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At least 1,135 records · Page 63Linked to original sources
PubMed1997

Laser scanning cytometric analysis of cyclin B1 in primary human malignancies.

Cyclins are key components of the cell cycle progression machinery. They activate their partner-dependent kinases (CDKs) and target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. During unperturbed growth of normal cells, the timing of expression of several cyclins is discontinuous, occurring at discrete and well-defined periods of the cell cycle. Immunocytochemical detection of cyclins in relation to cell cycle position (DNA content) by multiparameter flow cytometric techniques has provided a new approach to cell cycle studies. This approach, like no other method, can be used to detect the "unscheduled" expression of cyclins, namely, the presentation of G1 cyclins by cells in G2/M and of G2/M cyclins by G1 cells, without the need for cell synchronization. By use of multiparameter flow cytometric and laser scanning cytometric analysis, we correlated the expression of cyclin B1 with cell cycle position in normal lymphocytes stimulated to proliferate by the mitogen phytohemagglutinin and in 28 primary human tumors of different organ and type. Eighteen of the 28 tumors expressed the cyclin B1 in more than 5% of cells (B1 positive), and the rest showed cyclin expression from 2.1 to 5% (B1 negative). In normal lymphocytes, the expression of cyclin B1 was restricted to very late S and G2 + M phases of the cell cycle. In 15 of 18 primary tumors studied, the expression of cyclin B1 was "unscheduled" (unrestricted to particular phases of the cycle). The data suggest that the "unscheduled" expression of cyclin B1 might be a common defect in neoplasia.

Adrenal Gland Neoplasms↗
PubMedDate not supplied

Multiple synchronous colorectal carcinomas: a ploidy study by image analysis.

Cytometry could represent an ancillary technique to morphology in order to understand whether multiple synchronous colorectal carcinomas arose independently. Twenty-eight multiple synchronous tumours, assessed by means of a computerized image analysis system for DNA ploidy, were categorized as diploid (4) or not diploid (24). The ploidy classes were: DNA-diploid, DNA-tetraploid, and DNA-aneuploid. The DNA Index (DI) ranged from 0,90 to 2,66. The overall concordance rate for ploidy and DI class with synchronous tumours was 69% and 31%, respectively. The high concordance rate in ploidic categories suggests the metastatic origin of our multiple synchronous tumours. Moreover, they showed site, Dukes' classification, degree of differentiation, percentage of ploidy class, and DI distribution comparable to the single colorectal carcinomas. In conclusion, image analysis is a reliable technique to determine the independent clonality or the common origin of multiple colorectal tumours when the evaluation based on the simple histopathological criteria is not satisfactory.

Aged↗
PubMed2002

Use of fluorescence ratio imaging microscopy and flow cytometry for estimation of cell vitality for Bacillus licheniformis.

For Bacillus licheniformis SJ4628, an organism widely used in the enzyme industry, methods for determination of cell vitality at a single cell level using 5(6)-carboxyfluorescein diacetate succinimidyl ester in combination with fluorescence ratio imaging microscopy and flow cytometry were developed. Immediately after inoculation and during growth, changes in intracellular pH values determined by fluorescence ratio imaging microscopy and in green fluorescence intensities determined by flow cytometry were observed. Correlations between the capacity to multiply and intracellular pH or green fluorescence intensity were demonstrated. Populations of cells not having a pH gradient or exhibiting low fluorescence intensities had significantly longer lag phases than populations of cells with a pH gradient and high fluorescence intensities.

Bacillus↗
PubMed2001

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold.

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

Cyanobacteria↗
PubMed2003

Flow and image cytometric DNA ploidy, including 5c exceeding cells, of serous borderline malignant ovarian tumors. Correlation with clinicopathologic characteristics.

OBJECTIVE: To analyze DNA ploidy of serous borderline ovarian tumors by flow cytometry (FCM) and image cytometry (ICM), with 5c exceeding cells also analyzed, and to evaluate their correlation with clinicopathologic characteristics of patients and tumors. STUDY DESIGN: Cell suspensions were prepared according to a modified Hedley method from formalin-fixed, paraffin-embedded tissue blocks of 43 tumors. One part of the suspension was used for flow cytometric measurement; from the other part, filter slides were prepared for ICM. RESULTS: FCM and ICM found 2 aneuploid (peridiploid) serous borderline ovarian tumors, and FCM found 1. ICM found 3 tumors with 5c exceeding cells and 2 tumors with octaploid cells. There was no correlation between DNA aneuploidy and presence of 5c exceeding cells with tumor size, International Federation of Gynecology and Obstetrics stage or survival. CONCLUSION: The results confirm a good correlation between FCM and ICM DNA ploidy and the ability of ICM to detect 5c exceeding cells. The prognostic value of DNA ploidy and 5c exceeding cells in serous borderline malignant ovarian tumors warrant further evaluation.

Aneuploidy↗
PubMed2005

Cytomics, the human cytome project and systems biology: top-down resolution of the molecular biocomplexity of organisms by single cell analysis.

A large amount of structural and functional information is obtained by molecular cell phenotype analysis of tissues, organs and organisms at the single cell level by image or flow cytometry in combination with bioinformatic knowledge extraction (cytomics) concerning nuclei acids, proteins and metabolites (cellular genomics, proteomics and metabolomics) as well as cell function parameters like intracellular pH, transmembrane potentials or ion gradients. In addition, differential molecular cell phenotypes between diseased and healthy cells provide molecular data patterns for (i) predictive medicine by cytomics or for (ii) drug discovery purposes using reverse engineering of the data patterns by biomedical cell systems biology. Molecular pathways can be explored in this way including the detection of suitable target molecules, without detailed a priori knowledge of specific disease mechanisms. This is useful during the analysis of complex diseases such as infections, allergies, rheumatoid diseases, diabetes or malignancies. The top-down approach reaching from single cell heterogeneity in cell systems and tissues down to the molecular level seems suitable for a human cytome project to systematically explore the molecular biocomplexity of human organisms. The analysis of already existing data from scientific studies or routine diagnostic procedures will be of immediate value in clinical medicine, for example as personalized therapy by cytomics.

Cell Biology↗
PubMed2006

Significance of AgNOR measurement in thyroid lesions.

OBJECTIVE: To evaluate the discriminating potential of AgNOR area measurement and count in thyroid tumors using static cytometry equipment. STUDY DESIGN: Slides were analyzed by a computerized system for image analysis, CAS 200 (Becton & Dickinson, U.S.A.), using the Cell Measurement computer program (CAS 200, Becton & Dickinson). The argyrophilic reaction (NORs) was evaluated with a 400-fold amplification directly from the computer monitor. RESULTS: Thirty-three cases were analyzed for AgNOR staining. The cases studied included 3 goiters, 10 follicular adenomas, 6 Hürthle adenomas, 4 follicular carcinomas, 7 papillary carcinomas, and 3 Hürthle carcinomas. A total of 6,600 nuclei were evaluated. For statistical purposes, lesions were classified as benign and malignant, and both the number and the area of counted NORs showed very similar values. The NORs median among 19 benign tumors was 1.484 (SD +/- 0.265) and of 14 malignant tumors was 1.436 (SD +/- 0.414); the NORs areas were 2.6584 (SD +/- 1.0653) and 2.3643 (SD +/- 0.6320), respectively. CONCLUSION: Our results showed that AgNOR evaluation was not a significant parameter to discriminate between malignant and benign thyroid lesions.

Humans↗
PubMed2006

Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti-infectious or anti-shock therapy as well as curative chemotherapy in combination with stem cell transplantation may provide better survival chances for patient at concomitant cost containment. Predictive medicine-guided optimization of therapy could lead to individualized medicine that gives significant therapeutic effect and may lower or abrogate potential therapeutic side effects. The 10th Leipziger Workshop combined with the 3rd International Workshop on SBC aimed to offer new methods in Image- and Slide-Based Cytometry for solutions in clinical research. It moved towards practical applications in the clinics and the clinical laboratory. This development will be continued in 2006 at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.

Cytophotometry↗
PubMed1996

Malignant mesothelioma: immunohistochemistry and DNA ploidy analysis as methods to differentiate mesothelioma from benign reactive mesothelial cell proliferation and adenocarcinoma in pleural and peritoneal effusions.

OBJECTIVE: To determine whether malignant mesotheliomas can be differentiated from adenocarcinomas and benign reactive mesothelial cells in pleural and peritoneal fluids using immunohistochemical analysis in conjunction with DNA ploidy analysis. DESIGN: Sixteen cases of malignant mesothelioma, including epithelial, sarcomatous, and biphasic types, were collected. DNA analysis using flow cytometry and/or image analysis was performed on paraffin-embedded tissue from 15 of the mesothelioma cases, as well as on cytospin cell preparations from samples of pleural and peritoneal fluids from cases with either cytologically proven adenocarcinoma (seven cases) or benign reactive mesothelial cells (seven cases). Immunohistochemical studies were done in 15 mesotheliomas, 5 adenocarcinomas, and 4 benign reactive mesothelial cell effusions. RESULTS: All malignant mesotheliomas tested (100%) stained positively for prekeratin, whereas stains for carcinoembryonic antigen, B72.3, Leu-M1, and Ber-EP4 were negative. Stains vimentin, epithelial membrane antigen, and CA125 were positive in 75%, 75%, and 25% of cases tested, respectively. Benign reactive mesothelial cell cases stained similarly. Adenocarcinomas were more likely to react positively with B72.3, Ber-EP4, and carcinoembryonic antigen, and negatively with vimentin. DNA analysis showed that all benign cases were diploid, while all adenocarcinomas were nondiploid. Fifty-three percent of the malignant mesotheliomas were nondiploid. Sensitivity for detection of nondiploidy was greater for image analysis than for flow cytometry (100% vs 75%). CONCLUSIONS: B72.3, Ber-EP4, carcinoembryonic antigen, and vimentin are useful immunohistochemical markers in differentiating malignant mesotheliomas from adenocarcinomas, whereas immunohistochemistry does not reliably distinguish malignant from benign hyperplastic mesothelial cells. The addition of DNA ploidy studies is useful for differentiating the latter two groups.

Adenocarcinoma↗
PubMed2004

Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.

BACKGROUND: Here we demonstrate the ability of the ImageStream 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features. METHODS: Live, necrotic, and early and late apoptotic cells were prepared and analyzed by immunofluorescence microscopy, conventional flow cytometry, and imaging flow cytometry, both as single populations and as a heterogeneous mixture of cells. RESULTS: Live (annexin V(-), 7-AAD(-)) and early apoptotic (annexin V(+), 7-AAD(-)) cells were readily identifiable using either conventional or ImageStream based flow cytometric methods. However, inspection of multispectral images of cells demonstrated that the annexin V(+), 7-AAD(+) population contained both necrotic and late-stage apoptotic cells. Although these cells could not be distinguished using conventional flow cytometric techniques, they were separable using unique combinations of photometric and morphometric measures available using ImageStream technologies. CONCLUSIONS: Using multispectral imagery, morphologically distinct cell populations can be distinguished using features not available with conventional flow cytometers. In particular, the ability to couple morphometric with photometric measures makes it possible to distinguish live cells from cells in the early phases of apoptosis, as well as late apoptotic cells from necrotic cells.

Cell Death↗
PubMed1997

Evaluation of DNA content in epithelial hyperplastic lesions of the larynx.

Quantification of cellular DNA by image or flow cytometry has achieved acceptance as an objective and reproducible component of diagnostic pathology. Although the majority of investigations have focused on the prognostic utility of DNA content measurements in malignant neoplasms, the detection of abnormal DNA content can also be useful for the detection of early neoplasia or premalignant change. Several studies of laryngeal epithelial hyperplastic lesions by image cytometry have shown that a proportion of these lesions exhibit abnormal DNA content and that the incidence of this finding correlates with the degree of dysplasia or atypia. Similar results were obtained by flow cytometry, which has only rarely been used for investigation of these lesions. Although there is some evidence that lesions with abnormal DNA content are more likely to progress to carcinoma, additional studies are needed to define the clinical importance of DNA cytometry in hyperplastic epithelial lesions of the larynx.

DNA↗
PubMed1990

Quantitation of oncogene products by computer-assisted image analysis and flow cytometry.

The use of antibodies permits the study of oncogene product expression in cells and tissues. However, quantitation of the levels of expression in immunohistochemical preparations is beset by difficulties, and the available scoring system provide semiquantitative data at best. Here we describe the use of computer-assisted image analysis for determination of oncoprotein levels in a model system and compare the results with those generated by flow cytometric analysis. The oncogene products measured are located in the nucleus (c-myc p62 and c-fos p55), the inner surface of the membrane (c-ras p21), and both sides of the membrane (c-erbB-2 p185). In each instance, both analytic modalities yielded concordant results. Our data indicate that computer-assisted image analysis is a useful tool for quantitating cell components in immunohistochemical preparations.

Breast Neoplasms↗
PubMed2002

Biotechnology apprenticeship for secondary-level students: teaching advanced cell culture techniques for research.

The purpose of this article is to discuss small-group apprenticeships (SGAs) as a method to instruct cell culture techniques to high school participants. The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems. Participants designed and completed experiments using both flow cytometry and laser scanning cytometry during the 1-month summer apprenticeship. In addition to effectively and efficiently teaching cell biology laboratory techniques, this course design provided an opportunity for research training, career exploration, and mentoring. Students participated in active research projects, working with a skilled interdisciplinary team of researchers in a large research institution with access to state-of-the-art instrumentation. The instructors, composed of graduate students, laboratory managers, and principal investigators, worked well together to present a real and worthwhile research experience. The students enjoyed learning cell culture techniques while contributing to active research projects. The institution's researchers were equally enthusiastic to instruct and serve as mentors. In this article, we clarify and illuminate the value of small-group laboratory apprenticeships to the institution and the students by presenting the results and experiences of seven middle and high school participants and their instructors.

Animals↗
PubMed1992

A comparative study of DNA ploidy in 115 fresh-frozen breast carcinomas by image analysis versus flow cytometry.

BACKGROUND: Qualitative and quantitative analysis of cellular DNA content may be clinically useful in the prognostic evaluation of certain types of malignant tumors, including breast carcinoma. Flow cytometric (FCM) analysis has been the most frequently used procedure for DNA analysis, but it requires a reasonably large tissue sample. Computer-based image analysis (IA) now allows imprint, cytospin, and needle aspiration smear preparations and other small tissue samples to be used. METHODS: To resolve concern about the diagnostic efficacy of small tissue samples in the use of IA, the authors performed a comparative study of FCM analysis and IA using 115 fresh-frozen breast carcinomas. Feulgen-stained imprint preparations for IA and single-cell suspensions from the same fresh-frozen tissue for FCM analysis were used, and the respective histograms were compared. RESULTS: The results were concordant in 90.4% (104 of 115) of the cases, but 11 specimens yielded discordant data. IA provided histograms with a somewhat lower resolution and a relatively high coefficient of variation for the G0/G1 peak, thus rendering occasional tumors, which were near-diploid aneuploid by FCM analysis (four cases), not amenable to diagnosis by aneuploid characterization. In three additional cases, FCM analysis showed aneuploid hyperdiploid (two cases) and multiploid (one case) histograms, but IA only demonstrated a diploid peak. Conversely, in four other cases, aneuploid peaks were recognized only by IA. CONCLUSIONS: Computerized IA has significant advantages over FCM analysis, including lower cost, the ability to analyze very small specimens, the capability of detecting rare high ploidy cells, the capacity to classify cellular populations according to specific morphologic type, and the fact that no destructive enzyme or chemical digestion is required for specimen preparation, thereby preserving the integrity of fragile cells.

Breast Neoplasms↗