Type-2 Phosphatidylserine (PS)-Positive Erythrocytes and Their Association with Markers of Hemolysis and Hemostatic Activation in Children with Sickle Cell Disease (SCD)

Introduction: Type-2 phosphatidylserine (PS)-positive red cells are a subpopulation of erythrocytes that are highly positive for PS, contain low levels of fetal hemoglobin, are specific for sickle cell disease (SCD) and have been identified in the dense red cell fraction. Studies have implicated PS-positive red cells in enhancing anemia due to phagocytosis and hemolysis. Shielding of red cell PS by diannexin, a synthetic homodimer of human annexin-V, has been demonstrated to provide protection against hemolysis and prevent activation of prothrombinase.

Methods: Using flow cytometry, we measured the levels of type-1 (red cells with low PS positivity) and type-2 PS-positive red cells in 50 children with SCD (31 with HbSS and 19 with HbSC), and assessed their association with various markers of hemolysis and hemostatic activation. Markers of hemolysis evaluated included plasma lactate dehydrogenase (LDH), reticulocyte count, and hemoglobin. Whole blood tissue factor (WBTF), pro-thrombin fragment F₁₊₂, and D-dimer were evaluated as markers of hemostatic activation.

Results: We demonstrate that the levels of type-2 PS-positive red cells are significantly increased in HbSS patients (1.37 ± 0.97%, p<0.01) compared to children with HbSC disease (0.32 ± 0.21%) and age- and race-matched controls (0.15 ± 0.15%, n=19). WBTF and D-dimer showed significant associations with both type-1 and -2 red cells with no significant differences in the strength of their association. However, significantly greater correlations were noted between type-2 PS red cells and hemolytic markers compared to those noted with type-1 (Steiger's Z=3.05 to 4.59, p<0.01). In addition our in vitro studies demonstrate increased osmotic fragility of these red cells.

Conclusions: Type-2 PS-positive red cells are elevated in SCD and the number of these cells correlates significantly with both markers of hemolysis and hemostasis. These findings provide a patho-physiologic link between the intravascular hemolytic milieu of SCD and the hemostatic perturbations previously noted in this disease.