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

通过成像微流控单细胞测量镰状血细胞的循环应激

• 批准号: 10605208
• 负责人: Ming Dao
• 金额: $ 60.35万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605208
• 关键词: Affect; Affinity; Biological Assay; Biological Markers; Biomechanics; Biophysics; Blood; Blood Cells; Cell Cycle; Cell Fraction; Cell Nucleus; Cells; Cellular Assay; Circulation; Clinical; Color; Complication; Defect; Detection; Development; Dose; Extinction; Fatigue; Fetal Hemoglobin; Foundations; Functional disorder; Hemoglobin; Image; In Vitro; Intervention; Joints; Kinetics; Measurement; Measures; Mechanical Stress; Mechanics; Membrane; Methods; Microfluidics; Microscopy; Morbidity - disease rate; Morphology; Outcome; Oxygen; Patient Monitoring; Patient-Focused Outcomes; Patients; Periodicity; Permeability; Pharmacologic Substance; Phase; Polymers; Population; Property; Publications; Research Personnel; Residual state; Resolution; Rheology; Risk; Role; Severity of illness; Shapes; Sickle Cell; Sickle Cell Anemia; Sickle Hemoglobin; Specimen; Speed; Stress; Techniques; Testing; Validation; Viscosity; Water; Work; bioimaging; biomechanical test; cellular imaging; design; fitness; hemoglobin polymer; hydroxyurea; in vivo; indexing; inhibitor; innovation; mechanical load; microscopic imaging; mortality; next generation; novel; oxygen transport; patient response; polymerization; risk stratification; sickling; small molecule; technological innovation; treatment response; vaso-occlusive crisis

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.

血管闭塞性危象（VOC）是镰状细胞贫血患者中大多数发病率和死亡率的最终原因。 疾病（SCD）。挥发性有机化合物的产生还不完全清楚。对于具有镰状血红蛋白（HbS）的RBC， 除其他生物物理作用外，脱氧会诱导聚合，降低细胞机械变形性 变化，增加VOC风险。通过利用最近开发的干涉相位和振幅 通过使用iPAM显微镜技术，我们在SCD患者的血液中发现了一个“不合适”的红细胞亚群， 材料特性，包括形状和粘度。在一项平行研究中， 动力学（MASK），我们发现细胞缺陷似乎积累后，无论是反复镰刀形或机械 应力循环，导致镰状细胞更快的镰状化，降低的变形性和显著的形状变化。 这些观察结果表明一个总体假设，即镰刀形红细胞的机械疲劳是由反复的 循环中的镰状化或机械负荷导致“缺陷”积累，产生“不适合”亚群 负责VOC引发的RBC。这种“不合适”的RBC亚群可以通过iPAM区分。 该提案将通过设计与MASK集成的下一代iPAM平台来验证这一假设， 阐明了重复的机械应力如何影响镰状红细胞特性和影响VOC倾向。我们 已成立一个由具备相关专业知识的调查人员组成的小组来处理这一问题。其中包括苏博士 是生物成像专家，Dao博士是微流体和生物力学专家，Higgins博士是 镰状细胞病病理生理学专家这个调查小组已经合作了五年多 多年来，与多家出版社合作。本提案中的工作分为四个目标。目标1侧重于 开发一种基于预防的iPAM，除了健身之外，还可以量化镰状红细胞流变学 指数.来自镰状病人的红细胞将在一个新的微流体平台上进行研究， 调制电变形以及重复脱氧-氧循环的细胞研究。 这些技术创新将使我们能够评估不合适的RBC是否受到机械损害 这是由于机械缺陷的积累以及这些不合适的细胞是否在脱氧时更快地镰状化。 在目标2中，我们将在iPAM中增加测量氧合血红蛋白和脱氧血红蛋白浓度的能力，使我们能够探索 机械循环是否影响通过RBC膜的氧转运及其对HbS的影响 聚合法在目标3中，iPAM将增加偏振分辨能力，使我们能够检测是否 残余的多聚HbS可以在常氧状态下持续存在于不健康的细胞内，作为细胞核来促进 聚合法我们将评估这种可能性作为一种补充机制以外的积累膜 缺陷来解释为什么不健康的细胞会更快地镰状化。最后，目标4将与基线患者临床结局相关 不健康细胞的水平。为此，我们将进一步评估羟基脲和voxelotor治疗的效果 对SCD患者的不合格细胞分数的影响。