Non Invasive Evaluation of Bone Marrow Activity in Patients with Sickle Cell Disease: Correlation with Disease Features, Genotype, Markers of Erythropoiesis, Iron Metabolism and Hydroxyurea Treatment
Authors: Papassotiriou Ioannis , Flevari Pagona , Poziopoulos Christos , Zaliou Sofia , Tsaousis Vasilis , Larissi Katerina , Dimopoulou Maria , Komninaka Veroniki , Politou Marianna , Terpos Evangelos , Voskaridou Ersi 
Published: November 2019

Background: Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by pathological polymerization of hemoglobin, increased red cell rigidity and poor microvascular blood flow with consequent tissue ischemia and infarction. Thus, hemolytic anemia, vaso-occlusion and vasculopathy are the hallmarks of its clinical presentation. The transferrin receptor (TfR) mediates the transport of iron into cells and the circulating TfR can be measured as soluble transferrin receptor (sTfR). sTfR levels are frequently used to establish the diagnosis of iron deficiency anemia, especially in the context of inflammation, but they also reflect bone marrow erythropoietic activity (BMA) and mass. Erythropoietic activity has been found to be the most important determinant of sTfR levels. In this context, we aimed to study and evaluate bone marrow activity in patients with compound heterozygous HbS and beta-thalassemia (HbS/βthal) based in sTfR measurements and explore possible correlations with of key features of the disease such as: the hemolytic component, vaso-occlusive crises (VOC), acute chest syndrome, venous thrombosis, arterial thrombosis including stroke, avascular necrosis, pulmonary hypertension, hydroxyurea therapy, inflammation and renal injury. along with other biomarkers of erythropoiesis and iron metabolism such as Placental Growth Factor (PlGF), Growth Differentiation Factor-15 (GDF-15), Ferritin and Hepcidin-25.
Patients and Methods: Ninety adult Caucasian patients with HbS/βthal [49 patients under hydroxyurea (HU+) treatment and 41 patients without hydroxyurea (HU-) treatment], were included in this study, while 22 apparently healthy individuals of similar age and gender served as controls. None of the patients has received any transfusions at least 6-monthes before enrollment in the study. Along with hematologic and blood chemistry parameters determination, levels of circulating sTfR, PlGF, GDF-15 and Hepcidin-25 were measured in patients with HbS/βthal and controls using RUO and IVD immunoenzymatic techniques. BMA activity was calculated from the established formula: patient-sTFR/meanControl-sTFR.
Results: We found that: sTfR levels were markedly elevated in all patients with HbS/βthal compared to controls (4.8±2.2 vs. 1.0±0.2 mg/L, p<0.001), resulting in a 1.6-11.9 fold increase of BMA. No correlation was found between BMA and disease features as well as regarding hydroxyurea treatment BMA (p>0.434). BMA correlated significantly with the markers of the erythropoietic and hemolytic component such as: Hemoglobin (r=-0.434, p<0.001); Reticulocyte Production Index (r=0.645, p<0.001); LDH (r=0.570, p<0.001); Billirubin (r=0.540, p<0.001), PlGF (r=0.597, p<0.001) and Hb A levels (r=-0.493, p<0.001), while no correlation was found between BMA and Hb F levels. Furthermore, BMA values correlated significantly only with GDF-15 (r=0.466, p<0.001), while interestingly no correlation was found between BMA and Ferritin and Hepcidin-25 levels alone (r=0.101, p>0.351 and r=-0.043, p>0.710, respectively), but a negative correlation was found between BMA and Hepcidin-25/Ferritin ratio, (r=-0.330, p=0.005).
Conclusions: Our findings demonstrate that all patients with HbS/βthal studied have a significantly increased degree of erythroid BMA as assessed by measurements of sTfR levels. Erythroid BMA correlated significantly with Hepcidin/Ferritin ratio, which is an index of the degree of Hepcidin expression relative to iron overload. The correlation of erythroid BMA with Hb A levels, indicate the important role of βthal genotype in HbS/βthal disease. Furthermore, BMA is not related to hydroxyurea therapy and/or iron metabolism parameters in these patients. This implicates a likely complex action of hydroxyurea, which causes intermittent cytotoxic suppression of erythroid progenitors and cell stress signaling. The latter affects erythropoiesis, leading to recruitment of erythroid progenitors with increased HbF levels, although the number of erythroid progenitors -the main source of sTfR- remains stable.

Disclosures
Voskaridou: Genesis: Consultancy, Research Funding; Protagonist: Research Funding; Celgene Corporation: Consultancy, Research Funding; Acceleron: Consultancy, Research Funding; Addmedica: Membership on an entity's Board of Directors or advisory committees.