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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A042
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.
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