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PubMed2000

Current and future applications of flow cytometry in aquatic microbiology.

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

Animals↗
PubMed1989

The streaming liver. V: Time and age-dependent changes of hepatocyte DNA content, following partial hepatectomy.

Two processes contribute to the change in hepatocyte ploidy following partial hepatectomy: one is age-dependent, while the other depends on the time which has elapsed since hepatectomy. While in the normal liver hepatocyte DNA content increases with the age of the cell, following hepatectomy the average DNA content (or ploidy) for the entire population rises as well. Hepatocyte age was derived from the cell's distance from the portal tract. We have previously shown that hepatocytes are formed adjacent to the portal tract and stream toward the terminal hepatic vein, advancing 2 microns/day. This finding enables the estimation of hepatocyte age from its location, since the older a cell the more remote it is from the portal tract. Twenty-four young male random-bred rats were partially hepatectomized and killed in groups of four animals at the following times: 1 h and 1, 2, 3, 7 and 14 days. Liver sections and nuclear suspensions which were collected by fine needle aspiration were stained with Feulgen and measured with the aid of computerized image cytometry. Part of the suspension which was aspirated with a fine needle was stained with propidium iodide and measured by flow cytometry. Both nuclear area and the DNA content increase with the age of the cell. The older a cell, the higher its ploidy. Generally, young hepatocytes which synthesized DNA did also divide, while older cells tended to accumulate DNA and became polyploid. The average nuclear DNA content of the entire population increased by 14% in 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

DNA content and expression of PCNA and p53 in Hodgkin's disease and Hodgkin's-like B-cell lymphoma.

DNA ploidy (by image cytometry) and expression of proliferating cell nuclear antigen (PCNA) and p53 tumor suppressor gene product (by immunohistochemistry) were investigated in 15 cases of Hodgkin's disease (HD) and 12 cases of HD-like B-cell lymphoma (HD-like NHL). Reed-Sternberg (RS) cells and their variants were DNA aneuploid in all cases. However, the fraction of hyperoctaploid tumor cells was higher in HD than in HD-like NHL. PCNA expression was high in neoplastic cells (> 50%) and variable (5-40%) in reactive lymphocytes in both HD and HD-like NHL. p53 positivity was found in RS cells and their variants in 64% of HD cases, but only in 25% of cases of HD-like NHL. Our results support the suggestion that HD-like B-cell lymphomas should be considered as highly malignant non-Hodgkin's lymphomas rather than Hodgkin's disease.

Adult↗
PubMedDate not supplied

Measurement of orientation and distribution of cellular alignment and cytoskeletal organization.

Endothelial cells elongate and align with the direction of applied fluid shear stress. Previously, automated methods for analysis of cell orientation distribution have used Fourier- or fractal-based methods. We used intensity gradients in images of control and sheared endothelial cells to measure orientation distributions. Automated measurements of mean orientation and angular deviation compared favorably with manual measurements. There was a significantly greater angular deviation in images of control cells compared with sheared cells. Automated methods were also used to quantify organization of cytoskeletal fibers using the local angular deviation and a measure of the local coalignment of fibers called the coalignment ratio. The local angular deviation of microtubules and microfilaments was significantly smaller in sheared cells compared with control. The coalignment of cytoskeletal fibers was significantly greater in sheared cells. We conclude that image intensity gradients can be used rapidly, accurately, and objectively to measure cell orientation distributions and cytoskeletal filament organization.

Algorithms↗
PubMed2006

Colocalisation of intraplaque C reactive protein, complement, oxidised low density lipoprotein, and macrophages in stable and unstable angina and acute myocardial infarction.

BACKGROUND: C reactive protein (CRP), an important serum marker of atherosclerotic vascular disease, has recently been reported to be active inside human atherosclerotic plaques. AIMS: To investigate the simultaneous presence of macrophages, CRP, membrane attack complex C5b-9 (MAC), and oxidised low density lipoprotein (oxLDL) in atherectomy specimens from patients with different coronary syndromes. METHODS: In total, 54 patients with stable angina (SA; n = 21), unstable angina (UA; n = 15), and myocardial infarction (MI; n = 18) underwent directional coronary atherectomy for coronary lesions. Cryostat sections of atherosclerotic plaques were immunohistochemically stained with monoclonal antibodies: anti-CD68 (macrophages), anti-5G4 (CRP), aE11 (MAC), and 12E7 (oxLDL). Immunopositive areas were evaluated in relation to fibrous and neointima tissues, atheroma, and media. Quantitative analysis was performed using image cytometry with systematic random sampling (percentage immunopositive/total tissue area). RESULTS: Macrophages, CRP, MAC, and oxLDL were simultaneously present in a higher proportion of fibrous tissue and atheroma of atherectomy specimens from patients with UA and MI compared with SA (p<0.05). Quantitative analysis showed significantly higher mean percentages of macrophages in plaques from patients with MI (44%) than UA (30%; p<0.01) and SA (20%; p<0.001). Significantly higher mean percentages of CRP were also seen in MI (25%) and UA (25%) compared with SA (12%; p<0.05). CONCLUSIONS: The presence of CRP, complement, and oxLDL in a high proportion of plaque tissue from patients with unstable coronary artery disease implies that these surrogate markers have important proinflammatory effects inside atherosclerotic plaques. This may increase vulnerability to plaque rupture and thrombosis, with subsequent clinical sequelae.

Angina Pectoris↗
PubMed1999

Predictive value of malignancy grading systems, DNA content, p53, and angiogenesis for stage I tongue carcinomas.

AIM: To assess the clinical value of malignancy grading systems compared with nuclear DNA content, protein p53, and angiogenesis for predicting recurrence of stage I (UICC, 1987) tongue carcinomas. METHODS: Histopathological malignancy grading according to Jakobsson and tumour front grading according to Bryne et al were performed on haematoxylin and eosin slides. DNA analysis was performed by image cytometry. Protein p53 and angiogenesis were evaluated by immunohistochemical analysis using antibody CM1 and antibody against factor VIII related antigen, respectively. RESULTS: 49 patients with stage I carcinomas of the mobile tongue were included, all treated by local surgical excision alone. Eight patients (16%) suffered from local recurrence during follow up, and 13 (27%) had regional recurrence. Both Jakobsson's malignancy grading system and p53 immunoreactivity proved to be useful predictors of regional recurrence in a Cox multivariate regression analysis. CONCLUSIONS: Histopathological malignancy grading systems provide valuable prognostic information and can still compete with current biological markers in this respect.

Adult↗
PubMed2003

Proliferation markers and DNA content analysis in urinary bladder TaT1 urothelial cell carcinomas: identification of subgroups with low and high stage progression risks.

AIMS: To evaluate whether in situ biomarkers Ki67, mitotic activity index (MAI), p53, mean area of the 10 largest nuclei (MNA10), and whole genome DNA ploidy by flow and image cytometry (FCM and ICM, respectively) have independent prognostic value in urinary bladder urothelial cell carcinomas (UCs). METHODS: Ki67 and p53 immunoquantitation was performed in TaT1 consensus diagnosis UCs. MAI and MNA10 were also determined. Single cell suspensions were stained (DAPI for FCM; Feulgen for ICM). There was enough material for all measurements in 171 cases. Kaplan-Meier curves and multivariate survival analysis (Cox) were used to assess the prognostic value of all features (including classic clinicopathological risk factors, such as stage, grade, multicentricity, carcinoma in situ). RESULTS: Thirteen (7.6%) patients progressed. Of the classic factors, grade was strongly prognostic in univariate analysis, as were all the biomarkers. In multivariate analysis, the strongest independent combinations for progression were MNA10 (threshold (T) = 170.0 micro m(2)) plus MAI (T = 30), or MNA10 (T = 170.0 micro m(2)) plus Ki67(T = 25.0%). p53 (T = 35.2%) plus Ki67 (T = 25.0%) also predicted progression well, with high hazard ratios, but p53 measurements were not as reproducible as the other features. The prognostic value of the quantitative biomarkers exceeded that of the classic risk factors and DNA ploidy. The sensitivity, specificity, positive, and negative predictive values of MNA10/MAI or MNA10/Ki67 at the thresholds mentioned were 100%, 79%, 57%, and 100%, respectively. These feature combinations were also strongest prognostically in the high risk treatment subgroup. CONCLUSIONS: The combined biomarkers MNA10/Ki67 or MNA10/MAI are more accurate and reproducible predictors of stage progression in TaT1 UCs than classic prognostic risk factors and DNA ploidy.

Adult↗
PubMed1998

Nuclear DNA content and p53 overexpression in stage I squamous cell carcinoma of the tongue compared with advanced tongue carcinomas.

AIMS: To evaluate the predictive value of the nuclear DNA content (image cytometry) and p53 overexpression (immuno-histochemistry using antibody CM-1) in uniformly treated stage I carcinomas of the mobile tongue. Also, to compare stage I carcinomas with advanced tongue carcinomas (stages II-IV). METHODS: Archival formalin fixed, paraffin wax embedded tumour specimens from 54 patients with stage I squamous cell carcinoma and 37 patients with advanced squamous cell carcinoma were analysed. Mean follow up time of the stage I carcinomas was 71 months (median, 62.5; range, 6-175). RESULTS: Twenty three patients (stage I) had recurring disease: 10 had local recurrence (in the tongue) and 13 had regional recurrence (cervical metastases). Locally recurring stage I carcinomas had a more pronounced DNA deviation than the other stage I carcinomas and this degree of deviation was comparable with the DNA content of advanced carcinomas. Stage I carcinomas that developed regional recurrences overexpressed p53 more frequently. In Cox multivariate regression analysis of time to recurrence, DNA deviation was a significant parameter in tumours that recurred locally (p = 0.032). p53 overexpression was the only parameter close to significance for regional recurrence (p = 0.065). CONCLUSIONS: Nuclear DNA content and p53 immunostaining are of value for the prediction of recurrence of stage I squamous cell carcinomas of the mobile tongue. Stage I tongue carcinomas that are prone to local recurrence show the same DNA content as do advanced tongue carcinomas.

Adult↗
PubMed2004

Paradoxical relationship between protein content and nucleolar activity in mammalian cardiomyocytes.

It was recently demonstrated that polyploidization of the avian myocardium is associated with a reduction of cardiac aerobic capacity evaluated by the heart mass to body mass ratio (heart index). To investigate possible cellular correlates of polyploidization, the protein content and nucleolar activity per cell and per genome were examined by image cytometry in 21 mammalian species, differing in the degree of heart polyploidization and heart index. We found that average cardiomyocyte ploidy level correlates negatively with the animal heart index (r = -0.75, p < 10(-4)), i.e., the large heart of athletic mammals is polyploidized to a lesser degree than the relatively smaller heart of sedentary species, which confirms the picture observed in birds. The protein content per genome decreased with the elevation of cardiomyocyte ploidy level. This inverse correlation was especially pronounced with the removed effect of body mass (r = -0.79, p < 10(-4)). Surprisingly, these changes were accompanied by the increase of nucleolar activity per genome (r = 0.61, p < 10(-3)). In the two species, for which the microarray gene expression data were available (human and mouse), this increase was paralleled by the elevated expression of ribosomal protein genes (but there was no increase in the expression of tissue-specific genes). Thus, in the polyploid cardiomyocytes there is a misbalance between protein content per genome and ribosome biogenesis. The reduction of protein content (per genome) of polyploid cardio my ocytes should further curtail heart functionality (in addition to reduction of heart index), because it is known that cardio myocyte protein content consists of more than 90% contractile proteins. This finding makes doubtful a widespread notion that polyploidization is necessary for cell function. Because somatic polyploidization is associated with stressful conditions and impaired energetics, we suppose that additional genomes can serve for cell regeneration and as a defense against oxidative damage in the organs that work at the limit of their metabolic capacity.

Animals↗
PubMed1997

Topographical analysis of spatial patterns generated by a cellular automaton model of the proliferation of a cancer cell line in vitro.

A well-suited model to simulate cellular population dynamics is the two-dimensional cellular automaton model, which consists of a lattice of sites, the value ai,j of each site being updated in discrete time steps according to an identical deterministic rule depending on a neighbourhood of sites around it. A cellular automaton is described which mimics cell population proliferation by replacing the site values by the age and the cycle phase of cells. The model takes into account the size of the cells. It is used to simulate the proliferation of the human breast cancer cell line MCF-7 and the results of the simulation are compared with experimental data obtained from a light microscopic image analysis of the proliferation process. The initial configuration of the cellular automaton is obtained from the discretization of the results of the initial stage of the image processing. After each day of proliferation the pattern obtained from the simulation is compared to the experimental result of the corresponding image analysis. The comparison is made from a topographical point of view through the concept of the minimal spanning tree graph. The agreement between experiment and model is a good starting point to complex models such as cell proliferation under growth effectors or drugs.

Breast Neoplasms↗
PubMed1997

Correlation between p53 status, DNA ploidy, proliferation rate and nuclear morphology in breast cancer. An image cytometric study.

The study was designed to detect differences in the nuclear morphology of tumours and tumour cell populations with different p53 expression in correlation with DNA ploidy and proliferation rate. The paraffin sections from routinely processed samples of 88 breast cancers were immunostained with the monoclonal p53-antibody DO-1. After localization and evaluation with a scoring system the sections were destained and stained by the Feulgen method. The nuclei were relocated automatically and measured by means of the image cytometry workstation. Significant differences between the tumours and tumour cell populations with different p53 expression were found in the euploid tumours as well as in the aneuploid tumours and in the breast cancers with a high proliferation rate. The breast cancers with a low immunoreactive score (IRS 1-4) differ from the negative cancers as well as from the cancers with a higher immunoreactive score (IRS 5-12). Evaluating the nuclear populations of the p53 positive cancers, there were differences in the features of the chromatin amount and distribution in the groups of the euploid breast cancers and in cancer with a high proliferation rate. In contrast, the nuclear populations of the aneuploid cancers did not show any differences in their nuclear morphology. The results showed the different impacts of the p53 expression, DNA ploidy and the proliferation rate on the nuclear morphology in breast cancer.

Adult↗
PubMed1998

Chromatin texture from hematoxylin stained thyroid lesions.

Quantitative aspects of cytology and histology should be considered in diagnostic standardisation processes. The present paper summarises the cytological differences detected in 75 thyroid lesions using a computerized textural analysis. Cells stained with progressive hematoxylin and taken from paraffin blocks were overlaid with the extracted texture. This technique was based on the lineal detection of a grey level gradient of the common logarithm of the integrated optical density (IOD) of each nucleus. Diffuse and nodular goiters (36 cases) were demonstrated to be composed of small cells containing high density texture that, on microscopical visual inspection, gave a "salt and pepper" appearance. The adenomatous goiters (2 cases) and adenomas (26 cases) were composed of low texture cells with a visual "blurry or smudgy" chromatin, while the atypical adenomas with capsular invasion (4 cases) were characterised by a "woodworm" nuclear appearance that produced the highest texture of the series. Finally, encapsulated folliculo-papillary carcinomas (3 cases) were composed of large clear nuclei with high IOD, low texture, and scattered lines that resulted in an "empty grape skin" aspect. Our findings seam to confirm the suitability of computerized textural techniques that aid in recognizing cell microscopic features objectively. The one used in the present work, based on a mathematical function of the DNA content of each individual nucleus (IOD), fulfills all microscopy detection criteria.

Chromatin↗
PubMed2001

Automated detection of connective tissue by tissue counter analysis and classification and regression trees.

OBJECTIVE: To evaluate the feasibility of the CART (Classification and Regression Tree) procedure for the recognition of microscopic structures in tissue counter analysis. METHODS: Digital microscopic images of H & E; stained slides of normal human skin and of primary malignant melanoma were overlayed with regularly distributed square measuring masks (elements) and grey value, texture and colour features within each mask were recorded. In the learning set, elements were interactively labeled as representing either connective tissue of the reticular dermis, other tissue components or background. Subsequently, CART models were based on these data sets. RESULTS: Implementation of the CART classification rules into the image analysis program showed that in an independent test set 94.1% of elements classified as connective tissue of the reticular dermis were correctly labeled. Automated measurements of the total amount of tissue and of the amount of connective tissue within a slide showed high reproducibility (r=0.97 and r=0.94, respectively; p<0.001). CONCLUSIONS: CART procedure in tissue counter analysis yields simple and reproducible classification rules for tissue elements.

Algorithms↗
PubMed2001

Application of neural networks to the classification of pancreatic intraductal proliferative lesions.

The aim of the study was to test applycability of neural networks to classification of pancreatic intraductal proliferative lesions basing on nuclear features, especially chromatin texture. Material for the study was obtained from patients operated on for pancreatic cancer, chronic pancreatitis and other tumours requiring pancreatic resection. Intraductal lesions were classified as low and high grade as previously described. The image analysis system consisted of a microscope, CCD camera combined with a PC and AnalySIS v. 2.11 software. The following texture characteristics were measured: variance of grey levels, features extracted from the grey levels correlation matrix and mean values, variance and standard deviation of the energy obtained from Laws matrices. Furthermore we used moments derived invariants and basic geometric data such as surface area, the minimum and maximum diameter and shape factor. The sets of data were randomly divided into training and testing groups. The training of the network using the back-propagation algorithm, and the final classification of data was carried out with a neural network simulator SNNS v. 4.1. We studied the efficacy of networks containing from one to three hidden layers. Using the best network, containing three hidden layers, the rate of correct classification of nuclei was 73%, and the rate of misdiagnosis was 3%; in 24% the network response was ambiguous. The present findings may serve as a starting point in search for methods facilitating early diagnosis of ductal pancreatic carcinoma.

Adenocarcinoma↗
PubMed2001

Prognostic classification of early ovarian cancer based on very low dimensionality adaptive texture feature vectors from cell nuclei from monolayers and histological sections.

In order to study the prognostic value of quantifying the chromatin structure of cell nuclei from patients with early ovarian cancer, low dimensionality adaptive fractal and Gray Level Cooccurrence Matrix texture feature vectors were extracted from nuclei images of monolayers and histological sections. Each light microscopy nucleus image was divided into a peripheral and a central part, representing 30% and 70% of the total area of the nucleus, respectively. Textural features were then extracted from the peripheral and central parts of the nuclei images.The adaptive feature extraction was based on Class Difference Matrices and Class Distance Matrices. These matrices were useful to illustrate the difference in chromatin texture between the good and bad prognosis classes of ovarian samples. Class Difference and Distance Matrices also clearly illustrated the difference in texture between the peripheral and central parts of cell nuclei. Both when working with nuclei images from monolayers and from histological sections it seems useful to extract separate features from the peripheral and central parts of the nuclei images.

Cell Nucleus↗
PubMed2003

Application of multilevel models to morphometric data. Part 1. Linear models and hypothesis testing.

Morphometric data usually have a hierarchical structure (i.e., cells are nested within patients), which should be taken into consideration in the analysis. In the recent years, special methods of handling hierarchical data, called multilevel models (MM), as well as corresponding software have received considerable development. However, there has been no application of these methods to morphometric data yet. In this paper we report our first experience of analyzing karyometric data by means of MLwiN - a dedicated program for multilevel modeling. Our data were obtained from 34 follicular adenomas and 44 follicular carcinomas of the thyroid. We show examples of fitting and interpreting MM of different complexity, and draw a number of interesting conclusions about the differences in nuclear morphology between follicular thyroid adenomas and carcinomas. We also demonstrate substantial advantages of multilevel models over conventional, single-level statistics, which have been adopted previously to analyze karyometric data. In addition, some theoretical issues related to MM as well as major statistical software for MM are briefly reviewed.

Adenoma↗