P106: EARLY PREVENTIVE DIAGNOSIS OF HEMOLYTIC ANEMIA IN SICKLE CELL PATIENTS BY DETECTING THE TRIGGERING OF ERYPTOSIS.

Abstract

Background/Aims: Sickle cell disease (SCD) also known as sickle cell anemia is one of the most worldwide-disseminated hereditary hemoglobinopathies. It is caused by a single amino acid substitution at the sixth residue of β globin gene (Glu6Val), which results in an abnormal hemoglobin called hemoglobin S (HbS). The acceleration of HbS polymerization induces rigid and dysfunctional erythrocytes that play a central role in acute and chronic clinical manifestations of SCD(Conran et al.,2009). Vaso-occlusion and hemolytic anemia are the hallmarks of SCD. We aim to explore the cellular environment of red blood cells to explain the physiopathology of SCD. In fact, the life span of circulating erythrocytes in healthy individuals vary from 100 to 120 days. In SCD, red blood cells undergo a form of cell death, namely, eryptosis before they reach their full life span. Eryptosis is triggered by a wide variety of factors as hyperosmolarity, oxidative stress and energy depletion. It is characterized by the presence of membrane blebbing, cell shrinkage, and phosphatidylserine (PS) exposure (Lang et al.,2012). In this study, we will explore the mechanism of triggering of eryptosis in Sickle cell disease. Methods: Following clinical diagnosis, 50 homozygous SCD patients and 30 healthy donors were identified for hematological and cellular assays. Flow cytometry was performed in order to determine the viability parameters of erythrocytes. The morphology of red blood cells and the externalization of phosphatidylserine was detected by labeling red blood cells with Annexine V. Moreover, we had identified intracellular calcium concentration and ceramide level by labeling erythrocytes with Fluo3-am and anti-ceramide antibodies. Finally, we had quantified reactive oxygen species (ROS) by labeling red blood cells with CM-H2DCFDA. Results and Discussion: Eryptosis in sickle cell patients is accelerated. In fact, PS (+) red blood cells are more present in patients than in healthy subjects. Therefore, eryptosis is triggered by oxidative stress, which stimulates the increase of calcium activity and subsequent externalization of PS and red blood cells shrinkage in sickle cell patients. However, the pathway of ceramide can also be considered a potential stimulating factor of eryptosis in SCD. Eryptosis ensures healthy erythrocyte quantity in circulation, whereas excessive eryptosis is the cause of acute anemia and may contribute to vaso-occlusive crisis in SCD patients.