Dynamic Sickling Assay: A Novel Approach for Assessing RBC Sickling Kinetics in SCD
Authors: Hines Patrick C , Goodrich Robert , Antongiorgi Jordan , Gao Xiufeng , Zaidi Aliya U , Liu Ke 
Published: June 2024

Abstract

Presentation Date: 6/9/2024
Presentation Start Time: 4:00:00 PM

Background
Sickle cell anemia has been considered a rare disease for a long time. Recently it has been estimated that the number of cases is constantly increasing and the number of deaths, many of which are children under the age of five, is constantly growing.

Dynamic sickling assay
a novel
Deoxygenated hemoglobin S (HbS) polymerization leads to red blood cell (RBC) sickling which drives the molecular pathogenesis of sickle cell disease (SCD). In recent decades the scientific community has invested enormous effort in finding new therapies and treatments, including hemoglobin (Hb) modifiers, fetal Hb (HbF) inducers, pyruvate kinase activators, and gene editing therapies, that reduce the HbS polymerization rate and prevent RBC sickling, thus targeting a fundamental mechanism of the disease. Effective monitoring biomarkers are therefore needed to objectively assess target engagement and pharmacodynamic impact.
Dynamic sickling assay (DSA) provides a novel approach to assess morphologic RBC sickling kinetics through induced hypoxia using a simple enzymatic reaction. The process of hypoxia-induced RBC sickling is captured by time lapse microscopy and analyzed with a machine learning algorithm to evaluate dynamic changes in RBC morphology. The resulting sickling profiles encompass several important characteristics, including area under the curve, calculated as the integral of the sickling profile (AUC15), delay of induced sickling, labeled as morphologic point of sickling at 5% (mPoSi@5%) and 50% (mPoSi@50%) and maximum rate of sickling (ratei@50%). This multi-faceted approach assures a comprehensive assessment of cell sickling behaviors and the resistance to low oxygen saturation.

Methods
Eight patient samples were treated with different doses of voxelotor, including 0%, 25%, 50%, 75%, 100% and 125% of modification. Data from sickling morphology (DSA) and deformability (ektacytometry), an important cell membrane biomechanical property and one determinant for vaso-occlusive crisis (VOC) in SCD, obtained using the commercially available ektacytometry (oxygenscan mode), were collected simultaneously.
To assess the ability of DSA to determine the therapeutic effects in a dose-response study of an FDA-approved Hb-modifier treatment (voxelotor) to SCD, we correlated sickling parameters with deformability data. Sickling parameters obtained from untreated samples have also been correlated to available hematological markers, typically associated with SCD severity.
Pearson correlation coefficients and linear regression analyses were obtained using GraphPad Prism. p-values &lt; 0.05 were considered statistically significant.

Results
Significant correlations were found between DSA and ektacytometry parameters (Figure 1A). Minimum elongation (Elmin) and point of sickling (PoS) from ektacytometry significantly correlated with AUC15 (r= -0.86 and r = 0.84, respectively; p &lt; 0.0001), mPoSi@5% (r = 0.76 and r= -0.65, respectively; p &lt; 0.0001), mPoSi@50% (r = 0.83 and r= -0.86, respectively; p &lt; 0.0001) (Figure 1B) and ratei@50% (r= -0.65 and r = 0.67, respectively; p &lt; 0.0001).
Hematological markers and DSA parameters in eight untreated patient samples showed significant correlations. The delay in the induced sickling (mPoSi@50%) correlates with HbF (r = 0.95, p &lt; 0.0001), HbS (r= -0.90, p = 0.002), RBC count (r = 0.96, p &lt; 0.0001), hematocrit (r = 0.75, p = 0.03), percentage of neutrophils (r = 0.97, p &lt; 0.0001), lymphocytes (r= -0.87, p = 0.005) and reticulocytes (r= -0.83, p = 0.01), IRF (r= -0.71, p = 0.048) and RET-He (r= -0.86, p = 0.006). Refer to figure 2 for the comprehensive correlation analysis.

Conclusions
Measurements of morphologic sickling kinetics using DSA showed statistically significant correlations with ektacytometry-based RBC deformability parameters in a dose-response study. This data demonstrates that DSA may be an important pharmacodynamic assessment tool for effectiveness of therapies.
Morphologic sickling kinetics biomarkers may also be used to characterize SCD phenotype and risk-stratification, as supported by the correlation between DSA and hematologic parameters.
Additional studies are ongoing to assess the utility of DSA as a pharmacodynamic assessment tool and to determine the clinical predictive value of morphologic sickling kinetics for outcomes such as VOCs, acute chest syndrome, stroke, pulmonary hypertension, and sickle nephropathy.