Assessing Real-Time Sickling Kinetics in Individual Cells from Erythrocyte Populations of Subjects with Different HbA, HbS and HbF Percentages

INTRODUCTION: Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Treatment in sickle cell disease (SCD) aims to reduce the risks of red blood cell (RBC) sickling and associated complications including, but not limited to vaso-occlusive crises (VOC). There is substantial promise in developing therapies targeting HbS polymerization that consequently decrease RBC sickling, a fundamental pathophysiological driver of SCD. Nonetheless, there are limited options available for reliable diagnostic tools for the assessment of disease status and therapeutic outcomes. A robust and easy-to-use system to assess population-scale sickling kinetics and to monitor responses to Hb-modifying therapies in clinical trials for the clinical management of patients may be beneficial. We have developed a dynamic sickling assay (DSA) that uses an enzymatic oxygen scavenging system to provide tight control over the rate and depth of induced-hypoxia. DSA is able to differentiate samples based on endogenous Hb expression and can be used to monitor sickling kinetics pre- and post HbS-modifying therapies, including hematopoietic stem cell transplantation (HSCT). METHODS: SCD and sickle cell trait (SCT) subjects were recruited under the RBC Health Initiative IRB protocol number FF-RBC-003. Four hematopoietic stem cell transplant (HSCT) SCD subjects were enrolled in NIH haploidentical HSCT protocol 17-H-0069. Diluted blood samples were supplemented with protocatechuic acid (PCA), followed by protocatechuate 3,4-dioxygenase (PCD) enzyme inducing hypoxia at a predetermined rate. The sample was then injected into a microfluidic chamber, and resulting RBC morphological changes were observed and captured with time-lapse photography. Image analysis identified and quantified RBC using an AI-trained SICKLE image analysis software. HbAS (n=3), HbSS with HbA (post-transfusion, n=3) and HbSS samples containing 3.1 to 34.5% HbF (n=9) were processed with DSA to assess the effects of hemoglobin distribution on sickling kinetics.Pre- and post-HSCT samples were run to ascertain the effect on sickling kinetics at baseline, 3M, 6M and 12M post-HSCT. Sickling profiles were constructed enabling a comprehensive assessment of RBC sickling and treatment outcomes. The reported parameters include: mPOS@5% (time to reach 5% induced sickling, min), mPOS@50% (time to reach 50% maximum induced sickling,min), rate of sickling (the maximum sickling rate, %/min), maximum induced sickling