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Biomedical subjects

G Brugal

Publications and source records attributed to G Brugal.

At least 37 records · Page 2Linked to original sources
PubMed1994

A new approach to man-machine communication for computerized microscopy.

HOME is a new computerized microscope designed to assist pathologists and cytotechnicians in routine examinations. The HOME workstation is composed of a standard light microscope fitted with objective and stage encoders, and a built-in high resolution computer display which superimposes dialog, drawing, and messages onto the optical microscope image. The software runs under Windows 3.x and provides interactive facilities such as accurate localization and relocation of zones of interest, morphometric measurements, patient data access, and quality control processes.

Cell Biology↗
PubMed1994

Relocation accuracy on HOME computerized microscopes.

A major practical advantage of the HOME (highly optimized microscope environment) computerized microscope is the facility for relocating cells or other microscopic objects. Features can be marked directly on the microscope image using a mouse-driven cursor, and an interactive finder can then be used to relocate the marked features. Tests on a prototype HOME microscope have shown that positions can be relocated with an accuracy of standard deviation (SD) < 7 microm. The marked features could also be relocated on a second HOME microscope, although with somewhat reduced accuracy (standard deviations of < 17 microm). The system provides a very user-friendly environment for tasks requiring relocation of microscopic objects.

Calibration↗
PubMed1994

Combination of DNA in situ hybridization and immunocytochemical detection of nucleolar proteins: a contribution to the functional mapping of the human genome by fluorescence microscopy.

The recent application of DNA cloning and non-radioactive in situ hybridization techniques has strengthened the hypothesis of an ordered chromatin structure in interphase nuclei. The arrangement of specific chromosomal regions is not random and is strongly suspected to vary with functional activity. The combination of in situ hybridization and immunocytochemistry, allowing simultaneous detection of nucleic acid sequences and specific antigens in the same nucleus, has already made significant contributions to the study of gene expression, to simultaneous karyotyping and phenotyping of tumor cells, and to in situ analysis of viral infections. This report emphasizes the considerable interest of such combined techniques for functional in situ mapping of the genome at the individual cell level. We propose a method that combines fluorescence immunocytochemical detection of nucleolar proteins and fluorescence in situ hybridization of centromeric and telomeric probes specific for chromosome 1 in two cultured human cell lines. The preparative constraints for a broad application of this procedure are defined so that the cell preparations can be further analyzed by fluorescence microscopic imaging techniques and confocal laser scan microscopy. The two selected sequences of the human chromosome 1 can be localized in the nucleus with respect to nucleolar proteins in a one-step fluorescence microscopic observation.

Cell Nucleolus↗
PubMed1993

Detection of chromosome 1 aberrations by fluorescent in situ hybridization (FISH) in the human breast cancer cell line MCF-7.

In situ hybridization using nucleic acid probes specific for a particular chromosome repetitive sequence makes it possible to determine the number of corresponding chromosomes and potential aberrations in each cell of the population under investigation. The number of in situ hybridization spots per nucleus, indicating chromosomal ploidy and/or aberrations, thus marks the possible cell subpopulations having an abnormal karyotype. Applying such an interphase cytogenetics approach on the MCF-7 human breast cancer cell line with centromeric (pUC 1.77) and telomeric (1p-79) probes specific for repetitive sequences on the chromosome 1, we investigated the heterogeneity of the cell population on the basis of the number of fluorescent in situ hybridization (FISH) spots per nucleus and their location with respect to the others. We also followed the chromosome 1 genetic drift as the cell population aged. Our results confirmed the already known trisomy for the chromosome 1 in this cell line and demonstrated a deletion and a translocation of its telomeric sequence. Moreover, a subpopulation of cells with six chromosomes 1 representing a few percent of the population was transiently detected.

Breast Neoplasms↗
PubMed1992

HOME: highly optimized microscope environment.

The Highly Optimized Microscope Environment (HOME) is a computerized microscope designed to assist pathologists and cytotechnicians in clinical routine tasks. The prototype system consists of a IBM-PC compatible computer and a light microscope in which a built-in high-resolution computer display image is superimposed on the optical image of the specimen. Also, a manually operated encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on a slide to be uniquely defined and makes it possible to measure interactively lengths and areas larger than the size of the microscope field. Software, written in the C language and operating under the MS-DOS/MS-Windows environment, is controlled by means of a mouse-driven cursor moving over menu light-buttons displayed on the microscope image. The HOME microscope workstation is potentially useful in a wide range of applications such as i) tagging information on particular cells and tissue structures that can thus be accurately located and relocated, ii) performing morphometric measurement, differential counting, and stereological assessment of biological specimens, and iii) training and educating laboratory personnel. Finally, HOME will offer in the near future a user-friendly interface for automatic image processing of cells and tissue entities in interactively selected specimen areas.

Animals↗
PubMed1992

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.

Animals↗
PubMed1992

The HOME microscope workstation. A new tool for cervical cancer screening.

The Highly Optimized Microscope Environment (HOME) is a computerized microscope design for assisting pathologists and cytotechnologists in routine clinical tasks. The prototype system consists of an IBM PC-compatible computer and a light microscope in which a built-in high-resolution computer display image is super-imposed on the optical image of the specimen. Also, an encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on the stage to be uniquely defined. Software, written in C language and running under the MS-DOS/MS-Windows environment, is controlled by means of a mouse-driven cursor. A specific application has been developed for cervical cancer screening, taking into account the needs and constraints of microscopists performing this task. Informatics tools offered by the HOME system provide them with precise flagging and relocation of objects on the slide, control of the scanning pathway, and ability to write and print the report directly through the microscope. The computer files generated by microscopic examination are stored and contain information available for quality control assessment and laboratory management.

Cytodiagnosis↗
PubMed1991

Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment.

Simultaneous quantification of DNA and Ki-67 proliferation-associated antigen was performed using fluorescence image cytometry. In the MCF-7 cell line, the Ki-67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki-67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki-67 antigen remains constant during G1 (Ki-67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki-67 (Ki-67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate-sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki-67 content (Ki-67 increase pathway) was observed from intermediate-sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki-67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki-67 decrease pathway and Ki-67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.

Animals↗
PubMed1991

Inhibitory effect of 17 beta-estradiol on thymocyte proliferation and metabolic activity in young rats.

Proliferation of 21-day-old rat thymus lymphocytes has been assessed after 7 daily injections of 17 beta-estradiol (E2). Two methods were used: 1) image cytometry of cell nuclei using the SAMBA 200 image processor, and 2) cell culture making it possible to design cell proliferation assay, mitochondrial activity evaluation and measurement of IL2 activity. Using image cytometry, we found a significant decrease in the proliferation index at the dose of 100 micrograms of E2 per day. When 200 micrograms and 400 micrograms per day were given, this decrease reached 70% of inhibition. 3H-thymidine incorporation was significantly reduced in cell culture even after 5 micrograms of E2 per day. On the other hand, with either 5 micrograms or 50 micrograms of E2, a significant increase of IL2 was found in the supernatant.

Animals↗
PubMed1990

Detection of S cells and evaluation of DNA denaturation protocols by image cytometry of fluorescent BrdUrd labelling.

This paper addresses the problem of detecting weak incorporation of BrdUrd and the related efficiency of the denaturation protocols used to unmask this thymidine analog. Evidence is presented that measuring the distribution of BrdUrd-tagged fluorescence intensities by image cytometry generates a standard deviation threshold that discriminates between positive and negative MRC5 cells in vitro. A comparison of the thresholding by standard deviation (SDT) with the usual thresholding by the nuclear total fluorescence intensity (FIT) demonstrated that SDT has a significantly higher sensitivity (99.4-100%, depending on the denaturation protocols) than FIT (94.7 and 74.3%, respectively), although both tests have a high specificity (93% and 100%, respectively) for detecting S cells. Since detecting the S cells is not only dependent on the test used, but also on the denaturation protocols, a quality index (QI) was derived from the standard deviation and the mean value of the non-specific fluorescence of negative cell population versus BrdUrd fluorescence of positive cell population. The following DNA denaturation protocols have been assessed according to QI: acidic denaturation, thermal denaturation in formamide, and thermal denaturation in distilled water. Each denaturation procedure was preceded or not by incubation in either proteinase K or Triton X-100. The results showed that thermal denaturation in formamide, especially when preceded by proteinase K incubation, revealed the largest difference between negative and positive cells. This work also demonstrated that image cytometry of BrdUrd-labelled cells can be suitable for clinical application because of the high sensitivity provided and the small samples needed.

Bromodeoxyuridine↗
PubMed1990

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line.

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.

Actins↗
PubMed1989

[Contribution of quantitative cytology and ploidy in the diagnosis and monitoring of tumors of the bladder. Study of 52 cases using the Samba analyser].

Image processors and present specific software applications have now placed scanning microcytometry in the ranks of other conventional technics for routine laboratory examinations, particularly for urinary cytology and DNA measurements (ploidy). Conventional urinary sediment smears of 52 patients with bladder tumor were digitized, processed and statistically analyzed by the image analyzer, Samba. They were automatically classified and ploidy measurements were made and compared with 6 conventional cytological diagnostics for each smear. The Samba program assigns the cell images to two classes, negative and positive. 100% of the cells were correctly classed with respect to the cytologist's diagnostics. Within the positive class the degree of malignancy established by the program was the same as the cytologist's classifications. For smears of class III, the comparison of the diagnostics of the cytologists for each patient permitted the validation of the differential classification, negative-positive, made using quantitative cytology. The interpretation of the two DNA histogram parameters, the degree of ploidy and the proliferation index, provided an excellent prognosis of which patients would show tumoral recrudescence, as verified by follow ups.

Cytophotometry↗
PubMed1988

Image cytometry of estrogen receptors in breast carcinomas.

A significant level of estrogen receptors (ER) in breast cancer cells is an indication of tumor differentiation and suggests that a homeostatic control of cell growth may persist in these cancers. In medical practice, the Dextran-coated charcoal assays (DCCA) are still the most frequently used test to characterize patients having ER-positive malignant breast tumors and for whom hormonal therapy is justified. Nevertheless, this routine biochemical technique is not satisfactory because it is a broad method unsuitable for revealing receptor tissue heterogeneity. However, immunocytochemical labeling, such as the ER-ICA method, which involves a monoclonal antibody linked to peroxidase, is a specific reaction for this purpose but which until now was not quantitative. The present study uses an original cell preparation technique combining the PAP reaction with toluidine blue counterstain for image analysis on the SAMBA system. Special software has been developed for the quantitative analysis of immunocytochemistry in cancers. Results obtained showed a high correlation between the DCCA values and the score derived from the mean ER concentration per positive tumor cell and the labeling index. In addition, intracell and intratumor heterogeneity can be displayed according to several parameters and were shown to vary according to tumor and to antiestrogen (Tamoxifen) presurgical therapy.

Breast Neoplasms↗
PubMed1987

Cytometry of breast carcinoma: significance of ploïdy balance and proliferation index.

Image cytometry of DNA distribution in fine needle biopsies of breast carcinomas at first diagnosis was performed to see if there were significant differences in DNA histograms between patients having very different outcome but same tumor histological typing and similar therapy. Two groups of patients were considered retrospectively: the first (20 patients) with survival time shorter than 5 years and the second (20 patients) with survival time longer than 10 years. Seven benign tumors were used as controls. Ten ploïdy classes were defined. The frequencies of cells in those classes were used as independent features in a supervised multivariate analysis. The advantages of this approach was pointed out with respect to the four-type classification of Auer. The scattering of DNA histograms within the feature space showed that a subgroup of patients with poor prognosis was clearly separated from a subgroup of patients with good prognosis but both long survival patients and short survival patients were scattered in between. In order to replace the multivariate classification of histograms by a simpler approach, two parameters were computed which explained most of the scattering in the feature space: the ploïdy balance (difference between the percentages of euploid and aneuploïd cells) and the proliferation index (percentage of cells between peaks). The scattergram of patients according to these parameters showed again that some DNA distributions were specific for either good or bad prognosis. But the separation was uncertain for seven short-survival patients and six long-survival patients. For six patients, the DNA distributions were very similar between long and short survival times. Those patients thus could not be separated even by means of discriminant analysis. The main conclusion of this study was that, for a significant number of patients, the objective multivariate classification of tumors DNA profiles is of little assistance to the pathologist who has to give a prognosis for the one patient under consideration.

Aneuploidy↗
PubMed1987

A quantitative analysis of the human bone marrow erythroblastic cell lineage using the SAMBA 200 cell image processor. I. The normal maturation sequence.

A quantitative image analysis of the normal maturation sequence for the human bone marrow erythroblastic lineage was performed using the SAMBA 200 cell image processor. The different image analysis steps (image acquisition, preprocessing, segmentation, parametrization and data analysis) are briefly described. Thirty-three parameters related to geometry, color, texture and densitometry were computed on 638 cell images belonging to the five erythroblastic maturation stages. The automated classification of these cells, based upon a stepwise linear discriminant analysis, resulted in 80% correctly classified cells. Acceptance of confusions between successive maturation stages enhanced the rate of correctly classified cells to 100%. Among the ten most discriminating parameters, the nuclear area showed the highest correlation with the changes throughout the maturation process. The projection of the maturation sequence onto the factorial plane resulting from the canonical analysis emphasizes the existence of three phases of the maturation process, a finding that correlates well with the cytologic evolution and the biochemical and functional events during the maturation. The trajectory of cells within this factorial plane is thus regarded as a differentiation path from which a measure of the maturation could be derived.

Bone Marrow Cells↗