bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry
Authors: Azzolina L. S. , Stevanoni G. , Tridente G. 
Published: August 2005

AbstractA microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1–3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide‐RNAse solution.The results, obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen‐stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli.This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H‐thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90° light scatter and double fluorescence labelling of PBL nuclei.
Flow cytofluorometric analysis of the uptake of the fluorescent fatty acid pyrene‐dodecanoic acid by human peripheral blood cells
Authors: Fibach Eitan , Giloh Haim , Rachmilewitz Eliezer A. , Gatt Shimon 
Published: August 2005

AbstractThe fluorescence activated cell sorter (FACS) was used for measuring the uptake of the fluorescent fatty acid derivative 12‐(1‐pyrene) dodecanoic acid (P12) by human peripheral blood cells. The results indicate that blood cells differ widely in their ability to take up P12, with polymorphonuclear cells showing the greatest uptake, followed by lymphocytes, platelets, and RBCs. These differences in P12 uptake provide a potential additional parameter for differential cell counting. Using the ability of the FACS to “gate out” nonrelevant cells, it was possible to measure the rate of P12 uptake by each respective cell type even when admixed with other cells. Thus elaborate physical separation procedures could be avoided, and contaminating cells did not influence the results. Differences in P12 uptake were also utilized to separate blood cells into pure subpopulations of specific cell types.
Five‐dimensional flow cytometry as a new approach for blood and bone marrow differentials
Authors: Terstappen Leon W. M. M. , Loken Michael R. 
Published: August 2005

AbstractWe have used five independent variables on a flow cytometer to discriminate and to quantify the cellular components within both blood and bone marrow aspirates. The signals were stored in list mode by which a five‐dimensional space was created. The cells–differentiated into: (1) erythrocytes, (2) reticulocytes, (3) nucleated erythroid cells, (4) platelets, (5) lymphocytes, (6) monocytes, (7) neutrophils, (8) eosinophils, and (9) immature leukocytes–had to meet unique criteria with regard to their characteristics in the created five‐dimensional space in order to be classified in a specific cell category.Forward and orthogonal light‐scattering signals were matched with three fluorescence variables to obtain discrimination without necessitating erythrocyte lysis.Thiazole orange (binding predominantly to RNA) and LDS‐751 (principally detecting DNA) were used to differentiate erythrocytes, platelets, reticulocytes, and nucleated cells. A monoclonal antibody, CD45, conjugated with phycoerythrin, was used to aid in discriminating between lineages of nucleated cells.
Comparison of absorption measurements of DNA stain content by utilizing video and scanning image cytometers
Authors: Allison David C. , Mayall Brian H. , Levin Jack 
Published: August 2005

AbstractAfter staining with the Feulgen reaction, the DNA stain contents of 155 mouse bone marrow cells and 22 adjacent chicken erythrocytes were measured by absorption image cytometry by utilizing two different systems—a scanning cytometer and a video cytometer. In the scanning cytometer (M85 microdensitometer, Vickers Instruments, Malden, MA), a spot of light was scanned across the cell. In the video cytometer (TAS Plus, E. Leitz, Rockleigh, NJ), the microscope field, which may contain several nuclei, was imaged onto a Plumbicon video camera. With each system, cells were scanned, digitized into their elementary pixels, and analyzed to determine their intergrated absorbance.Comparison of the DNA stain contents of the same G0/G1 bone marrow cells and chicken erythrocytes, as measured by video and scanning cytometry, showed that both techniques gave comparable results; scanning cytometry is more precise. The coefficients of variation of the measurements for the G0/G1 bone marrow cells and for the chicken erythrocytes were 5.9% and 7.0%, respectively, when measured by video cytometry at the absorption peak (584 nm), compared to 4.1% and 3.5%, respectively, for the same cells when measured by scanning cytometry off the absorption peak (615 nm). The video‐based measurements were relatively lower than the scanning measurements for darkly stained cells; this suggests that glare and other optical errors which increase with stain darkness caused greater systematic errors in the video cytometer than they did in the scanning cytometer.
Relationship between changes of the steroid receptor and synchronization in human endometrial adenocarcinoma cells in vitro
Authors: Nishiya Iwao , Kagabu Teruo , Saito Satoshi , Fujimoto Jiro , Okada Hiroji 
Published: August 2005

AbstractIt has been reported that the response of target cells to steroid hormone (SH) stimulation may depend on their position in the cell cycle. The DNA and RNA contents of malignant cells of the endometrium cultured in vitro were measured using flow cytometry (FCM). We also measured estrogen receptor (ER) and progesterone receptor (PR) levels of cells at different positions in the cell cycle.The G1 and S phases of the cell cycle were investigated using cells synchronized by sodium n‐butyrate (G1 block), methotrexate (S block), and excess thymidine (S block). For DNA measurements, the cells were stained with propidium iodide following RNase treatment. For RNA measurements (double‐straded RNA) the cells were treated with DNase.We found that S phase synchronization by methotrexate was 136.2% of control (100%). Using the excess thymidine block and release procedure, the S phase fraction was 185.1% of control. G1 phase synchronization by sodium n‐butyrate was 134% of control.The estrogen receptor level in G1 phase synchronized cells increased to 5.94 fmol/μg DNA in the cytosol and 12.35 fmol/μg DNA in the nuclear fraction. These levels represent a sevenfold total increase over that of the control estrogen receptor level. Cells in S phase showed no significant increase in estrogen receptor levels over control cells. Based on this study, the functional increase of the steroid receptor was most significant in the G1 phase.
Technical and statistical improvements for flow cytometric DNA analysis of paraffin‐embedded tissue
Authors: Sickle‐Santanello Brenda J. , Farrar William B. , Decenzo Jane F. , Keyhani‐Rofagha Sedigheh , Klein John , Pearl Dennis , Laufman Harry , O'Toole Robert V. 
Published: August 2005

AbstractFlow cytometric DNA analysis of paraffinembedded solid tumors has permitted review of large series of archival tissue in attempts to relate abnormal DNA content to prognosis. Limitations of the technique include: (1) a laborious, time‐consuming procedure; (2) variation in technique between laboratories; and (3) lack of an objective method of computing DNA indices. Critical evaluaiton of our technique has shortened the time involved in dewaxing and rehydration, selectively utilized patient's own normal tissue as the internal standard, proved reproducibility of stored specimens, standardized DNA index computation, and developed a statistical analysis to confirm aneuploidy. These technical improvements and the development of a statistical analysis provide a way to shorten the procedure time and standardize the data generated from flow cytometric DNA analysis so as to improve the quality of retrospective reviews of paraffin‐embedded tumors and accelerate the definition of flow cytometry's role as a prognostic indicator.
Comparison of automated and manual techniques for analysis of DNA frequency distributions in bladder washings
Authors: Wheeless Leon L. , Coon John S. , Deitch Arline D. , White Ralph W. DeVere , Koss Leopold G. , Melamed Myron R. , Reeder Jay E. , Robinson Roy D. , Weinstein Ronald S. , Wersto Robert P. 
Published: August 2005

AbstractQuantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant to the relatively low signal‐to‐noise ratios often found in DNA distributions obtained from clinical samples.Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S.A computer‐based method has been developed for extraction of the above‐mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight‐line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer‐based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer‐based method. Correlation of percent hyperdiploid cell fraction was also excellent. This computer‐based method for the analysis of DNA frequency distributions should have utility in the establishment of DNA flow cytometry of bladder irrigation specimens as a useful and routine laboratory procedure.
Comparison of S‐phase fractions measured by flow cytometry and autoradiography in human transplant tumors
Authors: Feichter G. , Czech W. , Haag D. , Goerttler K. , Futterman G. , Loehrke H. , Abel U. 
Published: August 2005

AbstractThe 3H‐thymidine labeling index (TLI) and the percentage of cells in the S‐phase have been determined by autoradiography and by flow cytometry, (FCM), respectively, in six malignant tumors of human origin transplanted on athymic nude mice. The Dean and Jett model and the graphical model were used to determine the percent of S‐phase cells by FCM. Cell cycle analysis was performed using (1) no correction for background; (2) an algebraic function for background correction; and (3) an exponential function for background subtraction. Each of these three data sets was evaluated using both the Dean and Jett model and a graphical model for the evaluation of DNA histograms. The S‐phase fractions (SPF) were compared to the corresponding labeling index results.SPF without background correction were 1.54 times higher than the TLI. SPF, after correction using the algebraic model, were 1.29‐fold higher than the TLI, whereas SPF obtained after background subtraction according to the exponential model were only 1.05‐fold higher than the TLI. Student's t‐test revealed significant differences between the mean TLI values (16.25 ± 9.06) and the mean SPF obtained by FCM without background correction (mean 25.0 ± 9.36, P < 0.01), but not between the mean TLI values and the mean SPF percentages after algebraic (mean 21.0 ± 10.29) and exponential background correction (mean 17.11 ± 11.59), P > 0.05 each. There was no difference between the results obtained using the Dean and Jett model and those obtained using the graphic evaluation. The SPF obtained by FCM are similar to the 3H‐thymidine labeling indices of the same sample of a solid tumor if the histograms first are cleaned from nonspecific background pulses.
Relevance of DNA ploidy as a measure of genetic deviation: A comparison of flow cytometry and cytogenetics in 25 cases of human breast cancer
Authors: Remvikos Y. , Gerbault‐Seurreau M. , Vielh P. , Zafrani B. , Magdelénat H. , Dutrillaux B. 
Published: August 2005

AbstractTwenty‐five human breast cancers, surgically resected, were studied by cytogenetic analysis and DNA flow cytometry (FCM). The establishment of karyotypes showed that multiple cell populations probably were derived from a single ancestor clone, because common marker chromosomes always could be demonstrated. Differences of up to 30% were observed when the estimates of DNA content by the two methods were compared. A general tendency toward the acquisition of large marker chromosomes should be at the origin of this discordance, as the proportion of markers for each case correlated significantly with the magnitude of the difference. Parallel use of the two methods revealed the existence of tumors with DNA diploid FCM profiles and highly abnormal hypodiploid karyotypes (35–40 chromosomes), which may explain the limited value of DNA ploidy as an independent prognostic factor in breast cancer.
A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation
Authors: Van Erp Piet E. J. , Brons Paul P. T. , Boezeman Jan B. M. , De Jongh Gijs J. , Bauer Franz W. 
Published: August 2005

AbstractThis report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achived by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.
Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining
Authors: Dolbeare Frank , Gray Joe W. 
Published: August 2005

AbstractWe describe an enzymatic procedure for exposure of single‐stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X‐100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti‐HdUrd antibody IU‐4 in Chinese hamster ovary cells labeled with 10 μM BrdUrd or 10 μM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU‐1, an anti‐HdUrd antibody with lower binding affinity. IU‐4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU‐4 binding.
Osmotic response of lymphocytes measured by means of forward light scattering: Theoretical considerations
Authors: Sloot Peter M. A. , Hoekstra Alfons G. , Figdor Carl G. 
Published: August 2005

AbstractRecent data provide solid experimental evidence that increase in cell size of human lymphocytes, caused by osmotic stress, is not directly proportional to forward scattering intensity but rather is inversely proportional. Here, we provide arguments that this phenomenon can be quantitatively explained by means of a model based on a modified Rayleigh‐Debye‐Gans theory. As a consequence, the current view that forward scattering intensities can be used as a measure of gross cell size needs to be reconsidered. In addition, we postulate that structural changes of the cytoplasm, imposed by an osmotic stress, reflect corresponding changes in the nucleus.
Cyanine dye labeling reagents for sulfhydryl groups
Authors: Ernst Lauren A. , Gupta Ravinder K. , Mujumdar Ratnakar B. , Waggoner Alan S. 
Published: August 2005

AbstractCyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from < 500 nm to > 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity.
Two statistical approaches to nuclei shape and size in a morphometric description of lymph node sections in non‐Hodgkin's lymphoma
Authors: Lesty Claude , Raphaël Martine , Nonnenmacher Luc , Binet Jacques‐Louis 
Published: August 2005

AbstractAn attempt is made to evaluate more clearly the potential contribution of quantitative nuclear profile shape and size measurements to lymph node section histologic description in 70 cases of non‐Hodgkin's lymphoma (NHL). The area of nuclear profiles and five nonredundant and size‐free shape indices were measured using the THECLA program on a Leitz Texture Analysis System (Leitz‐TAS). Two statistical approaches were applied, known respectively as “parametric,” (first statistical moments of variable distributions over samples of 200 nuclear profiles) and “nonparametric,” which are percentages of nuclear profiles distributed into five “cytological” classes that are defined by shape: round (A), elongated (B), kidney shaped (C), irregular (D) and cleaved (E) nuclear profiles.Both statistical approaches provide proper overall discrimination of the eight histological categories identified with reasonable reliability by pathologists. Above all, the present report discusses the ability of a set of parametric and nonparametric variables to describe NHL cell populations, in an objective and meaningful way, according to nuclei shape. A method of synthesizing multidimensional correlations (CORICO program) is proposed in support of the discussion. Also, the specific descriptive power of each of the variables is described; in particular, it is concluded that there is a close link between the shape and size of the nuclear profiles of the cells.
Quantitative morphological analysis of Adriamycin‐resistant human K562 leukemic cells
Authors: Dufer J. , Akeli M. G. , Jeannesson P. , Desplaces A. , Jardillier J. C. 
Published: August 2005

AbstractThe morphological changes associated with Adriamycin resistance in a human leukemic cell line have been investigated by image analysis. An Adriamycin‐resistant subline of the human erythroleukemic K562 cell line has been established. Three sets of cells have been analysed: sensitive cells, resistant cells cultured in the continuous presence of Adriamycin, and resistant cells cultured without the drug. Image analysis shows that Adriamycinresistant K562 cells display significant morphological changes as compared with sensitive cells, at both the nuclear and cytoplasmic levels. These changes make it possible to separate sensitive and resistant cells automatically and with a classification accuracy of 76% and only four cytological parameters. Image analysis may therefore offer an interesting tool for studying drug resistance in leukemic cells, from both an experimental and a clinical point of view.
The multivariate prognostic index and nuclear DNA content are independent prognostic factors in primary breast cancer patients
Authors: Van Der Linden J. C. , Lindeman J. , Baak J. P. A. , Meijer C. J. L. M. , Herman C. J. 
Published: August 2005

AbstractThe predictive value of a previously described Multivariate Prognostic Index (which incorporates weighted values of the mitotic activity index, tumor size, and the axillary lymph node status), and the nuclear DNA content (DNA) was evaluated in 156 patients with primary invasive ductal breast cancer, diagnosed between 1980 and 1983. The results were analysed with respect to the occurrence of distant recurrence and survival of the patients after at least 3 yr of follow‐up (range 36–73 months; median 44 months).Known prognostic factors such as lymph node status, tumor size, and the mitotic activity index correlated independently with distant recurrence. Furthermore, in respect to survival, the investigated prognostic factors (except DNA content) were significantly correlated.The results indicate that the predictive value of the Multivariate Prognostic Index (MPI) is stronger (P < 0.001) than of the nuclear DNA content (P < 0.005) with respect to distant recurrence.In a Cox multivariate regression analysis DNA ploidy turned out to be an independent prognostic factor once the MPI was selected. Furthermore, in Cox's analysis, DNA ploidy was the fourth selected variable after lymph node status, mitotic activity index, and tumor size in individual parameter analysis. The results of this study indicate that, with respect to breast cancer screening programs, it seems worthwhile to integrate morphometric features, the MPI, and DNA ploidy in a new prognostic model.
Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry
Authors: Robertson B. R. , Button D. K. 
Published: August 2005

AbstractFlow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high‐intensity UV excitation, condensing the laser beam onto a small area, using blemish‐free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high‐transmittance optical filters collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 μm3 as calibrated by coulter impedance. Plastic spheres down to 0.014 μm3, 0.3 μm in diameter, were resolved. Aquatic bacteria 0.05 μm3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA‐dependent because DNase treatment eliminated most fluorescence.These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA‐free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.