Single-cell measurement of cyclic stress on sickle blood cells by imaging-microfluidics

血管闭塞危象(VOC)是镰状细胞发病和死亡的主要原因。 疾病(SCD)。VOC的启动机制尚不完全清楚。对于具有镰刀状血红蛋白(HBS)的红细胞， 除其他生物物理作用外，脱氧作用可诱导聚合，降低细胞的机械变形能力 变化，并增加VOC风险。通过利用最近开发的干涉相位和幅度 显微镜(IPAM)技术，我们在SCD患者的血液中发现了一种“不适合”的红细胞亚群 材料特性包括形状和粘度。在一项使用新型微流控检测镰刀的平行研究中 动力学(MASK)，我们发现细胞缺陷似乎在反复镰刀或机械切割后积累 压力循环，导致镰状细胞更快地镰状，变形性降低，并显著改变镰状细胞的形状。 这些观察结果表明，一个重要的假设是，镰刀状红细胞的机械疲劳是通过重复 循环中的镰刀或机械负荷会导致“缺陷”累积，从而产生一个“不适合”的亚群。 负责VOC启动的RBC。这种“不适合”的红细胞亚群可以通过IPAM来区分。 该提案将通过设计与MASK集成的下一代IPAM平台来检验这一假设， 阐明重复机械应力如何影响镰刀状红细胞特性和影响VOC倾向。我们 已经组建了一支具有相关专业知识的调查小组来解决这个问题。这些人包括苏博士 是生物成像专家，道博士是微流体学和生物力学专家，希金斯博士是 镰状细胞病病理生理学方面的专家。这支调查团队已经合作了五年多 多年来，出版了多份联合出版物。这项提案中的工作分为四个目标。目标1侧重于 开发一种基于消光的IPAM，除了健康外，还将允许量化镰刀状红细胞流变学 指数。来自镰刀患者的红细胞将在一种新型微流控平台上进行研究，该平台将使幅度- 被研究细胞的调制电变形以及重复的脱氧-充氧循环。 这些技术创新将使我们能够评估不合适的红细胞是否受到机械损害 由于机械缺陷的积累，以及这些不适合的细胞在脱氧后是否会更快地镰刀。 在目标2中，我们将增加在IPAM中测量氧合和脱氧-Hb浓度的能力，使我们能够探索 机械循环是否影响红细胞膜的氧转运及其对高压氧的影响 聚合反应。在目标3中，将向IPAM添加偏振分辨能力，使我们能够检测 残留的聚合的HBs可能会持续存在于处于常氧状态的不适合的细胞内，作为核促进 聚合反应。我们将评估这种可能性作为膜堆积之外的补充机制。 缺陷来解释为什么不适合的细胞可能会更快地镰刀。最后，目标4将关联基线患者的临床结果。 不适合细胞的水平。为此，我们将进一步评估羟基脲和体素传导治疗的效果。 关于SCD患者中不适合的细胞比例。

Vaso-occlusive crises (VOC) are ultimately responsible for the majority of morbidity and mortality in sickle cell disease (SCD). The initiation of VOC is not fully understood. For RBCs with sickle hemoglobin (HbS), deoxygenation induces polymerization, reducing cellular mechanical deformability, among other biophysical changes, and increasing VOC risk. By utilizing a recently developed interferometric phase and amplitude microscopy (iPAM) technique, we found a subpopulation of “unfit” RBCs in the blood of SCD patients with altered material properties including shape and viscosity. In a parallel study using a novel microfluidic assay for sickling kinetics (MASK), we found that cellular defects appear to accumulate after either repeated sickling or mechanical stress cycles, resulting in faster sickling, reduced deformability, and significant shape changes in sickle cells. These observations suggest an overarching hypothesis that mechanical fatigue of sickle RBCs by repeated sickling or mechanical loading in circulation causes “defects” to accumulate, producing an “unfit” subpopulation of RBCs that is responsible for VOC initiation. This subpopulation of “unfit” RBCs can be distinguished by iPAM. This proposal will examine this hypothesis by designing a next-generation iPAM platform integrated with MASK, elucidating how repeated mechanical stress affects sickle RBC properties and influences VOC propensity. We have assembled a team of investigators with relevant expertise to tackle this problem. These include Dr. So who is an expert in bioimaging, Dr. Dao who is an expert in microfluidics and biomechanics, and Dr. Higgins who is an expert in sickle cell disease pathophysiology. This team of investigators has worked together for over five years with several joint publications. The work in this proposal is divided into four aims. Aim 1 focuses on developing an extinction-based iPAM that will allow quantification of sickle RBC rheology in addition to fitness index. The RBCs from sickle patients will be studied in a novel microfluidic platform that will enable amplitude- modulated electrodeformation as well as repeated deoxygenation-oxygenation cycles for the cells under study. These technological innovations will allow us to evaluate whether unfit RBCs are mechanically compromised due to the accumulation of mechanical defects and whether these unfit cells sickle faster upon deoxygenation. In Aim 2, we will add the ability to measure both oxy- and deoxy-Hb concentration in iPAM, allowing us to explore whether mechanical cycling affects oxygen transport through the RBC membrane and its effect on HbS polymerization. In Aim 3, polarization-resolved capability will be added to iPAM enabling us to detect whether remnant polymerized HbS may persist inside unfit cells in the normoxic state acting as nuclei to promote polymerization. We will evaluate this possibility as a complementary mechanism beside accumulated membrane defects to explain why unfit cells may sickle faster. Finally, Aim 4 will correlate baseline patient clinical outcome with the level of unfit cells. In this aim, we will further evaluate the effect of hydroxyurea and voxelotor treatment on unfit cell fraction in SCD patients.