Françoise Giroud

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.

Standardisation of DNA quantitation by image analysis: quality control of instrumentation.

BACKGROUND DNA image analysis is frequently performed in clinical practice as a prognostic tool and to improve diagnosis. The precision of prognosis and diagnosis depends on the accuracy of analysis and particularly on the quality of image analysis systems. It has been reported that image analysis systems used for DNA quantification differ widely in their characteristics (Thunissen et al.: Cytometry 27: 21-25, 1997). This induces inter-laboratory variations when the same sample is analysed in different laboratories. In microscopic image analysis, the principal instrumentation errors arise from the optical and electronic parts of systems. They bring about problems of instability, non-linearity, and shading and glare phenomena. METHODS The aim of this study is to establish tools and standardised quality control procedures for microscopic image analysis systems. Specific reference standard slides have been developed to control instability, non-linearity, shading and glare phenomena and segmentation efficiency. RESULTS Some systems have been controlled with these tools and these quality control procedures. Interpretation criteria and accuracy limits of these quality control procedures are proposed according to the conclusions of a European project called PRESS project (Prototype Reference Standard Slide). Beyond these limits, tested image analysis systems are not qualified to realise precise DNA analysis. CONCLUSIONS The different procedures presented in this work determine if an image analysis system is qualified to deliver sufficiently precise DNA measurements for cancer case analysis. If the controlled systems are beyond the defined limits, some recommendations are given to find a solution to the problem.

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.

Adaptive clustering for time series: Application for identifying cell cycle expressed genes

The biological problem of identifying the active genes during the cell division process is addressed. The cell division ensures the proliferation of cells, which is drastically aberrant in cancer cells. The studied genes are described by their expression profiles during the cell division cycle. Commonly, the identification process is a supervised approach based on an a priori set of reference genes, assumed as well-characterizing the cell cycle phases. Each studied gene is then classified by its peak similarity to one pre-specified reference gene. This classical approach suffers from two limitations. On the one hand, there is no consensus between biologists about the set of reference genes to consider for the identification process. On the other hand, the proximity measures used for genes expression profiles are unjustified and mainly based on the expression values regardless of the genes expression behavior. To identify genes expression profiles, a new adaptive clustering approach is proposed which consists of two main points. First, it allows in an unsupervised way the selection of a well-justified set of reference genes, to be compared with the pre-specified ones. Secondly, it enables the users to learn the appropriate proximity measure to use for genes expression data, a measure which will cover both proximity on values and on behavior. The adaptive clustering method is compared to a correlation-based approach through public and simulated genes expression data.

A random-periods model for the comparison of a metrics efficiency to classify cell-cycle expressed genes

This paper addresses the clustering and classification of active genes during the process of cell division. Cell division ensures the proliferation of cells, but it becomes increasingly abnormal in cancer cells. The genes studied here are described by their expression profiles (i.e. time series) during the cell division cycle. This work focuses on evaluating the efficiency of four major metrics for clustering and classifying genes expression profiles and is based on a random-periods model for the expression of cell-cycle genes. The model accounts for the observed attenuation in cycle amplitude or duration, variations in the initial amplitude, and drift in the expression profiles.

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.

Adaptive Dissimilarity Index for Gene Expression Profiles Classification

DNA microarray technology allows to monitor simultaneously the expression levels of thousands of genes during important biological processes and across collections of related experiments. Clustering and classification techniques have proved to be helpful to understand gene function, gene regulation, and cellular processes. However the conventional proximity measures between genes expression data, used for clustering or classification purpose, do not fit gene expression specifications as they are based on the closeness of the expression magnitudes regardless of the overall gene expression profile (shape). We propose in this paper an adaptive dissimilarity index which would cover both values and behavior proximity. The effectiveness of the adaptive dissimilarity index is illustrated through a classification process for identification of genes cell cycle phases.

Which Distance for the Identification and the Differentiation of Cell-Cycle Expressed Genes?

This paper addresses the clustering and classification of active genes during the process of cell division. Cell division ensures the proliferation of cells, but becomes drastically aberrant in cancer cells. The studied genes are described by their expression profiles (i.e. time series) during the cell division cycle. This work focuses on evaluating the efficiency of four major metrics for clustering and classifying gene expression profiles. The study is based on a random-periods model for the expression of cell-cycle genes. The model accounts for the observed attenuation in cycle amplitude or duration, variations in the initial amplitude, and drift in the expression profiles.

Image analysis of nuclear chromatin pattern

Recent studies suggest that the interface nucleus is organized into functional domains which may be involved in the control of DNA replication and transcription, transcript processing, ribosome biogenesis and gene regulation. This functional compartmentalization of the eukaryotic nucleus also involves chromatin and thus, it can be supposed that chromatin is specifically arranged in each of these domains. Such a relationship between organization of the chromatin and the functional state of a cell has been demonstrated by several in situ studies, suggesting that each step in proliferation and/or differentiation is characterized by the chromatin pattern of Feulgen-stained nuclei It I. in such a context, a large set of new nuclear descriptors was introduced. Although they seem to be rather sensitive probes, their structural and functional significance are still poorly understood. Our purpose is to address such questions coherently by utilizing proliferating cell systems such as the MRC-5 and the MCF-7 cell lineages.