bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of flow cytometric methods for diagnosis of chronic granulomatous disease variants under routine laboratory conditions
Authors: Emmendörffer A. , Nakamura M. , Rothe G. , Spiekermann K. , Lohmann‐Matthes M.‐L. , Roesler J. 
Published: June 2005

AbstractNeutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10 chronic granulomatous disease (CGD)‐patients (4 CGD+), and from 18 X‐linked carriers for CGD have been tested for their production of H2O2 using staining with dihydrorhodamine 123 and subsequent flow cytometry. Additionally, neutrophils from three patients with myeloperoxidase deficiency were assessed. Cells were activated to produce H2O2 by the phorbol ester phorbol‐myristate‐acetate (PMA) and by phagocytosis of Escherichia coli bacteria. To evaluate the sensitivity of the method, H2O2‐production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured. The results were compared to those from other methods (NBT‐testing, cytochrome c‐reduction, and especially chemiluminescence). Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, X = 840 ± 59 (S.D.), 97% < 890, for pediatric patients. Normal quantitative values also resulted from small blood samples of infants (< 1 year, n = 6, X = 830 ± 52). For CGD+ (n = 4) the results were clearly far below the normal range. In indicating decreased production of reactive oxygen intermediates the method was at least as sensitive as lucigenin enhanced chemiluminescence. Cytochrome b558‐expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the monoclonal antibody 7D5. The normal range was 97% > 485, 97% < 680, peak channel fluorescence. We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of cell‐surface and intracelluar antigens in leukemia diagnosis
Authors: Knapp Walter , Strobl Herbert , Majdic Otto 
Published: June 2005

AbstractNew technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro‐)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferntiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to he used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB‐1), CD79b (B29), CD87 (uPA‐R), and CD117 (c‐kit). © 1994 Wiley‐Liss, Inc.
Flow cytometric immunophenotyping of lymphocyte subsets in samples that contain a high proportion of non‐lymphoid cells
Authors: Pattanapanyasat Kovit , Kyle Dennis E. , Tongtawe Pongsri , Yongvanitchit Kosol , Fucharoen Suthat 
Published: June 2005

AbstractFlow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three‐color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71‐flurescein isothiocyanate (FITC), FSC/glyco A‐FITC, or FSC/CD45‐peridinin chlorophyl protein (PerCP) profiles. The CD71‐FITC negative cells, glyco A‐FITC negative cells, or CD45‐PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non‐lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two‐color antibodies. Of the three gating strategies, CD45‐PerCP and glyco A‐FITC methods are better than the CD71‐FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six‐tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of nonlymphoid cells. © 1994 Wiley‐Liss, Inc.
Radiation‐induced changes in nucleoid halo diameters of aerobic and hypoxic SF‐126 human brain tumor cells
Authors: Wang Jingli , Basu Hirak S. , Hu Lily , Feuerstein Burt G. , Nederlof Petra M. , Deen Dennis F. 
Published: June 2005

AbstractNucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF‐126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propidium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose‐dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony‐forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors. © 1995 Wiley‐Liss, Inc.
Can measurements of potential doubling time (Tpot) be compared between laboratories? A quality control study
Authors: Haustermans K. , Hofland I. , Pottie G. , Ramaekers M. , Begg A. C. 
Published: June 2005

AbstractThe purpose of this study was to investigate the reproducibility of potential doubling time measurements of human tumors in different laboratories and to distinguish which steps in the measurement procedure were subject to the greatest variation. This was achieved by comparing measurements on the same source material in two different laboratories in which three aspects of the technique were separately studied, namely, preparation and staining of the nuclear suspensions, running the samples on the flow cytometer (FCM), and analyzing the two‐parameter FCM data. This involved exchange between the two centers of fixed tumor material, stained nuclear suspensions, and FCM data on floppy disks. The analysis step was found to be the least variable step. For DNA synthesis time, Ts, and the labeling index, LI, the coefficients of determination (R2) ranged from 92% to 95.4%. A small systematic bias was observed, with one center measuring approximately 15% higher values for both LI and Ts. Different criteria for window placements were found to be a contributing factor. Variations in the FCM step were approximately equal to those for analysis, with no systematic deviations. Variations for the preparation and staining step were the largest (R2 = 60.5% and 38.1% for LI and Ts, respectively). However, this step was the only one subject to intratumoral variability, which was the largest contributing factor to the variations observed. In addition, however, LI was on average 41% higher in one center compared to the other, resulting in a systematic bias. Differences in the level of green fluorescence of the labeled cells implicated antibody differences as a possible cause. The variations found here for the three procedural aspects were significantly smaller than variations observed between tumors, a requirement for a predictive assay. Suggestions for implementation of quality control procedures include objective (computer‐assisted) data analysis on two‐parameter histograms and optimization of antibody combinations. © 1995 Wiley‐Liss, Inc.
Measurement of intracellular pH in mammalian sperm cells under physiological conditions
Authors: Jones Jeffrey M. , Lorton Steven P. , Bavister Barry D. 
Published: June 2005

AbstractThe goal of this study was to develop a technique for the measurement of intracellular pH (pHi) in living mammalian (bovine) sperm cells under physiological conditions. Like many other biochemical measurements, pHi measurements have typically been made under non‐physiological conditions on cells whose immediate functional status is not readily assessed and may even be non‐viable. Additionally, many pHi measurement techniques may themselves alter the pHi of the cells being measured. Such measurements could yield misinformation. The sperm cell is unique in that its functional status can be easily and continuously monitored by means of its motility, which is directly affected by pHi. In this respect, the sperm cell provides an ideal model system for evaluating pHi measurement techniques. In this article we summarize the validation of a ratiometric absorbance technique for the measurement of pHi of mammalian (bovine) sperm cells under physiological conditions which does not affect their functional status. The pHi of ejaculated bovine sperm cells was calculated to be 6.9 ± 0.05 (11 replicates). This approach may also be suitable for pHi measurements in other cell types. © 1995 Wiley‐Liss, Inc.
Image processing algorithms for the automated micronucleus assay in binucleated human lymphocytes
Authors: Böcker W. , Müller W.‐U. , Streffer C. 
Published: June 2005

AbstractThe frequency of micronuclei in binucleated lymphocytes (cytochalasin B assay) may serve as a biological dosimeter after radiation exposure. The automation of the micronucleus assay in binucleated human lymphocytes has been considerably advanced in recent years. In our studies for this purpose the detection of binucleated cells (BNCs) and the scoring of micronuclei (MN) was divided into two parts. First, detection of BNCs was feasible with low microscopic magnification (× 100). The positions of classified BNCs were stored. Second, after an automatic change of microscope objective, the stored BNCs were automatically analyzed in sequence at high microscopic magnification (× 630) for occurrence of MN. For both phases of image analysis we used empirical methods based on mathematical morphology. The system is able to recognize nearly 65% of BNCs with false positive decisions of 6% and about 75% of the MN with false positive decisions of 7%. © 1995 Wiley‐Liss, Inc.
Fluorescent erythrocyte ghosts as standards for quantitative flow cytometry
Authors: Doberstein S. K. , Wiegand G. , Machesky L. M. , Pollard T. D. 
Published: June 2005

AbstractWe report here a quick and inexpensive method for preparing standards of known fluorochrome content for calibration and quantitation of flow cytometry fluorescence signals. Erythrocyte ghosts prepared by hypotonic lysis are filled with solutions containing fluorescently labeled dextran. Standards prepared by this technique have a narrow range of fluorescence and a linear response of fluorescence to fluorochrome content up to 2 × 106 fluorochrome molecules/cell. The volume of ghost standard particles is roughly 70 femtoliters (fl)/cell. The fluorescence of ghost standards is nearly identical to that of commercially available microbead standards of similar fluorochrome content. Ghost standards have stable fluorescence for at least 3 weeks at 4°C. These standards can be made with any fluorochrome or combination of fluorochromes over a wide concentration range. © 1995 Wiley‐Liss, Inc.
Debris compensation of Dna histograms and its effect on S‐phase analysis
Authors: Wersto Robert P. , Stetler‐Stevenson Maryalice 
Published: June 2005

AbstractDebris compensation is an important variable affecting S‐phase fraction (SPF) analysis in flow cytometric DNA histograms. The SPF was estimated in fresh frozen breast carcinomas using the following four debris subtraction algorithms: modeling debris as an exponential curve (EXP); the incorporation of nuclei cut a single time into the exponential moel (EXP‐SC); the random cutting of nuclei into multiple pieces of varying sizes (MC); and a combination of both nuclear cutting models (SC‐MC). Comparison of SPF estimates indicated that the various debris subtraction models yielded differences in SPF, with SPF values obtained using the exponential model having considerable variation compared to SPF estimates from histograms where debris was modeled by algorithms based on nuclear slicing and fragmentation. However, SPF estimates could be affected by initial placement of the nuclear debris boundaries, the coefficient of variation of the G0/1 peak, and the relative amount of debris. Using the ratio of the height of the G0/1 peak to the height of the debris between the chicken red blood cells (CRBC) and G0/1 peaks as an objective measurement of nuclear debris, debris compensation was necessary in diploid DNA histograms where this ratio was as low as 1.5%. Taken in the context of SPF prognostic cutoff levels, variation in debris models and boundaries can change the classification of cases with borderline SPF levels into the poor prognostic high SPF categories, thereby making the comparison of SPF values derived from different studies difficult. © 1995 Wiley‐Liss, Inc.
New epi‐fluorescence optical system for independent analysis of two different fluorochromes in microscopy
Authors: Heiden Thomas , Tribukait Bernhard 
Published: June 2005

AbstractA new epi‐fluorescence optical system is described that uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. The optical system was constructed for analysis of two different fluorochromes, e.g., DAPI and TRITC. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm, 546 nm, 435–500 nm, and 590–750 nm), (2) wavelength switching without image displacement and blur by means of a light chopper alternating between ultraviolet‐excitation/blue‐detection and green‐excitation/red‐detection at frequencies of up to 140 Hz for observation by eye without image flicker, and (3) separate positioning of lenses for compensation of chromatic aberrations. This system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was obtained with good temporal resolution. It has been shown that it is feasable to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected. © 1995 Wiley‐Liss, Inc.
Flow cytometric analysis of apoptosis and BCL‐2 in human solid neoplasms
Authors: Steck Kim D. , McDonnell Timothy J. , El‐Naggar Adel K. 
Published: June 2005

AbstractApoptosis (programmed cell death) and the genes regulating this process (e.g., bcl‐2), have recently become a focus of interest in the study of cancer development and progression. We adapted flow cytometric techniques for measuring apoptosis and bcl‐2 protein in solid tissues and concomitantly determined both parameters in 65 malignant solid neoplasms. Four different fixation methods were evaluated for bcl‐2 analysis using a lymphoma cell line possessing a t(14;18), and the Daudi cell line as positive and negative controls, respectively; optimal fixation was achieved using 70% ethanol. Apoptosis was determined using the terminal deoxynucleotidyl transferase (TdT) end‐labeling method of (Gorczyca et al.: Cancer Res 53:1945–1951, 1993). Treated (5Gy radiation, 4 h) and untreated portions of a murine cell line were used as positive and negative controls for apoptosis induction. For tumor specimens, bcl‐2 positivity ranged from 0 to 89.5% (15.8 ± 22.9), and apoptosis (TdT end‐labeling) ranged from 0.4 to 84.3% (15.9 ± 17.0). Generally, tumors with high bcl‐2 expression (≥20.0%) showed significantly lower numbers of apoptotic cells than those with low (<20.0%) bcl‐2 (P = 0.05). A subset of tumors, however, exhibited low values for both parameters. We also observed that the proliferative fractions of tumors with high apoptosis (⩾15.0%) were significantly different from those with low (<15.0%) apoptosis (P = 0.005); higher proliferative rates were associated with high apoptosis. We conclude that optimal bcl‐2 analysis is achieved using ethanol fixation and that flow cytometry provides a rapid and reliable technique for the measurement of these parameters. Concurrent analysis of these markers provides detailed information on the biological characteristics of tumor subpopulations and may assist in categorizing tumors for different management strategies. © 1995 Wiley‐Liss, Inc.
Classification and regression trees for bone marrow immunophenotyping
Authors: Beckman Richard J. , Salzman Gary C. , Stewart Carleton C. 
Published: June 2005

AbstractMethods are needed to assist with automating three‐color flow cytometric immunophenotyping of bone marrow from leukemia patients. Described is a method in which a normal bone marrow data set is used as a template against which to compare leukemic bone marrow data sets. This template is obtained using techniques of cluster analysis and cluster editing. Leukemic cells often inappropriately express antigens and appear in a different part of the multivariate data space than normal cells. To recognize the cells exhibiting inappropriate antigen expression, an artificial cluster of “cells” is added to the normal template. The “cells” in this cluster fill the space not occupied by normal cells. Classification and regression tree (CART) analysis is used to train a classifier that can then be used to isolate the major cell types and the inappropriate expression cells in a leukemic bone marrow specimen. © 1995 Wiley‐Liss, Inc.
Flow cytometric assessment of the cellular pharmacokinetics of fluorescent drugs
Authors: Dordal Margaret S. , Ho Amy C. , Jackson‐Stone Marisa , Fu Yi Fan , Goolsby Charles L. , Winter Jane N. 
Published: June 2005

AbstractDevelopment of multidrug resistance (MDR) in cancer cells decreases net doxorubicin uptake as a result of either increased efflux, or decreased intracellular sequestration, or decreased membrane permeability. Kinetic parameters of drug uptake can distinguish among these forms of altered transport. Cellular uptake of fluorescent drugs was monitored by a flow cytometric assay using a rapid‐injection system and analyzed with a three‐compartment model in which rapid diffusion from extracellular fluid into the cell was followed by uptake into a nonexchangeable pool. In agreement with our recent studies of 14C‐doxorubicin distribution (Dordal et al.: J Pharmacol Exp Ther 271:1286–1290, 1994), sequestration of doxorubicin was decreased 2.7‐fold in P‐glycoprotein‐expressing SU‐4R lymphoma cells compared to drug‐sensitive SU‐4 cells (14.0 ± 4.8 vs. 5.0 ± 0.9 nl s−1) without a change in membrane permeability or evidence of active efflux. In contrast, sequestration of the highly fluorescent dye rhodamine 123 was decreased 20‐fold (17.1 ± 8.3 vs. 0.9 ± 0.8 nl s−1). Resistant cells were significantly less permeable to rhodamine than sensitive cells (3.8 ± 1.2 vs. 10.2 ± 2.6 × 105 cm2 s−1), and rhodamine efflux was increased by 24%. Thus, SU‐4R cells exhibit multiple alterations that cause decreased intracellular drug concentrations, of which decreased sequestration is quantitatively the most significant. © 1995 Wiley‐Liss, Inc.
Heterogeneity of the proliferative fraction and cyclin D1/CCND1 gene amplification in head and neck squamous cell carcinoma
Authors: El‐Naggar Adel K. , Steck Kim , Batsakis John G. 
Published: June 2005

AbstractHigh proliferative fraction and cyclin D1/CCND1 gene amplification have been associated with certain aggressive features of head and neck squamous carcinoma in some studies, but not in others. The differences may be related to the intratumoral heterogeneity of these factors. Moreover, the interrelationship between these seemingly related factors has not been determined. In order to determine the correlation between tumor proliferative fractions and CCND1 gene amplification, 3 spatially different samples from each of 32 primary head and neck squamous cell carcinomas (HNSCC) were separately analyzed by flow cytometry. A mixture of these specimens was also minced and snap frozen for molecular studies. DNA was extracted from patient lymphocytes, normal appearing squamous epithelium, and tumors. A genomic DNA probe containing the first exon of CCND1 was used for hybridization by Southern technique. A 5.6 kb genomic DNA probe of immunoglobulin heavy chain was used as an internal standard for quantification of CCND1 gene amplification. Our results showed that 30% of the tumor cases manifested intratumoral heterogeneity (>50% differences) of their proliferative activity. The highest value was used for correlation with gene amplification. Eleven (34.4%) of the 32 tumors showed CCND1 amplification (2–10‐fold). When the proliferative fraction was dichotomized into high and low groups based on the mean value (≥13%), a highly statistical correlation between CCND1 amplification and tumor proliferation was obtained (P < 0.001). No significant correlation between gene amplification and other clinicopathologic parameters was noted. Our study indicates that (1) intratumoral heterogeneity of the proliferative fraction is a feature of HNSCC and (2) CCND1 gene amplification correlates significantly with high proliferation and may be associated with the tumorigenesis of a subset of these neoplasms. © 1995 Wiley‐Liss, Inc.