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 mea sure red blood cell (RBC) stiffness (resistance to elongation) and rel axation time. It combines many of the advantages of flow channel studi es of point-attached RBCs with the simplicity, sensitivity and accurac y 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 fi ve thousand cells adhered as a monolayer to glass. Further, the time c ourse of physicochemical effects on cell membrane and wash-in/wash-out kinetics of interactions can be followed. An automated version of thi s technique was developed. A dense monolayer of point-attached RBCs wa s 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 p hotometrically. Photomicrographs compared with photometric results sho wed that the flow-induced bending and curvature change of RBC membrane was associated with the increase of light transmission. There was a l inear correlation between the photometric index of elongation and the elongation taken from photomicrographs for shear stresses up to 0.75 P a. 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 sudde n 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. Diamid e treatment, known to stiffen RBCs, did result in dose-dependent decre ases of elongation and relaxation time. With increasing temperature, t he relaxation time decreased at a rate of -2.96 ms/K; the stiffness in creased significantly at a rate of 0.0038 Pa/K, and the stiffness-rela xation time product decreased with -2.95 mPas/K, reflecting an inverse relationship between RBC viscosity and temperature. Using the automat ed version of this technique (Elias-c-) to test RBCs of 36 healthy sub jects, we found the inter-individual coefficients of variation to be 8 .6% for stiffness, 7.9% for relaxation time and 12.4% for stiffness-re laxation time product.