A Photometric Method to Analyze Induced Erythrocyte Shape Changes

Abstract

In this paper, a photometric method was introduced to quantify biochemically-induced red blood cell (RBC) shape changes when no shear force was acting on the cells. To obtain the photometric RBC shape parameter (RF1), a monolayer of point-attached RBCs was prepared on the floor of a flat flow chamber and the transmission of light perpendicular to the monolayer plane was measured: 1) in phosphate buffered saline with 0.1 % bovine serum albumin (PBS₊) and 2) in PBS₊, containing a shape changing compound (in both, the RBCs were not deformed due to shear flow). To normalize the data, a third transmission value at a shear stress of 3 Pa was measured in PBS₊ from the same RBC monolayer. To validate the photometric data, RF1 of RBCs exposed to shape changing agents was correlated by linear regression analysis with 1) data obtained with the tangent-counting technique (TC) and 2) the morphological index (MI). The coefficient of correlation was calculated at 0.95 for the TC data and 0.94 for the MI data, respectively. The sensitivity of the photometric method was tested with stomatocytogenic chlorpromazine (CP) and echinocytogenic sodium salicylate (SA). CP (2.5 µM) induced a significant decrease of RF1 to -0.045 (N = 6 donors, p < 0.01), whereas SA (2.5 mM) increased RF1 to +0.027 significantly (N = 6, donors, p < 0.01). Both the CP-induced and the SA-induced shape changes appeared less than 2 min after application of the shape changing agents, and changed gradually within another 30 min when the agent was present in PBS₊, partly disappearing within about 2 min after reincubation of the shape transformed RBCs in PBS₊ not containing the agent.