Reverse transcriptase-polymerase chain reaction (RT-PCR)-controlled immunomagnetic purging of breast cancer cells using the magnetic cell separation (MACS) system: a sensitive method for monitoring purging efficiency.

A modified reverse transcriptase-polymerase chain reaction (RT-PCR) technique was established with the aim of monitoring the tumor cell contamination in peripheral blood stem cells harvested from breast cancer patients. In an experimental approach, single cell suspensions of different breast cancer cell lines were mixed to normal peripheral blood mononuclear cells in order to 1) determine the sensitivity of tumor cell detection within PBMC and 2) compare polymerase chain reaction in its capacity of monitoring the efficiency of immunomagnetic purging using the magnetic cell separation (MACS) system to immunocytochemical staining. Several target sequences were assessed for their indicative potential and specificity allowing the detection of breast cancer cells by RT-PCR. Among the sequences evaluated, epithelial growth factor receptor (EGF-R) mRNA and Cytokeratin 19 mRNA were shown to be highly specific and sensitive markers for the detection of breast cancer cells within normal peripheral blood mononuclear cells and for the evaluation of the efficiency in immunomagnetic purging. In addition, we were able to show that the MACS is a potent and efficient tool for the selection of tumor cells from peripheral blood mononuclear cells, thus establishing its value for clinical scale immunomagnetic purging.