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

 产品说明：镰状细胞病（SCD）是一种毁灭性的遗传性疾病，影响全球1300多万人，仅在美国每年的医疗保健费用就超过10亿美元。SCD的起源是血红蛋白突变体（镰状血红蛋白或HbS）在脱氧时粘附成刚性纤维的能力。这些纤维降低了红细胞的变形能力，这导致血液流变学的巨大变化，并最终导致微血管的完全闭塞、组织梗死、器官损伤甚至死亡。临床上唯一批准的SCD治疗药物是羟基脲，被认为是通过诱导胎儿血红蛋白（HbF）的合成来起作用。已知HbF在体外抑制HbS聚合，但血液中的平均HbF水平与患者结果仅弱相关，并且羟基脲具有广泛可变的临床疗效。虽然已经提出或正在开发新的治疗方法，但如果没有在动物模型和人体中进行广泛的试验，这些潜在治疗方法对血管闭塞的影响很难预测。最终，缺失的环节是了解细胞HbF水平与血管闭塞可能性之间的定量关系。这将使我们能够预测新疗法的潜在疗效， 监测正在接受HbF诱导治疗（如羟基脲）的患者。在这些研究中，我们将量化血液HbF水平与血管闭塞风险之间的关系。我们的主要假设是HbF的治疗功效主要取决于HbF在红细胞（RBC）中的分布。如果所有RBC含有超过阈值百分比的HbF，则保护患者免于血管闭塞，但是将大部分HbF分离到仅少数细胞中并不能显著改善患者结果。我们提出使用流式细胞术直接检验该假设以量化单个RBC HbF分布（"HbF异质性"）。我们将患者血液的HbF异质性与血管闭塞的风险相关联。由于血管闭塞很难在体内研究或重现，我们开发了一个微流控平台来研究血管闭塞的体外。我们将使用该平台来量化血液样本中闭塞的可能性，并且我们将使用定量流式细胞术将该测量与从患者样本中测量的HbF异质性相关联。其结果将是一个临床检测，可用于监测患者对羟基脲的反应，并用于诱导HbF合成的新疗法的临床试验。此外，我们预计这些研究将阐明HbF是否是羟基脲的主要治疗机制。

 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.