Enhanced assessment of DNA/proliferative index by depletion of tumor infiltrating leukocytes prior to monoclonal antibody gated analysis of tumor cell DNA.

Flow cytometry has become an important tool for the analysis of breast tumors, and assessment of S phase fraction and DNA ploidy are potential indicators of tumor aggression. Due to masking or dilution of infrequent tumor cell events, the presence of normal cell types, such as inflammatory cells and fibroblasts, can interfere with accurate DNA analysis of solid tumor samples. MDA-MB-175-VII human breast carcinoma cells, WI-38 human lung fibroblast cells, and peripheral blood leukocytes were mixed, in varying proportions, in order to represent human breast tumor samples. The cells were subsequently treated with CD45 conjugated magnetic microspheres to deplete tumor infiltrating leukocytes, thus enriching for tumor cells. The tumor cell mixtures were then stained with a pan-cytokeratin specific monoclonal antibody or with a monoclonal antibody that reacts with breast epithelial membrane antigen (EMA). When used in combination with monoclonal antibody gating, utilization of this bead-based technology resulted in enhanced precision of DNA analysis.