Erythropoietic Stimuli and Response in Hereditary Spherocytosis
Authors: Rocha Susana , Rocha-Pereira Petronila , Castro Elisabeth Bayer , Cleto Esmeralda , Ferreira Fátima , Antunes Marika , Barbot José , Quintanilha Alexandre , Belo Luís , Santos-Silva Alice 
Published: April 2020

Abstract
Hereditary Spherocytosis (HS) is the most common non-immune hemolytic anemia in Europe. HS ranges from assymptomatic to transfusion-dependent hemolytic anemia and is clinically classified as mild, moderate or severe. Clinical, biochemical and molecular heterogeneous patterns are HS trademarks. In a previous work, we reported that in HS patients the erythropoietic response appears to be impaired in the more severe cases of the disease, since erythropoietin (EPO) levels did not correlate with reticulocyte count or with reticulocyte production index (RPI) [Rocha S. et al. (2005) Br J Haematol. 131(4):534]. It is known that spherocytes are removed by splenic macrophages. The activation of these cells is associated with cytokine release, namely, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), both known as EPO inhibitors. The aim of our work was to evaluate the erythropoietic stimuli and response according to HS severity and to clarify the role of these cytokines in HS. We studied 31 HS patients and 25 healthy individuals as the control group. The HS group included only unsplenectomised patients classified as presenting mild (n=19), moderate (n=9) or severe HS (n=3) according to the Guidelines for the diagnosis and management of hereditary spherocytosis [Bolton-Maggs et al. (2004) Br J Haematol. 126(4):455]. The basic hematological study was performed in all samples, as well as the reticulocyte count and RPI calculation; the plasma levels of EPO and soluble transferrin receptor (sTfR) (as markers of erythropoietic stimuli), of bilirubin (as a marker of hemolysis) and of TNF-α and IFN-γ (known as EPO inhibitors) were also determined. Data was analysed according to the severity of HS and versus the control group. Erythrocyte count, hemoglobin concentration, hematocrit and mean cell volume were found to be significantly lower in patients than in controls and their values decreased with HS severity; for MCHC, RDW, reticulocytes, RPI, bilirubin, EPO and sTfR patients presented significantly higher values than controls, and their values increased with HS severity. However, the increment in EPO and sTfR levels was more marked than for reticulocytes and RPI. As for TNF-α and IFN-γ no differences were found between patients and controls or between HS severities; no correlations with the other studied variables were observed. In mild HS patients, sTfR was found to be statistically and positively correlated with reticulocytes (r=0.783; p&lt;0.001), RPI (r=0.748; p&lt;0.001) and bilirubin (r=0.870; p&lt;0.001); in patients with more severe forms (moderate and severe HS) these correlations were lower and only a statistically and positively correlation between sTfR and reticulocytes (r=0.657; p&lt;0.05) was found. Our results confirm the inadequate erythropoietic response in the more severe cases of HS, given that, the erythropoietic stimuli (shown by higher EPO, sTfR and indirectly, by bilirubin levels) was not able to trigger an adequate erythropoietic response as shown by the reticulocyte number and RPI, as occured in mild HS patients. Our hypothesis, that this disturbance could be due to an increase in TNF-α and IFN-γ, was not confirmed, as we did not found any significant increase in these cytokines that could explain the non adequate erythropoietic response in moderate/severe HS patients. In conclusion, the erythropoietic stimuli seem to increase with HS severity; however, the response to that stimulus appears to be impaired in the more severe cases of HS. Further studies are needed to clarify this erythropoietic disturbance in HS patients, which may contribute to the severity of the clinical presentation of the disease.