6th ESACP Congress, Heidelberg, April 7-11, 1999

A042
DNA CONTENT BY FLUORESCENCE IMAGING
Chassoux D 1, Linares-Cruz G 2, Debey P 1

1) INRA 806, IBPC, Paris, 2) Laboratoire de Pharmacologie Expérimentale, IGM Saint Louis, Paris, France

DNA ploidy is one of the parameters considered in pathology for diagnosis and prognosis purposes. The measure of DNA content considered as reference and a reliable method in cytopathology involves an acidic denaturation of DNA. It implies displacement of associated nuclear proteins and limits multi- parametric approaches. The fluorescence approach is increasingly recognised as a quantitative tool that also allows to consider several biological parameters concomitantly with DNA content on the same preparation. We have used fluorescence microscopy with a CCD camera and automated image analysis to measure DNA content on whole cycling adherent cells in situ avoiding DNA denaturation. Cells were fixed with paraformaldehyde and DNA stained with Hoechst 33342. The latter stochiometrically bounds DNA with the fixative used. The precision of the measure was demonstrated by comparison, on a per cell basis, with an specific S phase label, also achieved in conditions preserving nuclear structure and associated proteins. Multiparametric analysis of biological markers (i.e. topological distribution and compartmentalisation) and DNA content may be envisaged.