Sickle cell disease (SCD) is a recessive genetic blood disorder exhibiting abnormal blood rheology. Polymerization of sickle hemoglobin, due to a point mutation in the β-globin gene of hemoglobin, results in aberrantly adhesive and stiff red blood cells (RBCs). Hemolysis, abnormal RBC adhesion, and abnormal blood rheology together impair endothelial health in people with SCD, which leads to cumulative systemic complications. Here, we describe a microfluidic assay combined with a micro particle image velocimetry technique for the integrated in vitro assessment of whole blood viscosity (WBV) and RBC adhesion. We examined WBV and RBC adhesion to laminin (LN) in microscale flow in whole blood samples from 53 individuals with no hemoglobinopathies (HbAA, N = 10), hemoglobin SC disease (HbSC, N = 14), or homozygous SCD (HbSS, N = 29) with mean WBV of 4.50 cP, 4.08 cP, and 3.73 cP, respectively. We found that WBV correlated with RBC count and hematocrit in subjects with HbSC or HbSS. There was a significant inverse association between WBV and RBC adhesion under both normoxic and physiologically hypoxic (SpO 2 of 83%) tests, in which lower WBV associated with higher RBC adhesion to LN in subjects with HbSS. Low WBV has been found by others to associate with endothelial activation. Altered WBV and abnormal RBC adhesion may synergistically contribute to the endothelial damage and cumulative pathophysiology of SCD. These findings suggest that WBV and RBC adhesion may serve as clinically relevant biomarkers and endpoints in assessing emerging targeted and curative therapies in SCD. 1 | INTRODUCTION Sickle cell disease (SCD) is one of the most common inherited diseases in the world. 1,2 It is caused by a single-point mutation in the β-globin gene of hemoglobin. Sickle hemoglobin (HbS) polymerizes into long and stiff chains within the red blood cell (RBC) under low-oxygen conditions. 3,4 As a result, HbS-containing RBCs (sickle RBCs) become abnormally stiff and adherent (impairing microcirculatory blood flow), and fragile (resulting in hemolysis). Microvascular occlusion causes episodic and unpredictable vaso-occlusive events (VOE), pain, and Erdem Kucukal and Yuncheng Man contributed equally to this study.
A point-of-care diagnostic technology and approach is presented to perform both anemia detection and hemoglobin variant identification in a single test using paper-based microchip electrophoresis.
Sickle cell disease, a genetic disorder affecting a sizeable global demographic, manifests in sickle red blood cells (sRBCs) with altered shape and biomechanics. sRBCs show heightened adhesive interactions with inflamed endothelium, triggering painful vascular occlusion events. Numerous studies employ microfluidic-assay-based monitoring tools to quantify characteristics of adhered sRBCs from high resolution channel images. The current image analysis workflow relies on detailed morphological characterization and cell counting by a specially trained worker. This is time and labor intensive, and prone to user bias artifacts. Here we establish a morphology based classification scheme to identify two naturally arising sRBC subpopulations—deformable and non-deformable sRBCs—utilizing novel visual markers that link to underlying cell biomechanical properties and hold promise for clinically relevant insights. We then set up a standardized, reproducible, and fully automated image analysis workflow designed to carry out this classification. This relies on a two part deep neural network architecture that works in tandem for segmentation of channel images and classification of adhered cells into subtypes. Network training utilized an extensive data set of images generated by the SCD BioChip, a microfluidic assay which injects clinical whole blood samples into protein-functionalized microchannels, mimicking physiological conditions in the microvasculature. Here we carried out the assay with the sub-endothelial protein laminin. The machine learning approach segmented the resulting channel images with 99.1±0.3% mean IoU on the validation set across 5 k-folds, classified detected sRBCs with 96.0±0.3% mean accuracy on the validation set across 5 k-folds, and matched trained personnel in overall characterization of whole channel images with R2 = 0.992, 0.987 and 0.834 for total, deformable and non-deformable sRBC counts respectively. Average analysis time per channel image was also improved by two orders of magnitude (∼ 2 minutes vs ∼ 2-3 hours) over manual characterization. Finally, the network results show an order of magnitude less variance in counts on repeat trials than humans. This kind of standardization is a prerequisite for the viability of any diagnostic technology, making our system suitable for affordable and high throughput disease monitoring.
Abnormal erythrocyte adhesion due to polymerization of sickle hemoglobin is central to the pathophysiology of sickle cell disease (SCD). Mature erythrocytes constitute >80% of all erythrocytes in SCD, and the relative contributions made by erythrocytes to acute and chronic vasculopathy in SCD are not well understood. Here, we show that the bending stress exerted on the erythrocyte plasma membrane by the sickle hemoglobin polymerization under hypoxia enhances sulfatide-mediated abnormal mature erythrocyte adhesion. We hypothesized that sphingomyelinase activity that is upregulated by accumulated bending energy leads to elevated membrane sulfatide availability and thus hypoxic mature erythrocyte adhesion. We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856±481 vs. 78±23, mean±SEM), while sickle reticulocyte (early erythrocyte) adhesion, high to begin with, does not change (1281 ±299 vs. 1258±328, mean±SEM). We show that greater mean accumulated bending energy of adhered mature erythrocytes is associated with higher acid SMase activity and increased mature erythrocyte adhesion (p=0.022, for the acid SMase activity and p=0.002 for the increase in mature erythrocyte adhesion with hypoxia, N=5). In addition, hypoxia results in sulfatide exposure on the erythrocyte membrane, and sphingomyelinase increases while anti-sulfatide inhibits the enhanced adhesion of erythrocytes. These results suggest that lipid components of the plasma membrane contribute to the complications in SCD. Therefore, sulfatide and the components of its upregulation pathway, particularly sphingomyelinase should be further explored as potential therapeutic targets to inhibit sickle erythrocyte adhesion.
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