Erythrocyte deformability as assessed by nuclepore filters
Velker, John Albert
McIntire, Larry V.
Master of Science
Erythrocyte deformability characteristics were assessed by passing red cell suspensions through Nuclepore polycarbonate filters having pore diameters of 3 ym. with pore lengths of 12 ym. During flow through these filters, at a constant flow rate of 2.17 cc/min., the pressure drop across the filter was monitored as a function of time. This measurement was the primary index of altered deformability. These filters serve as a reproducible technique for measuring erythrocyte deformability, and are a physical model of the splenic microcirculation. Shear stresses of 15 dynes/cm or greater applied to erythrocytes for two minutes in a concentric cylinder viscometer result in red cell fragmentation and hemolysis. In this study the effects of lower shear stresses on cell deformability were measured by observing the relative ability of erythrocytes to traverse Nuclepore filters. Cell morphology was examined immediately after shear stress, and after subsequent passage through the filter to assess cell damage. Shear stresses from 25-14 dynes/cm, applied with a concentric cylinder viscometer, were studied. A good correlation was found between the magnitude of the applied shear stress, and both the pressure drop produced at constant flow rate and the extent of morphologic damage due to filter passage. Behavior indistinguishable from unsheared cells was observed only when the applied shear stress was at or below a lower threshold value of approximately 25 dynes/cm^. This indicates that altered erythrocyte deformability results from application of shear stresses considerably less than those required to produce red cell fragmentation and overt hemolysis. The shear stress induced cell alterations in deformability were determined not to be time recoverable. Incubations with glucose, dibutyryl cyclic AMP and cyclic GMP, and adenosine triphosphate following shear were made in an attempt to reverse the effects of shear stress. These had little or no effect. Incubation with adenosine, however, was capable of returning sheared cells to their behavior before shear stress. The effects of adenosine incubation were so complete that the deformability of the sheared and incubated cells was virtually the same as fresh unsheared cells as assessed by Nuclepore filter passage. Aging erythrocytes for periods of up to 24 hours, produced cells that caused much higher pressure drops during filtration. A cell, however, retained most of its deformability characteristics if the incubation time was less than six hours. Loss of deformability was rapid after ten hours. The effects of incubation with the protein denaturing compounds colchicine, vinblastine and cytochalasin B to produce deformability alterations were tested using the filtration technique. No consistently measurable differences in deformability were seen between the incubated cells and the control cells. The results imply that cell ATP levels play an important role in maintenance of deformability. Application of shear stress appears to induce a membrane defect similar to that seen in metabolically depleted cells. Presumably the shear stress forces cause a drastically increased energy demand within the cell, which depletes the intracellular ATP, resulting in a more rigid, less deformable cell. The in vivo survival of such a cell may be reduced, due to greater likelihood of sequestering in the spleen.