Electrorotation measurements were used to demonstrate that the dielectric properties of the metastatic human breast cancer cell line MDA231 were significantly different from those of erythrocytes and T lymphocytes. These dielectric differences were exploited to separate the cancer cells from normal blood cells by appropriately balancing the hydrodynamic and dielectrophoretic forces acting on the cells within a dielectric affinity column containing a microelectrode array. The operational criteria for successful particle separation in such a column are analyzed and our findings indicate that the dielectric affinity technique may prove useful in a wide variety of cell separation and characterization applications.Cell separation has numerous applications in medicine, biotechnology, and research and in environmental settings. For example, the use of autologous bone marrow transplants in the remediation of advanced cancers requires the removal of cancer cells from the patient's marrow (1), the study of signaling between blood cells requires purified cell subpopulations (2), and the purification of contaminated water supplies necessitates elimination of parasites such as Giardia and Cryptosporidium (3,4). Current sorting technologies usually exploit differences in cell density, immunologic targets, or receptor-ligand interactions. These techniques are often inadequate, producing insufficiently pure cell populations, being too slow, or being too limited in the spectrum of target cells. Identification of novel properties by which different cell types may be discerned and of new ways for their selective manipulation are clearly fundamental components for improving sorting methodologies.A particle suspended in a medium of different dielectric characteristics becomes electrically polarized when subjected to an alternating electrical field. Interaction between this induced polarization and the field gives rise to various electrokinetic effects. For example, a spatially inhomogeneous field will exert a lateral dielectrophoretic (DEP) force on the particle, directing it toward the minimum of dielectric potential (5-9), while a rotating field will induce particle electrorotation (ROT; refs. 10-12). The frequency dependencies of the magnitude and direction of these forces are functions of the intrinsic electrical properties of the particle that depend on the particle constitution and structural organization (13-15). For living cells, these characteristics are defined by composition, morphology, and phenotype (16, 17). Thus, DEP and ROT have been used to study bacteria (18), yeasts (11,19), plant cells (10), and mammalian cells (20,21) and to investigate cellular alterations accompanying physiological changes such as mitotic stimulation (16) and induced differentiation (7,17,21 MATERIALS AND METHODS Cells. Peripheral blood was collected by venipuncture into 90 parts Ca2+/Mg2+-free phosphate-buffered saline containing 5 mM hemisodium EDTA to prevent clotting. T lymphocytes were obtained from (human immunodeficiency virusa...