain labeling by in situ hybridisation of the X chromosome centromeres. Metaphases are located, digitised at high resolution, and segmented fully automatically. A three Stage adaptive classification scheme for labeled X chromosomes is then applied. This consists of a simple box classifier to identify plausible X and false positive X chromosomes, followed by a quadratic discriminant classifier that is re‐trained for each sample. The modal number of X chromosomes is then determined for each sample and used to refine the classification. A simple fragile site detector is applied to the distal portion of the detected X chromosome long arms. From the results we estimate computer and operator time requirements for a screening system in which the operator reviews only the apparently fragile X chromosomes detected by the computer.
Flow cytometric detection of β‐globin mRNA in murine haemopoietic tissues using fluorescent in situ hybridization
Authors: Bayer Jan A. , Bauman Jan G. J.
Published: August 2005

The novel method for flow cytometric detection of cellular RNA species in suspended cells by fluorescent in situ hybridization (FC‐FISH) was applied in the evaluation of β‐globin expression in murine haemopoietic tissues. Normal murine bone marrow cells and regenerating bone marrow cells obtained after lethal irradiation and bone marrow transplantation as well as murine 15 d fetal liver were examined. Furthermore, spleens and bone marrow of phenylhydrazine‐induced anaemic mice were studied. Biotinylated sense‐ and antisense single strand RNA probes, obtained by transcription of a 510 nucleotides murine β‐globin cDNA sequence subcloned into the pGEM1 plasmid were used as hybridization probes. For detection of the hybrids formed, avidin‐FITC was used.

Only the antisense β‐globin probe gave strongly positive fluorescence signals in a defined population of cells in each of the tissues examined, whereas the sense probe did not give signals higher than control samples. Melting characteristics of the hybrids showed the specificity of the in situ hybridization reaction. Forward light scatter distributions, reflecting cell size of the positive cells were as expected from erythroid cells. Within the erythrocyte subpopulation both β‐globin‐negative and ‐positive cells were detected. The percentages of positive cells determined flow cytometrically correlated with the percentages observed in May‐Grünwald/Giemsa stained preparations. Differences observed in fluorescence intensity between positive cells of different organs were no larger than about a factor of two, indicating a rather constant β‐globin mRNA content over the entire differentiation range. An exception was 15 d fetal liver, which was shown biochemically to contain about eight times more β‐globin RNA and which had a 2.4 times higher fluorescence intensity. We estimate that the sensitivity of the present method is such that as little as 500 copies per cell of a specific mRNA of 1 kb length would be detectable.
Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping
Authors: Trask Barbara , van den Engh Ger , Nussbaum Robert , Schwartz Charles , Gray Joe
Published: August 2005

Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide‐based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3–5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X‐linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.
Construction of gene libraries for each human chromosome
Authors: Van Dilla Marvin A. , Deaven Larry L.
Published: August 2005

We describe the construction of two complete sets of small insert, complete digest DNA libraries for each of the 24 human chromosomal types by the National Laboratory Gene Library Project. Flow sorting was used to purify the chromosomes which provided the DNA for cloning. One set of libraries was cloned into the HindIII site of the lambda vector Charon 21A, and the other set was cloned into the EcoRI site of the same vector. Characterization information from both in‐house experiments and user feedback is presented. These chromosome‐specific libraries are available to the general scientific community from a repository at the American Type Culture Collection, Rockville, MD. The second phase of the project, the construction of large insert, partial digest libraries in both lambda and cosmid vectors, is underway.
Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry
Authors: Kirkhus Bente , Clausen Ole Petter Fraas
Published: August 2005

Hairless mice were injected intraperitoneally with bromodeoxyuridine (BrdUrd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate‐buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cyle progression of a pulse‐labeled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H‐thymidine labelling studies. The percentage of cells in S phase was highest at night and‐lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indiications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation‐keratin K10 was turned on only in G1 phase and after the last division.
Flow cytometric analysis of erythrocyte populations in tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen
Authors: Bigbee William L. , Langlois Richard G. , Stanker Larry H. , Vanderlaan Martin , Jensen Ronald H.
Published: August 2005

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, GalNAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu‐NAcα2‐3Galβ1‐3(NeuNAcα2‐6)GalNAcα1‐O‐Ser(Thr) tetrasaccharide or to fixed neuraminidase‐digested cells presenting the Gal‐GalNAc disaccharide. The percentages of Tn‐positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A‐specific monoclonal antibodies showed that the erythrocytes composing the Tn‐negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn‐positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 X 107 erythrocytes, suggesting that the frequency of such cells in normal individuals is > 1 X 10−6.
Changes in neutrophil right‐angle light scatter can occur independently of alterations in cytoskeletal actin
Authors: Kraus Eliyahu , Niederman Richard
Published: August 2005

Forward‐angle light scatter (FALS) and right‐angle light scatter (RALS) are commonly employed to discriminate between leukocyte subclasses. Recently the application of RALS has expanded, and it is now also used as an indicator of neutrophil actin polymerization. In this communication we critically examine the relationship of RALS to changes in cytoskeletal actin. The data indicate that agonists which stimulate an increase, a decrease, or no change in F‐actin content can all stimulate a biphasic change in RALS. We therefore conclude that changes in RALS can occur independently of changes in F‐actin content. This leads us to suggest that caution must be taken when interpreting RALS data in relation to changes in F‐actin. Furthermore, the data also support the idea originally proposed by Yuli and Snyderman (J Clin Invest 73:1408–1417, 1984), that RALS may be an exceptionally sensitive indicator of cell activation.
Partition of protein and DNA during cytokinesis in human breast cancer cell lines
Authors: Sennerstam Roland , Auer Gert
Published: August 2005

Three human breast cancer cell lines (HTB‐126, MDA‐231, and HTB‐122) with DNA index (DI) values between 1.26 and 1.72 were analysed together with a diploid mouse embryonal carcinoma cell line (PCC3) by a TV‐video time‐lapse technique (pedigree analysis). Cytochemical parameters (DNA and proteins) were studied in individual cells in a rapid scanning microspectrophotometer. Postmitotic sister cell pairs were analysed after Feulgen‐naphthol‐yellow staining. The DI values of the cell lines were selected to reflect various well‐known clinical ploidy entities differing in malignancy potentials. A mitotic disturbance of the partition of DNA and protein to daughter cells was found in particular in MDA‐231 closest to the triploid DNA modal value (DI = 1.37). Duration of mitosis was considerably longer in the near triploid line compared to the other lines. The MDA‐231 line was also least sensitive to suboptimal growth conditions.

This report calls attention to a possible causality between mitotic error and intraclonal genotype and cell mass heterogeneity.
Interfacing a flow cytometer computer with a PC‐based patient information system
Authors: Blume Philip
Published: August 2005

We have established an interface between our flow cytometer's computer and the personal computer (PC) which supports our patient database system. The PC has been equipped with a commercially available IEEE‐488 bus interface board which is connected to the interface bus of the cytometer's Hewlett‐Packard 9000/300 computer (HP). The PC is set as a bus device with the same address as that of the HP's printer. It is programmed to examine the stream of data sent to the printer and extract from it and store in an MS‐DOS text file selected information which subsequently may be transferred to the database system.
Flow cytometric competitive binding assay for determination of actinomycin‐D concentrations
Authors: Saunders George C. , Martin John C. , Jett James H. , Perkins Ann
Published: August 2005

A single step, separation free competitive binding reaction between the fluorescent antibiotic mithramycin and actinomycin‐D for common binding sites on DNA coated 10 μm diameter microspheres is described. The fluorescence of the microspheres is measured with a flowcytometer. In the presence of a constant amount of mithramycin, the microsphere fluorescence is inversely proportional to actinomycin‐D concentration.
Improved method for computing potential doubling time from flow cytometric data
Authors: White R. Allen , Terry Nicholas H. A. , Meistrich Marvin L. , Calkins Dennise P.
Published: August 2005

Relative movement methods use the timed progression of the mean fluorescence of cells which have heen labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, Ts. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity vis introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to In(2)T8/Tpot so that Tpot (equal to In(2)T8/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.
Cytofluorometric determination of protein‐bound thiols and DNA in cell nuclei
Authors: Schabronath J. , Streffer C. , Zamboglou N.
Published: August 2005

The aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein‐bound sulfhydryl‐groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N‐iodoacetyl‐N(5‐sulfo‐1‐naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction with sodium borohydride or thioglycollic acid. The method was established by using nuclei of human lymphocytes, which then served as a biological standard for further investigations of the nuclei of different mammalian cell types: nuclei from mouse liver cells and nuclei from the cells of two human melanoma cell lines. For non‐proliferating lymphocytes distinct DNA‐ and PSH‐values could be measured. The PSH‐values detected in the nuclei of the other cell types were higher by comparison and varied within the cell cycle; i.e., PSH increased during the S‐phase and was almost doubled during the cell generation cycle from G1‐ to G2‐phase. Cell line and cell cycle‐dependent variations of nuclear disulfides could also be detected. These results are discussed with respect to their radiobiological implications. In conclusion, thiol groups may represent one factor determining the radiosensitivity of cells, but they are not the only decisive one.
Intracellular pH, esterase activity, and DNA measurements of human lung carcinomas by flow cytometry
Authors: Liewald F. , Demmel N. , Wirsching R. , Kahle H. , Valet G.
Published: August 2005

An important intention of flow cytometric investigations is to obtain biochemical and biophysical information about cells which is suitable for automated tumor diagnosis. In this study, the ploidy status, the intracellular pH value, the intracellular esterase activity, and the cell volume of vital cells and the DNA and cell volume of dead cells were measured in cancerous tisse and normal lung tissue of 30 patients by flow cytometry. The cell samples were simultaneously stained with the pH and esterase indicator dye 1.4‐diacetoxy‐2,3‐dicyano‐benzene (ADB) and propidium iodide (PI). The flow cytometric measurements were performed in three‐parameter list mode. The data were evaluated on an AT‐compatible personal computer with the DIAGNOS1 program system for automated diagnosis of flow cytometric list mode data.

Significant differences were found between normal and malignant tissue in DNA ploidy, in the intracellular esterase activity, in the cell, volume and in the percentage of inflammatory cells and parameters of necrosis. DNA‐aneuploidy was observed in 38% of the lung carcinomas. The simultaneous detection of DNA‐aneuploidy and tumor‐associated properties in a multifactorial analysis led to correct automatic tumor diagnosis in 85% of cases. DNA‐aneuploidy was found at a significant higher frequency in advanced tumors. Adenocarcinomas displayed DNA‐aneuploidy more often (80%) than squamous cell carcinomas (33%).
Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry
Authors: Johnson N. F. , Wilson J. S. , Habbersett R. , Thomassen D. G. , Shopp G. M. , Smith D. M.
Published: August 2005

Basal and secretory cells have been separated as highly enriched viable populations from single‐cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2° and 90° light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony‐forming efficiency than secretory cells (3.4%). Significant differences in blue auto‐fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents.
Relationship of DNA ploidy to chemoresistance of tumors as measured by in vitro tests
Authors: Volm Manfred , Efferth Thomas
Published: August 2005

To examine whether patients with aneuploid tumors might derive more benefit from chemotherapy than would patients with diploid tumors, predictive tests for determining resistance in human tumors were carried out and the test results compared with the DNA ploidy of the corresponding tumors. Multidrug‐resistance in 15 kidney carcinomas grown as primary cultures was determined by immunofluorescence by Mab C219, which is specific for the plasma membrane glycoprotein P‐170, and by the use of tritiated nucleotide incorporation after addition of doxorubicin. Aneuploid tumors had a higher tendency to be more sensitive than diploid tumors, but the correlation was not significant. This was confirmed by reanalyzing our earlier data on ovarian and lung cancers. In conclusion, DNA measurement using flow cytometry does not appear to be a suitable tool for prediction of resistance of human tumors to chemotherapy.
Cyanine dye labeling reagents—carboxymethylindocyanine succinimidyl esters
Authors: Southwick Philip L. , Ernst Lauren A. , Tauriello Erica W. , Parker Stephen R. , Mujumdar Ratnakar B. , Mujumdar Swati R. , Clever Hester A. , Waggoner Alan S.
Published: August 2005

Ten carboxymethylindocyanine dyes which from the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino‐containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water‐soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previosly (Mujumdar et al.: Cytometry 10:11–19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two‐color immunofluorescent imaging of internal structures in a mammalian cell and for two‐color flow‐cytometry experiments. The use of values of chromophore‐equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.
Cytolytic activity of human natural killer cell subpopulations isolated by four‐color immunofluorescence flow cytometric cell sorting
Authors: Srour Edward F. , Leemhuis Tom , Jenski Laura , Redmond Robert , Jansen Jan
Published: August 2005

The natural killer activity and phenotypic properties of six different subpopulations of normal human peripheral blood lymphocytes obtained by four‐color immunofluorescence cell sorting were examined. Phycoerythrin‐conjugated CD16 and CD56 were used simultaneously to identify (CD16+CD56+) NK cells. The cells most effective in mediating NK cytolysis against K562 target cells were CD3−(16+56+)57‐8+. Although most of the K562 killing was found in the CD3−(16+56+) groups of cells, a substantial degree of NK activity was detected in the CD3+(16+56+) subpopulations of some individuals. The level of expression of CD57 and CD8 was significantly higher on CD3+(16+56+) than on CD3−(16+56+) cells.
Logarithmic amplifiers
Authors: Gandler William , Shapiro Howard
Published: August 2005

Logarithmic amplifiers (log amps), which produce an output signal proportional to the logarithm of the input signal, are widely used in cytometry for measurements of parameters that vary over a wide dynamic range, e.g., cell surface immunofluorescence. Existing log amp circuits all deviate to some extent from ideal performance with respect to dynamic range and fidelity to the logarithmic curve; accuracy in quantitative analysis using log amps therefore requires that log amps be individually calibrated. However, accuracy and precision may be limited by photon statistic and system noise when very low level input signals are encountered.
Effect of focus on cell detection and recognition by the cell analyzer
Authors: Spadinger Ingrid , Poon Steven S. S. , Palcic Branko
Published: August 2005

The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 μm below to 25 μm above the optimal focus position. These limits were asymmetrical about ideal focus due to a pronounced asymmetry in the effects of positive and negative defocusing on the cell signal.
Comparison of the Papanicolaou and Feulgen staining methods for DNA quantification by image analysis
Authors: Gurley A. M. , Hidvegi D. F. , Bacus J. W. , Bacus S. S.
Published: August 2005

The possibility of using archival cytology material to study the evolution of neoplastic disease with regard to DNA content abnormalities was investigated. The accuracy of measuring the integrity optical density (OD) of nuclei that correlates to DNA amounts of those nuclei, on slides stained by the Papanicolaou method, was assessed and compared with a standard Feulgen method. Our data on rat liver nuclei peritoneal washings from patients with ovarian cystadenofibromas and ovarian cystadenocarcinomas suggested that analysis of cytological material using the Papanicolaou method is not reliable and that destaining the slides followed by Feulgen staining provides an optimal and reliable method of DNA quantification.
Variations in feulgen stainability of epithelial parenchymal cells extracted from paraffin‐embedded salivary gland specimens
Authors: Schimmelpenning Hendrik , Falkmer Ursula G. , Hamper Klaus , Seifert Gerhard , Auer Gert U.
Published: August 2005

The variations of Feulgen stainability of cells extracted from paraffin‐embedded archival specimens for DNA assessment by means of image cytometry (ICM) were investigated in normal salivary gland parenchyma. The Feulgen stainability of the deparaffinized, rehydrated, and disaggregated preparations was found to exhibit variations of up to 300%, expressed by the mean of integrated optical density (IOD), when a routine procedure was applied to a first series of Cytospin preparations of disaggregated specimens. When measured in nondisaggregated tissue sections, only negligible variations were observed. After minimization of the mechanical strains to the cellular material in the Cytospin preparations in a second series, the variations in Feulgen stainability were found to be considerably lower. The findings indicate that the main reason for variations in the Feulgen stainability of extracted cells is, most likely, the disaggregation procedure itself. Factors such as initial treatment of the specimens, duration and kind of formalin fixation, and length of storage time periods seem to be of minor importance. Retrospective studies on paraffin‐embedded specimens require a carefully controlled tissue type‐adapted disaggregation procedure. In addition, we concluded that the interpretation of histograms, obtained by means of ICM DNA assessments in Cytospin preparations of archival material, requires a well‐defined internal specific standard.
An improved flow cytometric assay for somatic mutations at the glycophorin A locus in humans
Authors: Langlois Richard G. , Nisbet Barbara A. , Bigbee William L. , Ridinger David N. , Jensen Ronald H.
Published: August 2005

An improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. The new assay, named the “BR6” assay, can be performed on a commercially available, single‐beam flow cytometer, in contrast to the previously described 1W1 assay that required a dual‐beam flow sorter. A modified cell labeling method developed for the BR6 assay results in improved separation of normal and mutant phenotype cells, as well as eliminating some cellular artifacts that affected the 1W1 assay. Parallel measurements on samples from 17 normal donors showed that the BR6 assay yields comparable variant cell frequencies and improved measurement precision compared with the 1W1 assay. A detailed analysis of three individuals who showed large differences in background variant frequency with the 1W1 assay confirmed that these differences could also be detected with the BR6 assay. A dramatically elevated variant cell frequency was seen with the BR6 assay of an individual exposed to a high level of ionizing radiation in an accident at Goiania, Brazil.
Isolation of giant DNA fragments from flow‐sorted human chromosomes
Authors: Minoshima Shinsei , Kawasaki Kazuhiko , Fukuyama Ryuichi , Maekawa Masahiko , Kudoh Jun , Shimizu Nobuyoshi
Published: August 2005

We have established a method using a conventional cell sorter equipped with a single argon laser to sort intact human chromosomes that can be used as a source for the production of giant DNA fragments. Various improvements were made to both the equipment and sorting method to enhance the sorting resolution and avoid destruction of chromosomal DNA. Using this improved method chromosomes 21 and 22 were sorted from the B‐lymphoblastoid line GM00130B, digested with the rare cutting restriction endonuclease NotI, and analyzed by pulsed field gel electrophoresis followed by Southern hybridization using the Alu repetitive sequence as a probe. More than 25 discrete NotI giant DNA fragments ranging from 50 kb to longer than 2.5 Mb were separated and the size distribution pattern was unique for each chromosome, indicating successful sorting of intact chromosomes. The cumulative size of these Alu‐positive NotI DNA fragments were 22.7 Mb and 25.5 Mb for chromosomes 21 and 22, respectively. These values are 47% and 49% of the estimated size of chromosomes 21 (48 Mb) and 22 (52 Mb).
Clinical applications of cytometry: 4th annual meeting
Authors: Giorgi J. V. , Hurtubise P. E. , Parker J. W. , Cram L. S. , La Via M. F.
Published: August 2005

Quantitative cytology is undergoing rapid evolution because of dramatic advances in optics, lasers, photochemistry, computers and digital imaging. Novel clinical applications utilizing the latest flow and imaging technology as well as exciting new findings using more conventional methods are the focus of an annual meeting in Charleston, South Carolina. The 4th Annual Meeting, which featured sessions on image cytometry, fluorescence intensity, quantitation, leukemia/lymphoma analysis and transplantation, is summarized here.
Instrument for real‐time pulse‐shape analysis of slit‐scan flow cytometry signals
Authors: van Oven C. , Aten J. A.
Published: August 2005

An instrument is described which analyses shapes of fluorescence profiles generated by particles passing through the focussed laser beam of a flow cytometer. The output signal of this pulse‐shape analyzer is used as input for the signal processing electronics of a commercial flow cytometer system. The instrument detects dips in pulse‐profiles; a shape parameter named Pulse Dip Index (PDI) is defined as the ratio of the integrated signal from the beginning of the pulse until the first dip, relative to the integrated signal of the complete profile. This PDI is similar to the Centromeric Index of chromosomes. The composition of aggregates in mixtures of fluorescent particles of different sizes was evaluated by PDI analysis. In our experiments the PDI was determined within 30 μs from the onset of the pulse‐profile and particles with a specified morphology of interest were selected for on‐line registration of their profiles as digitized pulse‐shapes. In a cell sorter system, the PDI can be used as a parameter for sorting.
Effects of cellular fixatives on human immunodeficiency virus production
Authors: Cory Joan M. , Rapp Fred , Ohlsson‐Wilhelm Betsy M.
Published: August 2005

Effects of cell fixation procedures appropriate for flow cytometric analysis on the infectivity of human T lymphoblastoid H9 cells infected with human immunodeficiency virus‐1 (HIV‐1) were evaluated to provide guidelines for choosing cell treatments for potentially infectious samples. H9 cells experimentally infected with HIV‐1 were treated by the test fixation procedure, washed, and cocultured with equal numbers of live, uninfected H9 cells. To estimate the reduction in infectivity due to the fixation procedure, dilution series of live infected H9 cells in uninfected H9 cells were simultaneously established in culture. Cell cultures were incubated 8–10 d, harvested, and evaluated for evidence of HIV‐1 infection by the presence of cell‐associated HIV‐1 antigens and/or by the presence of particle‐associated reverse transcriptase activity in cell culture supernatants. Thirty‐minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehyde (0.5%) reduced the infectivity of HIV‐1‐infected H9 cells by greater than 99.99%. To the same degree, a multi‐step fixation procedure utilizing formaldehyde and ethanol was effective in reducing HIV‐1 infectivity. Conversely, the erythrocyte fixative dimethylsuberimidate at 3 μg/ml was ineffective in reducing HIV‐1 infectivity.
Flow‐cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets
Authors: Tschoepe D. , Spangenberg P. , Esser J. , Schwippert B. , Kehrel B. , Roesen P. , Gries F. A.
Published: August 2005

Occlusive vascular diseases are promoted by a “prethrombotic state” with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow‐cytometric bio‐assay and the related change in the actin status by using the DNase‐I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F‐actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein “actin” and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow‐cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.
A new principle of cell sorting by using selective electroporation in a modified flow cytometer
Authors: Schut Tom C. Bakker , de Grooth Bart G. , Greve Jan
Published: August 2005

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill.

We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 μs and resulting field strengths of 2.0 and 3.2 X 106 V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.).

The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.
Flow cytometric identification of microorganisms by dual staining with FITC and PI
Authors: Miller Jane S. , Quarles John M.
Published: August 2005

The identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.
Fixation‐Associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs
Authors: Holtfreter Hiroko B. , Cohen Nicholas
Published: August 2005

We have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.
Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse
Authors: de Launoit Yvan , Kiss Robert , Danguy André
Published: August 2005

We compared cytomorphonuclear parameters–related to DNA histogram and chromatin distribution–on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen‐stained nuclei. The spectrophotometric results–assessed by integrated optical density values–indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol‐formalin‐acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well‐defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.
Histomorphometric study of the lower urogenital tract in pre‐ and post‐menopausal women
Authors: Semmelink H. J. F. , de Wilde P. C. M. , van Houwelingen J. C. , Vooijs G. P.
Published: August 2005

In this study 22 histomorphometric parameters were used to quantify the histologic changes in the vagina, trigone of the bladder, and the proximal and distal urethra of 37 women aged between 18 and 82 years.

The results of this quantitative study revealed a hormonally induced, age‐independent atrophy of smooth muscle in the lamina propria of the mucosa and the longitudinal part of the muscular layer in the aforementioned anatomical sites in post‐menopausal women. In the lamina propria of the mucosa of the lower urogenital tract in post‐menopausal women hormonally induced quantitative changes of blood vessels were not observed. Senile atrophy of smooth muscles was only observed in the deeper part of the muscular layer of the vagina and the trigone of the urinary bladder and was less prominent than the hormonally induced smooth muscle atrophy.
Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes
Authors: Dietzel Andreas H. , Hain Jens , Virsik‐Peuckert R. Patricia , Harder Dietrich
Published: August 2005

CHO‐K1 cells were irradiated in G0/G1 phase with 150 k V X‐rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose‐dependent changes in the flow karyotypes, they were split into peak‐ and background‐portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose‐dependent reduction in peak contents. The number of radiation‐induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non‐chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose‐effect relations and split‐dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.
Flow cytometric method to monitor the destruction of CD4+ cells following their fusion with HIV‐infected cells
Authors: Schols Dominique , Pauwels Rudi , Desmyter Jan , De Clercq Erik
Published: August 2005

Syncytium formation between HUT‐78 cells persistently infected with human immunodeficiency virus type 1 (HIV‐1) and uninfected CD4‐bearing MOLT‐4 or CEM cells results in a rapid destruction of the MOLT‐4 or CEM cells. This syncytium formation is due to the interaction between the gp120 glycoprotein expressed by the persistently HIV‐1‐in‐fected HUT‐78 cells and the CD4 receptor present on MOLT‐4 or CEM cells. A flow cytometric method has been applied to separate the infected (HUT‐78) from the uninfected (MOLT‐4, CEM) cell populations. This method is based on a modified DNA staining protocol which clearly shows the differences in DNA content between HUT‐78 cells, on the one hand, and MOLT‐4 or CEM cells, on the other hand. Using this flow cytometric method we have demonstrated that those compounds (i.e., sulfated polysaccharides, aurintricarboxylic acid) that interact with gp120 (of the HIV‐infected cells) or CD4 (of the uninfected cells) suppress syncytium formation and concomitant destruction of the CD4+ cells.