Detection of the red blood cell adhesion to the vascular endothelium using microfluidic chip in beta thalassemia patients

Beta thalassemia is a hereditary genetic disorder affecting the hematopoietic system, primarily due to mutations in the beta globin gene. The disease is characterized by ineffective erythropoiesis resulting from reduced or absent beta globin production, leading to low or no production of hemoglobin A. This deficiency causes an accumulation of free alpha globin chains in the cytoplasm of erythroid cells, which disrupts their physiology. The free α-globin chains aggregate and precipitate, causing cytotoxic effects, oxidative stress, and decreased survival of erythroid cells, leading to high levels of hemolysis. The hemolysis of red blood cells (RBCs) releases hemoglobin and heme into plasma. Extracellular hemes are highly proinflammatory, activating immune and endothelial cells (ECs), contributing to the disease's pathogenesis. EC activation leads to the overexpression of adhesion molecules and increased RBC adhesion to ECs. Beta thalassemia patients exhibit altered RBC flow characteristics, causing microcirculatory issues. However, the mechanisms and molecules involved in RBC adhesion in beta thalassemia remain not fully understood. This study investigated RBC adhesion in beta thalassemia major (BTM) patients under flow conditions using a microfluidic chip with microchannels functionalized with 40 μM heme-activated HUVECs, fibronectin, and laminin. Increased RBC adhesion in BTM patients in microchannels functionalized with 40 μM heme-activated HUVECs and fibronectin was demonstrated compared to laminin. There were significant positive correlations between hemoglobin, WBC, platelet values, and adherent RBC number in microchannels functionalized with fibronectin. Application of Forskolin (activator of adenylyl cyclase) and SQ22536 (inhibitor of adenylyl cyclase) indicated that RBC adhesion could be altered by targeting the AC-cAMP-PKA signaling pathway. RBC deformability was also assessed, showing a slight decrease after shear stress at high shear stress levels for untreated patient blood, with Forskolin and SQ22536 impairing deformability compared to the vehicle after shear stress. Lastly, plasma hemoglobin concentration was higher in beta thalassemia major patients' plasma samples than healthy controls. These data highlight the importance of VCAM-1 and fibronectin for RBC adhesion in BTM patients and the effects of targeting the AC-cAMP-PKA signaling pathway on RBC deformability and adhesion. This study has contributed to understanding the pathophysiology of beta thalassemia major patients and importance of targeting AC-cAMP-PKA signaling pathway. This underscores the need for these studies to be conducted with a larger patient cohort to gain more thorough insights.