The effect of frequency on electric field induced surface force in red blood cell membrane

The use of electric field induced surface force acting on a red blood cell membrane to explain the observed sphering and hemolysis of the cell when exposed to the field is discussed. The frequency of the applied sinusoidal field inversely affects the field strengths at which sphering and hemolysis occur. Increase in frequency decreases the magnitude of the surface force. The effect appears to be the result of an /spl alpha/-dispersion of the dielectric property of the cell membrane.<<ETX>>


Introduction
When an external electric field (EF) was applied to red blood cells (RBC), the RBC were observed to change from the flaccid biconcave shape to a spheroidal state. This sphering action is different from swelling in that there is no change in the RBC membrane surface area. Sphering may or may not subsequently lead to hemolysis depending on the EF strength and the time cif exposure. The transmembrane potential (AV,,,) was found to be an order of' magnitude lower than the accepted threshold of 1 volt [ 11 necessary to cause RRC membrane breakdown and lead to hemolysis. EF induced surface traction (surface force density, S,) acting on the RBC membrane was derived using the Maxwell's stress tensor [2] and was used to explain the observed sphering and hemolysis of RBC [3]. Table 1 summarizes the experimental observations and the radial surface tractions obtained a t an applied EF frequency of 1 kHz [3]. Surface traction, S,, being always outward pointing, forced the flaccid RBC membrane outward to give the sphering action at E, = 60 V/cm. The membrane was not stretthed. When EF persisted, S, eventually stretched the membrane led to pore formation, water influx and hemolysis (at E, = 120 V/cm). A t E,, = 140 V/cm, S, (at 506 dynes/cm2) wiis sufficiently large to t car the RBC membrane apart, hemolyris resulted quickly. This study examines the effect of the applied frequency. If IS, is maintained constant, but the frequency is increased, do sphering and hemolysis still occur at the same field btrength levels?

Method
The E F was generated between a pair of parallel platinum-iridium electrodes (0.5 microns in diameter) set 0.5 millimeter apart on a microscope glass slide. Blood sample was obtained by venipuncture, diluted with saline to give a 2% hematocrit solution. The 2% solution was placed between the parallel electrodes for EF irradiation.
The sinusoidal EF was applied continuously. Sphering and hemolysis was observed visually under a microscope. Figure 1 shows that the threshold of sphering (E,,,) increases as a function of applied EF frequency. The frequency axis (x-axis) is logarithmic, and the E , axis (y-axis) is linear. The curve displays a sigmoidal shape.  Table 2 shows the result when E, was maintained at 140 Vlcm, but the frequency was increased from 1 kHz to 5 kHz. Whereas hemolysis occurred quickly at 1 kHz, RBC only sphered but did not hemolyze a t 5 kHz.

Discussion
The sigmoidal shaped data curve in Figure 1 is characteristic of a dielectric dispersive behavior. However, this frequency dependency appears more like an adispersion than the much better known Maxwell-Wagner R-dispersion [4]. The applied frequency range (100 Hz -10 kHz) is much lower than where &dispersion of erythrocyte suspension resides. The existence of an a-dispersion for RBC has been described. Schwan Table 2. Note in Table 2, with E, at 140 V/cm and 1 kHz, S& was 506 dynedcm2. When the frequency was increased t o 5 kHz, E, at 140 V/cm only yielded an S, of 366 dynedcm'.
The value of 366 dynes/cm2 is within the range of expected values. It was expected that the S, (at 140 V/cm and 5 kHz) should be comparable to 372 dynedcm'. At 372 dynedcm', from E, = 120 V/cm and f = 1 kHz (see Table  11, RBC sphered and eventually hemolyzed after a sustained exposure to EF. The observation at 140 Vlcm and 5 k H z was sphering but no hemolysis. Hence, S, a t 5 kHz should be, at maximum, comparable to 372 dynedcm', and certainly much smaller than 506 dynedcm', in order to explain the observation that RBC sphered but did not hemolyze. The applied frequency of the EF has a significant effect on the RBC membrane. Frequency inversely affects the magnitude of the membrane surface traction, S, . Thus, increase in EF frequency delays or even prevents RBC from sphering andor hemolysis.
The frequency dependency can best be described as the result of an adispersion of the dielectric property of the RBC membrane.