Sensitive and Simple Methodologies for Measuring of Red Blood Cell (RBC) Electrical Properties and Cell Aggregation.

The red blood cell membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that confers viscoelastic behaviour. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface, which creates a repulsive electric zeta potential (ζ) between the cells.

The compact layer of charge or Stern consists of ions rigidly bonded to the cell and the double layer includes ions diffusely distributed around the cell. Measurement of RBC layer of charge and zeta potential can be use as a marker of RBC membrane injury. Study of membrane electrical properties and forces and bindings between cells, can provide a better comprehension of membrane injury presented in RBC unit storage, after RBC unit irradiation, cell senescence process and in addition, also to improve the sensitivity and specificity of the hemoagglutination reaction. The aim of this study was to measure the RBC double layer thickness of the charge (DLC) around the cell, zeta potential (ζ) and cell aggregation force, using optical tweezers.

To measure DLC (d) and zeta potential (ζ) a special chamber was built consisting of 2 platinum electrodes separated by a channel. An external electrical field (E) was applied with a voltage power supply connected to the electrodes. The optical tweezers consisted of Nd:YAG laser beam focused through the 100x objective of a microscope equipped with a minicam, which registered the trapped cell image in real time. These images were recorded in VHS and captured in a computer where they could be analyzed using Image Pro Plus software. Optical tweezers utilizes the light momentum transfer to trap the silica beads with pico Newtons forces (10⁻¹²). Ten RBC units from healthy blood donors were collected in CPDA-1. Samples were diluted (1:1000μl) in AB plasma. Silica beads were added to a RBC solution (10μl). DLC thickness (d) was obtained measuring, in the special chamber, the force on the silica bead attached to a single RBC in response to an applied voltage (50–100V). Zeta potential (ζ) was obtained by measuring the RBC terminal velocity (Tv) after being released from the optical trap at a constant voltage applied supply (100V) Tv=(εE/η) ζ; ε= medium dielectric constant; η= viscosity). The DLC was calculated by the equation Fop= (Aεζ/d)E (Fop= optical force; A= RBC area). DLC median size (d) was 0,85 μm (min 0.4/max 1.3) and zeta potential was ζ=−12,5 mV (min- 9.3/max −15). These results were in accordance with the data in literature, where other methods to measure the same property were used.

For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities (2,5,7,12,18,32X10⁻⁷m/s) was determined. RBC O Rh(D) positive were diluted (1:1000μl) in plasma AB (control group) and in agglutination potentiator solutions, used in immunohematological tests (dextran 0.15%, low ionic strength solution-LISS, bromelain and papain enzymes). Aggregation was also analyzed using anti-D antibodies serum (5μl). The median aggregation force obtained of 20 RBC control samples was 1x10⁻³ (0.1–2.5) poise.cm. Samples analyzed with dextran 0.15%, LISS and bromelain lacking antibodies showed no agglutination. RBCs treated with papain enzyme presented high adherence preventing the capability of separating cells. The 20 samples analyzed with anti-D serum showed median aggregation force 2x10⁻³ (1.0–4.0) poise.cm. These results obtained from RBC with antibodies were statistically higher (p<0.001) when compared to control. The method revealed to be sensitive and results were in accord with serological tests used in transfusionroutine, where false positive results could be frequently found in RBC treated with papain, however rarely obtained with dextran and bromelain. In conclusion, the methodologies proposed are simple and may provide specific information on RBC interaction in addition to cell aggregation. Individual cell analyze methods are always more sensitive to small differences than those based on average values. This is the first time that the size of the double layer was measured using optical tweezers and the ability of the optical trap also allowed us to obtain the zeta potential and DLC thickness for the same RBC.