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Background: COVID-19 has raised special concern for patients with β-thalassemia major (β-TM) due to frequent comorbidities, regular blood transfusions, and iron overload. However, the exact implications of COVID-19 for patients with β-TM remain uncertain. We aimed to explore the COVID-19 incidence and severity, and the serological response to SARS-CoV-2 infection and vaccination in patients with β-TM., Methods: Patients with β-TM ( n  = 105) and age-matched healthy controls, all individuals of all control groups were health care workers of the hospital, were prospectively enrolled at the haematology department of Al-Shifa hospital in the Gaza Strip from January 1st, 2021 to December 31st, 2021. Data on COVID-19 incidence and severity were analysed, with Alpha, Beta, and Delta SARS-CoV-2 variants dominating at that time. Anti-SARS-CoV-2 IgG antibody levels were measured and compared between study groups., Findings: Patients with β-TM showed a higher incidence of SARS-CoV-2 infection than the general population (61.9% vs. 7.1%, p < 0.0001). Most patients with β-TM had asymptomatic (70.8%) or mild disease (26.1%), with no fatalities recorded. COVID-19 illness was more severe among female than male patients with β-TM. Anti-SARS-CoV-2 IgG antibodies were significantly higher in symptomatic patients with β-TM than controls post-infection (geometric mean ÷ geometric standard deviation 1299.0 ÷ 3.3 vs. 555.7 ÷ 2.4 AU/mL, p = 0.009) and post-vaccination (8404.0 ÷ 3.9 vs. 2785.6 ÷ 5.0 AU/mL, p = 0.015). Similar responses were observed when comparing splenectomised to non-splenectomised (both asymptomatic and symptomatic) patients with β-TM post-infection (595.4 ÷ 3.9 vs. 280.7 ÷ 3.5 AU/mL, p = 0.005) and post-vaccination (13,778.2 ÷ 3.2 vs. 4961.8 ÷ 4.1 AU/mL, p = 0.045)., Interpretation: This distinctive β-TM cohort exhibited a high susceptibility to SARS-CoV-2 infection but mild disease course. Our findings support favourable serological responses to SARS-CoV-2 infection and to vaccination in patients with β-TM, indicating a potential interplay between iron availability and COVID-19-related immunity., Funding: This study was funded by Mr. Hosam and Wasim s. El Helou., Competing Interests: There are no competing interests to disclose., (© 2023 The Authors.)

Increasing evidence suggests that free haem and iron exert vasculo-toxic and pro-inflammatory effects by activating endothelial and immune cells. In the present retrospective study, we compared serum samples from transfusion-dependent patients with β-thalassaemia major and intermedia, hereditary spherocytosis and sickle cell disease (SCD). Haemolysis, transfusions and ineffective erythropoiesis contribute to haem and iron overload in haemolytic patients. In all cohorts we observed increased systemic haem and iron levels associated with scavenger depletion and toxic 'free' species formation. Endothelial dysfunction, oxidative stress and inflammation markers were significantly increased compared to healthy donors. In multivariable logistic regression analysis, oxidative stress markers remained significantly associated with both haem- and iron-related parameters, while soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble endothelial selectin (sE-selectin) and tumour necrosis factor α (TNFα) showed the strongest association with haem-related parameters and soluble intercellular adhesion molecule 1 (sICAM-1), sVCAM-1, interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) with iron-related parameters. While hereditary spherocytosis was associated with the highest IL-6 and TNFα levels, β-thalassaemia major showed limited inflammation compared to SCD. The sVCAM1 increase was significantly lower in patients with SCD receiving exchange compared to simple transfusions. The present results support the involvement of free haem/iron species in the pathogenesis of vascular dysfunction and sterile inflammation in haemolytic diseases, irrespective of the underlying haemolytic mechanism, and highlight the potential therapeutic benefit of iron/haem scavenging therapies in these conditions., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Patients with thalassemia exhibit an increased risk of thrombotic events that is augmented after splenectomy. Heparanase protein enhances cancer progression, angiogenesis, and inflammation; it also activates the coagulation system through direct interaction with tissue factor (TF). Additionally, erythropoietin, which is elevated in anemic patients, up-regulates heparanase expression via the Janus kinase 2 (JAK-2) pathway. This study aimed was to explore the heparanase profile in thalassemia. Coagulation factors were analyzed via immunostaining, enzyme-linked immunosorbent assay, and heparanase procoagulant activity assay. In spleen specimens of thalassemia major patients, a higher level of heparanase staining was observed compared with control spleens resected after trauma (P < 0.001). Higher heparanase levels, heparanase and TF procoagulant activity, and erythropoietin levels were found in the plasma of 67 thalassemia major patients compared with 29 control subjects. No difference was found in pediatric patients (23 of 67) compared with adults or splenectomized versus nonsplenectomized patients. Higher levels of heparanase, TF, TF pathway inhibitor, and TF pathway inhibitor-2 were observed in liver, spleen, heart, and kidney tissues of thalassemia intermedia mice (Hbb th3/+ ). These protein levels significantly reduced when mice were treated with the JAK-2 inhibitor ruxolitinib (P < 0.0001). In summary, heparanase levels are elevated in thalassemia, which may contribute to thrombotic phenomena in these patients. Inhibition of heparanase or the JAK-2 pathway may reduce thrombotic risk in thalassemia., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

β-Thalassemia (β-thal) is a very common disease in the Palestinian population of the Gaza Strip. We studied their mutation frequency and clinical features. Thirteen different mutations were identified. The most common mutation was IVS-I-1 (G>A) (HBB: c.92+1G>A), which was prevalent in 31.5% of the thalassemia alleles studied. The IVS-I-110 (G>A) (HBB: c.93-21G>A) mutation was found in 25.0% of the alleles. Homozygotes for the IVS-I-1 mutation had higher mean hemoglobin (Hb) levels, required less blood transfusions, and lower transferrin saturation than the homozygotes for the IVS-I-110 mutation. This milder phenotype was, most likely, the result of the persistent production of Hb F; it was 9-fold higher in absolute terms (g/dL) and 7.7-fold higher in relative terms (percentage of total Hb). About half of our IVS-I-1 patients carried the XmnI polymorphism, which is known to be associated with elevated Hb F levels.

Background/aims: Erythropoiesis in long-term hemodialyzed (LTH) patients is supported by erythropoietin (rHuEpo) and intravenous (IV) iron. This treatment may end up in iron overload (IO) in major organs. We studied such patients for the parameters of IO in the serum and in major organs., Methods: Patients were treated with rHuEpo (6-8 x 10(3) units × 1-3/wk) and IV 100 mg ferric saccharate., Results: Of 115 patients, 21 had serum ferritin (SF) > 1000 ng/mL. This group was further analyzed. Their SF and transferrin saturation (TSAT) were 2688 ± 1489 ng/mL and 54.2 ± 32.7%, respectively (vs. 125-360 ng/mL and 20-50% in normal controls). Serum hepcidin was 60.1 ± 29.5 nm (vs. 10.61 ± 6.44 nm in controls) (P < 0.001). Nineteen patients had increased malonyldialdehyde, a product of lipid peroxidation, indicating oxidative stress. T2* MRI disclosed in 19 of 21 patients moderate to severe IO in the liver and spleen, in three of eight patients in the pancreas, but in no patient in the heart. After stopping IV iron for a mean of 12 months, while continuing rHuEpo, the mean SF decreased in 11 patients to 1682 ng/mL and the mean TSAT decreased to 28%, whereas hemoglobin did not change indicating that tissue iron was utilized., Conclusion: High SF correlates with IO in the liver and spleen, but not in the heart., (© 2012 John Wiley & Sons A/S.)

Background: In transfusional siderosis, the iron binding capacity of plasma transferrin is often surpassed, with concomitant generation of non-transferrin-bound iron. Although implicated in tissue siderosis, non-transferrin-bound iron modes of cell ingress remain undefined, largely because of its variable composition and association with macromolecules. Using fluorescent tracing of labile iron in endosomal vesicles and cytosol, we examined the hypothesis that non-transferrin-bound iron fractions detected in iron overloaded patients enter cells via bulk endocytosis., Design and Methods: Fluorescence microscopy and flow cytometry served as analytical tools for tracing non-transferrin-bound iron entry into endosomes with the redox-reactive macromolecular probe Oxyburst-Green and into the cytosol with cell-laden calcein green and calcein blue. Non-transferrin-bound iron-containing media were from sera of polytransfused thalassemia major patients and model iron substances detected in thalassemia major sera; cell models were cultured macrophages, and cardiac myoblasts and myocytes., Results: Exposure of cells to ferric citrate together with albumin, or to non-transferrin-bound iron-containing sera from thalassemia major patients caused an increase in labile iron content of endosomes and cytosol in macrophages and cardiac cells. This increase was more striking in macrophages, but in both cell types was largely reduced by co-exposure to non-transferrin-bound iron-containing media with non-penetrating iron chelators or apo-transferrin, or by treatment with inhibitors of endocytosis. Endosomal iron accumulation traced with calcein-green was proportional to input non-transferrin-bound iron levels (r(2) = 0.61) and also preventable by pre-chelation., Conclusions: Our studies indicate that macromolecule-associated non-transferrin-bound iron can initially gain access into various cells via endocytic pathways, followed by iron translocation to the cytosol. Endocytic uptake of plasma non-transferrin-bound iron is a possible mechanism that can contribute to iron loading of cell types engaged in bulk/adsorptive endocytosis, highlighting the importance of its prevention by iron chelation.

Non-transferrin bound iron (NTBI) is commonly detected in patients with systemic iron overload whose serum iron-binding capacity has been surpassed. It has been perceived as an indicator of iron overload, impending organ damage and a chelation target in poly-transfused thalassemia patients. However, NTBI is a heterogeneous entity comprising various iron complexes, including a significant redox-active and readily chelatable fraction, which we have designated as "labile plasma iron" (LPI). We found that LPI levels can be affected by plasma components such as citrate, uric acid, and albumin. However, the inclusion of a mild metal mobilizing agent in the LPI assay (designated here as "eLPI"), at concentrations that do not affect transferrin-bound iron, largely overcomes such effects and provides a measure of the full NTBI content. We analyzed three distinct groups of poly-transfused, iron overloaded thalassemia patients: non-chelated children (3-13 yrs, Gaza, Palestine), chelated adolescents-young adults (13-28 yrs, Israel), and chelated adults (27-61 yrs, Israel) for LPI and eLPI. The eLPI levels in all three groups were roughly commensurate (r(2) = 0.61-0.75) with deferrioxamine-detectable NTBI, i.e., DCI. In older chelated patients, eLPI levels approximated those of LPI, but in poly-transfused unchelated children eLPI was notably higher than LPI, a difference attributed to plasma properties affected by labile iron due to lack of chelation, possibly reflecting age-dependent attrition of plasma components. We propose that the two formats of NTBI measurement presented here are complementary and used together could provide more comprehensive information on the forms of NTBI in patients and their response to chelation., (Copyright © 2011 Wiley Periodicals, Inc.)

In the present study, we questioned the role of oxidative stress in hereditary spherocytosis (HS), where red blood cells (RBC) have a shortened survival due to primary deficiency in membrane proteins. Using flow cytometry techniques, we showed that RBC derived from 17 HS patients of seven families generate more reactive oxygen species, membrane lipid peroxides, and less reduced glutathione than normal RBC. Following in vitro incubation of HS-RBC from seven patients with a fermentation bioproduct of Carica papaya (fermented papaya preparation (FPP)) with known antioxidative properties, oxidative stress markers were significantly reduced. Similar results were obtained following treatment with FPP for 3 months of 10 adult HS patients, as well as decreased tendency to undergo hemolysis. The hemoglobin levels increased by >1 g/dl, mean corpuscular hemoglobin concentration decreased by >1 g/dl, and the reticulocyte count decreased by 0.93%. Concomitantly, lactic dehydrogenase decreased by 17% and indirect bilirubin by 50%. A significant decrease in malonyldialdehyde was also detected. These data indicate that oxidative stress plays an important role in the pathophysiology of HS which can be ameliorated by an antioxidant such as FPP. Additional clinical trials with FPP and other antioxidants are warranted.

Hepcidin is a major regulator of iron metabolism. We evaluated changes in serum hepcidin during 3 months of therapy with the iron-chelator deferasirox in patients with low-risk myelodysplastic syndrome and iron overload. Serum hepcidin was found to be high in these patients, correlated with their iron and oxidative status, and further increased by treatment with deferasirox. These findings support the concept that the hepcidin level represents a balance between the stimulating effect of iron overload and the inhibitory effects of erythropoietic activity and oxidative stress. These preliminary findings favour the rationale for iron chelation therapy in such patients., (© 2011 Blackwell Publishing Ltd.)

Iron overload (IO) in the heart is a life-threatening complication in transfusion-dependent patients with thalassaemia major (TM) and to a lesser extent in sickle cell disease (SCD), while no data are available in patients with sickle/beta(0)-thalassaemia. Iron deposition in the heart, liver and pancreas was assessed using T2* MRI sequences, as well as free iron species assays - non-transferrin bound iron (NTBI) and labile plasma iron (LPI), in addition to serum ferritin, percentage transferrin saturation and serum hepcidin, in 10 multitransfused patients (>30 yr) with sickle/beta(0)-thalassaemia. None of the patients had iron deposition in the heart. Three patients had mild, one had moderate, and two had severe liver IO. Two patients had mild iron deposition in the pancreas. In all the patients, serum hepcidin levels were normal - NTBI and LPI were not detected. Possible explanations of these findings are discussed.

Low-risk myelodysplastic syndrome (MDS) is characterized by cytopenia, mainly anemia, because of ineffective hematopoiesis. Some of the patients with ineffective erythropoiesis, with or without ring sideroblasts in their bone marrow, develop severe anemia requiring frequent blood transfusions and consequently develop iron overload. Excess free iron in cells catalyses the generation of reactive oxygen species (ROS) that cause cell and tissue damage. Using flow cytometry techniques, we compared the oxidative status of red blood cells (RBC), platelets and neutrophils in 14 MDS patients with those of normal donors. The results show that ROS were higher while reduced glutathione (GSH) was lower in their RBC and platelets compared with normal cells. In neutrophils, no difference was found in ROS, while the GSH levels were lower. A correlation (r = 0.6) was found between serum ferritin levels of the patients and the ROS in their RBC and platelets. The oxidative stress was ameliorated by a short incubation with the iron-chelators, the deferrioxamine and deferiprone or with antioxidants such as N-acetylcysteine, suggesting that MDS patients might benefit from treatment with iron-chelators and antioxidants.

The method of cardiovascular T2 magnetic resonance imaging (MRI) allows in vivo estimation of iron in the heart and liver and was used to measure the degree of iron overload in 10 transfused MDS patients (average 90 blood units) and in 3 patients with congenital hemolytic anemia. In all MDS patients iron overload was found in the liver but not in the heart. Patients with congenital anemias had iron in both organs despite iron chelation. It is possible that in MDS more time and more transfusions are required to induce iron accumulation in the myocardium. Therefore, cardiac MRI may serve as a diagnostic tool to assess if and when iron chelation is indicated., ((c) 2007 Wiley-Liss, Inc.)

Sickle cell disease (SCD) is basically a red blood cell (RBC) disorder characterised by sickling and haemolysis, but platelets and polymorphonuclear neutrophils (PMN) are also involved. Oxidative damage may play a role in the pathogenesis of SCD. Using flow cytometry, we measured oxidative-state markers simultaneously in RBC, platelets and PMN obtained from 25 normal donors, nine homozygous (SS) patients and six SS/beta-thalassaemia patients. Reactive oxygen species (ROS) and reduced glutathione (GSH) were measured following staining of blood samples with fluorescence probes and gating on specific subpopulations based on size and granularity. Ten- to 30-fold higher ROS production and 20-50% lower GSH content were found in RBC, platelets and PMN from SCD patients versus those of their normal counterparts. This could in part account for the clinical manifestations, such as haemolysis, a hypercoagulable state, recurrent bacterial infections and vaso-occlusive incidences, in SCD. We further showed that exposure of SCD samples to antioxidants, such as N-acetyl-cysteine, vitamin C and vitamin E, decreased their oxidative stress. These results suggest that antioxidant treatment of patients with SCD could reduce oxidative damage to RBC, PMN and platelets, thereby alleviating symptoms associated with their pathology. The flow cytometry techniques presented herein could assist in monitoring the efficacy of such treatment.