P-061: de novo appearance of vacuoles in transfused red blood cells in sickle cell disease patients with defective splenic function

Purpose: Defective spleen function (hyposplenism) affects subjects with sickle cell disease (SCD) and is associated with complications. Red blood cell (RBC)-related markers are very accurate to assess spleen function. Vacuole-containing RBC (pocked RBC) are observed in high proportions (10 to 70%) of hyposplenic subjects whereas they do not exceed 5% of RBC in healthy subjects. Vacuoles in pocked RBC are considered by many as remnants of erythropoiesis-related processes. Materials and methods: Patients with SCD who benefit from an exchange transfusion program, have not only their own HbSS RBC but also transfused HbAA RBC in circulation. In vitro sickling “Emmel test” discriminates HbSS RBC, that sickle, from HbAA RBC, that do not. We perfused a mixture of HbSS RBC and HbAA RBC (from exchange transfusion left-overs) into human spleens ex-vivo. We then analyzed spleen sections and quantified vacuoles in RBC containing hemoglobin polymers or not. Results: Using Emmel test, we observed vacuoles in HbSS RBC but also in transfused HbAA RBC. Pocked HbAA RBC were more frequent in transfused SCD patients than in healthy subjects (25.1±4.41% vs. 2.8±2.1%). Into human spleen ex-vivo, we observed a high rate of vacuoles in either polymerized and non-polymerized RBC (29.8±3.7% vs. 17.9±5.8%). In our experimental set-up, HbAA RBC is deducted as the absence of polymerization. Conclusion: Taken together, these results strongly suggest that vacuoles are created de novo in transfused HbAA RBC.Counting vacuoles in RBC labeled with anti-HbS and anti-HbA antibody is ongoing to validate these observations. When the spleen is dysfunctional, these pocked RBC stay in circulation hence their validity as a spleen function marker. De novo appearance of vacuoles in transfused HbAA RBC likely impacts the kinetics of reappearance of pocked RBC in circulation following regular or exchange transfusion to hyposplenic subjects, and opens interesting prospects on cell processes involved in their genesis. M. CAVAZZANA declares a conflict of interest: Consultancy, Expert: consultant for Smart-Immune biotech