A Technique to Detect Reduced Mechanical Stability of Red Cell Membranes: Relevance to Elliptocytic Disorders

A method has been developed for determining the relative tendency of red cell membranes to fragment under shear stress. For these measurements, resealed ghosts were subjected to constant, high fluid shear stress in an ektacy-tometer. The deformability signal, which this instrument provides, progressively declined as the deformable cells were transformed into less deformable, more spherical cell fragments. The deformability signal for normal ghost preparations decayed over a narrowly defined time period; this decay was accompanied by the production of cell fragments, as shown by microscopic examination and analysis of particle volume distribution. The method was used to study cells from patients with various hemolytic disorders. Membrane preparations from patients with various types of nonhemolytic congenital elliptocytoses showed a reduction in the time required for fragmentation to about half the normal value. Membrane preparations from patients with hemolytic elliptocytosis associated with a severe deficiency of the membrane skeletal protein band 4.1 showed an even greater reduction in fragmentation time, distinct from the values for nonhemolytic samples. In elliptocytoses secondary to myelofibrosis, the fragmentation times were within the normal range. In hereditary spherocytosis (HS), heterogeneity in fragmentation times was observed; marginally decreased or normal fragmentation times were seen for cells from 8 patients, but a pronounced shortening in fragmentation time was found for one other HS patient. A defect was previously found in spectrin-band 4.1 binding for this same patient. These observations suggest that this membrane fragmentation technique may be useful for the identification of certain specific disorders or their clinical variants in which the integrity of membrane skeletal interactions has been perturbed in such a way that the mechanical stability of the membrane is reduced.