Dissecting the origins of fetal hemoglobin modulation of sickle cell vaso-occlusion

? DESCRIPTION: Sickle cell disease (SCD) is a devastating hereditary disorder that affects more than 13 million people worldwide with health care costs in the U.S. alone exceeding $1 billion per year. The origin of SCD is the ability of a hemo- globin mutant (sickle hemoglobin or HbS) to polymerize into rigid fibers upon deoxygenation. These fibers reduce red blood cell deformability, which leads to large changes in blood rheology and can ultimately result in complete occlusion of the microvasculature, tissue infarction, organ damage, and even death. The only clinically approved therapeutic for SCD, hydroxyurea, is thought to work by inducing synthesis of fetal hemoglobin (HbF). HbF is known to inhibit HbS polymerization in vitro, but average HbF levels in blood correlate only weakly with patient outcomes, and hydroxyurea has widely variable clinical efficacy. Although new therapies have been proposed or are in development, the effect of these potential therapies on vaso-occlusion is difficult to predict without extensive trials in animal models and humans. Ultimately, the missing link is to understand the quantitative rela- tionship between cellular HbF levels and the likelihood of vaso-occlusion. This would allow us to predict the potential efficacy of new therapies and to clinically monitor patients who are receiving HbF inducing therapies such as hydroxyurea. In these studies, we will quantify the relationship between blood HbF levels and the risk of vaso-occlusion. Our primary hypothesis is that the therapeutic efficacy of HbF depends primarily on the distribu- tion of HbF among red blood cells (RBCs). If all RBCs contain more than a threshold percentage of HbF, patients are protected from vaso-occlusion, but having the majority of HbF segregated into only a few cells does not significantly improve patient outcomes. We propose to directly test this hypothesis using flow cytometry to quan- tify single RBC HbF distributions (HbF heterocellularity). We will correlate the HbF heterocellularity of patient blood with the risk of vaso-occlusion. Because vaso-occlusions are difficult to study or reproduce in vivo, we have developed a microfluidic platform to study vaso-occlusion in vitro. We will use this platform to quantify the likelihood of occlusion in a blood sample, and we will correlate this measurement with HbF heterocellularity measured from patient samples using quantitative flow cytometry. The result will be a clinical assay that can be used to monitor patien response to hydroxyurea and an assay for clinical trials of new therapies that induce HbF synthesis. Additionally, we anticipate that these studies will elucidate whether HbF is the primary therapeutic mechanism for hydroxyurea.