Gérard Brugal

Digital Imagery/Telecytology

ISSUES Optical digital imaging and its related technologies have applications in cytopathology that encompass training and education, image analysis, diagnosis, report documentation and archiving, and telecommunications. Telecytology involves the use of telecommunications to transmit cytology images for the purposes of diagnosis, consultation or education. This working paper provides a mainly informational overview of optical digital imaging and summarizes current technologic resources and applications and some of the ethical and legal implications of the use of these new technologies in cytopathology. CONSENSUS POSITION Computer hardware standards for optical digital imagery will continue to be driven mainly by commercial interests and nonmedical imperatives, but professional organizations can play a valuable role in developing recommendations or standards for digital image sampling, documentation, archiving, authenticity safeguards and teleconsultation protocols; in addressing patient confidentiality and ethical, legal and informed consent issues; and in providing support for quality assurance and standardization of digital image-based testing. There is some evidence that high levels of accuracy for telepathology diagnosis can be achieved using existing dynamic systems, which may also be applicable to telecytology consultation. Static systems for both telepathology and telecytology, which have the advantage of considerably lower cost, appear to have lower levels of accuracy. Laboratories that maintain digital image databases should adopt practices and protocols that ensure patient confidentiality. Individuals participating in telecommunication of digital images for diagnosis should be properly qualified, meet licensing requirements and use procedures that protect patient confidentiality. Such individuals should be cognizant of the limitations of the technology and employ quality assurance practices that ensure the validity and accuracy of each consultation. Even in an informal teleconsultation setting one should define the extent of participation and be mindful of potential malpractice liability. ONGOING ISSUES Digital imagery applications will continue to present new opportunities and challenges. Position papers such as this are directed toward assisting the profession to stay informed and in control of these applications in the laboratory. Telecytology is an area in particular need of studies of good quality to provide data on factors affecting accuracy. New technologic approaches to addressing the issue of selective sampling in static image consultation are needed. The use of artificial intelligence software as an adjunct to enhance the accuracy and reproducibility of cytologic diagnosis of digital images in routine and consultation settings deserves to be pursued. Other telecytology-related issues that require clarification and the adoption of workable guidelines include interstate licensure and protocols to define malpractice liability.

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.

Correlation between silver-stained nucleolar organizer region area and cell cycle time.

BACKGROUND The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. METHODS After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. RESULTS Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. CONCLUSIONS The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units.

A proliferation control network model: The simulation of two-dimensional epithelial homeostasis

Despite the recent progress in the description of the molecular mechanisms of proliferation and differentiation controls in vitro, the regulation of the homeostasis of normal stratified epithelia remains unclear in vivo. Computer simulation represents a powerful tool to investigate the complex field of cell proliferation regulation networks. It provides huge computation capabilities to test, in a dynamic in silico context, hypotheses about the many pathways and feedback loops involved in cell growth and proliferation controls.Our approach combines a model of cell proliferation and a spatial representation of cells in 2D using the Voronoi graph. The cell proliferation model includes intracellular (cyclins, Cyclin Dependent Kinases - CDKs, Retinoblastoma protein - Rb, CDK inhibitors) and extracellular controls (growth and differentiation factors, integrins). The Voronoi graph associates a polygon with every cell and the set of these polygons defines the tissue architecture. Thus, the model provides a quantitative model of extracellular signals and cell motility as a function of the neighborhood during time dependent simulations.The 2D simulations illustrate the influence of the microenvironment on cell proliferation in basal layers of stratified epithelia and of differential adherence in keratinocytes differentiation and related upward migration. Our results particularly show the role of CDK inhibitors (mainly the protein p27) in the Rb dependent control pathway of the transition from the G1 to S phase of the cell cycle.

Identifying cytologic characteristics and grading endocervical columnar cell abnormalities. A study aided by high-definition television.

OBJECTIVE To test the ability of cytotechnologists to recognize and accurately interpret selected architectural, cellular and nuclear features presented on a high-definition television (HDTV) and to make a reliable diagnosis with HDTV. STUDY DESIGN A total of 1,122 features considered diagnostic of different endocervical columnar cell abnormalities were selected from 50 smears from 48 women with the help of a motor-driven-stage microscope by five observers who had knowledge of the final diagnosis. The selected and stored features were presented on an HDTV and evaluated in five successive sessions without knowledge of the final diagnosis. RESULTS Specific types of features were correctly identified in a high number of cases. Considerable interobserver variability was demonstrated in the scoring of grades of expression of features. Overrated and under-rated monitor diagnoses were related to overvalued and undervalued features. From a group of 437 images that were correctly diagnosed by four or five observers, five features proved to be highly related to the correct diagnosis. CONCLUSION Observers were capable of making a reliable diagnosis on features, selected by other observers, when presented on an HDTV. An overall correct diagnosis was made in 93% of cases.

Quantitation of AgNORs by Flow Versus Image Cytometry

AgNORs are nucleolar proteins that interact specifically with silver salts. The size of silver precipitates measured by image analysis (ICM) in cycling cells proved to be inversely proportional to the cell cycle time and provided a significant correlation with prognosis for a large spectrum of cancers. Because ICM is time-consuming and poorly reproducible among laboratories using different imaging settings, this article presents a new approach to AgNOR quantitation based on flow cytometry (FCM). We report that silver precipitates caused a great decrease in the forward scattered light and that this effect was correlated with the AgNORs relative area as measured by ICM. These results were confirmed by measuring cell lines having different cell cycle durations. Moreover, double staining using APase–Fast red fluorescence to reveal the Ki-67/MIB 1 antigen of cycling cells and silver nitrate to stain the AgNORs was successfully analyzed by FCM. The procedure makes it possible, for the first time, to validly and rapidly compare the growth fraction and cycling speed of partially proliferating cell populations, such as tumors.

GUIDED TOUR IN THE CELL NUCLEUS

GUIDED TOUR IN THE CELL NUCLEUS BRUGAL G6rard Equipe de Reconnaissance des Formes et de Microscopie Quantitative, Universitd Joseph Fourier, 38041 GRENOBLE, France The number of cell probes and markers is rapid ly increasing. During the last few years it has become possible to label total nuclear DNA, synthesized DNA, specific A / T versus G / C rich DNA, Z-DNA, nuclear matrix proteins, cell cycle proteins (PCNA, Ki67), hormonal receptors, DNA sequences, genes and gene pr imary transcripts.. .Taking advantage of this roster of cellular probes implies not only quantitating their amount per cell but also ana lys ing their intra-nuclear respective dis t r ibut ion and extra-nuclear variation.

POTENTIAL USE OF FLUORESCENT IN SITU HYBRIDIZATION (FISH) FOR THE CONTROL OF THE GENETIC STABILITY AND HOMOGENEITY OF CULTURED CELLS: APPLICATION TO THE HUMAN BREAST CANCER CELL LINE MCF-7

Simultaneous visualization of nuclei and hybridization signals is a rapid and efficient method for counting the number of chromosomes of interest in each nucleus from a population of cells in culture and thus 8ires information on potential aneuploidy for the cell line under investigation. We applied this procedure on MCF-7 breast.cancer stemline whose chromosome number has been determined to be ranged from hypertriploidy to hypotetraploidy. The nucleic acid probe that was used, pUC !.77, designates a clone of the plasmid vector pUC9 containing a 1.77 kb long human Eco R ! fragment as an insert. The insert represents a tandemly organized repetitive sequence in the region qh of chromosome I. The plasmid was nicktranslated with either biotin 11-dUTP or digoxigenin 11-dUTP. Following in situ hybridization, the hybridized probes were detected by immunofluorescence with avidin-fluorescein isothiocyanate (FITC) or anti-digoxigenin antibody linked to FITC. The nuclei were conterslained with propidium iodide, The slides were examined with a fluorescent microscope : at least 600 cells were counted per slide. The results suggest the presence of a minor clone subpopulation with 6 chromosomes ! in a major population of tnsomtc MCF-7 cells for this chromosome. This procedure, which does not require the preparation of metaphases, can be used as a substitute for whole cytogenetic analysis for laboratories without special competence. The future development of image analyser resident software will allow the automation of the counting phase, thus leading tc a routine procedure. Moreover. the attempts to develop in silu hybridization procedures compatible with a DNA quantification will give information on the cell cyO phase of each hybridized cell in the population. USE OF NONYL A C R I D I N E O R A N G E AND RHODAMINE 123 TO ASSESS STRUCTURAL AND FUNCTIONAL MODIFICATIONS OF MITOCHONDRIA IN MULTIDRUG RESISTANT CELLS. DENIS-GAY Michelle, MAFTAH Abderrahman. ~ATINAU~ JULIEN Raymond. inszitut de biotechnologie, 123 Avenue Albert Thomas, 87060 LIMOGES C~dex.