A Novel High-Throughput Method for Evaluating deoxy-Hb S Solubility.

The distortion of erythrocytes in sickle cell disease (SCD) is a direct consequence of self-assembly of deoxy-Hb S heterotetramers into low-solubility polymers. Despite the broad recognition that Hb S solubility can be used to predict the efficacy of potential antipolymerization (antisickling) therapies, currently available methods require significant time and effort that detract from their utility by limiting assay as well as the reliability of any relevant results. To this end, we have developed an uncomplicated, rapid, and highly reproducible high-throughput assay for measuring the solubility of deoxy-Hb S in vitro. In contrast to existing methods that remove insoluble deoxy-Hb S polymers by centrifugation, we utilize a highly efficient filtration approach. Briefly, the solubility of deoxy-Hb S is determined in a defined phosphate buffer by filtering insoluble polymers and determining the concentration of filtered Hb spectrophotometrically. We tested the utility of this method by assessing the effects of four parameters known to affect deoxy-Hb S polymerization in vitro, including the concentration of Hb S, the molarity of the phosphate buffer, and the presence of antipolymerization agents including Hb F and the previously reported anti-gelling agent INN 312. Eight different concentrations of Hb S (0.01 – 0.07 g/dL) were prepared in each of three phosphate buffers (1.60 M, 1.65 M, and 1.70 M; pH 7.4). Each reaction was prepared in quadruplicate to facilitate evaluation of assay reproducibility. Soluble oxy-Hbs were prepared in a 96-well 0.45 μm Multiscreen® Solvinert filter plate, sealed with oxygen-impervious optically inert mineral oil, then deoxygenated by supplementation with 30 mM sodium dithionite. The filter plate was incubated at 30°C for 60 min and then vacuum-filtered into a collection plate where the filtrate was maintained under deoxygenated conditions. The absorbances of the filtrates (corresponding to soluble heterotetrameric Hb) were determined at 556 nm. Parallel analyses were carried out using Hb S samples containing 5, 10, 15, or 20% Hb F; 10, 20, 30, or 40% Hb A; or 0.5, 1.0, or 2.0 mM of the known anti-gelling agent INN 312. We observed that the solubility of Hb S in the filtrate varied in direct proportion to the concentration of phosphate buffer, Hb F concentration, or the concentration of INN 312, consistent with the known effect of each parameter thus validating the utility of the method. In addition, the assay demonstrated high reproducibility with a standard deviation of ±3.2% for all samples. Consequently, the reliability, rapidity (< 2 h), and high reproducibility of this novel assay make it a suitable option for high-throughput screening of potential anti-polymerization agents. To further validate this assay, we tested hemolysates from eight de-identified blood samples obtained from patients with SCD or sickle trait. The solubility of the samples determined by our novel high-throughput method correlated exactly with the globin phenotypes of each sample determined by HPLC. These results confirm the potential utility of the microfilter assay as a tool for assessing the solubility of deoxy-Hb S under a variety of experimental and clinical conditions. We conclude that this simple, rapid, and highly reliable high-throughput method—, which may be adapted to automated systems—, may be highly valuable for the primary screening of sickle cell therapies that act by inhibiting the polymerization of deoxy-Hb S.