Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Sickle Cell Disease, Translational Research, Sickle Cell Trait, assays, Hemoglobinopathies, Diseases, emerging technologies, Technology and Procedures
Sickle cell disease (SCD) is a genetic disorder caused by a single mutation in the beta-globin gene resulting in sickle hemoglobin (HbS). Upon deoxygenation, HbS polymerizes in red blood cells (RBCs) resulting in RBC sickling, and the irreversibly sickled RBCs provoke complex pathophysiology of acute and chronic organ damage . Cycles of oxygenation/deoxygenation of RBCs in blood circulation results in sickling/unsickling cycles which makes the sickle RBC more fragile with half life cycle and hemolysis compared to healthy RBCs [1,2]. Previous studies have focused on reporting the kinetics of sickling of HbS containing RBCs. However, the kinetics of RBC’s sickling/unsickling cycles in SCD genotypes remains unclear . Here we developed a microfluidic assay to investigate the sickling/unsickling dynamics among the common sickle phenotypes including homozygous HbSS (SCD) and heterozygous HbSC (hemoglobin S-C disease), and HbAS (sickle cell trait, SCT).
Methods: Venous blood samples were collected in EDTA-coated tubes from subjects with HbSS, HbSC, HbAS, and Healthy controls (HbAA) following IRB-approved protocol. The microfluidic chip was fabricated with polydimethylsiloxane (PDMS) using a standard soft lithography protocol . Following the fabrication of the microfluidic channel, the microfluidic chip was then assembled and incubated with 1% poly-d-lysine overnight at 4°C, then rinsed and left to dry at 4°C for 5 hours. RBCs were isolated from whole blood, re-suspended in PBS at 0.5% hematocrit, and passed through the microchannels with a constant inlet pressure. The microfluidic devices were connected to a gas exchanger that introduced a gaseous mixture containing 5 % CO2 and 95 % N2 inducing an oxygen saturation level of 83% at the microchannel's inlet. Microscope images of the sickling microchannel were recorded for 20 minutes at 2 frames per second. Using a neural network algorithm, the sickling time for half the number of cells, the number of sickled cells, the duration of sickling, and the sickling/ unsickling cycles until they become irreversibly sickled cells were determined for every sample. Data were reported as SEM (Standard mean error) in this study
We non-selectively immobilized red blood cells from 3 HbAA, 10 HbSS, 5 HbSC, and 3 HbAS subjects using the microfluidic chip. Morphological changes of single RBCs were analyzed with the neural network tools and used to determine the sickling time, the duration of the sickling process, and the number of sickling/unsickling cycles until the cells were irreversibly sickled. We observed that the number of sickled RBCs increased in successive sickling cycles while the number of the non-sickle cell was reduced. No sickling was observed in HbAA samples while SCD genotypes HbSS, HbSC, and HbAS exhibited unique sickling trends (Figure 1A). The sickling time was – mean ± SEM = 2 ± 0.9, 11 ± 2.3, and 15 ± 1.7 (minutes) for HbSS, HbSC, and HbAS respectively. This parameter was significantly different across all three SCD genotypes with p = 0.02, 0.001 ,and 0.03 (Mann–Whitney) for HbSS vs HbSC, HbSS vs HbAS ,and HbSC vs HbAS respectively (Figure 1B). Further, we obtained the sickling duration based on the steepness of the sickling curves (Figure 1A) and found that it was 4 ± 0.9 minutes for HbSS and 2 ± 1.5 minutes for HbSC. HbSC samples took longer to initiate sickling but once the sickling process started, they achieved maximum sickling 2x faster than HbSS samples. The number of sickling-unsickling cycles before irreversible sickling varied between subjects with a range of 3 – 8 cycles for HbSS and 3- 4 cycles for HbSC. The refractory period was reduced by 2 minutes (on average) for each subsequent cycle in both HbSS and HbSC samples.
These results suggest that the sickling dynamics differ across SCD phenotypes and across patients. A clear understanding of the mechanisms of sickling especially for HbSC may be beneficial in understanding the variations between patients and in therapeutic development. Prospective studies are required to understand how the sickling/unsickling cycles could relate to well-known SCD physiological parameters such as deformability, endothelial activation, and blood rheology.
Disclosures: Gurkan: Xatek Inc.: Patents & Royalties; Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Dx Now Inc.: Patents & Royalties.
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