bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bivariate cytokeratin/DNA flow cytometric analysis of paraffin‐embedded samples from colorectal carcinomas
Authors: Leers M. P. G. , Theunissen P. H. M. H. , Schutte B. , Ramaekers F. C. S. 
Published: June 2005

AbstractAdmixture of normal and neoplastic cells is a serious problem in the evaluation of tumor cell kinetic parameters by flow cytometry, in particular for DNA diploid tumors. The admixture of non‐neoplastic cells, such as stromal cells and inflammatory cells, can disturb the estimation of the proliferative tumor fraction. This problem has been addressed in fresh tumor samples by applying bivariate flow cytometric analyses for DNA and cytokeratin. We have adapted this approach for formalin‐fixed and paraffin‐embedded tissue samples of colorectal carcinomas. After preparation of a single cell suspension from paraffin blocks by means of an enzymatic digestion step, the cells of epithelial origin were selectively stained with a panel of subtype specific cytokeratin antibodies. DNA analysis could thus be performed on the cytokeratin‐positive cells. The proliferative fractions of the paraffin‐embedded samples could be compared with those of the fresh tissue samples and a very good correlation was seen between DNA indices from fresh and paraffin‐embedded material. As expected, after gating on the cytokeratin‐positive cells an enrichment of the S‐phase fraction was seen compared with the ungated cell population. However, this enrichment was more pronounced in the cell suspensions derived from the paraffin‐embedded part of the tumor compared with the fresh disaggregated, ethanolfixed part of the tumor.
In situ hybridization to chromosomes stabilized in gel microdrops
Authors: Nguyen Bao‐Tram , Lazzari Kristina , Abebe Joseph , Mac Ivan , Lin James B. , Chang Albert , Wydner Karen L. , Lawrence Jeanne Bentley , Cram L. Scott , Weier Heinz‐Ulrich , Weaver James C. , Bradley David W. 
Published: June 2005

AbstractConventional chromosome in situ hybridization procedures rely on fixation to glass slides followed by microscopic evaluation. This report describes the development of a microdrop in situ hybridization (MISH) method which facilitates hybridization to chromosomes in suspension. Chromosomes encapsulated in gel microdrops (GMDs) composed of an agarose matrix withstood stringent hybridization and denaturation conditions. Because of the increased stability, hybridization to encapsulated chromosomes was detected by flow cytometry as well as conventional microscopy. Thus, the MISH method offers a means for chromosome hybridization without slides and may enable identification and isolation of chromosomes using hybridization rather than nucleic acid binding dyes. © 1995 Wiley‐Liss, Inc.
Characterization of N‐(6‐methoxy‐8‐quinolyl)‐p‐toluenesulfonamide for the detection of zinc in living sperm cells
Authors: Andrews Jane C. , Nolan John P. , Hammerstedt Roy H. , Bavister Barry D. 
Published: June 2005

AbstractZinc stabilizes membranes and DNA and inhibits respiration in somatic cells. It is present in high concentrations in the male reproductive tract and may stabilize spermatozoa prior to fertilization. Herein, we evaluate N‐(6‐methoxy‐8‐quinolyl)‐p‐toluenesulfonamide (TSQ) for analysis of Zn2+ in phosphatidylcholine (PC) vesicles and living spermatozoa using spectrofluorometry and flow cytometry. TSQ: Zn fluorescence in decanol or PC vesicles was compared to that in aqueous buffer. Evaluation of cation specificity, kinetics of TSQ: Zn binding, quenching of TSQ by dithionite and Zn2+ chelation by D‐penicillamine established that TSQ is more fluorescent in decanol or PC vesiles than in aqueous buffer, has a high affinity for lipid bilayers and is specific for Zn2+ compared to Mg2+ and Ca2+. Fluorescence measurement of vesicles with and without pretreatment with Zn2+ indicated that, in the absence of Zn2+, 90% of the residual TSQ fluorescence was destroyed by dithionite but >50% was protected by the presence of Zn2+. When D‐penicillamine was added the remaining fluorescence was quenched (T1/2 = 10 s) indicating that TSQ remains in/on the membrane. These results established that TSQ can be used to effectively evaluate Zn2+ in artificial membranes and sperm cells. Additional experiments will be necessary to explain the dynamics of TSQ: Zn: membrane interactions. © 1995 Wiley‐Liss, Inc.
Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content
Authors: Schutte Bert , Tinnemans Monique M. F. J. , Pijpers Gio F. P. , Lenders Marie‐Hélène J. H. , Ramaekers Frans C. S. 
Published: June 2005

AbstractThe analysis of the cell cycle distributions by univariate flow cytometric DNA measurement has been widely applied in the clinic to determine kinetic parameters of human malignancies. A common problem with measurements of cell cycle phase distributions in tumor biopsy material is the presence of non‐malignant diploid cells. Furthermore, such a static measurement might not be accurate enough to describe the dynamic process of cell proliferation. For this purpose alternative methods have been developed to include BrdUrd incorporation or the presence of intrinsic proliferation associated markers such as PCNA or Ki67‐Ag into the analysis. However, the presence of nonmalignant diploid cells will influence also these bivariate analyses, especially in case of DNA‐diploidy of the tumor cells.Here we present a three parameter flow cytometric assay based on the simultaneous detection of cytokeratin, DNA and a proliferation associated marker, such as BrdUrd, PCNA or Ki67‐Ag. Based on the presence of cytokeratin, epithelial cells can be selected for a detailed cell cycle analysis. This method can be applied to frozen tissue, which makes this assay useful for multicentre clinical studies. © 1995 Wiley‐Liss, Inc.
S‐Phase cells of the lymphoplasmocytic compartment in hyperdiploid multiple myeloma are diploid cells
Authors: Haraldsdóttir Vilhelmina , Haanen Clemens , Kalsbeek‐Batenburg Ellen , Olthuis Fred 
Published: June 2005

AbstractIn vivo S‐phase cell labeling with iododeoxyuridine (IdUrd) was performed in six multiple myeloma (MM) patients. Myeloma cells from four patients were hyperploid. In three out of four patients, DNA/IdUrd flow cytometry revealed that most of the labeled cells, which had divided during the period, elapsed between flash labeling and sampling, had returned to the diploid G0/G1 compartment and not to the hyperdiploid peak. To eliminate contaminating cells belonging to the normal hematopoiesis, plasmocytic and lymphocytic cells were fractionated and analyzed separately. Cell enrichment was performed with use of murine monoclonal antibodies (MoAbs) against plasmocytic and lymphocytic cell markers and subsequent magnetic activated cell sorting with immunobeads, i.e., polysterene magnetic particles coated with sheep anti‐mouse IgG. The purified plasmocytic cells were mainly hyperdiploid and largely unlabeled. The lymphocytic cells appeared to belong chiefly to the diploid cell population. The IdUrd‐labeled cells were predominantly lymphocytic cells, returning after mitosis to the diploid G0/G1 peak. Although this pattern of S‐phase cells in hyperdiploid MM, belonging to the diploid cell compartment, was observed in three out of four hyperploid cases and although the number of observations is small, S‐phase cells may demonstrate an aspect of tumor cell kinetics in hyperploid MM, which has been debated for many years and which indicates the existence of a non‐plasmocytic stem cell compartment that feeds the plasmocytoma. The behavior of the labeled cells as observed in a few cases of MM provides another, hitherto undescribed, argument that, at least in some MM patients, a part of the proliferating tumor cells may be diploid lymphocytic (precursor) cells. These findings should be considered when targeting and monitoring treatment of MM and also in purging procedures of bone marrow in patients to be treated by ablative cytotoxic therapy and autologous bone marrow transplantation. © 1995 Wiley‐Liss, Inc.
Discrimination of apoptotic thymocytes by forward light scatter
Authors: Ormerod M. G. , Paul F. , Cheetham M. , Sun X.‐M. 
Published: June 2005

AbstractThe discrimination of apoptotic and normal rat thymocytes by forward‐angle light scatter in the UV dependend on the angle over which the scattered light was collected. To achieve good separation, the light needed to be collected over a narrow angle. It was also observed that apoptotic thymocytes gave less forward light scatter than normal cells, whereas, under identical conditions, apoptotic cells from a murine cell line gave more light scatter than their normal counterparts. We suggest that the forward light scatter of apoptotic cells is affected by several factors, including changes in cell size and in the internal structure of the cell. © 1995 Wiley‐Liss, Inc.
Aequorea green fluorescent protein analysis by flow cytometry
Authors: Ropp J. Dezz , Donahue Christopher J. , Wolfgang‐Kimball David , Hooley Jeffrey J. , Chin James Y. W. , Hoffman Robert A. , Cuthbertson R. Andrew , Bauer Kenneth D. 
Published: June 2005

AbstractThe isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263:802–805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663–664, 1995) have generated a GFP mutant (serine‐65 to threonine; S65T‐GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild‐type GFP (wt‐GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild‐type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T‐GFP relative to all other laser line/GFP pairs. The wt‐GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt‐GFP and S65T‐GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm.
Reutilization of previously hybridized slides for fluorescence in situ hybridization
Authors: Epstein Lucy , DeVries Sandy , Waldman Frederic M. 
Published: June 2005

AbstractApplication of fluorescence in situ hybridization (FISH) to clinical material is sometimes limited by sample size. In addition, heterogeneity among slides prepared from a single sample may lead to variation in FISH analyses. Reutilization of material for repeated FISH analyses would help to alleviate these problems. We have developed a simple procedure for repeated FISH analyses with directly conjugated probes. Previously hybridized probes are removed by incubation in denaturing solution, and slides can then be rehybridized without residual signals remaining. Several cycles of this procedure allow a full complement of chromosomal loci to be analyzed on the same population of cells. Advantages of this protocol include gaining more cytogenetic information from small samples and eliminating the problem of intratumor variability. © 1995 Wiley‐Liss, Inc.
Idiopathic CD4 + T‐lymphocytopenia: Analysis of a patient with selective IgA deficiency and no evidence of HIV infection
Authors: Vertes Dianne , Linden Michael D. , Carey John L. 
Published: June 2005

AbstractIdiopathic CD4+ T‐lymphocytopenia (ICL) in HIV‐seronegative patients is a newly described, rare entity. The common underlying abnormality is a usually stable depletion in CD4+ lymphocytes in patients, some of which have unexplained opportunistic infections. We present a previously unreported condition of an asymptomatic individual with CD4+ T‐lymphocytopenia and a selective lgA deficiency. The subject is a 35‐year‐old healthy white male with a documented 5‐year history of low CD4+ T cell counts. He has been repeatedly HIV seronegative and has no risk factors for HIV infection. Data were obtained from several laboratories over a 5‐year period and include standard WBC differentials, HIV testing, serum immunoglobulin quantitation, mitogen stimulation assays, diphtheria and tetanus antitoxin titers, and flow cytometric immunophenotyping. The composite results show a subject with a normal white blood cell count, an absolute lymphopenia, a slight granulocytosis, and a selective lgA deficiency. Leukocyte subset analyses show essentially normal B but significantly altered T cell phenotypes. The normal CD4:CD8 ratio shows extreme inversion, primarily due to CD4 T‐lymphocytopenia. © 1995 Wiley‐Liss, Inc.
Assessment of aerosol containment on the elite flow cytometer
Authors: Ferbas John , Chadwick Karen R. , Logar Alison , Patterson Anne E. , Gilpin Richard W. , Margolick Joseph B. 
Published: June 2005

AbstractBiohazardous aerosols generated during cell sorting have been of increased concern recently because of interest in sorting specimens containing human immunodeficiency virus type 1 (HIV‐1). Current flow cytometers have features designed to contain such aerosols within the sorting chamber, but the efficacy of these features has not been established. Therefore, we tested aerosol containment by two ELITE flow cytometers (Coulter Cytometry, Inc., Hialeah, FL) during sorting of specimens containing high titers of bacteriophage. Agar plates confluent with susceptible Escherichia coli were used to detect infectious units released from the sorting chamber. Under recommended operating conditions very few infectious units were released from the sorting chambers. Release increased when the center stream was not optimally collected in a vacuum‐exhausted tube or the chamber door was not completely closed. Failure of the negative pressure and high efficiency particle air (HEPA) filtration features had less of an effect. The data indicate that these standard safety features provide a rational expectation of safety for the flow cytometry operator. © 1995 Wiley‐Liss, Inc.
Comparison of monoclonal antibodies for flow cytometric analysis of HLA‐B27 antigen
Authors: Ward Anne M. , Nikaein Afzal 
Published: June 2005

AbstractHLA‐B27 is an antigen associated with the disease ankylosing spondylitis. Ninety percent of Caucasians with ankylosing spondylitis possess the HLA‐B27 antigen. However, only 20% of Caucasians with the HLA‐B27 antigen will develop the disease. Defining the presence or absence of the HLA‐B27 antigen can be helpful in differentiating ankylosing spondylitis from juvenile rheumatoid arthritis. In this study, we evaluated the application of two monoclonal antibodies (MoAbs), using flow cytometric analysis for the detection of HLA‐B27 antigen. After an initial comparison of HLA‐B27 analysis by flow cytometry to the standard microlymphocytotoxicity assay, cutoffs were established to differentiate HLA‐B27 positive from HLA‐B27 negative. One MoAb showed very reliable results with >99% accuracy in discriminating HLA‐B27 positive from HLA‐B27 negative samples. Various parameters were investigated to obtain the optimum results and showed that incubation time, reagent lot, and the flow cytometric instrument can affect the results. We concluded that a reliable MoAb and flow cytometry are valuable for the rapid and inexpensive determination of HLA‐B27 typing in the clinical setting. However, testing conditions can affect the accuracy of results; therefore, adequate parallel testing in various conditions must be performed in order to establish the proper standards. © 1995 Wiley‐Liss, Inc.
Reproducibility in DNA flow cytometric analysis of breast cancer: Comparison of 12 laboratories' results for 67 sample homogenates
Authors: Baldetorp Bo , Bendahl Pär‐Ola , Fernö Mårten , Alanen Kalle , Delle Ulla , Falkmer Ursula , Hansson‐Aggesjö Britt , Höckenström Thomas , Lindgren Anders , Mossberg Lotta , Nordling Stig , Sigurdsson Helgi , Stål Olle , Visakorpi Tapio 
Published: June 2005

AbstractFlow cytometric (FCM) DNA analysis yields information on ploidy status and the S‐phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra‐ and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non‐diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples when tetraploidy was considered [i.e., euploid (diploid + tetraploid) vs. aneuploid (the remaining non‐diploid)]. For the SPF, pairwise comparison of the results of all 12 laboratories yielded a mean Spearman's rank correlation of 0.78 (range: 0.54–0.93). For those 39 samples being categorized in low or high SPF by all laboratories, all agreed in 14 samples (36%). Similar patterns were obtained with kappa measures, agreement being good for ploidy status (diploid vs. non‐diploid; overall K = 0.87 and 0.74 for euploid vs. aneuploid), but moderate for the SPF [overall k = 0.47 (for low SPF vs. high SPF vs. “no SPF reported”)]. Discrepancies were chiefly attributable to differences in the categorization of the S‐phase values, rather than in FCM procedures, other critical differences being in the detection and interpretation of near‐diploid and small non‐diploid cell populations, the definition of tetraploidy, and the choice and execution of the method used for S‐phase estimation. Based on the observations of this study, detailed guidelines for FCM analysis and interpretation of data are proposed in the Appendix. Some issues remain, however, e.g., to standardize a method for S‐phase calculation and tetraploid definition.© 1995 Wiley‐Liss, Inc.
Measurement of apoptotic cells in peripheral blood
Authors: Carbonari Maurizio , Cibati Marina , Fiorilli Massimo 
Published: June 2005

AbstractThe measurement of apoptosis in peripheral blood might represent a useful tool in acquired immunodeficiency syndrome (AIDS) and cancer research. Among the many assays that are currently used to identify apoptotic leukocytes, flow cytometric methods are the most valuable in terms of rapidity, simplicity, and level of analytical detail. Some flow cytometric assays may also offer the additional advantage of detecting the earliest phases of apoptosis, which is of paramount importance for measuring apoptotic cells in vivo before they are destroyed by phagocytes. © 1995 Wiley‐Liss, Inc.
DNA analysis of in situ ductal carcinoma of the breast via flow cytometry
Authors: Ottesen Gyda Lolk , Christensen Ib Jarle , Larsen Jørgen K. , Christiansen Jette , Hansen Bodil , Andersen Johan A. 
Published: June 2005

AbstractFlow cytometric (FCM) DNA analysis of 41 clinical cases of ductal carcinoma in situ (DCIS) of the breast was performed on fine‐needle aspirates of unfixed, frozen tissue. Based on analysis of a single tissue sample, abnormal DNA content was found in 35 (85%) of the cases, and 15 (37%) disclosed heterogeneity. If five or more samples were analyzed, then heterogeneity was found in eight of ten cases. No conclusive correlation could be found between DNA index (DI) and histopathology. Our results on DCIS demonstrate a very high concordance with invasive breast cancer with respect to the frequency of DNA nondiploid cases, distribution of DI, occurrence of multiple clones, and S‐phase fraction. This indicates that major genetic alterations and DNA heterogeneity are early events in carcinogenesis that are already established at the preinvasive stage. © 1995 Wiley‐Liss, Inc.
Instrument‐Dependent fluorochrome sensitivity in flow cytometric analyses
Authors: Chance John T. , Larsen Sandra A. , Pope Victoria , Measel John W. , Cox David L. 
Published: June 2005

AbstractFlow cytometry has become the preferred technique by which critical clinical evaluations are made such as CD4 counts and aneuploid analyses. Mounting concern has arisen over the numerous techniques, reagents, and different flow cytometers employed to determine these data. Several studies have documented significant differences in results when different flow cytometers are utilized to analyze the same sample. Fluorochrome‐dependent instrument sensitivity also has been reported by numerous investigators. As more and more procedures are performed by cytometric analysis, light scatter and fluorescence limitations, which appear to be instrument dependent, demonstrate that not all flow cytometers have the same capabilities. Attempts were made to calculate molecules of equivalent soluble fluorochrome (MESF) values on nine different flow cytometers using fluorescein isothiocyanate (FITC) and R‐phycoerythrin (R‐PE) labeled microsphere reference standards produced by Flow Cytometry Standards Corporation (FCSC). Dramatic differences were observed in the ability of some cytometers to resolve these microspheres. The diminished resolution appeared to be instrument model and fluorochrome dependent. We propose that diminished fluorescence resolution in certain flow cytometers could be responsible for significant variability in clinical values reported from laboratories utilizing different flow cytometers. © Wiley‐Liss, Inc.
Amplification and detection of a Y‐chromosome DNA sequence by fluorescence in situ polymerase chain reaction and flow cytometry using cells in suspension
Authors: Timm Earl A. , Podniesinski Ed , Stewart Carleton C. , Duckett Larry , Cardott John 
Published: June 2005

AbstractA procedure for amplifying and detecting nucleic acid sequences in situ using cells in suspension and flow cytometry has been developed. The process involves the use of the polymerase chain reaction (PCR) and a fluorescent in situ hybridization (FISH) protocol developed in our laboratory to detect the amplified PCR product. For these studies, a Y‐chromosome specific repeat DNA sequence was amplified. Daudi cells, a B‐cell lymphoma culture line established from a male, was used as a positive control and HL‐60, a promyelocytic leukemia culture line established from a female, was used as a negative control. During the in situ PCR process cellular autofluorescence (noise) increases causing markedly reduced detection sensitivity of the probe (signal) bound to the amplified product within the positive cells. An autofluorescence reduction circuit was applied which was integrated into a standard bench top flow cytometer to reduce this noise, thereby producing a 10‐fold increase in detection sensitivity of the signal. Without the application of the autofluorescence reduction circuit, the positive control histogram distribution was virtually indistinguishable from the negative control sample distributions. After autofluorescence reduction, the data showed that the Y‐chromosome DNA was only amplified in the Daudi cells subjected to the complete in situ PCR protocol. This increased sensitivity also provided direct detection of the Y‐chromosome repeat sequence, albeit exhibiting less signal compared to the amplified target after the in situ PCR. © 1995 Wiley‐Liss, Inc.