Dependence of Membrane Antigen Expression On Cross-Over Frequency of Human Erythrocytes and Corresponding Theoretical Explanation

Since Pohl's initial exploration of dielectrophoretic force to explain the behavior of polarizable particles in a non-uniform electric field, adjustments have been made to the Clausius-Mossotti factor to include non-spherical particles and particles of a core-shell composition.  This work has become increasingly important since dielectrophoresis has been applied to biological cell systems because it allows for the separation and sorting of cells of similar origin but slightly varying dielectric properties.  Knowing the frequency at which the Clausius-Mossotti factor is equal to zero allows the researcher to determine the conductivity and permittivity of the cell of interest.  However, current equations describing the Clausius-Mossotti factor ignore slight membrane differences such as antigen expression despite previous results showing that small membrane changes make appreciable changes to the Clausius-Mossotti factor.  This work builds upon the previous adjustments made to the dielectrophoretic force equation to account for not only the shelled ellipsoidal shape of human erythrocytes but also the differences in membrane antigen expression.  The common ABO-Rh typing system consists of 4 different membrane antigens and provides the basis for these studies.  The Rhesus factor (Rh) is a group of transmembrane antigens that indicate positive blood type (presence of D antigen) or negative blood type (absence of D antigen).  The ABO blood grouping system consists of three surface antigens, whose presence or absence dictate blood type.  A and B antigen chains differ from the O backbone by a single sugar molecule, however this small difference gets magnified because there are 1.5 million copies of the antigen on the erythrocyte surface.  In order to determine the affect of the ABO antigens on the dielectrophoretic behavior, erythrocytes were treated with β(1-3) galactosidase, which cleaves polysaccharide antigens at a galactose bond.  Human erythrocytes of known ABO-Rh blood type were suspended in a 0.1S/m dextrose buffer solution and subjected to an electric field of 0.1V/μm and varying frequency from 100kHz to 80MHz in order to determine their cross-over frequencies.  Experiments were repeated with β(1-3) galactosidase treated erythrocytes. It was found that the lower cross-over frequency is ABO blood type dependent and that galactosidase modification causes a shift in the cross-over frequency.  The higher cross-over frequency was Rh blood type dependent and did not change after β(1-3) galactosidase modification.  The change in the lower cross-over frequency post-modification proved that the polysaccharide antigen chain influences the dielectric properties of the erythrocyte and therefore the dielectrophoretic behavior.  The shelled ellipsoidal Clausius-Mossotti factor model was fit to each ABO blood type response to quantify membrane conductivity differences due to antigen expression.  This work will provide insight into molecular level contributions to conductivity and permittivity –driven cell polarizations in dielectrophoretic fields.