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At least 1,297 records · Page 72Linked to original sources
PubMed2002

Detection and relocation of cord blood nucleated red blood cells by laser scanning cytometry.

BACKGROUND: Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. METHODS: CD71-enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb-gamma) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO-PRO-3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. RESULTS: In CB, 59% of events double positive for Hb-gamma and TO-PRO-3 were identified as CB-NRBC. In contamination studies, male fetal CB-NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. CONCLUSIONS: Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well-defined and located target cells may be feasible.

Cell Nucleus↗
PubMedDate not supplied

Simultaneous measurement of nucleolin and estrogen receptor in breast cancer cells by laser scanning cytometry.

BACKGROUND: The purpose of the study was to test the feasibility of laser scanning cytometry (LSC) to simultaneously measure estrogen receptor (ER) and nucleolin (NU) expression in the nuclei of the same individual breast cancer cells. MATERIALS AND METHODS: Cancer cells from 64 breast tumors were labeled with anti-NU and biotinylated anti-ER antibodies, then with secondary FITC-conjugated antibody and streptavidin-APC conjugate, respectively, and measured by LSC. The expression of NU in the nucleus and NU aggregates (NUA), number of NUA, nuclear and NUA areas and ER expression were assessed for aspiration each cell. RESULTS: ER-bound APC fluorescence correlated with nuclear NU (r=0.65; p<0.001) and NUA-bound FITC fluorescence (r=0.59; p<0.001). Good correlation was found between percentages of ER-positive cells in LSC and by image analysis in paraffin-embedded sections (r=0.59, p<0.001). CONCLUSION: ER and NU expression can be measured simultaneously in the same nuclei of breast cancer cells.

Adult↗
PubMed1991

Optical design in a flow system for imaging cells.

The imaging flow cytometer is an instrument that acquires the images of cells in a flow system. These images are analyzed for real-time cell classification and sorting. The optical units in the system perform the functions of laser beam focusing, cell detection, and cell imaging. The glass nozzle in which the cell stream flows introduces air-glass optical interfaces, resulting in optical aberrations that impair performance. This report describes the analysis of the optical aberrations in nozzles fabricated from a cylindrical glass capillary tube. Flat surfaces could be machined on the originally cylindrical surface to reduce the severity of these aberrations. The analysis shows that a nozzle with three flat surfaces is a feasible low-cost solution.

Equipment Design↗
PubMed2003

DNA ploidy and cell cycle analysis in breast cancer.

During the past 10 years there has been considerable interest in the application of new technologies to identify human malignancy and predict disease outcome. Markers of cell proliferation and the technologies of flow cytometry and image analysis for the determination of DNA total content in human tumor cells have been studied in breast cancer for 20 years. In this review, the uses and limitations of these technologies for the determination of ploidy status are discussed. This review also considers the prognostic significance and potential clinical utility of ploidy measurements, S phase calculation, and individual cell cycle regulatory biomarker expression levels.

Breast Neoplasms↗
PubMed1996

Molecular cytometry of cancer.

The application of molecular probes to diagnosis and prognosis of malignancies has redefined our perceptions of disease, allowing diagnosis by genotypic rather than phenotypic criteria. DNA analysis is especially useful when applied to pathological material in situ, because this allows the pathologist to combine information from both morphological and molecular observations. DNA in situ hybridization is a useful approach for the molecular pathologist, especially when combined with cytometric analysis. Potential clinical applications for in situ hybridization and the recently described technique of comparative genomic hybridization in tumor diagnosis and prognosis are described.

Chromosome Aberrations↗
PubMed1996

Immunophenotypic analysis of hematologic malignancy by laser scanning cytometry.

The authors tested a newly-developed computerized laser scanning cytometer (LSC) as a means of performing immunophenotypic analysis of hematologic specimens within their community hospital. Results were compared on a case-by-case basis with parallel flow cytometric and immunohistochemical data. A total of 71 specimens analyzed include 22 excised lymph nodes or other tissue biopsies, 18 peripheral bloods, 17 bone marrow aspirates, 7 body fluids, and 7 fine-needle aspiration biopsies of lymphoid tissue. The LSC proved to be a useful instrument capable of generating simultaneous two-color immunofluorescent data directly analogous to that obtained via conventional flow cytometry. However, laser scanning cytometric analysis provides advantages over flow cytometric analysis, because the LSC measures cells on a slide rather than in a fluid stream. Specifically, cells can be microscopically examined at any time--before, during, or after automated immunofluorescent analysis. In addition, specimen preparation techniques are less restricted and more cost efficient. Lastly, even extremely small and/or hypocellular specimens (such as body fluids and fine-needle aspiration biopsies) can be successfully analyzed.

Diagnosis, Computer-Assisted↗
PubMed2003

Mapping mechanical strain of an endogenous cytoskeletal network in living endothelial cells.

A central aspect of cellular mechanochemical signaling is a change of cytoskeletal tension upon the imposition of exogenous forces. Here we report measurements of the spatiotemporal distribution of mechanical strain in the intermediate filament cytoskeleton of endothelial cells computed from the relative displacement of endogenous green fluorescent protein (GFP)-vimentin before and after onset of shear stress. Quantitative image analysis permitted computation of the principal values and orientations of Lagrangian strain from 3-D high-resolution fluorescence intensity distributions that described intermediate filament positions. Spatially localized peaks in intermediate filament strain were repositioned after onset of shear stress. The orientation of principal strain indicated that mechanical stretching was induced across cell boundaries. This novel approach for intracellular strain mapping using an endogenous reporter demonstrates force transfer from the lumenal surface throughout the cell.

Animals↗
PubMed2001

Local proliferation contributes to lymphocyte numbers in normal lungs.

Lymphocytes in the bronchoalveolar lavage fluid (BAL) are increased in many lung diseases, which might be an indicator for protective reactions or pathology. Higher lymphocyte numbers at distinct organ sites may be due to a number of reasons such as increased entry, increased proliferation, reduced apoptosis or reduced exit. It is not known whether lymphocyte numbers are influenced by local proliferation in the healthy lung. Therefore, the proliferation of lymphocytes was studied in vivo in different lung compartments of healthy rats: the marginal vascular pool, the interstitial pool and the bronchoalveolar pool. Bromodeoxyuridine (BrdU) was used as an S-phase proliferation marker. The cells were obtained 1 h and 24 h after i.v. injection of BrdU. The labeled cells and their phenotypes were determined by immunocytochemistry, since it was not possible to use flow cytometry because of the low numbers. In the lung compartments 0.7-1.5% of all nucleated cells were found to be BrdU+, whereas 24 h later this increased to 3.2-5.7%. The frequency of BrdU+ T cells was significantly higher in the lung compartments compared to the blood, with the maximum in the marginal vascular pool. Local proliferation of lymphocytes involved mainly CD8+ T cells. Thus, local proliferation plays a role in the number and composition of lymphocytes in the healthy normal lung.

Animals↗
PubMed1983

Nondiscrete heterogeneity of human erythrocytes: comparison of Coulter-principle flow cytometry and Soret-hemoglobinometry image analysis.

In the blood of normal subjects, the volumes of single erythrocytes are distributed with a coefficient of variation (CV) of 10.8 +/- 1.8%; while in hemoglobinopathies, CV increases proportionately to the degree of anemia produced. Using single cell Soret-band hemoglobinometry and focused-aperture impedance counting, we compared the distribution of red cell volume, area, hemoglobin content, and hemoglobin concentration in normals and subjects with anemic disorders. The CV, nondiscrete heterogeneity, is first, a general characteristic of biologic measurement, second, a sensitive indicator of abnormality of erythropoiesis, and third, consistently less for hemoglobin concentration than for volume, area, or hemoglobin content of the same cells.

Analysis of Variance↗
PubMed2004

Biomarker discovery by comprehensive phenotyping for autoimmune diseases.

There is a well-recognized but unmet need for biological markers to characterize disease type, status, progression, and response to therapy in autoimmune diseases. We are developing and applying an integrated bioanalytical platform and clinical research program to facilitate comprehensive differential phenotyping of patient samples and enable the discovery of biomarkers. Our measurement platform includes microvolume laser scanning cytometry for the quantification of hundreds of cellular parameters in whole blood and other samples, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry for the quantification of proteins and low molecular weight biomolecules in serum and other fluids or tissues, and specific immunoassays for the quantification of trace proteins in serum. We describe the technologies and discuss initial applications to the analysis of subjects with rheumatoid arthritis (RA) and healthy controls.

Arthritis, Rheumatoid↗
PubMed1995

In vitro digital cell image analysis of morphonuclear modifications induced by natural DNA-interacting anticancer drugs in three neoplastic cell lines.

The effects of seven natural anticancer drugs acting through interaction with DNA were characterized at three levels: cell proliferation, cell cycle kinetics and chromatin texture. These drug-induced effects were monitored in vitro by means of the digital cell image analysis which provides 15 morphonuclear parameters. In terms of cell proliferation, results show that the drug-induced effects ranged from 10(-11)M to 10(-6)M. With respect to the cell cycle kinetics, the results show that the drugs induced an accumulation of the cells in the G2 phase. Concerning the chromatin levels, the results show that it is possible to classify the drugs according to their mechanism of action on the basis of the multivariate analysis of the 15 parameters.

Animals↗
PubMed2000

[Molecular biological diagnosis].

Biological behavior of lung cancer was evaluated by basic study. Malignancy Associated Change is the concept that the nuclear features of normal cells in the vicinity of cancer show subtle morphological difference from those of healthy individuals. The difference was recognized by high resolution cytometry and the expression of MAC cells was correlated with the degree of abnormality of chest diseases. Comparative genomic hybridization and fluorescence in situ hybridization were performed to investigate genetic abnormality of. Multiple genetic abnormalities and chromosomal instability showed poor prognosis. Two dimensional electrophoresis was employed to detect the expression of the specific protein of lung cancer. TAO2 was proved to be specific to well differentiated adenocarcinoma. Also, metabolic analysis will be employed for cell analysis.

Biomarkers, Tumor↗
PubMed2005

Analysis and enumeration of T cells, B cells and NK cells using the monoclonal antibody fluorescence capability of a routine haematology analyser (Cell-Dyn CD4000).

This communication details a method for the quantitative and qualitative analysis of blood T-, B- and NK-cell populations using the Abbott Cell-Dyn CD4000 haematology analyser. A series of 66 ethylenediaminetetraacetic acid (EDTA)-anticoagulated samples with lymphocyte counts between 0.2 and 33.3 x 10(9)/l were selected and analysed with CD3, CD19, Ia and CD56 monoclonal reagents. The flow cytometry reference method utilized a lymphocyte gate defined by optical scatter, with phenotypic analyses referencing to this gate and the absolute lymphocyte count. The CD4000 method analysed all leucocyte events, set primary gates for specific immunophenotypic fractions, and then determined population counts by reference to the white blood cell (WBC) count. Comparisons of CD3+ T-cell and CD19+ B-cell numbers showed high coefficients of correlation (R(2) > 0.95) and agreement (y = 1.01x) between the CD4000 and flow cytometry reference methods. Lower coefficients of correlation were obtained for CD3-CD56+ (R(2) = 0.52) and CD3+CD56+ (R(2) = 0.83) components. No major discrepancies were observed, and the CD4000 procedures additionally provided qualitative insights into the possibility of T-cell activation. The potential to undertake immediate analysis of EDTA-anticoagulated blood samples to determine the nature of abnormal lymphocyte morphology or numbers represents a considerable advance in the capability of haematology laboratories.

Antibodies, Monoclonal↗
PubMed2004

Increase of melanogenesis by retinoic acid: an ultrastructural and morphometric study.

Melanin is a dark pigment protecting the skin against UV radiation in some organisms. Studies on invasion and metastasis using retinoic acid as inhibitor agent are well known, but its role in melanin production (melanogenesis), especially at ultrastructural level and using morphometry were not well studied. In the present study, we analyzed the effects of retinoic acid on the melanosomes in B16F10 melanoma cells. These organelles were identified and quantified using routine electron microscopy and the specific HMB45 antibody. Other approaches such as immunofluorescence, and flow cytometry were also used. Our results indicated that retinoic acid increased the melanogenesis process in B16F10 melanoma cells. Furthermore, this work also provided evidence that this substance interferes at the subcellular level altering the numerical density of melanosomes, as well as the relative volume of the nucleus and nucleolus. In addition, the cells displayed altered morphology and an increase in the percentage of the relative volume of melanosomes, mainly the stages II-III and IV, leading to melanin formation. Furthermore, a decrease in the cells number after retinoic acid treatment was also observed.

Animals↗
PubMed2000

Immunophenotyping of peripheral blood leukocytes by laser scanning cytometry.

Many clinical situations demand repeated analyses of blood parameters but permit only minimal amounts of peripheral blood to be taken, e.g., in neonates with low birth weight, during extensive operations of young children, or in patients with restricted bone marrow function. In these cases laser scanning cytometry is the ideal tool to determine the distribution of different leukocyte-subsets. The purpose of this protocol is to describe stepwise a new method of immunophenotyping by laser scanning cytometry. In this assay nuclear DNA is stained by 7-aminoactinomycin-D (7-AAD) and surface antigens are detected by direct three-colour immunofluorescence. For data acquisition, measurements are triggered on the 7-AAD-fluorescence. Data are obtained for forward scatter, green, orange, and long red fluorescence by excitation with the argon-laser, and for far red fluorescence by excitation with the helium-neon-laser. Using this protocol the amount of peripheral blood needed is minimised to 10 microl. Specimens can be stained a second time in a different way and analysed repeatedly and archived.

Antigens, CD↗
PubMed1994

Reversing the effect of formalin on the binding of propidium iodide to DNA.

Formalin, an excellent preservative of cellular morphology, is a commonly used fixative for tissue specimens in hospital pathology laboratories. This preserved material is a potential source of tissue for diagnostic and retrospective research studies on DNA using flow cytometry. Unfortunately, formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA, thus altering the measurement of DNA content by flow cytometry or image analysis. This interference has been attributed to the cross-linking of histones by formalin. Since formalin alters the measurement of DNA content in formalin-fixed and formalin-fixed, paraffin-embedded tissues, this study was designed to explore the use of various physicochemical methods to reverse the effect of the formalin on the binding of PI to DNA. This study demonstrates that resuspending formalin-fixed cells in PBS and heating them at 75 degrees C for at least 1 h prior to staining with PI restores the staining of the DNA to approximately the same fluorescence intensity as that of fresh tissue.

Artifacts↗
PubMed2003

Detection of Aspergillus fumigatus hyphae by solid phase cytometry.

A rapid method based on solid phase cytometry (SPC) for the detection of Aspergillus fumigatus hyphae is described. With an enzymatic "viability" staining procedure, fungal hyphae can be detected non-specifically within the hour. By combining this procedure with an immunofluorescence labelling, a distinction between Aspergillus spp. and other clinically important fungi is possible, except for Penicillium spp. due to cross-reactivity. To differentiate both genera, microcolonies are generated by incubation at 45 degrees C prior to viability staining. The latter approach in conjunction with immunofluorescence labelling allows a quasi-specific detection of A. fumigatus hyphae and has shown its applicability to samples of bronchoalveolar lavage liquid (BAL).

Aspergillosis↗
PubMed2003

Cell filtration-laser scanning cytometry for the characterisation of circulating breast cancer cells.

BACKGROUND: Epithelial cells may be detected in the circulation of the majority of patients with metastatic breast cancer. Quantification of such presumptive cancer cells might allow for the monitoring of patients with early or late stage disease as an early index of relapse. Additionally, biomarker analysis may allow a more rational approach to therapeutics. We have developed a new method for the detection and characterisation of these cells. METHODS: Blood was filtered through polycarbonate membranes containing cylindrical pores, 8 microm in diameter. All the red cells and a large majority of the white blood cells passed through the filter while the larger epithelial cells were trapped. Cells on the membrane were fixed in ethanol, stained with propidium iodide and anti-pan-cytokeratin-FITC (to identify epithelial cells). The filters were then examined by laser scanning cytometry (LSC), which allowed enumeration and localisation of cells. RESULTS: With normal blood spiked with cells from breast carcinoma cell lines, 99.9% of the leukocytes passed through the membrane, while close to 100% of the epithelial cells were trapped, with a detection limit of less than one epithelial cell/ml of blood. All of 20 samples from patients with widespread metastatic disease contained cytokeratin-positive cells with the morphological characteristics of carcinoma cells, the number of cells ranging from 0.2 to 5.7/ml of blood. CONCLUSIONS: Cell filtration-LSC is a viable technique for detecting and studying breast carcinoma cells in peripheral blood.

Breast Neoplasms↗