bluuebunny/crossref_metadata_2025_split huggingface dataset:
Factors underlying the variability of lipid droplet fluorescence in MA‐10 leydig tumor cells
Authors: Gocze Peter M. , Freeman Dale A. 
Published: June 2005

AbstractNeutral lipids accumulate in cellular lipid droplets. These droplets vary remarkably in number and amount between cells. In the present studies, the variability in lipid content was quantified by comparing the coefficient of variation of fluorescence histograms of nile red lipid‐stained cells to the variability of cell size or cell protein distributions. This measure of lipid droplet variability persisted through a wide range of cell lipid content and averaged 4.4‐fold more variability than cell size and 2.6‐fold more variability than cell protein content. While looking for possible explanations for this variability, it was determined that cell to cell variability could not be explained by multiple clonal populations of cells or the confluence of the cell monolayer. Analysis of lipid variability using a more droplet‐specific fluorescent dye, bodipy, reduced variability by about 44%. Cell cycle analysis revealed that G2 + M cells contained more lipid than S‐phase cells, which in turn contained more lipid than G0 + G1 cells, but that variability was equally large throughout the cell cycle. The cholesteryl ester hydrolase inhibitor, diethylumbelliferyl phosphate, inhibited hydrolysis of both cholesteryl esters and triglycerides. Lipid content of diethylumbelliferyl phosphate‐treated cells increased while the variability in lipid staining decreased by an average of 72%. Thus, the excess lipid fluorescence variability compared to cell size or protein fluorescence could in part be explained by variability in cellular hydrolysis of triglyceride and cholesteryl ester. Excess lipid fluorescence variability could be reduced by an average of 44% when a more lipid droplet‐specific stain was used instead of nile red. © 1994 Wiley‐Liss, Inc.
Classification of red blood cells as normal, sickle, or other abnormal, using a single image analysis feature
Authors: Wheeless Leon L. , Robinson Roy D. , Lapets Oleg P. , Cox Christopher , Rubio Ana , Weintraub Michael , Benjamin Lennette J. 
Published: June 2005

AbstractSickle cell anemia is a disease for which there is currently no effective treatment. One method of evaluating clinical status is the counting of cell types based on morphology. There is a need for a rapid, reproducible method, superior to human inspection, for classification of these cells. Quantitative digital‐image analysis is being applied to this need. Blood from 24 patients with sickle cell anemia (SS) and SC disease and ten hematologically normal volunteers (AA) was stressed by bubbling with nitrogen. One hundred fifty cells were analyzed from each sickle specimen, and 100 were analyzed from each nonsickle specimen. Expert observers classified each cell as normal (N), sickle (S), or other abnormal (A). Cells were analyzed with a custom, high‐resolution image‐analysis instrument. A total of 42 features including metric, optical density‐derived, and textural features were extracted. The metric feature Form Factor (4πArea/Perimeter2) was selected by recursive partitioning analysis as the sole feature needed for segregating cells into the classes of N, A, and S. The agreement of automated classification (using cutpoints determined by recursive partitioning analysis) with a human expert for specimens from individuals with sickle cell anemia was 89% for N‐, 73% for A‐, and 92% for S‐classified cells. For specimens from individuals with sickle cell anemia, rates of agreement between two human experts were compared and found to be 86% for N, 84% for A, and 80% for S. For specimens from AA individuals, the agreement was 90% for N and 87% for A. © 1994 Wiley‐Liss, Inc.
Flow cytometry distinction between adherent and phagocytized yeast particles
Authors: Giaimis Jean , Lombard Yves , Poindron Philippe , Muller Christian D. 
Published: June 2005

AbstractOur laboratory recently developed a light microscopy staining technique that provides a mean to distinguish between yeast that are simply bound to the surface of macrophages and yeast that have actually been phagocytized by macrophages (7). We adapted this technique by using fluorescent probes in order to test phagocytic activity by flow cytometry. Thus we are able to distinguish unambiguously extracellular from intracellular yeast during phagocytosis with the fast rate of flow cytometry (∼200 cells/s). The fluorescence quenching induced by a 1% tannic acid solution (w/v) can be applied to any FITC‐labeled, heat‐killed yeast cell or bacteria. The yeast cells already engulfed in the macrophage remain with their native fluorescence (internal and external pH equilibrated by 50 μ monensin 30 min/4°C) protected from the action of tannic acid, a nonmembrane permeable molecule. The results presented here validate this new technique. An application is presented showing the inhibition of endocytosis by cytochalasin‐B. © 1994 Wiley‐Liss, Inc.
Detection of immunocytochemically stained rare events using image analysis
Authors: Mesker Wilma E. , v.d. Burg Marja J. M. , Oud Peter S. , Knepfle Cecile F. H. M. , Ouwerkerk‐v. Velzen Maria C. M. , Schipper Nellie W. , Tanke Hans J. 
Published: June 2005

AbstractThe detection of rare‐event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation.SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS‐red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counterstaining based on ethyl green. Slides were automatically screened for cytokeratin‐positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 × 106 negative cells. A comparison between manual screening and automated screening for cytokeratin‐positive cells showed a high level of correlation (0.9998).For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study.It is concluded that optimization of preparation and staining procedures for the detection of rare‐event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events. © 1994 Wiley‐Liss, Inc.
Methodologies for the preservation of proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry
Authors: Mikulka Warren R. , Bolton Wade E. 
Published: June 2005

AbstractThe retention of antigen expression of PCNA, p120, and p105 in two tumor cell lines (MOLT‐4 and MDA‐MD‐175‐VII) under various conditions of fixation was investigated using flow cytometric analysis. Four currently utilized procedures for fixation/permeabilization of intracellular antigens were compared for their ability to stain the nuclear antigens. A procedure using a brief incubation in a solution of lysolecithin in paraformaldehyde followed by fixation in ice‐cold methanol prior to antibody staining was selected to evaluate reagent protocols aimed at preserving antigen expression. Holding samples overnight at 4°C in 2.5% fetal bovine serum after the lysolecithin/paraformaldehyde and methanol fixation steps prior to staining with monoclonal antibodies resulted in no decline in the percentage of cells positively stained for all three markers with little decrease in intensity of fluorescence and no increase in DNA coefficient of variation (c.v.). Fixed/permeabilized MOLT‐4 cells held longer than 24 h before staining were lower in PCNA fluorescence than freshly stained cells; holding samples of either cell line longer than 48 h resulted in decreased PCNA and p120 staining. Prefixing and holding cells in 50% methanol or 50% ethanol overnight before processing and staining severely depressed PCNA and p120 fluorescence. Prefixing either cell line in a range of concentrations (0.25–1.0%) of paraformaldehyde also resulted in reduced intensity of PCNA and p120 fluorescence along with increased DNA c.v. P105 staining appeared to be relatively unaffected by all prefixation/storage conditions tested, except for a decline of fluorescence when MDA cells were prefixed in 50% ethanol. Cells cryopreserved in liquid nitrogen for 1 week before processing showed <5% loss of PCNA and p120 fluorescence compared to freshly processed cells, but p105 fluorescence dropped 29% in cryopreserved MDA cells. These results underscore the fact that specific protocols for the fixation and storage of biological samples prior to staining and analysis must be determined for the specific nuclear antigen marker under investigation. © 1994 Wiley‐Liss, Inc.
Bladder irrigation specimens assayed by fluorescence in situ hybridization to interphase nuclei
Authors: Wheeless L. L. , Reeder J. E. , Han R. , O'Connell M. J. , Frank I. N. , Cockett A. T. K. , Hopman A. H. N. 
Published: June 2005

AbstractBladder irrigation specimens provide a sampling of the entire bladder urothelium and are the most practical sample for longitudinally monitoring patients. This study presents cross‐sectional fluorescence in situ hybridization (FISH) analyses with correlated DNA cytometry data on 76 patients monitored for recurrent bladder tumors. FISH probes complementary to centromeric satellite sequences for chromosomes 1, 7, 9, 11, 15, and 17 were used. Aberrations in copy number were observed for chromosomes 1, 7, 11, and 17 principally in patients with aneuploid tumors. Monosomy of chromosome 9 was observed in 39% of the diploid and 31% of the specimens with high hyperdiploid fraction. Significantly, 24% of patients with a history of bladder cancer but with no clinical evidence of disease exhibited monosomy of chromosome 9. This suggests a persistent and significantly large population of abnormal cells in the absence of clinical evidence of disease. Loss of chromosome 9 relative to DNA ploidy was observed in 24% of patients with no evidence of disease, in 59% of patients with tumor, and in 79% of patients with histologically confirmed transitional cell carcinoma, grades 1–3. Loss of chromosome 15 was also observed in a large percentage of patients. Loss of chromosome 15 was observed in 41% of specimens from patients in whom no tumor was seen, in 38% of specimens from patients with tumor, and in 67% of specimens from patients with histologically confirmed transitional cell carcinoma. Results of this study document the use of bladder irrigaton specimens as a specimen source for FISH analyses. The ability to perform FISH analyses on bladder irrigation specimens from patients followed for recurrent bladder cancer is important in that it offers the potential of studying early genetic events as well as correlating specific aberrations with progression and recurrence. © 1994 Wiley‐Liss, Inc.
A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique
Authors: Orfão Alberto , García‐Sanz Ramón , López‐Berges M. Consuelo , Vidriales M. Belén , González Marcos , Caballero M. Dolores , San Miguel Jesüs F. 
Published: June 2005

AbstractIn the present paper a CD38/propidium iodide double staining technique is described which separately assesses the cell cycle distribution of myelomatous plasma cells from that of the residual normal hemopoietic cells. For this purpose, bone marrow (BM) cells from a group of 42 untreated multiple myeloma patients were analyzed. Of these, 23 cases were aneuploid (55%) and 19 diploid (45%). The use of the CD38/propidium iodide double staining method allowed a clear separation between CD38 strong positive cells from the remaining bone marrow populations, cells sorting experiments confirming that plasma cells were almost exclusively contained in the former fraction where they represented 97 ± 2% of the total cells sorted. In all cases, the S‐phase in plasma cells and in the remaining normal hemopoietic bone marrow cells was assessed, being higher in normal hemopoietic cells (8.0 ± 6.3%) than in plasma cells (3.3 ± 2.6%, P < 0.002). In addition, there was no correlation between the S‐phase of the neoplastic and normal bone marrow cells (r = 0.22; P > 0.10); this work therefore shows that the assessment of the total proliferative rate of bone marrow samples does not reflect either the proliferation of normal cells or that of neoplastic plasma cells but will depend on the proliferative rate and the percentage of each population within the sample, which can be assessed by the technique described here. © 1994 Wiley‐Liss, Inc.
Constitutive expression of P‐glycoprotein as a determinant of loading with fluorescent calcium probes
Authors: Brezden Christine B. , Hedley David W. , Rauth A. Michael 
Published: June 2005

AbstractDetermination of intracellular calcium levels in Chinese hamster ovary (CHO) cells using the fluorescent calcium probe indo‐1AM was hindered by the low level of accumulation of indo‐1 in these cells. CHO cells are known to express basal levels of the multidrug resistance efflux pump P‐glycoprotein (P‐gp). Rhodamine‐123, which is a known substrate of P‐gp, was used to confirm the presence of P‐gp in CHO cells. Verapamil and cyclosporin (CsA), both inhibitors of P‐gp, enhanced accumulation of indo‐1 in these cells and therefore allowed for improved intracellular calcium measurements. P‐gp overexpressing colchicine‐resistant CHO cells (CHRC5) also displayed enhanced indo‐1AM loading with P‐gp inhibitors. Nondetectable levels of P‐gp activity were found in wild‐type CEM‐CCRF cells (human T lymphoblasts), and these cells did not show any difference in indo‐1AM loading in the presence or absence of P‐gp inhibitors. Loading of a second calcium fluorescent probe fluo‐3AM was improved in CHO cells by P‐gp inhibition, whereas the structurally related pH probe BCECF‐AM was minimally affected. Because low levels of P‐gp may be expressed by a range of cell lines and normal tissues, it is suggested that this be considered if difficulties are encountered in loading fluorescent calcium probes. © 1994 Wiley‐Liss, Inc.
Cytogenetic analysis by chromosome painting
Authors: Carter Nigel P. 
Published: June 2005

AbstractChromosome painting is a term used to describe the direct visualisation using in situ hybridisation of specific chromosomes in metaphase spreads and in interphase nuclei. Chromosome painting, coupled with fluorescence in situ hybridisation (FISH), is now used routinely to enhance the identification of chromosomal rearrangements, the assignment of breakpoints, and the determination of the origin of extra chromosomal material. Amplification of small numbers of flow‐sorted chromosomes by the polymerase chain reaction allows labelled chromosome paints to be generated in a matter of days. These technologies have enabled the development of reverse chromosome painting, in which the paint is produced from sorted aberrant chromosomes and hybidised back onto normal metaphase spreads to identify directly the composition of the aberrant chromosome. Reverse chromosome painting is able to identify not only the chromosomal origin of marker chromosomes but also the regions and breakpoints involved. In some cases, such as interstitial translocations and complex marker chromosomes, the combination of conventional (forward) chromosome painting and reverse chromosome painting combine to provide a definitive analysis of the rearrangement. With the availability of chromosome paints and painting kits from a variety of commercial sources, multicolour chromosome painting has now become a routine method of analysis in the clinical cytogenetic laboratory. © 1994 Wiley‐Liss, Inc.
Immunophenotyping specimens from HIV‐infected persons: Laboratory guidelines from the Centers for Disease Control and Prevention
Authors: Nicholson Janet K. A. 
Published: June 2005

AbstractThe Centers for Disease Control and Prevention has published laboratory guidelines for enumerating CD4+ T‐cells from human immunodeficiency virus (HIV)‐infected persons to help improve the quality of lymphocyte immunophenotyping. New data that clarify some of the variables in the process of immunophenotyping have been obtained. These include information relevant to both hematology and immunophenotyping procedures as well as quality control parameters that are important in producing reliable results. © 1994 Wiley‐Liss, Inc.
Applications of cytometry to study acute leukemia: In vitro determination of drug sensitivity and detection of minimal residual disease
Authors: Campana Dario 
Published: June 2005

AbstractModern clinical applications of cytometry include the determination of the most powerful antileukemic drugs in each patient at the time of diagnosis and the monitoring of residual disease during and off treatment. The precision of in vitro assays to test the susceptibility of cancer cells to cytotoxic drugs depends on the ability to maintain the cells' viability in culture. We found that bone marrow‐derived allogeneic stromal cells are critical to prevent death by apoptosis of acute lymphoblastic leukemia (ALL) cells. Thus, we devised an in vitro drug sensitivity assay in which ALL cells are seeded onto stromal cells and viable leukemic cells are counted at the end of cultures by flow cytometry. Our preliminary results indicate that this assay is suitable for evaluating the drug sensitivity of leukemic lymphoblasts and testing the antileukemic activity of potentially effective compounds which have not yet been administered to patients with ALL. The identification of immunophenotypes expressed on leukemic cells but absent or extremely rare among normal hematopoietic progenitors allows close monitoring of the effects of drug treatment in vivo. Phenotypes that afford a detection level of 1 leukemic cell among 10,000 normal bone marrow cells have been identified in 90% of cases of T‐ALL, 25% of B‐lineage ALL, and 40% of acute myeloid leukemia (AML). In several studies, residual disease emerging during continuation therapy or off treatment almost invariably anticipated overt relapse by 1–7 months. These data indicate the reliability of immunologic techniques to detect occult leukemia. The combined application of drug sensitivity testing and residual disease monitoring may improve the accuracy of clinical management practices, and, ultimately, lead to improved cure rates. © 1994 Wiley‐Liss, Inc.
Cell subset (CS) parameter to record the identities of individual cells in flow cytometric data
Authors: Redelman Doug , Coder David M. 
Published: June 2005

AbstractThe Flow Cytometry Standard (FCS) for cytometric data (Dean et al.: Cytometry 11:323–332, 1990) provides for appending an ANALYSIS section to a data file, but it does not explicitly provide for recording the identities of individual cells. We propose an extension to the FCS definition in order to record the identity of each individual cell in list mode data. In order to do so, one first defines the subpopulations of cells to be identified and one then assigns a number to each defined population. For example, in an analysis in which peripheral blood mononuclear cells are labeled with antibodies to CD3, CD4, and CD8, the “negative” lymphocytes that are labeled with none of the antibodies could be identified as population 1, the CD3−CD4−CD8+ lymphocytes as population 2, etc. As the measured values from each cell are analyzed, the number that identifies the population to which that cell belongs is assigned as the value of an additional parameter. Since this procedure merely adds a new parameter, the only necessary extension to the FCS specification is that a particular name is recognized for this parameter. We propose that CS (abbreviation for cell subset) be recognized as the name and that CS be used as the value for the $PnN (parameter name) keyword in the FCS file TEXT. The CS values that are created can be used to aid in data analysis and can be permanently recorded in a conventional FCS file. A data file that is saved with CS values includes an explicit and integral record of the complete analysis, regardless of the complexity of the analysis. We describe how to create and record the CS parameter, provide examples of how the CS parameter can be used to assist in data analysis, and propose ways in which the CS parameter can be used to monitor quality control © 1994 Wiley‐Liss, Inc.
Detection of intracellular lymphoid differentiation antigens by flow cytometry in acute lymphoblastic leukemia
Authors: Sartor Mary , Bradstock Kenneth 
Published: June 2005

AbstractThe value of flow cytometric detection of the intracellular lymphoid differentiation antigens CD3 and CD22 in the differential diagnosis of acute leukemia was assessed in cases of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and leukemic cell lines. Cells were fixed in 0.25% paraformaldehyde at 4°C for 60 min, permeabilized with 0.2% Tween 20 at 37°C for 15 min, then stained with CD3 or CD22 monoclonal antibodies by indirect immunofluorescence. Cytoplasmic CD22 was detected on greater than 20% (mean 55%; range 20–87%) of blasts from all 20 cases of precursor‐B ALL analyzed. The percentage of cells with cytoplasmic CD22 was greater than that with membrane CD22 in all except 2 cases of precursor‐B ALL. Cytoplasmic CD22 was not detected in 8 cases of precursor‐T ALL, 4 T‐leukemia cell lines, or in 7 cases of AML. In contrast, cytoplasmic CD3 was detectable by flow cytometry in all 8 cases of precursor‐T ALL, but not in precursor‐B ALL, pre‐B leukemia cell lines, or in AML. These results confirm that cytoplasmic CD3 and CD22 are excellent markers of the early T and B lineages in ALL and can be reliably detected by flow cytometry. This technique should be a valuable addition to routine immunophenotyping for classification of acute leukemia. © 1994 Wiley‐Liss, Inc.
Hardware and software requirements for quantitative analysis of comparative genomic hybridization
Authors: du Manoir Stanislas , Kallioniemi Olli‐P. , Lichter Peter , Piper Jim , Benedetti P. A. , Carothers A. D. , Fantes J. A. , García‐Sagredo J. M. , Gerdes T. , Giollant M. , Hemery B. , Isola J. , Maahr J. , Morrison H. , Perry P. , Stark M. , Sudar D. , van Vliet L. J. , Verwoerd N. , Vrolijk J. 
Published: June 2005

AbstractRecommendations are made for hardware and software capabilities that will permit a level of performance of comparative genomic hybridization (CGH) analysis on metaphase chromosomes that is comparable to the best current practice. Guidelines for interpreting the results of CGH analysis in terms of chromosomal gains or losses are also presented. © 1995 Wiley‐Liss, Inc.