Deformation of the erythrocyte membrane by the micropipette technique permits analysis of intrinsic material characteristics of the membrane and provides a means to differentiate purely membrane factors from such extrinsic factors as surface area-to-volume ratio. Using small micropipettes (less than 0.5 microns radius) to deform cells, it is evident that the red cell membrane behaves like a solid for periods of time up to 5–10 min of sustained deformation; for long periods of strain, permanent deformations occur, indicative of the semi-solid structural character. In the time range in which the membrane behaves like a solid, the material is linearly elastic up to strains of 400%, implying a loose network structure in the membrane plane, and evaluation of the elastic parameter mu (mu for normal discocytes equals 7 x 10(-3) dynes/cm) suggests that the elements comprising the network may have a molecular weight of approximately that of the water-soluble membrane protein spectrin. Whether the network system is cross-linked or simply a polymer solution remains unanswered. Experimental data indicate that plastic flow of the membrane under conditions of protracted strain may lead to permanent deformation of the membrane, whereas uniform dilation of the membrane, requiring over 1000 times more energy than for plastic flow, results in membrane failure and lysis. Analyses of the data from larger micropipettes of limiting mean cylindrical diameter show their utility in evaluating extrinsic factors, e.g., surface area-to-volume relationships, which are related to the capability of the whole cell to form a new configuration with implicit resistance to total surface area change, as the cell enters narrow channels of the microcirculation. Thus, micropipettes with diameters in the 2.7–3.0-microns range can provide sensitive comparisons of cellular deformability of erythrocytes.

The deformability of stored erythrocytes decreases progressively with duration of storage. This change can be measured in terms of mean pressure required to deform the cells sufficiently to cause them to enter and traverse a micropipette analogue of the microcirculation. The disc shape, with relatively higher surface area to volume ratio, is more deformable than the spherical form; and mean deformability at specific time intervals correlates with reported posttransfusion survival values. Only 76 per cent of red blood cells stored for three weeks were able to pass through a pipette with a minimal dimension of 2.85 μ, a dimension through which all fresh cells pass. This dimension is of magnitude similar to that previously reported for the microcirculation of the spleen. Regeneration of cellular ATP by incubation in adenosine is associated with restoration of mean deformability toward the normal range; and the majority of cells, having regained the biconcave disc shape, has values for individual cell deformability similar to fresh cells. The remaining spherical cells retain their relatively decreased deformability.

Excess calcium binding affects the shape and dynamics of cellular deformation of human erythrocytes. It may be hypothesized that incorporation of calcium may modify cellular deformability by processes which include specific cross-linking of membrane proteins with resultant changes in cell shape and deformability. Since previous studies indicate that accumulation of calcium ions causes development of gamma-glutamyl-epsilon-lysine bridges in membrane proteins, under control of a membrane transamidating enzyme which specifically requires calcium ions for activation, experiments were devised to examine the relationship between cross-linking and deformability and to determine the effects of specific inhibitor of membrane protein cross-linking on the calcium-dependent modification of erythrocyte to the echinocytic shape. The elastic shear modulus of the membrane was not significantly affected by calcium-induced cross-linking, indicating that induced shape change, not altered elasticity, causes the observed reduction in cellular deformability. These findings support the interpretation that Ca++-induced and transamidase-catalyzed cross-linking of membrane proteins contributes to fixation of altered cellular shape and decreased cellular deformability.

Mechanical measurements of intrinsic membrane material properties are used to characterize the defect in hereditary spherocyte membrane at a continuum level. The value of the surface elastic shear modulus is two-thirds as large as normal values, and the value of the yield shear resultant is one-third as large as normal values. The viscosity of the surface above the elastic-plastic transition appears normal. Under similar geometric conditions, the force required to fragment a hereditary spherocyte is about one-third as large as the force required to fragment a normal cell.

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