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PubMed2000

Intranuclear localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma.

OBJECTIVE: To investigate, with laser scanning cytometry (LSC), proliferating cell nuclear antigen (PCNA) expression during the cell cycle in renal cell carcinoma. STUDY DESIGN: DNA ploidy and intracellular localization of PCNA in renal cell carcinoma were determined using LSC and immunohistochemistry. The subjects were nine patients who had received surgery for renal cell carcinoma. After DNA ploidy analysis, the glass slides were restained by immunohistochemistry of PCNA. LSC allowed direct observation of PCNA localization during the cell cycle because we could obtain immunohistochemical staining of PCNA as a function of cell cycle phase for individual cells. RESULTS: PCNA was not demonstrated in the nuclei of G0/G1 cells. PCNA expression increased from the S phase of the cell cycle. PCNA rapidly degraded at the end of the G2 phase. In the late G2 and M phase, PCNA was not detected in almost any nucleus. CONCLUSION: LSC allows morphologic observation of the intracellular distribution of PCNA during the cell cycle in renal cell carcinoma.

Adult↗
PubMed1997

Simultaneous effects of aneuploidy and oncogenic human papillomavirus on histological grade of cervical intraepithelial neoplasia.

OBJECTIVE: To investigate whether ploidy and oncogenic human papillomavirus types can be correlated with the histological grade of cervical intraepithelial neoplasia (CIN) in the same tissue sections. DESIGN: Histological data were obtained from 292 dysplastic lesions of the cervix and classified according to the CIN and the Bethesda terminologies. The samples were analysed using Feulgen-stained image analysis cytometry for ploidy and Human papillomaviruses DNA typing by in situ hybridisation, respectively. SETTING: Colposcopy Clinic at Alfred Fournier Institute, Paris. POPULATION: Three hundred and forty women referred for an abnormal cervical smear. RESULTS: The ploidy data strongly segregate high grade from low grade squamous intraepithelial lesions (aneuploidy 78% versus 21%; P < 0.0001). There was a significant association between aneuploidy and the severity of the lesions (94% for CIN 3, 55% for CIN 2 and 14% for CIN 1; P < 0.0001). Both classifications showed a significant association of histological grade with oncogenic human papillomavirus types (HPV 16-18-33; 20% in low grade and 78% in high grade squamous intraepithelial lesions, 31% and 75% in CIN 1 and CIN 3, respectively; P < 0.0001). The simultaneous effects of these viruses and ploidy demonstrate an association in aneuploid cells between the presence of oncogenic human papillomavirus types and histological grade (76% and 18% in high and low grade squamous intraepithelial lesions, respectively; P < 0.0001). Such an association was not observed in diploid cells (20% in both low and high grade squamous intraepithelial lesions). CONCLUSIONS: High risk human papillomavirus types do not exert and an independent effect on the histological grade of cervical intraepithelial neoplasia.

Aneuploidy↗
PubMed2004

A suitable method for identifying cell aggregates in laser scanning cytometry listmode data for analyzing disaggregated cell suspensions obtained from human cancers.

BACKGROUND: The presence of cell aggregates in cell suspensions obtained from human solid tumors can interfere with the measurement of cell DNA content of cell singlets, and can confound multiparameter analysis of other measurements on the same cells. Flow cytometric corrections for cell aggregates based on signal pulse shape have not proven to be reliable. Mathematical models have been developed to correct for cell aggregates in binned DNA histogram data, but they are not suitable for the correction of correlated non-DNA measurements obtained on the same cells. METHODS: A total of 21 samples representing a variety of normal and malignant human cell types, including normal lymphocytes, normal sputum, human breast cancer cell lines, and mechanically disaggregated cell suspensions from primary breast cancers and nonsmall cell lung cancers, were studied by laser scanning cytometry (LSC) using the CompuCyte laser scanning cytometer (Cambridge, MA). Nuclear area, nuclear perimeter, and an LSC-based cell texture parameter were measured on approximately 400 cells in each sample, using an air-cooled, violet laser emitting at a wavelength of 405 nm for DAPI excitation, and each cell was classified as a singlet or aggregate by its appearance under direct observation. A "saddle function" provided by CompuCyte was used, together with an algorithm based on the measurements of nuclear area, perimeter, and cell texture (the APT algorithm), to identify cell aggregates and exclude them from the listmode data file. RESULTS: Proportions of cell aggregates in the uncorrected samples ranging from 6 to 56% (mean, 20%) were reduced to proportions ranging from 0 to 7% (mean, 2.4%) after correction. The discriminant function was "tuned" to maintain both average cell singlet purity and average cell singlet yield at >70% over a broad range of cell DNA contents. CONCLUSIONS: A combined approach to cell aggregate detection, which utilizes both the saddle function and the APT algorithm, produces list mode data files that exclude >80% of cell aggregates from samples of disaggregated cell suspensions of human tumors and other sources of clinical material. Such data files are suitable for multiparameter analysis.

Algorithms↗
PubMed2000

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Animals↗
PubMed1996

Interlaboratory comparison of DNA image analysis.

Interlaboratory quality assurance studies have been conducted for DNA flow cytometry, but not for DNA image analysis systems. The purpose of this study was to investigate if concordance of DNA image analysis systems existed with respect to classification and staining of standardized material. In three separate rounds, human liver cells were measured randomly by means of the image cytometry system present in each participating laboratory. The features integrated optical density (IOD) and AREA were reported. The relationship between the coefficient of variation (CV) of the 2c and 4c peak were compared with three models. In the three rounds the number of participating laboratories was 11, 14 and 11, respectively. Sequential plotting of normalized IOD values yielded useful information about intra-measurement variation. Comparison of measurements in specimens stained in the participating and central laboratory revealed similar CV values. In general, the precision of the instruments, expressed as the 4c/2c and 8c/2c ratios was good. The accuracy of the different laboratories expressed as the CV of IOD for the three rounds varied from 2-17%. The relation of the CVs of the 2c and the 4c peaks was best fit with the model of the addition of two normal distributions. We conclude that interlaboratory comparison of DNA measurements performed on different instruments is certainly feasible and could facilitate improvement in quality standards.

DNA↗
PubMed2006

Cytomics and drug discovery.

Pharmaceutical companies try to develop new drugs that have a high success rate of reaching the market. However, current disease models lack a strong correlation to clinical reality, because of the underestimation of the complexity and variability of clinical disease processes. This leads to high attrition rates late in drug development and soaring costs. Improvement of disease models is an important issue to reduce the high attrition rates in drug development. Using cell-based disease models, which should take into account the molecular diversity of the human cytome, will improve the predictive value of drug discovery.

Biomedical Research↗
PubMed2006

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.

BACKGROUND: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. METHODS: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. RESULTS: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an engagement of the minor groove. In vivo spectral analysis of DRAQ5 demonstrated shifts to longer wavelengths upon binding with DNA. Analysis of spectral windows of the dual emission peaks at 681 and 707 nm in cells showed that cell cycle compartment recognition was independent of the far red-near IR emission wavelengths monitored. CONCLUSIONS: The study provides new clues to modes of DNA binding of the modified anthraquinone molecule in vivo, and its AT base-pair selectivity. The combination of low quantum yield but high DNA affinity explains the favorable signal-to-noise profile of DRAQ5-nuclear fluorescence. The robust nature of cell cycle reporting using DRAQ5, even when restricted spectral windows are selected, facilitates the analysis of encroaching spectral emissions from other fluorescent reporters, including GFP-tagged proteins.

Anthraquinones↗
PubMed2005

Szilard's dream.

Explore the source record for details and available documents.

Bioreactors↗
PubMed2005

A microfluidic chemostat for experiments with bacterial and yeast cells.

Bacteria and yeast frequently exist as populations capable of reaching extremely high cell densities. With conventional culturing techniques, however, cell proliferation and ultimate density are limited by depletion of nutrients and accumulation of metabolites in the medium. Here we describe design and operation of microfabricated elastomer chips, in which chemostatic conditions are maintained for bacterial and yeast colonies growing in an array of shallow microscopic chambers. Walls of the chambers are impassable for the cells, but allow diffusion of chemicals. Thus, the chemical contents of the chambers are maintained virtually identical to those of the nearby channels with continuous flowthrough of a dynamically defined medium. We demonstrate growth of cell cultures to densely packed ensembles that proceeds exponentially in a temperature-dependent fashion, and we use the devices to monitor colony growth from a single cell and to analyze the cell response to an exogenously added autoinducer.

Bioreactors↗
PubMed2005

Application of a static fluorescence-based cytometer (the CellScan) in basic cytometric studies, clinical pharmacology, oncology and clinical immunology.

The CellScan apparatus is a laser scanning cytometer enabling repetitive fluorescence intensity (FI) and polarization (FP) measurements in living cells, as a means of monitoring lymphocyte activation. The CellScan may serve as a tool for diagnosis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) as well as other autoimmune diseases by monitoring FP changes in peripheral blood lymphocytes (PBLs) following exposure to autoantigenic stimuli. Changes in FI and FP in atherosclerotic patients' PBLs following exposure to various stimuli have established the role of the immune system in atherosclerotic disease. The CellScan has been evaluated as a diagnostic tool for drug-allergy, based on FP reduction in PBLs following incubation with allergenic drugs. FI and FP changes in cancer cells have been found to be well correlated with the cytotoxic effect of anti-neoplastic drugs. In conclusion, the CellScan has a variety of applications in cell biology, immunology, cancer research and clinical pharmacology.

Allergy and Immunology↗
PubMed2002

CD4 lymphocyte enumeration in patients with human immunodeficiency virus infection using three-colour and four-colour dual-platform flow cytometry: an inter-laboratory comparative evaluation.

INTRODUCTION: This study aims to evaluate the correlation and agreement between 2 methodologies of CD4 lymphocyte enumeration. MATERIALS AND METHODS: Fifty-two blood samples from patients with human immunodeficiency virus (HIV) infection were sent for CD4 lymphocyte enumeration at 2 major hospitals using dual-platform flow cytometry where the absolute lymphocyte counts were determined on separate haematology analyzers. CD4 cell enumeration was accomplished using 3-colour flow cytometry on the FACSCalibur cytometer (Becton Dickinson) in Hospital A and 4-colour flow cytometry on the Coulter Epic XL cytometer (Beckman Coulter) in Hospital B. The percentages and absolute counts of CD4 lymphocytes obtained were analysed using both linear regression and Bland-Altman plots. RESULTS: On linear regression plots, the total white cell counts, absolute lymphocyte counts, CD4 lymphocyte percentages and absolute CD4 counts from the 2 hospitals correlated well with correlation coefficients, r > 0.95. On the Bland-Altman plots, there was a mean difference of 0.13 x 10(9)/L and 0.06 x 10(9)/L in the total white cell and absolute lymphocyte counts from the 2 hospitals respectively. The CD4 lymphocyte percentages revealed a mean difference of only 0.05% (95% limits of agreement, -3.6 to 3.7%) but there was a mean difference of 14/uL in the absolute CD4 lymphocyte counts (95% limits of agreement, -113 to 142/uL). CONCLUSIONS: The CD4 lymphocyte percentages obtained using the 3-colour and 4-colour flow cytometry correlated and agreed well. However, the absolute CD4 lymphocyte counts obtained using the dual-platform technique in both the hospitals did not agree well. Hence, absolute CD4 lymphocyte counts from the 2 hospitals cannot be used interchangeably in clinical practice due to poor inter-laboratory comparability.

CD4 Lymphocyte Count↗
PubMed2003

Carcinoma in situ of the female breast. A clinico-pathological, immunohistological, and DNA ploidy study.

Carcinoma in situ of the breast (CIS) comprise a heterogenous group of lesions, covering a wide spectrum of clinical conditions and histopathological changes. With respect to biological behavior, CIS range from biologically aggressive lesions with a substantial risk of progression into invasive carcinoma (IC), to lesions with a very low malignant potential. Two main types of CIS are described--ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). Previous studies of CIS indicate that approximately a third will subsequently develop IC. Autopsy studies indicate that CIS is frequently occurring and it was estimated that about 20% of all women will develop CIS during lifetime. Only a minor fraction is ever diagnosed, although the incidence of DCIS is increasing, especially related to mammography screening. The lack of knowledge about the biological significance of the histopathological subtypes was the background of the present study. In 1982, a nationwide, prospective study of CIS (protocol DBCG 82-IS) was initiated by the Danish Breast Cancer Cooperative Group (DBCG). From this protocol, the group of patients treated with breast conservation surgery (BCS) constituted the material for clinico-histological investigation. A total of 275 women were included in the period 1982-89. Follow-up studies showed that recurrence rate was significantly related to nuclear size of the primary lesion. Since nuclear changes might be related to DNA content and, furthermore, many invasive breast carcinomas were shown to be DNA aneuploid, flow cytometric (FCM) DNA ploidy analysis was performed in a series of DCIS lesions. More than 80% of these lesions were DNA aneuploid, with a distribution similar to that found in invasive carcinomas. This finding raised the hypothesis that the DNA pattern of an invasive carcinoma was already established at the preinvasive stage of DCIS. Therefore, FCM DNA analysis was performed on a series of ICs with predominance of DCIS. Partial or complete concordance in DNA ploidy between DCIS and IC within the individual case was found in most cases, except for the additional presence in the IC component of DNA hyperdiploid clones that might possibly be of importance for the process of invasion. In order to further characterize CIS lesions and, possibly, to discriminate biologically different groups, immunohistochemical markers were investigated in a consecutive series of CIS and IC with predominance of DCIS. The results were correlated to the histopathological and DNA ploidy findings. In DCIS, significant correlation was shown between large nuclear size and comedonecrosis, both of which showed also strong association to DNA aneuploidy, high proliferation activity, low steroid receptor content, and overexpression of c-erbB-2 and p53--factors that may indicate an aggressive behavior. Small nuclear CIS, whether LCIS or DCIS, on the contrary, were DNA diploid with low proliferation, and no cases showed overexpression of c-erbB-2 and p53. In IC, comparison of the DCIS and the invasive component showed similar patterns. No significant differences, in neither morphology, immunohistochemistry, nor DNA ploidy, were shown between DCIS without and with invasion. These findings may indicate that none of the parameters in question may on its own be essential for the decisive event of invasive growth.

Adult↗
PubMed1997

Solid phase cytometry for detection of rare events.

We describe a simple and rapid laser scanning device for solid phase cytometry. This system can detect and count fluorescent cells over a 22 mm diameter surface (membrane or glass circle) as well as quantify the fluorescence that they emit. A comprehensive discrimination package includes optical and software parameters and accurately distinguishes between valid signals (e.g., labelled cells) and nonspecific signals (e.g., auto-fluorescent particles and debris). Any event detected may also be easily confirmed by visual observation after transfer of the sample to an epifluorescence microscope fitted with a motorized stage driven by the system. We show a linear relationship between the amount of fluorescein coupled to the cells and the fluorescence signal of the cells detected. This approach is not destructive and further characterization of the sample may be carried out. We have been able to detect rare cellular events at a frequency of 10(-7) in 3 min. Potential applications include monitoring of residual disease in oncology and detection of virus-infected cells circulating at very low frequencies.

Animals↗