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At least 1,153 records · Page 64Linked to original sources
PubMed1993

Deoxyribonucleic acid ploidy studies in choroidal melanomas.

In several tumors of different organ sites, the amount of DNA in a cell (ploidy) is associated with malignancy. We performed DNA quantitation in 21 choroidal melanomas and compared flow cytometry with image analysis in 11 of these melanomas. We modified our preparation technique to overcome problems with pigment and control cell populations in the image analysis group. Fifteen tumors were diploid and two tumors were tetraploid. Four tumors were unprocessable by flow cytometry, but two of these tumors were diploid by image analysis. Image analysis also detected tetraploidy in two tumors that were diploid by flow cytometry. During image analysis, cells were classified according to the Callendar classification and histograms were plotted for each cell type. All spindle A cells were diploid and most tetraploid peaks were formed by epithelioid cells. The use of image analysis on small samples of choroidal melanomas may be of value both in confirmation of diagnosis and prognosis of these lesions, and perhaps therapeutically, for example, in the monitoring of radiation treatment.

Choroid Neoplasms↗
PubMed2006

Personal cytometers: slow flow or no flow?

BACKGROUND: Although some manufacturers have optimistically described instruments with prices in the 40,000 US dollars range as "personal cytometers", analogy with the personal computer suggests that the target price for a true "personal" cytometer should be under 5,000 US dollars. Since such an apparatus could find a wide range of applications in cytomics in both developing and developed countries, it seemed desirable to consider its technical and economic feasibility. METHODS: Using resolution targets and a variety of fluorescent bead standards immobilized on filters and/or slides, we evaluated high-intensity LEDs as fluorescence excitation sources, relatively inexpensive CCD cameras as detectors, and 35 mm camera lenses and plastic low-power microscope optics for light collection in a simple, inexpensive low-resolution imaging cytometer. RESULTS: The components tested could be combined toproduce an instrument capable of detecting fewer than 10,000 molecules of cell-associated fluorescent label, and thus applicable to a broad range of cytometric tasks. CONCLUSIONS: Given the requirements for light sources, detectors, optics, mechanics, electronics and data analysis hardware and software, and the components presently available, it should be easier to reach the desired 5,000 US dollars price point with an image cytometer than with a flow cytometer.

Flow Cytometry↗
PubMed2005

DNA ploidy and immunomarking of bladder urothelial tumors before and after intravesical bacillus Calmette-Guérin treatment.

OBJECTIVE: To investigate DNA ploidy and immunoexpression of Ki-67 and p53 as predictivefactors in cases of superficial urothelial cell carcinoma (UCC) treated with bacillus Calmette-Guérin (BCG). STUDY DESIGN: Samples were obtained from 66 patients with UCC (pTa grade 3 or high grade and pT1 independent of grade or with concomitant carcinoma in situ) before and after intravesical BCG treatment. DNA ploidy analysis (ploidy balance, degree of hyperploidy and aneuploidy, proliferation index) was done by static cytometry. Ki-67 and p53 were analyzed immunohistochemically in paraffin-embedded tissue, and their quantification was carried out using an image analysis system. RESULTS: During a mean follow-up of 63.8 months, 31 of the 66 patients developed recurrent tumors (46.9%). DNA ploidy analysis showed that ploidy balance as well as degree of hyperploidy and aneuploidy were not statistically different between recurrent and nonrecurrent tumors. Only proliferation index was statistically significant between recurrent and nonrecurrent tumors. No statistically significant difference was observed in the percentage of Ki-67- and p53-positive cells between primary tumors that recurred and those that did not. CONCLUSION: These findings suggest that only proliferation index has predictive value for recurrence and progression in UCC treated with BCG.

Adult↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed1997

Effects of intermittent or continuous gravitational stresses on cell-matrix adhesion: quantitative analysis of focal contacts in osteoblastic ROS 17/2.8 cells.

The relationship between cell morphology and cell metabolism and the role of mechanical load in bone remodeling is well known. Mechanical stimulation induces changes in the shape of osteoblasts, probably mediated by reorganization of focal contacts. We studied the influence of gravity (Gz) variations occurring during parabolic flight on osteoblast focal adhesion of ROS 17/2.8 osteosarcoma cells subjected to 15 or 30 parabolic flights. Significant flight-induced shape changes consisted of decreased cell area associated with focal contact plaque reorganization. Identical durations of continuous mechanical stress induced by centrifugation (2 Gz) or clinorotation (Gz randomization) had no major effect on cell focal adhesion. ROS 17/2.8 G2/M synchronization by treatment with nocodazole inhibited the flight-induced decrease in adhesion parameters. We concluded that ROS 17/2.8 cells are sensitive to Gz switches and that their adaptation is at least dependent on microtubule function.

Animals↗
PubMed1998

Histopathological assessment of localized proliferation in cases of Bowen's disease using immunostaining and a laser cytometer.

In order to evaluate the localized proliferative activity of intratumor cells in Bowen's disease using tissue sections, skin specimens from ten patients were compared with skin samples from seven normal individuals for their expression of proliferating cell nuclear antigen (PCNA), Ki-67 immunostaining and intranuclear DNA contents, quantitated with a laser cytometer (LCM). In normal epidermis, the largest proportion of PCNA- and Ki-67-positive cells was observed in the basal cell layer, with the amounts decreasing through the suprabasal cell layer towards the prickle cell layer. Examination by LCM also revealed the highest average fluorescence intensity of individual nuclei in the basal cell layer and, as with the immunohistological parameters, reducing towards the upper layer of the epidermis. In the Bowen's disease tissue sections, the largest proportion of PCNA- and Ki-67-positive cells was found in contact with the basement membrane (base of the tumor), with lower amounts in the center of the tumor nest and in the marginal epidermis. The average fluorescence intensities of individual nuclei were in line with these results. These results show that tumor cells distributed in Bowen's disease tumor nests have different proliferative activities depending on their location.

Bowen's Disease↗
PubMed2006

DNA and kinetic heterogeneity during the clonal evolution of adrenocortical proliferative lesions.

Monoclonal adrenocortical lesions show inverse correlation between proliferation and apoptosis, with proliferation being the single most important criterion of malignancy in adrenal lesions. No study yet has evaluated the variability of proliferation regarding the clonal pattern and diagnosis in adrenocortical nodular hyperplasias (ACNHs), adrenocortical adenomas (ACAs), and adrenocortical carcinomas (ACCs). We studied 69 ACNHs, 64 ACAs, and 23 ACCs (World Health Organization criteria) from 156 females. Clonality HUMARA test (from microdissected DNA samples), DNA content and proliferation analysis (slide and flow cytometry), and mitotic figure (MF) counting/50 high-power fields (HPFs) were performed in the same areas. Heterogeneity was assessed by 5cER (percentage of nonoctaploid cells with DNA content exceeding 5c) and standard deviation of MF/HPF. Statistics included analysis of variance/Student t tests regarding the clonal patterns and diagnosis. Polyclonal patterns were observed in 48 of 62 informative ACNHs and 7 of 56 informative ACAs, and monoclonal in 14 of 62 ACNHs, 49 of 56 ACAs, and 21 of 21 ACCs, with all hyperdiploid lesions (14 ACCs and 13 ACAs) being monoclonal. The standard deviation of MF/HPF progressively increased in ACNH-ACA-ACC (0.048 +/- 0.076, 0.110 +/- 0.097, 0.506 +/- 0.291, respectively; P = .0023), but did not differentiate ACNH/ACA. Only tetraploid percentage (P = .0496) and 5cER (P = .0352) distinguished polyclonal (3.64 +/- 2.20 and 0.14 +/- 0.15) from monoclonal (7.25 +/- 7.52 and 1.00 +/- 1.74) benign lesions. Malignancy significantly correlated with a low diploid percentage and high tetraploid percentage. Cell kinetic heterogeneity is the hallmark of adrenocortical neoplasms: tetraploid/hypertetraploid cell accumulation characterizes monoclonal lesions (suggesting nondisjunctional mitoses), whereas heterogeneously distributed mitotic figures and decreased diploid percentage define ACCs.

Adrenal Cortex Neoplasms↗
PubMed2003

Measuring the mitotic index in chemically-treated human lymphocyte cultures by flow cytometry.

In the human lymphocyte chromosome aberration assay, the mitotic index (MI) is the standard cytotoxic parameter for determining which test concentrations will be evaluated for chromosome aberrations. Assessment of the MI is performed microscopically by determining the frequency of mitotic cells in a population of 1000 cells. With the commercial availability of antibodies to the mitosis-specific marker, phosphorylated-histone H3 at serine 10, automating the assessment of the MI using flow cytometry is now possible [Cytometry 32 (1998) 71]. Our laboratory has utilized and validated this technology to measure the mitotic index of chemically-treated human lymphocyte cultures. Comparisons between the microscopic and flow MI frequencies from 24h treatments with mitomycin-C, aphidicolin, eugenol, etoposide, hydroxyrurea, potassium cyanide, staurosporine, ethyl alcohol, noscapine and colcemid((R)) are presented. Our results show that the mitosis specific H3-P marker is excellent for measuring the MI frequency in human lymphocyte cultures treated up to toxic concentrations. In addition, this study demonstrates that automation of analysis by flow cytometry is an excellent alternative to the microscopic method of analysis producing less variability than the microscopic scoring and a more complete dose response curve.

Adult↗
PubMed2003

Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy.

A flow cytometric technique for scoring the incidence of micronucleated reticulocytes in rat peripheral blood was compared to a standard microscopy-based procedure. For these studies, groups of five male Sprague-Dawley rats were treated with vehicle or a broad range of chemical genotoxicants: 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, vincristine, methylaziridine, acetaldehyde, methyl methanesulfonate, benzene, monocrotaline, and azathioprine. Animals were treated once a day for up to 2 days, and peripheral blood was collected between 24 and 48 h after the final administration. These samples were processed for flow cytometric scoring and microscopy-based analysis using supravital acridine orange staining, and the percentage of reticulocytes and micronucleated reticulocytes was determined for each sample. The resulting data demonstrate good agreement between these scoring methodologies, although careful execution of the flow cytometric method was found to enhance the micronucleus assay by reducing both scoring time and scoring error. These data add further support to the premise that the peripheral blood compartment of rats can be used effectively to detect genotoxicant-induced micronuclei.

Acridine Orange↗
PubMed2001

Detection and quantification of small numbers of circulating tumour cells in peripheral blood using laser scanning cytometer (LSC).

The detection of circulating tumour cells disseminated from solid tumours requires extremely sensitive methods. Molecular genetic methods, which are most sensitive, are not applicable to solid tumours because no tumour-specific genetic markers are available. Detection of disseminated tumour cells by immunocytochemistry is time-consuming, whereas fluorimetry is fast and quantitative. The laser scanning cytometer (LSC) provides an automated microscopic procedure for screening up to 5x10(4) cells in suitable time. Using this system together with an enrichment procedure which allows up to ten thousand-fold enrichment, we have quantified minimal numbers of tumour cells. In a model system, breast cancer cell line cells diluted into peripheral blood mimicked seeding of tumour cells into the periphery. After staining with fluorochrome-conjugated anti-epithelial antibody, slides were screened for positive events directly or after enrichment with antibody-coated magnetic beads. One positive cell was unequivocally detectable in 10(4) cells and 50 out of 60 tumour cells were reliably recovered from a 20 ml blood volume, equal to 1-2 cells per 10(7), after magnetic bead enrichment. This method allows quantitation of tumour cells in peripheral blood and bone marrow in reasonable time and will, for the first time, enable extensive investigation of the seeding behaviour of tumours.

Breast Neoplasms↗
PubMed2006

Quantitative assay for quality assurance of human cells for clinical transplantation.

Transplantation of human cells after isolation and culture has become an important alternative for treatment of acute or chronic skin wounds. To increase the efficacy and reduce cost for transplantation of skin cells, more efficient and accurate techniques for evaluation of cell proliferation are needed. Hemocytometer counts provide a valid assessment of cell proliferation and viability, but they are very labor intensive and require removal of the cells from their substrate. In this study, hemocytometer counts were compared with a fluorometric assay (n = 21 per condition) that uses the commercially available reagent alamarBlue, which is reduced to a fluorescent substrate by cellular dehydrogenases. Human epidermal keratinocytes were inoculated at 200, 600, 2000, and 6000 cells/cm2 incubated for 6 days in modified MCDB 153 medium. Alamar Blue was incubated with cells for 2 h at 37 degrees C, and fluorescence was measured with a microplate reader at 590 nm. Hemocytometer counts (x10(-4)) from the respective cell inoculation densities were 0.30 +/- 0.04, 1.07 +/- 0.10, 6.37 +/- 0.62, and 16.99 +/- 0.96. Fluorescence values (x10(-3)) for the respective inoculation densities were 0.14 +/- 0.01, 0.34 +/- 0.02, 1.20 +/- 0.09, and 1.79 +/- 0.12. Regression analysis showed a statistical significant (p < 0.0001) correlation (r2 = 0.87) between cell counts and optical density from the alamarBlue assay. These data demonstrate that alamarBlue provides a valid substitute for cell counts to assess cell proliferation before clinical transplantation of engineered skin. AlamarBlue also allows repeated, nondamaging assessment of living cells over time. These advantages are expected to increase the validity and reliability of quality assurance standards for transplanted skin cells, and to increase the efficacy of healing of cutaneous wounds.

Biological Assay↗
PubMed2000

Neural network prediction of prostate tissue composition based on magnetic resonance imaging analysis. A pilot study.

OBJECTIVE: To develop a neural network model that estimates prostate histology using magnetic resonance imaging (MRI). STUDY DESIGN: Fifty-three men with lower urinary tract symptoms (average age = 63.8 +/- 8.9 years) underwent a prostate MRI (T2) and sextant biopsy of the prostate. Masson Trichome and immunohistochemical prostate-specific antigen staining of the biopsy material were used to calculate the amount of stroma and epithelium in the inner gland (central plus transition zone). MRIs were normalized to the mean intensity of the obturator internus muscle for comparative analyses. Gray scale and texture features were extracted from the inner gland in the midsection transverse MRI slice. Clinical and image variables were used in two neural networks predicting a high amount of stroma and a high amount of epithelium, respectively. RESULTS: The positive and negative predictive values of the stroma and epithelium neural networks were 95%, 69% and 65%, 92%, respectively. CONCLUSION: These data suggest that the combined use of these neural networks may predict patient response to medical therapy targeting prostatic stroma or epithelium.

Adult↗
PubMed1990

[DNA cytometry and morphometry, based on TV image analysis, in the diagnosis of gastric carcinoma].

Cytological smears from gastric biopsies were studied by two types of TAS. Böcking's algorithm and a great number of form factors were used to separate the intact cells from the tumourous ones. The numerically expressed density features and morphometric parameters are able to indicate the possibility of malignant transformation even in dysplasia. These values become much higher in tumours and lead to unequivocal diagnosis. Both types of TAS render valuable help in assessing cases not reliably diagnosed by routine staining procedures.

Biopsy↗
PubMed2001

Combined confocal and wide-field high-resolution cytometry of fluorescent in situ hybridization-stained cells.

BACKGROUND: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. METHODS: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode. RESULTS: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems. CONCLUSIONS: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance.

Cell Nucleus↗