Background: Cellular expression of receptors for the hormones estrogen and progesterone in human mammary tumors is of diagnostic and prognostic value. Ligand binding assays have been replaced by immunohistochemical analysis of receptor expression. However, both of these techniques are slow, and in the ligand-binding assay it is difficult to measure heterogeneity of receptor expression in individual cells. Flow cytometry has been used extensively for monitoring the expression of cellular receptors in hematopoietic tumors but has been of limited value in the analysis of mammary tumors, which are difficult to disaggregate into single cells for flow analysis. Hormone receptors have a predominant nuclear localization, and it is relatively easy to isolate nuclei from paraffin-embedded archival tissues for flow cytometric analysis of receptor expression.
Methods: Thick sections from formalin-fixed paraffin-embedded archival mammary tumors were digested by different enzyme solutions for the isolation of single nuclei. Different fixatives were used to compare the results on subsequent staining of the nuclei for estrogen receptor (ER) expression. Double staining with propidium iodide and fluorescein isothiocyanate labeled secondary antibodies for ER expression was used for multiparametric analysis of ER and DNA content.
Results: Digestion of paraffin sections with low concentration of pepsin and detergents was ideal for isolation of single nuclei. Fixation with paraformaldehyde in the presence of Triton X-100 improved staining of the cells. Isolated nuclei had enhanced immunoreactivity compared with the whole cells, and subpopulations differing in reactivity could be identified in the nuclear fractions. Double staining of nuclei for ER expression and DNA content could allow for multiparametric analysis of these two important parameters.
Conclusions: The procedures described can be used for processing of archival paraffin-embedded mammary tumors for monitoring of ER expression and aneuploidy. These two parameters have important diagnostic and prognostic significance in mammary tumors. Laser flow cytometry by providing multiparametric analysis can allow for correlation of these cellular markers with other important cellular and clinical parameters.