Sample preparation for bivariate flow cytometric analysis of breast tumor specimens

The availability of monoclonal antibodies (MAbs) raised against functional or phenotypic cell markers can improve the suitability of flow cytometry (FCM) in assessing and validating prognostic parameters of human neoplasia. The major problems encountered in this type of study, when performed on solid tumors, are the recovery of a large and representative number of tumor cells and the possibility of performing a DNA analysis on antigen-selected populations. A panel of MAbs was chosen on the basis of the recognized membrane antigens important to the biologic characterization of human breast cancer cell populations. The possibility of performing bivariate FCM analysis of cell surface antigens and DNA content was investigated on 4 breast tumor cell lines and 10 fresh breast biopsies. Different fixations and tumor disaggregation procedures were considered. The results indicate that ethanol fixation is the most effective procedure for performing biparametric FCM analysis and that a combined enzymatic-mechanical disaggregation procedure applied on breast tumor specimens allows great recovery of intact cells without affecting antigen detection or DNA distribution.