bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative analysis of mitotic and early‐G1 cells using monoclonal antibodies against the AF‐2 protein
Authors: Di Vinci Angela , Geido Elio , Pfeffer Ulrich , Vidali Giorgio , Giaretti Walter 
Published: August 2005

AbstractWe have recently described a novel protein (AF‐2), conserved between fission yeast and man, and we have shown by flow cytometry (FCM) that AF‐2 is highly accessible to specific monoclonal antibodies (MoAbs) in mitotic and postmitotic early‐G1 phase cells. The aim of the present study was to optimize the FCM methodology using MoAbs against AF‐2 and to show that the evaluation of the mitotic cells, using different cell lines, was quantitative and reproducible. We found that a method based on fixation with ethanol, instead of formalin, resulted in improved DNA histogram coefficients of variation and implemented separation of early‐G1 cells from late‐G1 cells. In addition, by eliminating several cell permeabilization and protein salt extraction steps, the method became straightforward, conserved a clear‐cut separation of the green fluorescence of M‐ with respect to G2‐phase cells, and did not significantly affect cellular integrity. The coefficient of correlation among the mitotic index values evaluated by this FCM method using MoAbs against AF‐2 and by microscopic visual counting was R=0.94. When the FCM/AF‐2 method was tested against an independent FCM method, which allows clear separation of M‐ and G2‐phase cells according to 90° scattering, we found R=0.93. We conclude that MoAbs against the AF‐2 protein may be used in FCM for quantitative analysis and for isolation of M‐phase cells, providing as well, the identification of the early‐Gl cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF‐2 antigen. © 1993 Wiley‐Liss, Inc.
Flow cytometric DNA content and clinical outcome in patients with non‐small cell lung cancer given postoperative radiation therapy
Authors: Yu Jinming , Shaeffer James , Zhu An , Kuban Deborah A. , El‐Mahdi Anas M. , Philput Christine B. 
Published: August 2005

AbstractDNA content by flow cytometry was assessed in 47 cases from a series of 130 patients with non‐small cell lung carcinoma (NSCLC) given radiation therapy postoperatively. This was done in an attempt to identify which patients might benefit, or not benefit, from postoperative radiotherapy. From archival formalin‐fixed paraffin‐embedded specimens, 16 of the 47 cases (34%) had DNA diploid tumors while 31 cases (66%) were nondiploid. A diploid DNA content was significantly related to improved overall survival (P = 0.0061) and tumor‐free survival (P = 0.0167) but not with frequency of tumor recurrence within the irradiated field. Histological grade was not significantly related to overall survival, tumor‐free survival, or frequency of in‐field tumor recurrence. DNA content was found in this study of NSCLC patients irradiated postoperatively to be a useful marker for predicting survival but not for predicting local recurrence. © 1993 Wiley‐Liss, Inc.
Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry
Authors: Landay Alan , Jennings Cheryl , Forman Meryl , Raynor Robert 
Published: August 2005

AbstractDual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CD8+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA‐1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CD8+ cells. The method effectively quantitated TIA‐1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system. © 1993 Wiley‐Liss, Inc.
Separation of rooster spermatogenic nuclei by means of centrifugal elutriation
Authors: Boix Jacint , Roca Joaquim 
Published: August 2005

AbstractBy application of minor variations in the usual method of centrifugal elutriation, we have been able to separate a nuclear suspension from rooster testes. We have obtained a population of elongated or late spermatid nuclei (98% purity), a population of round or early spermatid nuclei (90% purity), and, finally, a mixed population of meiotic and pre‐meiotic nuclei. © 1993 Wiley‐Liss, Inc.
Detection of rare cells at a frequency of one per million by flow cytometry
Authors: Gross Hans‐Joachim , Verwer Ben , Houck David , Recktenwald Diether 
Published: August 2005

AbstractIn a model study we seeded the pre‐B‐cell line REH into 250 million peripheral blood mononuclear cells (PBMC) at frequencies of 10−4, 10−5, and 10−6. By flow cytometry we could detect the REH cells and found a background of about one event per 100 million PBMC. This sensitivity was achieved by removing four sources of false positive events, including nonspecific immunofluorescence, autofluorescence, background particles from previous experiments, and bursts of events during acquisition. To overcome limits to rare event detection imposed by nonspecific staining and autofluorescence, we used positive and negative selection for the REH cells. Another fluorochrome was added to stain the background cells and particles. In order to remove particles and background from previous experiments, a cleaning technique was developed and event bursts were removed from the analysis by developing an algorithm that screens the list‐mode data for events that were not Poisson distributed. © 1993 Wiley‐Liss, Inc.
Determining Tpot in heterogeneous systems: A new approach illustrated with multicell spheroids
Authors: Durand Ralph E. 
Published: August 2005

AbstractLike many tumors, multicell spheroids from Chinese hamster V79‐171b fibroblasts display karyotypic heterogeneity as they enlarge. This creates significant problems for analysis of univariate DNA content histograms of cells from spheroids, and even for bivariate bromodeoxyuridine (BrdUrd)/DNA flow histograms. While attempting to routinely measure spheroid cell kinetics with the latter approach, a simple alternative that overcomes many of the inherent problems emerged. The exit rate of BrdUrd‐la‐belled cells from the S‐phase compartment(s), determined using standard DNA analysis software, is used to define the duration of S‐phase and ultimately the potential doubling time of the clonogenic cell population. This approach appears to offer several advantages over the conventional “relative movement” technique for heterogeneous samples, particularly since the subjective distinction between labelled/divided tetraploid G1 cells vs. labelled/undivided diploid G2 is no longer required. Further, the technique avoids some potential artifacts that might be expected for heterogeneous tumors where cell subpopulations proliferate with quite different cell kinetics. © 1993 Wiley‐Liss, Inc.
Comparison of pepsin and trypsin digestion on paraffin‐embedded tissue preparation for DNA flow cytometry
Authors: Tagawa Yutaka , Nakazaki Takayuki , Yasutake Toru , Matsuo Satoshi , Tomita Masao 
Published: August 2005

AbstractPreparation with enzymes plays an important part in obtaining good results in cellular DNA content measurements from paraffin‐embedded tissue using flow cytometry. Therefore, we have compared two groups of DNA histograms obtained with pepsin (Hedley method) and trypsin digestion (Schutte method). Samples of five tumor types were compared: eight lung, seven breast, five thyroid, seven stomach, and six colon cancer cases. All samples were stained with propidium iodide (PI).The incidence of DNA aneuploidy determined by the Schutte method indicated a higher ratio in four tumor types than that determined by the Hedley method.Concerning the coefficient of variation (CV) used to estimate the quality of the DNA histogram, the diploid G1 peak determined by the Schutte method produced a smaller CV in five tumor types than did the Hedley method. This Schutte method had especially good results in lung and thyroid cancer. Furthermore, the amount of debris (background) determined by the Schutte method indicated a decrease in four tumor types compared with the Hedley method.In cell‐cycle analysis of ten DNA diploid cases, DNA histograms by the Hedley method showed an increased S‐phase fraction due to an overlapping of debris and aggregated cells.These results indicate that preparation by trypsin digestion is a method superior to pepsin digestion when the sample from paraffin‐embedded tissue is stained with PI for the purpose of DNA content measurement. © 1993 Wiley‐Liss, Inc.
Variations in DNA aneuploid cell content during tumor dissociation in human colon and head and neck cancers analyzed by flow cytometry
Authors: Ensley John F. , Maciorowski Zosia , Hassan Mohammad , Pietraszkiewicz Haline , Sakr Wael , Heilbrun Lance K. 
Published: August 2005

AbstractExperimental research involving human solid tumors often requires single cell suspensions of high yield that are representative of the tissue of origin and in which the cellular property of interest is preserved. This is particularly necessary for the determination of DNA ploidy by flow cytometry. Mechanical dissaggregation and proteolytic enzyme digestion are the most commonly employed dissociation techniques for solid tumors. Comparative testing of techniques is often not performed. Mechanical and proteolytic enzyme dissociation techniques were comparatively tested in 77 human squamous cell cancers of the head and neck (SCCHN) and 25 human colon cancers for cellular yield, dye exclusion viability, quality, and morphology of DNA histograms, and the presence and proportion of DNA aneuploid subpopulations. Significant and consistent DNA aneuploid subpopulation losses were noted in mechanical preparations of SCCHN and enzymatic preparations of colon cancers. The frequency of SCCHN specimens with DNA aneuploid subpopulations was underestimated by 52% in mechanical cell suspensions, and the proportion of DNA aneuploid cells was diminished in an additional 30% of the specimens. Conversely, the frequency of specimens with DNA aneuploid subpopulations was underestimated by 38% in cell suspensions from enzymatically dissociated human colon cancer and their proportion diminished in an additional 50% of the specimens. Incubations of human colon cancers with three commonly employed proteolytic enzymes demonstrated a progressive loss of DNA aneuploid subpopulations as a function of enzyme concentration and incubation time. This is a serious potential source of error in the flow cytometric determination of DNA ploidy in human solid tumors, and may contribute to the diversity of results obtained and occasional contradictory conclusions reached in such studies. © 1993 Wiley‐Liss, Inc.
An improved assay for the detection of Toxoplasma gondii antibodies in human serum by flow cytometry
Authors: Cozon Grégoire , Roure Chantal , Lizard Gérard , Greenland Timothy , Larget‐Piet Dominique , Gandilhon Françoise , Peyron François 
Published: August 2005

AbstractThe diagnosis of toxoplasmosis relies primarily on the demonstration of specific antibodies to Toxoplasma gondii. We describe a flow cytometry method for the determination of antibodies to whole fixed tachyzoites by indirect immunofluorescence. Fixed tachyzoites in suspension have characteristic light scattering properties. The amount of IgM, IgG, and IgA antibodies from patients' sera bound to the tachyzoites can be estimated from the mean fluorescence intensities observed using class‐specific conjugates for different Ig heavy chains. Appropriate serum dilutions for the estimation of specific IgG titres and for the discrimination between sera positive and negative for IgA and IgM antibodies were established using 40 random sera from pregnant women. The method proved to be quantitative and highly sensitive as compared with currently used assays. Coefficients of variation between series ranged from 6.4% to 12.2% and could be controlled by the inclusion of positive and negative standard sera in the test series. The method might represent a major improvement in cost effectiveness as compared with routine immunofluorescence assay by fluorescent microscopy when the volume of testing is sufficient. It can be fully automated, and its large scale application is currently under investigation. © 1993 Wiley‐Liss, Inc.
White blood cell differentiation using a solid state flow cytometer
Authors: Doornbos R. M. P. , Hennink E. J. , Putman C. A. J. , De Grooth B. G. , Greve J. 
Published: August 2005

AbstractA flow cytometer using a solid state light source and detector was designed and built. For illumination of the sample stream two types of diode lasers (670 nm and 780 nm) were tested in a set‐up designed to differentiate human leukocytes by means of light scattering. The detector is an avalanche photodiode, which was used to detect the weak scattered light in the orthogonal direction. The new flow cytometer set‐up is very small, relatively cheap and yields similar results as a standard flow cytometer set‐up using a helium‐neon laser and photomultipliers. © 1993 Wiley‐Liss, Inc.
Heterochromatin study demonstrating the non‐linearity of fluorometry useful for calculating genomic base composition
Authors: Godelle Bernard , Cartier Delphine , Marie Dominique , Brown Spencer C. , Siljak‐Yakovlev Sonja 
Published: August 2005

AbstractA novel procedure for calculating basepair frequencies in whole genomes is reported. This has been developed during a study of the role of heterochromatin in microevolution. Closely related species of the Crepis praemorsa complex have similar karyotypes but for their heterochromatin. The changes in relative AT frequency between species have been attributed to heterochromatin sequences by in situ banding of chromosomes with two base‐specific fluorochromes. The absolute genome size of species, measured by cytofluorometry, correlated positively with increased karyotypic heterochromatin, as did the proportion of AT bases in the DNA. However, the determination of base content has called for a curvilinear interpretation of data obtained with two base‐specific fluorochromes (bisbenzimide Hoechst 33342 and mithramycin), in contrast to the commonly assumed but erroneous direct relationship between fluorescence intensity and base content. Essentially, the fluorochromes' requirements for a sequence of certain basepairs lead to the notion of Coefficients or' Overspecificity: the result is a simple for mula for calculating the AT proportion in a genome relative to a reference species from cytometric data, taking account of ligand binding statistics. These statistics and probabilities of oligonucleotide binding are essentially the same. © 1993 Wiley‐Liss, Inc.
Cluster analysis of flow cytometric list mode data on a personal computer
Authors: Schut Tom C. Bakker , De Grooth Bart G. , Greve Jan 
Published: August 2005

AbstractA cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS‐DOS personal computer. It uses k‐means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce the large number of subclusters. Thus we combine the advantage of the k‐means (speed) with that of the nearest neighbor technique (accuracy). In order to achieve a rapid analysis, no complex data transformations such as principal components analysis were used.Results of the cluster analysis on both real and artificial flow cytometric data are presented and discussed. The results show that it is possible to get very good cluster analysis partitions, which compare favorably with manually gated analysis in both time and in reliability, using a personal computer. © 1993 Wiley‐Liss, Inc.
False aneuploidy in flow cytometric DNA analysis of paraffin embedded tissue: Effects of carnoy's fixation
Authors: Carr Robert F. , Abaza Ann M. 
Published: August 2005

AbstractFalse aneuploidy was detected on flow cytometric DNA analysis of paraffin embedded axillary lymph nodes negative for tumor. It was hypothesized that “clearing” of axillary fat in Carnoy's solution to facilitate lymph node dissection might be responsible for false aneuploidy. Various tissues fixed overnight in Carnoy's were compared to formalin fixed paraffin embedded controls. Under these conditions no false aneuploid peaks were detected, but Carnoy's fixation did shift the G0/G1 histogram peak to the left, increase the G0/G1 CV and increase the S phase fraction relative to formalin fixed controls. It was then hypothesized that partial fixation of nodes in Carnoy's followed by formalin fixation might result in false aneuploid peaks. Twenty‐two lymph nodes were partially fixed in Carnoy's for periods ranging from 5 to 60 min followed by complete fixation in formalin. Seven of these nodes did show false aneuploid peaks. By contrast, no aneuploidy was detected in formalin fixed controls. It was concluded that tissues in contact with Carnoy's solution may be a source of false aneuploidy and/or false elevation of S phase fraction. This reinforces the need for matched negative tissue controls for DNA analysis of paraffin embedded specimens whenever possible. © 1993 Wiley‐Liss, Inc.
Improved dna content histograms from formalin‐fixed, paraffin‐embedded liver tissue by proteinase K digestion
Authors: Albro James , Bauer Kenneth D. , Hitchcock Charles L. , Wittwer Carl T. 
Published: August 2005

AbstractAn improved method for the enzymatic digestion of formalin‐fixed, paraffin‐embedded liver tissue for DNA content analysis by flow cytometry is presented. Forty samples of histologically normal liver were alternately digested by the traditional pepsin method or a new method utilizing proteinase K and heat. Sixteen (40%) of the pepsin‐digested samples had apparent DNA aneuploid peaks by flow cytometry. False DNA aneuploid peaks were not present in any of the histograms obtained after proteinase K digestion. Microscopy showed that the pepsin‐digested samples had residual cytoplasmic remnants which contained fluorescent material. Samples digested with proteinase K had few cytoplasmic remnants. The average G0/G1 coefficient of variation after proteinase K treatment was lower (41%) and the fluorescent intensity higher (128%) than the pepsin‐treated samples. The apparent mean S‐phase (a combination of S‐phase cells and underlying debris) after proteinase K digestion was 35% of the pepsin‐treated samples. Primary and secondary tumors of the liver that were DNA aneuploid after pepsin treatment were also DNA aneuploid after proteinase K treatment. A modified digestion protocol utilizing proteinase K and heat can provide superior results for DNA content analysis of formalin‐fixed, paraffin‐embedded liver tissue. © 1993 Wiley‐Liss, Inc.
CD4 T‐lymphocyte determinations on whole blood specimens using a single‐tube three‐color assay
Authors: Nicholson Janet K. A. , Jones Bonnie M. , Hubbard Marjorie 
Published: August 2005

AbstractWe evaluated a flow cytometrïc method for determining the proportion of CD4‐positive T lymphocytes in whole blood using a single three‐color tube containing fluorochrome‐labeled CD45, CD3, and CD4. Various ways of gating this sample were evaluated and results were compared with data obtained in our standard six‐tube, two‐color assay gated on light scatter parameters. Excellent correlation was found between the three‐color analysis using a CD45/side scatter gate and the standard two‐color analysis with a light scatter gate. This single‐tube three‐color test is a promising assay for monitoring CD4+ T‐lymphocytes. © 1993 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
Determination of the percentage of thiazole orange (TO)‐positive, “reticulated” platelets using autologous erythrocyte TO fluorescence as an internal standard
Authors: Bonan Jayne L. , Rinder Henry M. , Smith Brian R. 
Published: August 2005

AbstractDetermination of the percentage of thiazole orange (TO)‐positive or “reticulated” platelets by flow cytometry has been advocated as an aid in the diagnosis of thrombocytopenic disorders. However, a reproducible method for determining control fluorescence and setting threshold values on a routine clinical basis has not been described. We used erythrocyte TO fluorescence in whole blood as an internal standard to set threshold markers for TO fluorescence of autologous, purified platelets. Since platelets have approximately a threefold higher TO fluorescence than erythrocytes, multiplying the erythrocyte threshold marker by 3 allowed a determination of the percentage of “reticulated” platelets in 29 normal controls. This value was 3.64% ± 2.10% (S.D.) in men and 5.79% ± 2.22% (S.D.) in women; there was a significant (P < 0.05) difference between normal men and women for this measurement. Applying this method to our earlier studies of normals and patients with immune thrombocytopenic purpura confirmed its ability to discriminate between these populations with similar sensitivity and specificity as previously reported. This method controls for fluctuations in TO labeling and fluorescence and does not require daily determination of a new normal control value. © 1993 Wiley‐Liss, Inc.
Three‐deimensional visualization of multi‐channel volume data: The amSFP algorithm
Authors: Messerli J. M. , van der Voort H. T. M. , Rungger‐Brändle E. , Perriard J. ‐C. 
Published: August 2005

AbstractIn this paper we present a three‐dimensional visualization technique for multi‐channel volume data. The technique simulates the physical process of fluorescence, hence its name: achromatic multi‐channel simulated fluorescent process (amSFP). The data set is simulated as 3D distribution of different fluorescent dyes, where each channel is represented by a particular type of dye. Apart from the spatial density map, no additional characteristics about the data set have to be defined; no image segmentation is needed prior to visualization. The degree of interaction among the channels in the fluorescence process can be adapted to optimally render specific structures in the image. 3D multi‐channel data can be obtained by a three‐dimensional imaging device that is able to measure a number of physical quantities at a given location within a specimen. The fluorescence principle, the algorithm, and its implementation are presented. We have used the technique to investigate the relative spatial arrangement of blood vessels and astrocytes in the cat retina. The two components have been stained with different fluorescence dyes and recorded in a confocal light microscope to form a two‐channel 3D data set. © 1993 Wiley‐Liss, Inc.