bluuebunny/crossref_metadata_2025_split huggingface dataset:
Highly sensitive, specific, and stable new fluorescent DNA stains for confocal laser microscopy and image processing of normal paraffin sections
Authors: Tekola Paulos , Baak Jan P. A. , Beliën Jeroen A. M. , Brugghe Jane 
Published: June 2005

AbstractThe area, volume, shape, DNA content, and chromatin pattern of nuclei can be important for the diagnosis and prognosis of cancers. Confocal laser scan microscopy can be useful to obtain such information by optical slicing and three‐dimensional (3‐D) reconstruction of nuclei in thick paraffin sections. To retrieve individual quantitative features from the section, highly sensitive, specific, and stable fluorescent stains are required. Two new nuclei acids stains (TOTO‐1 iodide and YOYO‐1 iodide) can detect picogram amounts of nucleic acids in gels. Despite their high sensitivity to detect DNA, they have not been used to stain nuclei in paraffin embedded tissue sections (as routinely applied in surgical cancer pathology). We have developed a technique to stain nuclei in 4% buffered formaldehyde fixed paraffin tissue sections using TOTO‐1 iodide and YOYO‐1 iodide. The technique developed gives bright specific staining of the nuclei (with nearly zero background intensity, much less than with acriflavine). Moreover, TOTO‐1 iodide and YOYO‐1 iodide stains both give fluorescent signals only when they interact with DNA. Thus washing off the excess stain left on the stained specimen is not necessary (washing of excessive stain is necessary with acriflavine). Care has to be taken that the deparaffinizing liquids (xylol etc.) are not polluted with eosin (a frequently used counterstain in surgical pathology specimens), as this gives undesired fluorescence of cytoplasm and connective tissue at the same wavelength as TOTO‐1 iodide and YOYO‐1 iodide. Contrary to acriflavine, bleaching of TOTO‐1 iodide and YOYO‐1 iodide fluorescence is minimal, even after 30 min continuous laser light excitation. Morevover, integrated TOTO‐1 iodide and YOYO‐1 iodide fluorescence shows good linearity with the DNA content of diploid, tetraploid, and octaploid liver nuclei. The sections stained with TOTO‐1 iodide and YOYO‐1 iodide are particularly suitable for two‐dimensional morphometric and cytometric assessments of nuclei in sections, and also for the 3‐D reconstruction and visualization of nuclei by confocal microscopy. The high intensity and stability of the fluorescent image of the nuclei and the low uniform intensity of the background will decrease the work needed in image segmentation of nuclei and help to increase the accuracy of cytometric assessments. © 1994 Wiley‐Liss, Inc.
Simultaeous detection of β‐galactosidase activity and surface antigen expression in viable haematopoietic cells
Authors: Berger Christoph N. , Tan Seong‐Seng , Sturm Karin S. 
Published: June 2005

AbstractThe quantitation of intracellular β‐galactosidase activity has been described for viable cells. By using the fluorogenic substrate fluorescein‐di‐β‐D‐galactopyranoside (FDG)in conjunction with flow cytometry, the proportion of positive cells as well as the level of expression can be determined. In this paper we describe β‐galactosidase expression in lymphoid and myeloid cells from transgenic mice that widely express β‐galactosidase from an inserted lacZ transgene. Both foetal and adult haematopoietic tissues are able to express β‐galactosidase. The intracellular fluorescene reflecting β‐galactosidase activity can be readily combined with fluorescently labelled antibodies against cell surface antigens. Thus, β‐galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells. © 1994 Wiley‐Liss, Inc.
Reliable method for the simultaneous detection of cytoplasmic and surface CD3ε expression by murine lymphoid cells
Authors: Franek Karl J. , Wolcott R. Michael , Chervenak Robert 
Published: June 2005

AbstractDuring their development, T‐cell precursors (pre‐T cells) undergo a variety of changes with respect to their expression of specific surface proteins. Among the most critical of the surface markers acquired by developing T cells is the T‐cell receptor (TCR)/CD3 complex. Prior to the assembly and transport of complete TCR/CD3 multimeric complexes to the plasma membrane, the individual constituent subunits are expressed in the cytoplasm (ER‐Golgi). In order to study the expression of the T‐cell receptor TCR/CD3 complex during pre‐thymic T‐cell differentiation, we have developed a flow cytometric technique for the simultaneous detection of surface (sCD3ϵ) and cytoplasmic CD3ϵ (cCD3ϵ). This technique, which employs fixation in 1% paraformaldehyde and permeabilization with 1% saponin and 0.025% digitonin, features reliable internalization and low nonspecific binding of anti‐CD3ϵ in murine lymphoid cells, as well as tissue culture cell lines. The combination of saponin and digitonin treatment was also compatible with the staining of sCD3 and other lymphocyte surface antigens such as Thy1, CD4, CD8, B220, and IgM. In contrast, permeabilization of cells with the detergents Tween 20 and Triton X‐100 was shown to remove surface‐bound anti‐CD3ϵ. The present technique permitted the detection of discernible sCD3ϵ and cCD3ϵ double and single positive lymphocytes and may prove useful in defining bone marrow‐resident pre‐T cells. © 1994 Wiley‐Liss, Inc.
Flow cytometric quantitation of C‐myc and P53 proteins in bovine papillomavirus type 1‐transformed primary mouse fibroblasts
Authors: Agrawal Reitu S. , Agrawal Yash P. , Mäntyjärvi Rauno A. 
Published: June 2005

AbstractBovine papillomavirus type 1 (BPV‐1)‐transformed mouse fibroblast cell lines were analyzed via flow cytometry (FCM) for expression of p53 and c‐myc proteins along with their DNA content. In comparison to the nontransformed control cell line, significantly elevated levels of both the p53 and the c‐myc protein were present in some but not all of the transformed cell lines. Quantitation of p53 and c‐myc proteins in cell lines containing BPV‐1 DNA revealed that the tumorigenic cell lines expressed higher levels of both the p53 (P = 0.0034; Mann‐Whitney U test) and the c‐myc protein (P = 0.0039; Mann‐Whitney U test) as compared to the nontumorigenic cell lines. On average, at least 9,000–10,000 p53 or c‐myc protein molecules per cell were detected in the transformed tumorigenic cell lines. These results show that quantitative FCM can be reliably used to detect very low levels (3,000 molecules per cell) of specific protein, and FCM is a useful tool to study the virus‐induced changes in the levels of nuclear proteins within a cell population and in tumorigenesis. © 1994 Wiley‐Liss, Inc.
A knowlewldge‐based system for the interpretation of flow cytometry data in leukemias and lymphomas
Authors: Diamond Lawrence W. , Nguyen Doyen T. , Andreeff Michael , Maiese Russell L. , Braylan Raul C. 
Published: June 2005

AbstractFlow cytometry immunophenotyping and analysis of DNA ploidy and proliferative activity have become extremely helpful techniques for diagnosing and subclassifying hematopoietic cell populations in a modern, full‐service hematopathology laboratory. The number of physicians with special training in the interpretation of these studies is limited. A knowledge‐based computer system has been designed to aid in the interpretation of immunophenotyping and DNA flow cytometry results in hematopoietic disease. The system, known as “Professor Fidelio,” is a heuristic classification system that reasons on the basis of defined diagnostic patterns. In this study, Fidelio was tested as a stand‐alone system on 366 specimens from two large tertiary medical centers. Fidelio's interpretation was considered to be appropriate in all cases. In 300 of 366 (82%) specimens, the system's interpretation agreed with the diagnosis of record. Many of the disagreements could be traced to errors in the recording of the original diagnosis and minor differences in diagnostic criteria between Fidelio's knowledge base and the criteria in use at the medical centers. When used in a stand‐alone mode, Fidelio's interpretation was less specific than the diagnosis of record in certain lymphoproliferative disorders that require morphologic information for subclassification. Professor Fidelio is one module in a workstation for the diagnostic hematology laboratory. This workstation is designed for interpretive reporting, education, and database functions for clinical research. Clinical and morphologic information are shared between Fidelio and the other modules for peripheral blood analysis, bone marrow morphology, and lymph node interpretation by means of a relational database. The system will be useful in hospitals that lack individuals specially trained in flow cytometry. The consistency of interpretation from case to case will also be valuable for education and research. © 1994 Wiley‐Liss, Inc.
Optical and mathematical corrections of micropipette measurements of red blood cell geometry during anisotonic perifusion
Authors: Engström K. Gunnar , Meiselman Herbert J. 
Published: June 2005

AbstractAccurate knowledge of red blood cell (RBC) membrane surface area and cellular volume is critical to understanding the geometric factors that affect RBC deformability. A useful method to determine RBC geometry is via micropipette aspiration in which the cell partially enters the tip of a glass pipette with a diameter of about 2 μm. Measurement of the cell while in the pipette allows calculation of cell geometry, but such measurements are subject to several artifacts. Herein, we analyze the effects of three artifacts on calculated RBC geometry during experimental osmotic manipulation. (1) The pipette internal diameter, as measured optically, requires correction for refractive index and focusing errors; (2) the pipette barrel is tapered rather than cylindrical; (3) the observed RBC outline is affected by a diffraction phenomenon. These optical and mathematical errors were all found to influence the calculated RBC geometry; both absolute values and relative changes during osmotic manipulation were affected. For RBC in isotonic medium, use of the above corrections significantly (p < 0.001) reduced the calculated area by 3.7%, the volume by 7.0%, and the minimum cylindrical diameter by 3.9%. The calculated membrane area dilation for cells exposed to hypotonic media was also significantly reduced; at 200 mOsm, the dilation was 2.8% ± 0.2% without correction vs. 1.5% ± 0.2% when all corrections were employed (p < 0.01). We therefore suggest that micropipette aspiration of RBC requires use of these correction techniques to obtain accurate values for RBC geometry and that such correction methods are of particular importance when RBC of different sizes are compared. © 1994 Wiley‐Liss, Inc.
Display and correction of flow cytometry time‐dependent fluorescence changes
Authors: Kusuda Leo , Melamed M. R. 
Published: June 2005

AbstractThe resolution of measurements of cellular attributes by flow cytometry, based on differences in labeling by fluorescent markers, may be affected by variations in staining and sample stream flow that can occur during the course of data acquisition. We describe a method of display and correction of time‐dependent changes by looking at the list mode data in chronological order. © 1994 Wiley‐Liss, Inc.
Utilization of commercial antisera and flow cytometry in HLA‐B27 typing
Authors: Orr Ken , Thomson Glen T. D. , Alfa Michelle 
Published: June 2005

AbstractThe study of large groups of individuals (from reactive arthritis epidemics, for example) for epidemiologic and genetic data has been hampered by the techniques by which HLA typing is performed. Microlympho‐cytotoxicity is costly not only in materials but also in the time required to perform a given assay and thus does not lend itself to large scale testing. To study such epidemics, in the best way, one must be able to respond as soon as possible after the epidemic is recognized and on a large scale.We thus examined HLA‐B27 typing by flow cytometry and commercially available monoclonal antibodies. Two commercially available monoclonal HLA‐B27 antibodies were compared. We conclude that the utilization of monospecific HLA‐B27 antibodies and flow cytometry allows for the determination of HLA‐B27 and HLA‐B7 phenotype of a large number of individuals over a short period of time, thereby making the study of a large group both economical and feasible. © 1994 Wiley‐Liss, Inc.
Effects of whole blood lysis and fixation on the infectivity of human T‐lymphotropic virus type 1 (HTLV‐I)
Authors: Ericson Julie G. , Trevino Alex V. , Toedter Gary P. , Mathes Lawrence E. , Newbound Garret C. , Lairmore Michael D. 
Published: June 2005

AbstractWhole blood lysis and fixation methods for flow cytometric (FCM) analysis were tested for their ability to reduce the infectivity of human T‐lymphotropic virus type 1 (HTLV‐I). Our goals were to: (1) determine the effects of 1.0 and 2.0% paraformaldehyde (PF) fixation on HTLV‐I infected cell lines lines and (2) assess the infectivity of blood samples containing HTLV‐I‐infected cells following processing with 5 commercially available products (Immuno‐lyse, ImmunoPrep/Q‐Prep, FACS lysis solution, GenTrak lyse and fix reagent, and Ortho‐mune lysing reagent) compared to ammonium chloride lysis with either 0.1 or 1.0% PF fixation. Infectivity was determined by monitoring HTLV‐I p24 antigen production in cocultures of treated leukocytes with uninfected peripheral blood mononuclear cells (PBMC). Each method effectively reduced the viability of treated leukocytes. Commercial lysis/fixation methods significantly reduced HTLV‐I infectivity compared to prepared ammonium chloride/PF‐based methods. For all preparations, increasing the time of fixation (e.g., 60 min) effectively reduced viral infectivity. Taken together, these data suggest that commercially available fixatives greatly reduce, but do not eliminate the risk of HTLV‐I infection during processing of viral‐infected cells for FCM analysis. © 1994 Wiley‐Liss, Inc.
Computing the central location of immunofluorescence distributions: Logarithmic data transformations are not always appropriate
Authors: Coder David M. , Redelman Doug , Vogt Robert F. 
Published: June 2005

AbstractThe idea of the “average” intensity of immunofluorescence data is often poorly defined, with such terms as average, mean, and peak used interchangeably. In addition, the common use of logarthmic amplifiers with immunofluorescence data further complicates the problem. Log amplifiers permit the display of a wider range of fluorescence intensities. At the same time, they effect a log transformation of the data. This transformation decreases the variance resulting in narrower fluorescence distributions, which are assumed to approximate normal distributions. When the log transform is used, the distribution mean is the geometric mean of the untransformed data, which is computed simply as the mean of the channel values. This mean value serves as a simple indicator of the population center. Despite the prevalence of log transformations in flow cytometry, this transformation may not yield normally distributed immunofluorescence data, whereas the square root or other fractional power transformations can yield normal distributions. © 1994 Wiley‐Liss, Inc.
Low level CD20 expression on T cell malignancies
Authors: Warzynski Michael J. , Graham David M. , Axtell Richard A. , Zakem Michael H. , Rotman Richard K. 
Published: June 2005

AbstractAlthough initially thought to be a B lineage restricted antigen, low “density” or antibody binding capacity (ABC) CD20 has recently been detected on subset(s) of normal T lymphocytes (Hultin et al.: Cytometry 14:196–204, 1993). We report low ABC CD20 expression in three (two children, one adult) cases of T‐acute lymphoblastic leukemia (T‐ALL). CD20 and other pertinent antigens were detected using a direct dual color method with a Becton Dickinson FACScan® flow cytometer and Simulset® software. Only one cell population based on light scatter was noted in each case that immunophenotypically represented almost a pure population of malignant cells expressing T lymphocyte antigens (for example, CD7 98%, 92%, and 100%, respectively). A total of 95%, 87%, and 79% of the cells from the three cases expressed CD20 with an unusual low ABC compared to the customary “bright” CD20 expression on normal B lymphocytes. Other B lymphocyte associated antigens, such as CD19, CD22, Dr, and immunoglobulin light chains, were negative. Eleven other T lymphocytic malignancies from 1991 to 1993 were CD20 negative, including three other case of T‐ALL (one adult and two children). One unusual case of intestinal small lymphocytic non‐Hodgkin's lymphoma with a natural killer/T lymphocytic immunophenotype not described in this report appeared to be CD20“dim”+. Low ABC CD20 expression by T lymphocytic malignancies may provide a more unique immunophenotypic “fingerprint” to help support the diagnosis of T cell neoplasia vs. normal/reactive T cells (for example, low ABC CD20 cells represent only 2.4 ± 1.5% of normal peripheral blood lymphocytes). This characteristic might also facilitate monitoring patients for residual or recurrent disease. The prognostic significance of CD20 co‐expression is uncertain and must await further studies. © 1994 Wiley‐Liss, Inc.
Automated HLA‐B27 testing using the FACSPrep/FACScan system
Authors: Reynolds Wendy M. , Evans Philip R. , Lane Andrew C. , Howell W. Martin , Wilson Peter J. , Wong Raymond , Smith John L. 
Published: June 2005

AbstractTissue typing can help in the diagnosis of the seronegative arthropathy ankylosing spondylitis. Using an automatic sample preparation system and flow cytometry (FACSPrep/FACScan) we have developed a test for HLA‐B27 screening using whole blood which is both rapid, reproducible, and permits simultaneous screening of large numbers of samples. We have used both indirect (248 cases) and direct (126 cases) monoclonal antibody staining techniques. Results were assessed using median channel shift (CS) from the negative control and relative fluorescence intensity. There is known cross‐reactivity between HLA‐B27 and HLA‐B7 with the monoclonal antibody (MoAb) HLA‐ABC‐m3. Using this antibody and indirect staining, HLA‐B7 samples had a significantly lower CS value than HLA‐B27 samples. In 60% of these cases there was a clear distinction between HLA‐B27 and HLA‐B7. All HLA‐B27 and HLA‐B7 negative samples had a CS of 0 (P < 0.001). Using direct dual staining with CD3 and HLA‐B27, all HLA‐B7 negative samples had a low CS value (15 maximum), HLA‐B7 samples had an intermediate CS value (20–85), and HLA‐B27 samples had the highest CS values (70 upward). This system permits large scale rapid negative screening of samples with the elimination of over 60% as negatives (CS < 15). Limitations as a definitive test for HLA‐B27 are due to MoAb cross‐reactivity with HLA‐B7 which necessitates other confirmatory techniques. The substitution of the HLA‐ABC‐m3 with the recently available HLA‐B27 specific MoAb FD705 would substantially increase the value of this technique for routine HLA‐B27 typing. © 1994 Wiley‐Liss, Inc.
Quality control in flow cytometry for diagnostic pathology: II. A conspectus of reference ranges for lymphocyte immunophenotyping
Authors: McCoy J. Philip , Overton W. Roy 
Published: June 2005

AbstractImmunophenotyping, as many other clinical assays, is interpreted only in the context of reference values obtained from healthy control individuals. While the use of these reference values, or ranges, has been commonplace in the clinical flow cytometry laboratory for well over a decade, there has been little consensus in standardizing how these values should be obtained, analyzed, or expressed. This report reviews the variables to be considered in establishing reference ranges and statistical methods which can be used. Additionally, examples are given of previously published reference ranges for a variety of specimens often submitted for immunophenotyping. © 1994 Wiley‐Liss, Inc.
Semiautomated DNA probe mapping using digital imaging microscopy: I. System development
Authors: Mascio Laura N. , Verbeek Piet W. , Sudar Damir , Kuo Wen‐Lin , Gray Joe W. 
Published: June 2005

AbstractAlgorithms have been developed to help automate the mapping of DNA sequences along metaphase chromosomes using fluorescence in situ hybridization (FISH). Custom algorithm computationally define chromosome boundaries and compute chromosomal medial axes. A dynamic regional thresholding (DRT) algorithm is described that allows reliable detection of hybridization domains, even when they differ substantially in size and intensity. Chromosomal locations are calculated by determining the fractional location of each hybridization probe along the medial axis of a metaphase chromosome relative to the short arm (FLpter). These algorithms were tested on simulated data and by analysis of the location of probes that had been previously mapped by other techniques. These algorithms allow probes to be mapped rapidly along human chromosomes with a precision of 2–3 Mb. © 1995 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
Prognostic significance of DNA aneuploidy in diffuse malignant mesothelioma
Authors: Isobe Hiroshi , Sridhar Kasi S. , Doria Raul , Cohen Felicia , Raub William A. , Saldana Mario , Krishan Awtar 
Published: June 2005

AbstractDNA ploidy of pepsin digested preparations of 48 paraffin‐embedded specimens from 19 patients with histologically confirmed malignant mesothelioma was determined by laser flow cytometry. Eight of the 19 tumors (42%) were diploid and 11 (58%) were aneuploid. Of the aneuploid tumors, only one showed multiploidy. The median survival time of the patients with diploid tumors was 19, 16, and 14 months from the onset of symptoms, diagnosis, and treatment, respectively. The median survival in patients with aneuploid tumors was 8, 7, and 7 months from the onset of first symptoms, diagnosis, and treatment. Thus, patients with diploid tumors lived longer than patients with aneuploid tumors. These results suggest that DNA ploidy analysis may be of prognostic value in malignant mesothelioma. © 1995 Wiley‐Liss, Inc.
Is reduced accumulation of Hoechst 33342 in multidrug resistant cells related to P‐glycoprotein activity?
Authors: Lahmy Sylvie , Viallet Pierre , Salmon Jean Marie 
Published: June 2005

AbstractAlthough bisbenzimidazole‐DNA interactions have been studied in solution, little information has been available in living cells. The reduced accumulation of the nuclear dye Hoechst 33342 (H342) in cells with multidrug resistant (MDR) phenotype suggested its possible use in a functional test for detection of these cells. We performed experiments to elucidate the mechanisms involved in the H342‐exclusion from resistant cells. As contradictory results have been reported in literature, we compared the entire fluorescence spectra of H342 in solution and in intact living cells under different experimental conditions. The study was performed by fluorescence image cytometry. This technique allow accurate quantification of the amount of H342 bound to DNA in living cells. The dye uptake was followed in sensitive and resistant cells, a lymphoblastoid cell line, CCRF‐CEM, and its resistant variant selected with vinblastine CEM/VLB100 under conditions that could modulate H342‐cell binding. Competition experiments with sodium azide, verapamil, and vinblastine indicated that resistant cells did not differ in the number of possible binding sites for H342. The obtained results ruled out the possibility of discriminating cells on the basis of a spectral shift. Two modes of binding, differing in their affinity for the dye, seem to co‐exist in intact cells. Although it clearly appeared that the P‐glycoprotein expressed in MDR cells was mainly responsible for the H342‐exclusion, other mechanisms might also be involved. © 1995 Wiley‐Liss, Inc.