bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantification of low frequency white blood cells expressing human cytomegalovirus antigen by image cytometry
Authors: Jiwa N. M. , Mesker W. E. , Ploem‐Zaaijer J. J. , van Dorp W. , The T. H. , Raap A. K. 
Published: June 2005

AbstractHuman cytomegalovirus (HCMV) infections cause considerable morbidity and mortality in immunocompromized patients. During HCMV infection, leukocytes appear in the circulation in low frequencies that express the HCMV pp65 protein antigen. Since there is evidence that changes in the frequency of antigen‐positive cells in the early phase of the infection have prognostic value, we applied automated image cytometry to quantify these antigen‐positive cells. For this purpose weekly peripheral blood leukocyte samples of 80 kidney transplant recipients were visually examined for the presence of antigen‐positive cells using an immunocytochemical detection method. Seventeen patients, who reacted positive with this assay, were identified. Next, automated image cytometry was applied to quantitate the frequency of antigen‐positive cells in sequential blood samples from the 17 patients. Patients who developed a period of HCMV viremia had a significantly longer antigenemic period and a significantly higher frequency of antigen‐positive cells than patients with a HCMV infection who remained non‐viremic. Therefore, automated image immunocytometry based screening can be used to distinguish patients at risk for the development of a HCMV viremia. Moreover, automated quantitation reveals prognostic information about the HCMV infection at a more sensitive level than other HCMV detection techniques. © 1994 Wiley‐Liss, Inc.
Bovine papillomavirus E1 protein affects the host cell cycle phase fractions
Authors: Belyavskyi Michail , Miller Jane , Wilson Van 
Published: June 2005

AbstractC127 murine fibroblast cells were electroporated with a bovine papillomavirus E1 protein expression vector and examined by flow cytometry. E1 expressing cells (E1+) within the total cell population were distinguished from nonexpressing cells (E1−) by immunofluorescent staining with anti‐E1 serum and a fluorescein‐conjugated second antibody. Under conditions of saturation with the first and second antibodies, the specific green fluorescence reflected the level of intracellular E1 protein. Simultaneous staining with a DNA‐specific dye, propidium iodide (PI), enabled the cell cycle distributions for the E1+ and E1− cell populations to be determined. It was found that the E1+ subpopulation had a reduced percentage of cells in G1 phase and an increased percentage of G2+M phase cells, compared to the E1− subpopulation. There was no significant difference in overall doubling time or percentage of noncycling cells in the E1+ vs. E1− populations, indicating that the change in cell cycle distribution was not due to a general activation or inhibition of cell growth by E1. Direct measurement of cell cycle phase fractions confirmed that the G1 phase was decreased and the G2+M phase was increased in E1 expressing cells. As these observations were made in the absence of other viral proteins or viral DNA replication, it suggests that the E1 protein exerts an effect on the host cell independent of its direct role in viral DNA replication. Thus, E1 may interact directly with the host cell cycle regulatory machinery. © 1994 Wiley‐Liss, Inc.
Simultaneous analysis of c‐erbB‐2 expression and DNA content in breast cancer using flow cytometry
Authors: Stål Olle , Sullivan Siw , Sun Xiao‐Feng , Wingren Sten , Nordenskjöld Bo 
Published: June 2005

AbstractThe c‐erbB‐2 oncogene is frequently amplified and overexpressed in human breast cancer. We have studied the c‐erbB‐2protein in conjunction with DNA content in frozen samples from breast cancers using flow cytometry. The cell suspensions were obtained by mechanical disaggregation followed by a short fixation in 1% paraformaldehyde. The level of c‐erbB‐2 expression was calculated as a fluorescence index, taking into account the relative amount of total cellular fluorescence compared to non‐specific fluorescence. The flow cytometric value correlated with immunohistochemical results obtained with the same monoclonal antibody (c‐neu, clone 9G6). Overexpression of c‐erbB‐2 was significantly more frequent in DNA aneuploid tumors than in DNA diploid ones and correlated with increasing S‐phase fraction and estrogen receptor negativity. In 10 DNA multiploid tumors, the different aneuploid stemlines uniformly expressed c‐erbB‐2, supporting the hypothesis that overexpression is an early event in breast cancer. Of the 172 tumors, the 37 (22%) judged as positive with immunohistochemistry showed a somewhat higher rate of distant recurrence than others (P= 0.14). The fluorescence index was significantly associated with prognosis (P= 0.0012), as it was also among the immunohistochemically positive cases. If the degree of overexpression is important, then flow cytometry could be a feasible technique for classification. © 1994 Wiley‐Liss, Inc.
Bivariate flow karyotyping with air‐cooled lasers
Authors: Frey Tom , Houck David W. , Shenker Bruce J. , Hoffman Robert A. 
Published: June 2005

AbstractAn experimental flow cytometer was constructed using a quartz flow cell optically coupled to a 1.22 NA lens. A pair of crossed cylindrical quartz lenses allowed multilaser excitation. Two helium‐cadmium (HeCd) lasers, emitting 16 mW at 442 nm and 35 mW at 325 nm, were used to excite chromomycin A3 and Hoechst 33258 fluorescence, respectively. Bivariate flow karyotypes from normal human male chromosomes and from the Daudi cell line were obtained and were compared to those from a standard instrument using dual water‐cooled lasers. The new experimental instrument exhibited comparable resolution to that from the standard instrument. In further experiments with Daudi chromosomes, the 35 mW HeCd laser was replaced with a 10 mW HeCd laser, and the system still gave good, though slightly decreased, resolution. © 1994 Wiley‐Liss, Inc.
Quantitative precision of an automated image cytometric system for the measurement of DNA content and distribution in cells labeled with fluorescent nucleic acid stains
Authors: Poulin N. , Harrison A. , Palcic B. 
Published: June 2005

AbstractAn automated image cytometric system is described for the measurement of DNA content and distribution in cells stained with fluorescent DNA binding or intercalating compounds. The quantitative precision of integrated optical intensity (IOI) measurements using this system was estimated to be 2.0%, based on the coefficient of variation (cv) of the IOI of DNA check beads. Using peripheral blood lymphocytes stained with 4′‐6‐diamidino‐2‐phenylindole (DAPI), the cv of IOI was found to be 3.5%. Slide to slide variability of the IOI for peripheral blood lymphocytes (cv over 10 slides of the mean IOI) was found to be 2%. The most important sources of error in these measurements were glare, illumination instability, image calibration instability, and staining non‐uniformity. These errors were investigated and minimized. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of the cell cycle in polyamine‐depleted cells
Authors: Pohjanpelto Pirkko , Nordling Stig , Knuutila Sakari 
Published: June 2005

AbstractPolyamines are found in all cells but their function is not fully understood. We have studied the effect of polyamines on the passage of cells through the cell cycle using a polyamine auxotrophic mutant, CHO‐P22, which has no detectable ornithine decarboxylase activity. The ability of these cells to grow without serum allows efficient polyamine depletion. A flow cytometric analysis of DNA content and bromodeoxyuridine labeling showed that without added polyamines the cells accumulated in S‐phase, the rate of DNA synthesis was retarded, and the entry into mitosis was blocked. Addition of polyamines to cultures deprived of polyamines induced cells in all phases of the cell cycle to reinitiate cycling. Earlier studies have shown that cells with damaged DNA are blocked from entering into mitosis but caffeine can partly overcome this block and induce premature chromosome condensation. Polyamine‐depleted CHO‐P22 cells responded to caffeine in the same way as cells with damaged DNA. These results show that polyamine depletion in CHO‐P22 cells primarily affects DNA synthesis. The finding that polyamine‐starved cells continuously take up bromodeoxyuridine without a corresponding increase in the amount of DNA is compatible with extensive repair of erroneous and/or damaged DNA. Polyamine auxotrophic Chinese hamster ovary (CHO) cells might be useful in studies on the regulation of mitosis in mammalian cells. © 1994 Wiley‐Liss, Inc.
Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands
Authors: Ziegler O. , Cantin C. , Germain L. , Dupuis M. , Sekaly R. P. , Drouin P. , Chiasson J. L. 
Published: June 2005

AbstractThe binding of insulin to cultured IM‐9 human lymphocytes was studied by flow cytometry using FITC‐insulin and biotinylated insulins coupled to streptavidin‐phycoerythrin (NαB1‐biotinylinsulin (B‐insulin) and NαB1‐(biotinyl‐ϵ‐aminocaproyl)insulin (NBC‐insulin)). The reference methods were 125I‐insulin binding and the insulin‐antiinsulin antibody complexes for flow cytometry. There was a close correlation between 125I‐insulin binding and increase in fluorescence for B‐insulin, NBC‐insulin, and insulin‐antiinsulin antibody complexes, but not for FITC‐insulin. NBC‐insulin gave the largest increase in fluorescence (79 ± 9 channels) and the the insulin‐antiinsulin antibody complexes the smallest (34 ± 2 channels) (P < 0.05). FITC‐insulin and B‐insulin gave similar results: 47 ± 6 and 59 ± 6 channels. The concentration reducing 125I‐insulin binding by 50% was 1.1 × 10−9 M for native insulin, 2.7 × 10−9 M for B‐insulin, 3.3 × 10−9 M for NBC‐insulin, and 6.6 × 10−9 M for FITC‐insulin (P < 0.05). Nonspecific binding was low for B‐insulin and NBC‐insulin but reached 75% for 10−6 M FITC‐insulin. These results suggest that B‐insulin and NBC‐insulin are suitable ligands for insulin binding studies using flow cytometry. This two‐step procedure is easier than the insulin‐antiinsulin antibody complex technique. Its poor affinity, specificity, and sensitivity make FITC‐insulin less suitable. © 1994 Wiley‐Liss, Inc.
Evaluation of interspecific DNA content variations and sex identification in Falconiformes and Strigiformes by flow cytometric analysis
Authors: De Vita R. , Cavallo D. , Eleuteri P. , Dell'Omo G. 
Published: June 2005

AbstractA high interspecific karyotype variability has been evidenced in birds especially in Falconiformes and Strigiformes. Avian cytogenetic analysis, conventionally used for this study, presents several difficulties. We used flow cytometric analysis in order to obtain further information on the DNA patterns of different species of birds belonging to the above‐mentioned orders. Our study was performed on blood samples while chicken erythrocytes and human lymphocytes, with known cytometric DNA content, were used as reference cells. The blood samples of the birds under study were stained, simultaneously to the reference cell, with a lysis‐staining buffer containing propidium iodide. The nuclear DNA content of the bird samples was calculated as DNA index in relation to reference cells, and was expressed as nuclear DNA mass in picograms (pg) with respect to the standard value of 7.0 pg per human lymphocyte nucleus. The results obtained showed an interspecific variability of DNA content and evidenced the usefulness of FCM analysis as a rapid and easy tool for studying the DNA pattern of different species of birds. Moreover, our results have confirmed and extended the possibility of sex identification in species of birds characterized by sexual monomorphism by evaluating the small DNA content difference which exists between males and females. © 1994 Wiley‐Liss, Inc.
Rapid fluorescence in situ hybridization with repetitive DNA probes: Quantification by digital image analysis
Authors: Celeda Dino , Aldinger Klaus , Haar Frank‐Martin , Hausmann Michael , Durm Markus , Ludwig Horst , Cremer Christoph 
Published: June 2005

AbstractFluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported using a non‐enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA probes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH‐signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC‐antidigoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra‐nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation on the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9. © 1994 Wiley‐Liss, Inc.
Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique
Authors: Deka Chiranjit , Sklar Larry A. , Steinkamp John A. 
Published: June 2005

AbstractWe have developed a method for fluorescence lifetime measurements in a flow cytometer based upon the amplitude demodulation of the fluorescence signals using digital data acquisition techniques. Amplitude demodulation is one of the two methods by which excited state life‐times may be investigated in the frequency domain. The other method involves the phase‐shift measurements. In frequency‐domain measurement techniques, the amplitude‐demodulation and phase‐shift data serve mutually complementary roles to enhance the analytical capabilities of the measurements. The purpose of having amplitude demodulation measurement capability is to obtain information that supplements, rather than replaces, that obtained by the phase‐shift method alone. Application of amplitude demodulation measurements has been widely explored in static, cuvette‐based, frequency domain systems. However, due to time dependence of the amplitude of the modulated fluorescence signal in a flow cytometer, the amplitude demodulation measurements in flow turns out to be more complicated than similar measurements in a static system. The goal of the present work is to explore the problems involved in amplitude demodulation measurements in flow (using digital method), through detailed theoretical modeling and use the model to develop a practical method that can be incorporated into a flow cytometer to measure amplitude modulation lifetimes. We experimentally verify the amplitude demodulation measurement capability of this method using fluorescent micro‐spheres. The experimental measurements show good agreement with static frequency‐domain measurements on microspheres in bulk suspensions. © 1994 Wiley‐Liss, Inc.
Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo‐3/Fura Red fluorescence ratios
Authors: Novak Erik J. , Rabinovitch Peter S. 
Published: June 2005

AbstractMeasurment of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo‐1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo‐3. Both Fura Red and fluo‐3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo‐3/Fura Red ratio is greater than that achieved with indo‐1, while the intercellular variation in measurement is similar to that seen with indo‐1. Analyses can be simultaneously combined with immunofluorescent detection of PE‐labeled antibodies to enable [Ca2+]i measurement within cell subsets. © 1994 Wiley‐Liss, Inc.
Evaluation of a method for quantitative immunohistochemical analysis of cisplatin‐DNA adducts in tissues from nude mice
Authors: Johnsson Anders , Olsson Christina , Anderson Harald , Cavallin‐Ståhl Eva 
Published: June 2005

AbstractThe reproducibility of an immunohistochemical method for visualization and quantitation of the cytotoxic drug cisplatin (CDDP) in its active position, bound to nuclear DNA, was investigated in tissues from CDDP‐treated nude mice. Kidney, liver, and tumor sections were stained with a PAP technique using an antiserum elicited against CDDP‐DNA adducts. The resulting brown nuclear precipitate was quantitated using a CAS 200 image analyzer. The variance components of the errors in the staining procedure and in the image analysis were estimated. The method was found to be feasible for comparisons between slides of the same type of tissue, stained in the same batch, and measured by one observer on one occasion. The method should be a valuable tool for studies of CDDP pharmacodynamics, sensitivity prediction, and the effects of interactions between CDDP and other drugs and chemomodulators. © 1994 Wiley‐Liss, Inc.
Fully automatic chromosome segmentation
Authors: Ji Liang 
Published: June 2005

AbstractThis paper is concerned with automatic segmentation of high resolution digitized metaphases. This includes automatic detection and rejection of interphase nuclei, stain debris, and other “noise”; automatic detection and segmentation of touching and overlapping chromosome clusters; and automatic rejection of cells which are evaluated as being incomplete, or incorrectly segmented, or where the cell is otherwise unsuitable for further analysis.In this paper, a rule‐based approach is described which treats the cell as a whole rather than as a series of individual chromosomes or clusters. The rules adapt classification and segmentation parameters for each cell. Initially, different sets of parameters are chosen according to the staining method of the cells, and the goal of the segmentation. A chromosome number predictor is used to guide the adaptation of the parameters and to estimate the performance. The adaptation is iterative, and the self‐adjustment will stop when either a satisfactory result is achieved or if the cell is rejected. The method was implemented on both a Sun workstation and a Cytoscan, a commercial machine for chromosome analysis. Seven hundred and thirteen cells from real data have been tested. A success rate of 90–95% has been achieved. The procedure has been implemented in an automatic aberration scoring system for routine use. © 1994 Wiley‐Liss, Inc.
Comparative flow cytometric analysis of Ki‐67 and proliferating cell nuclear antigen (PCNA) in solid neoplasms
Authors: Steck Kim , El‐Naggar Adel K. 
Published: June 2005

AbstractThe proliferation associated antigens, Ki‐67 and proliferating‐cell nuclear antigen (PCNA), have been widely used in studies assessing the growth fraction in human malignancies. It remains unclear, however, whether these markers yield similar assessments of proliferative activity in any given neoplasm. In this study, we compared Ki‐67 and PCNA expression in 93 malignant solid neoplasms using bivarite flow cytometric analysis of these antigens and DNA content. The growth fractions measured by Ki‐67 and PCNA were compared and correlated with acridine orange (AO) analysis and tumor grade. Our results indicate a significant difference between Ki‐67 and PCNA values in neoplasms of low and intermediate grade (P = 0.002); Ki‐67 values were significantly lower than those obtained by PCNA in this group. No statistical difference between Ki‐67 and PCNA values was found in high grade neoplasms (P = 0.38). Analysis of different cell cycle compartments indicates that the observed difference in the positivity of these markers was due to their differential expression in the G0/1 segment of the cell cycle. We conclude that Ki‐67 may better reflect the proliferative activity in solid neoplasms than does PCNA. © 1994 Wiley‐Liss, Inc.
Development of a method for measuring cell number: Application to CNS primary neuronal cultures
Authors: Volonté Cinzia , Ciotti Maria Teresa , Battistini Luca 
Published: June 2005

AbstractIn the present work we further develop a method for counting cell number that is totally independent from the permeability or uptake conditions of the cells, from the state of activation of intracellular enzymes, and from cellular metabolism. We provide a visual characterization of the method and show that it is highly suitable for cells not growing as monolayers as well as for cultures containing numberous aggregates. We also extend the applicability of this method to CNS primary neuronal cultures and show its direct comparison with alternative means for cellular quantification. The technique is fast, does not require tedious procedures or long washes, and offers advantages such as a high sensitivity and no background. © 1994 Wiley‐Liss, Inc.