bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integration of a barcode reader with a commercial flow cytometer
Authors: Robinson J. Paul , Maguire D. , King G. , Kelley S. , Durack G. 
Published: August 2005

AbstractThis report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time.The system uses a standard “off‐the‐shelf” bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tribe reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.
Quantification of an in vitro cell‐cell adhesion assay using interactive laser scanning cytometry
Authors: Newton Sean C. , Millette Clarke F. 
Published: August 2005

AbstractWe are interested in identifying cellcell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cellspermatogenic cell adhesion. Numerous cell‐cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225—500 μm2, spermatids were 100—225 μm2 and residual bodies were < 100 μm2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub‐classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell‐cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell‐cell adhesion assay in which one cell type is adherent to a substrate.
Three‐dimensional component labeling of digital confocal microscope images enumerates replication centers in BrdUrd labeled fibroblasts
Authors: Baumann Peter H. , Schormann Thorsten , Jovin Thomas M. 
Published: August 2005

AbstractAn algorithm (3Dlabel 1) for labeling three dimensional binary data sets has been developed. Serial optical sections are acquired using a confocal laser scanning microscope. After filtering and thresholding operations, contiguous elements in the three dimensional data sets are identified and labeled. The results are used to calculate the number and size of objects. Additionally, the labeled data are displayed. by applying an algorithm (3Ddisp1) to generate stereo pairs, in which label numbers are color coded and depth is cued by intensity. These procedures have been applied in investigations of the temporal and spatial distribution of replication centers throughout S phase in BrdUrd pulse labeled mouse fibroblasts.
One or multiple samplings for flow cytometric DNA analyses in breast cancer‐prognostic implications?
Authors: Fernö Mårten , Baldetorp Bo , Ewers Sven‐börje , Idvall Ingrid , Olsson Häkan , Sigurdsson Helgi , Killander Dick 
Published: August 2005

AbstractFlow cytometric assessments of DNA ploidy status and the S‐phase fraction (SPF) have been shown to yield prognostic information in breast cancer. The aim of the present investigation was to elucidate the reproducibility of results with regard to tumor DNA heterogeneity, and to ascertain whether the prognostic value of DNA measurements might be enhanced by analyzing two pieces of a tumor instead of one.Agreement with regard to ploidy status (diploid versus non‐diploid) was obtained in 90% of cases (71/79) when two adjacent sections of the tumor were investigated, and in 77% of cases (10/13) when four biopsies from different quadrants of the tumor specimen were investigated. The corresponding figures for agreement in SPF (divided into three categories, < 7.0%, 7.0–11.9%, and > 112%) were 75% (59/79; 2‐sample series) and 55% (7/13; 4‐biopsy series).The main reason for variance in ploidy results was the difficulties in distinguishing near diploid cell populations. Discrepancies in SPF categories could be explained by minor fluctuations in SPF values near the cut‐off levels, or by variance in ploidy status, the fraction of nondiploid nuclei, and background noise due to cell debris.There was a stepwise increase in recurrence rate (RR) among patients with increasing SPF category (RR:20%, 41%, and 53%). Patients whose SPF categories varied, from low or intermediate in one part of the tumor to high in another, seemed to have a poor prognosis (RR = 57%).We conclude that the variation in ploidy status and SPF between different parts of the same tumor specimen is largely to be explained by uncertainty in the interpretation of the DNA histograms and by difficulties inherent in the estimation of SPF, rather than by the existence of DNA tumor heterogeneity. Evaluation of a different part of the same tumor specimen may be of value, especially in the case of SPF, as this may yield additional prognostic information. These findings also emphasize the need for good quality control of FCM DNA analysis.
Detection of viral surface antigens on HIV‐2ben infected human tumor cell lines by flow cytometry
Authors: Böhm Dorothea , Nick Sigrid , Voss Gerald , Hunsmann Gerhard 
Published: August 2005

AbstractThe human monocytic cell line U‐937 clone 2 and two T‐cell lines CEM and MOLT‐4 clone 8 were infected with HIV2ben, a recent isolate of HIV‐2. Infection and subsequent antigen expression on the cell surface was monitored by flow cytometry using a rabbit‐anti‐serum against tween‐ether‐treated HIV‐2ben and a fluoresceinisothiocyanate‐conjugated IgG against rabbit‐IgG.The sensitivity of the three cell lines to infection with HIV‐2ben correlated with the percentages of CD4‐expressing cells but not with the levels of CD4‐expression on the cell. The appearance of viral surface antigens preceeded the formation of syncytia and correlated closely with the infecting virus dose. After 1–2 weeks in culture, 20–85% of the cells of each line expressed viral surface antigens. The variation depended on the cell type and cell culture conditions. The MOLT‐4 clone 8 and the U‐937 clone 2 cells died around 10 or 20 days, respectively, after HIV‐2ben infection. Only HIV‐2ben infected CEM cells grew permanently.Flow cytometry was an appropriate method to monitor the expression of viral proteins on the cell surface of HIVinfected cell lines. Flow cytometry proved to be more sensitive than determination of RT activity in supernatants of HIV‐infected cells and more precise than light microscopy examinations.
Multivariate flow cytometry of epidermal regeneration provoked by a skin irritant and a tumor promoter
Authors: Kirkhus B. , Glasø M. , Clausen O. P. F. 
Published: August 2005

AbstractThe DNA content and the changes in cellular and nuclear size of isolated regenerating mouse epidermal basal cells were studied after topical application of the skin irritant cantharidin and the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to the back skin of hairless mice. The DNA and protein contents of isolated basal cells were stained with propidium iodide and fluorescein isothiocyanate, respectively, and analysed by flow cytometry using the total protein fluorescence as an estimate of cell size and the DNA fluorescence pulse width as an estimate of nuclear size. Transmission electron microscopy was used to identify cells sorted from regions in the bivariate DNA/protein distributions.The results showed that both chemicals induced an increase in cellular as well as nuclear size of the basal cells. The increase in size was higher in TPA treated than in cantharidin treated animals, anti the bivariate DNA/protein distributions of TPA treated cells differed from those of cantharidin treated cells in that two subpopulations of cycling keratinocytes could be identified. These deviations are probably related to the higher proliferative response observed after TPA treatment and the possibility that proliferative subpopulations in epidermis respond differently to TPA. It may reflect mechanisms providing for a growth advantage of initiated cells, important in tumor promotion. About 8% of the cells in the suspensions from treated animals were non‐cycling non‐keratinocytes, probably infiltrating leukocytes.The results indicate a strong correlation between rapid regenerative cell cycle progression, i.e., reduced G1 transit time and increased cellular and nuclear size. The increase in cellular size was observed simultaneously in all cell cycle phases, well before the wave of cells entering S phase 16 h after application. This suggests that an increase in size may be essential for the subsequent initiation of DNA synthesis and the reduction in cell cycle time. The results support the hypothesis that the increase in cell size may be caused by genie activation or posttranscriptional processes rather than an increase due to an initial block in cell cycle progression.
Quantitation of human anti‐mouse antibody in serum by flow cytometry
Authors: Labus J. M. , Petersen B. H. 
Published: August 2005

AbstractClinical investigations utilizing murine monoclonal antibodies require techniques for the detection of the human anti‐mouse antibody (HAMA) response in patient serum. We report here a flow cytometric assay for the quantitation of HAMA. Commercially available beads conjugated with goat anti‐mouse antibody provide a solid phase matrix for a triple bridge immunoassay. The measurement of fluorescein labeled antibodies by flow cytometry allows accurate quantitation of the HAMA. The assay will detect antibody levels of approximately 1.0 ng/ml. Antibody recovery in serum samples with known amounts of antibody added was greater than 90% at levels ⩾ 10 μg/ml. Serum samples obtained from 41 patients prior to and after single or multidose infusions of KS1/4‐Desacetylvinblastine were analyzed. These results were compared with HAMA titers previously determined by ELISA. With few exceptions, patients with low titers as determined by ELISA demonstrated low HAMA potencies by flow cytometry and those with highest titers the highest potencies. Patients with no detectable HAMA by ELISA were also negative by flour analysis. The results of our studies demonstrate that HAMA levels can be accurately and quantitatively determined by flow cytometry.
Activated and memory T lymphocytes in human milk
Authors: Wirt Daniel P. , Adkins Linda T. , Palkowetz Kimberly H. , Schmalstieg Frank C. , Goldman Armond S. 
Published: August 2005

AbstractSince activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint‐a‐Gate program was used to optimize gating on the lymphocyte population. A mean ± 1 SD of 4 ± 3% of total HM leukocytes were lymphocytes and 96 ± 3% were macrophages and granulocytes (N=33 subjects). HM lymphocyte populations were further analyzed in five subjects. T calls (CD3+) represented 83 ± 11% and B cells (CD19+) were 6 ± 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40–1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P < 0.02) and postpartum women (P < 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P < 0.002) and postpartum women (P < 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 ± 7% of CD3+ cells in HM were HLA‐DR+ (controls, 10 ± 4%; P < 0.001), and 15 ± 6% of CD3+ cells in HM were IL‐2R+ (controls, 6 ± 2%; P < 0.001). Subpopulations of CD4+ and CD8+ cells in HM defined by the T200 isoforms CD45RA and CD45RO were markedly altered compared to PB, indicating a striking shift from virginal to antigen‐primed (memory) T cells in HM and suggesting certain functional capacities for these cells. Virtually all CD4+ cells (99.8 ± 0.4%) and 92 ± 5% of CD8+ cells in HM were CD45R0+ (vs. 71 ± 12% and 50 ± 10%, respectively, in the PB of postpartum women; P < 0.001). CD4+ and CDS8+ cells expressing CD45RA were correspondingly markedly reduced (P < 0.001). This phenotypic pattern of T cells in HM may result from T cell activating substances in HM and/or selective homing of T cells to the breast. Conversely, activated T cells in HM may be responsible for cytokines in HM. These activated and memory T cell populations may be transferred to the infant via breastfeeding.
Quantitative and qualitative DNA variations in Anabaena azollae strasb. living in Azolla filiculoides lam.
Authors: Canini Antonella , Caiola Maria Grilli , Ferrucci Luigi 
Published: August 2005

AbstractHeterocysts and vegetative cells of the filamentous nitrogen‐fixing Anabaena azollae isolated from the apex to the basal leaf cavities of Azolla filiculoides were examined by epifluorescent microscope after fluorochrome staining. Acridine orange (AO), DAPI, and chromomycin fluorochromes were used in order to evidence total DNA content and respectively, A + T and G + C bases.Measurements of fluorescence intensities were made on photographic prints by the automatic image analysis system Quantimet 970.Heterocysts contained higher amounts of DNA than did vegetative cells, and their content strongly increased in the basal leaf cavities. The heterocyst DAPI brightness was quite uniform, whereas in vegetative cells DAPI brightness increased from the apex to the basal groups. In vegetative cells from the apex to the median group, the percentage of DAPI brightness was 60–85% with respect to AO brightness, whereas in heterocysts of the same groups DAPI brightness was 40–50% with respect to AO brightness. In the basal group, brightness due to DAPI staining was comparable with those of previous group both in heterocysts and in vegetative cells, whereas chromomycin brightness increased strongly in heterocysts.These data slow that heterocyst changes its DNA content and composition in the basal leaf cavities, suggesting that its lifetime is not completely over.
Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer
Authors: Kaern Janne , Wetteland Jarle , Tropé Claes G. , Farrants George W. , Juhng Sang‐Woo , Pettersen Erik O. , Reith Albrecht , Danielsen Håvard E. 
Published: August 2005

AbstractThe DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by IM Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.
Calibration of a flow cytometric assay of glucose‐6‐phosphate dehydrogenase activity
Authors: Severin E. , Seidler E. 
Published: August 2005

AbstractThe reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo‐ and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazanbearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose‐6‐phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.
Time‐resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization
Authors: Seveus Lahja , Väisälä Mikko , Syrjänen Stina , Sandberg Minna , Kuusisto Ari , Harju Raimo , Salo Juha , Hemmilä Ilkka , Kojola Hannu , Soini Erkki 
Published: August 2005

AbstractFluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased.We modified a standard epifluorescence microscope for time‐resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD‐camera was used for detection and the images were digitally processed.A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix.The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehydefixed, wax‐embedded specimens.
Distribution of non‐heme porphyrin content of individual erythrocytes by fluorescence image cytometry and its application to lead poisoning
Authors: Sassaroli Massimo , Dacosta Rui , Väänänen Heikki , Eisinger Josef 
Published: August 2005

AbstractThe quantitation of the non‐heme porphyrin content of circulating erythrocytes is an important tool in the screening, diagnosis, and management of lead disease, iron deficiency anemia, and certain porphyrias. Useful information about these pathological conditions may be obtained from the distribution of the non‐heme porphyrin content of individual red blood cells. An image‐based cytometry system was developed and used to determine the cellular distributions of zinc protoporphyrin (ZPP) in the erythrocytes of normal and lead‐exposed human subjects. The fluorescence cytometry system described here lends itself to a wide range of statistical studies of cell populations, in which extrinsic fluorescent probes or antibodies may be used instead of porphyrin. Image‐based cytometry appears to offer a number of important advantages over the more conventional flow cytometry systems, including greater sensitivity, applicability to a wider range of cellular parameters, and the ability to retain images of each cell used in the survey for subsequent examination. Pairs of absorption and fluorescence images of many fields of dispersed immobilized red cells were acquired by means of a program which controls the microscope stage, focussing, shutters, filter wheels, and a cooled, slow‐scan CCD camera. The observed marked differences in the ZPP distributions of erythrocytes from donors with chronic and acute lead exposure are discussed in terms of the metabolism of internalized lead.
Comparison of strategies to detect and quantitate uniquely marked cells in intra‐ and inter‐species hemopoietic chimeras
Authors: Pallavicini M. G. , Langlois R. G. , Reitsma M. , Gonzalgo M. , Sudar D. , Montoya T. , Ul. Weier H. , Haendel S. 
Published: August 2005

AbstractEvaluation of the outcome of successful bone marrow transplantation and indepth studies of transplantation biology rely increasingly upon detection and enumeration of donor hemopoietic cells in the transplanted recipients. The ability to detect and enumerate low levels of donor engraftment in interphase cell subpopulations in hemopaietic chimeras is particularly important for studies of mixed lineage chimerism, early relapse manifestations, and engraftment of subpopulations present at low frequency. We describe and compare the sensitivity and specificity of DNA‐based detection strategies (fluorescence in situ hybridization, in vitro DNA amplification using the polymerase chain reaction) and flow cytometric analysis of cell surface markers to detect cells carrying marker DNA or proteins in syngeneic (mouse‐to‐mouse) and xenogeneic (mouse‐to‐human, monkey, sheep) backgrounds. DNA‐based detection strategies offer advantages of rapid analysis and enumeration of target cell frequencies with detection sensitivities approximating 10−4. The sensitivity of immunofluorescence‐linked flow cytometric‐based detection of nucleated leukocytes approached 10−3, whereas flow cytometric‐based detection of fixed human erythrocytes was feasible at cell frequencies of 10−5. Data described in this manuscript should facilitate selection of appropriate methodologies for assessment of hemopoietic chimerism following transplantation.