Miccrorheology of Sickle Hemoglobin Gels

Abstract

Sickle cell disease is a rheological disease, yet no quantitative rheological data exists on microscopic samples. We have developed a novel method for probing the microrheology of sickle hemoglobin gels, based on magnetically driven compression of 5-8 μm thick emulsions containing hemoglobin droplets of ∼100 μm diameter. By observing the expansion of the droplet area as the emulsion is compressed, our method can resolve changes in thickness of a few nm with temporal resolution of ms. Carbon monoxide bound to sickle hemoglobin was dissociated by laser illumination allowing the resulting deoxyhemoglobin to form gels in target droplets. The amount of polymer formed was determined by observing, in the target droplet, the residual concentration in a small region that was unilluminated by the laser. Thickness was monitored by observing a non-photolyzed reporter droplet adjacent to the target droplet.Gels were formed at different initial concentrations, temperatures and fractional saturation with CO. In addition, some gels were formed in small spatial regions which then were allowed to grow to the full extent of the target droplet, to contrast with the same sample gelled completely in the target droplet ab initio, thereby creating a different domain structure in the gel. We find that all the gels behave as Hookean springs with linear and repeatable dependence of thickness on force. This allowed us to determine Young's modulus, which ranged from 300 to 1500 kPa for the gels which varied in polymerized hemoglobin concentration from 6 g/dl to 12 g/dl. A highly simplified model for the gel, treating it as a simple lattice with fixed junctions, describes the observed quadratic concentration dependence of Young's modulus data. These measurements provide a quantitative rationale for pathophysiology in the disease.