Optical and mathematical corrections of micropipette measurements of red blood cell geometry during anisotonic perifusion

Abstract

Accurate knowledge of red blood cell (RBC) membrane surface area and cellular volume is critical to understanding the geometric factors that affect RBC deformability. A useful method to determine RBC geometry is via micropipette aspiration in which the cell partially enters the tip of a glass pipette with a diameter of about 2 microns. Measurement of the cell while in the pipette allows calculation of cell geometry, but such measurements are subject to several artifacts. Herein, we analyze the effects of three artifacts on calculated RBC geometry during experimental osmotic manipulation. 1) The pipette internal diameter, as measured optically, requires correction for refractive index and focusing errors; 2) the pipette barrel is tapered rather than cylindrical; 3) the observed RBC outline is affected by a diffraction phenomenon. These optical and mathematical errors were all found to influence the calculated RBC geometry; both absolute values and relative changes during osmotic manipulation were affected. For RBC in isotonic medium, use of the above corrections significantly (p < 0.001) reduced the calculated area by 3.7%, the volume by 7.0%, and the minimum cylindrical diameter by 3.9%. The calculated membrane area dilation for cells exposed to hypotonic media was also significantly reduced; at 200 mOsm, the dilation was 2.8% +/- 0.2% without correction vs. 1.5% +/- 0.2% when all corrections were employed (p < 0.01). We therefore suggest that micropipette aspiration of RBC requires use of these correction techniques to obtain accurate values for RBC geometry and that such correction methods are of particular importance when RBC of different sizes are compared.