Microscopic photometric quantification of stiffness and relaxation time of red blood cells in a flow chamber.

The Microscopic Photometric Monolayer Technique provides a tool to measure red blood cell (RBC) stiffness (resistance to elongation) and relaxation time. It combines many of the advantages of flow channel studies of point-attached RBCs with the simplicity, sensitivity and accuracy of photometric light transmission measurement. This technique allows the study of the effects of physicochemical factors on the elongation and relaxation time of the same cells within an average of four to five thousand cells adhered as a monolayer to glass. Further, the time course of physicochemical effects on cell membrane and wash-in/wash-out kinetics of interactions can be followed. An automated version of this technique was developed. A dense monolayer of point-attached RBCs was prepared at the bottom of a flow-chamber. A steady-state flow, with stepwise increases of flow rate, induced the RBC elongation. The light transmission perpendicular through the monolayer plane was measured photometrically. Photomicrographs compared with photometric results showed that the flow-induced bending and curvature change of RBC membrane was associated with the increase of light transmission. There was a linear correlation between the photometric index of elongation and the elongation taken from photomicrographs for shear stresses up to 0.75 Pa. A stiffness parameter, S (in Pa), was defined as the ratio of shear stress and elongation at a shear stress of 0.25 Pa. Following a sudden flow stoppage, the RBCs returned to their resting shape and the RBC relaxation time was measured. The stiffness-relaxation time product, V (in mPas), was calculated to provide an estimate of viscosity. Diamide treatment, known to stiffen RBCs, did result in dose-dependent decreases of elongation and relaxation time. With increasing temperature, the relaxation time decreased at a rate of -2.96 ms/K; the stiffness increased significantly at a rate of 0.0038 Pa/K, and the stiffness-relaxation time product decreased with -2.95 mPas/K, reflecting an inverse relationship between RBC viscosity and temperature. Using the automated version of this technique (Elias-c-) to test RBCs of 36 healthy subjects, we found the inter-individual coefficients of variation to be 8.6% for stiffness, 7.9% for relaxation time and 12.4% for stiffness-relaxation time product.