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

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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.