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Adipocyte fatty acid-binding protein (A-FABP; FABP4) plays a significant role in the pathogenesis and progression of metabolically driven low-grade inflammation and organ damage. This study aimed to evaluate the performance of circulating FABP4 as a predictive and diagnostic biomarker for thalassemia-associated cardiometabolic events. This case-control study enrolled 50 adults with β-thalassemia and 30 age-, sex-, and body mass index-matched controls. Participants underwent a comprehensive evaluation, including complete blood count, liver and kidney function tests, serum blood glucose, lipid profile, and ferritin levels, pelviabdominal ultrasound, ECG, and echocardiography after taking a full medical history and conducting a clinical examination. Serum levels of FABP4 were measured using an Enzyme-Linked-Immunosorbent-Assay. The diagnostic performance of FABP4 was assessed using receiver operator characteristic (ROC) curve analysis to determine optimal values for excluding and confirming cardiometabolic metflammation. The thalassemia cohort exhibited a statistically significant higher concentration of FABP4 compared to the control group (p-value < 0.001). Positive correlations were found between FABP4 and ferritin serum levels above 800 or 1000 ug/L, as well as with ALT, TGS, and LDL (p-value < 0.05). Circulating FABP4 was identified as a statistically significant risk factor for thalassemia-associated cardiometabolic comorbidities (OR = 84.00, 95%CI:18.6-378.6, p-value < 0.001). ROC analysis determined that the FABP4 exclusionary cut-off value > 2.30 ng/ml could effectively discriminate between thalassemia-associated adverse metaflammation and controls, while the FABP4 confirmatory cut-off value was > 2.58 ng/ml. In conclusion, circulating FABP4 appears to be a potential risk factor for predicting progression to cardiometabolic events in thalassemia-associated adverse metaflammation. FABP4 holds promise as a diagnostic and prognostic biomarker for disease monitoring and risk stratification. Further validation through large-scale, multicenter, prospective studies is warranted., (© 2024. The Author(s).)

Data on iron overload status and change thresholds that can predict mortality in patients with transfusion-dependent β-thalassemia (TDT) are limited. This was a retrospective cohort study of 912 TDT patients followed for up to 10 years at treatment centers in Italy (median age 32 years, 51.6% female). The crude mortality rate was 2.9%. Following best-predictive threshold identification through receiver operating characteristic curve analyses, data from multivariate Cox-regression models showed that patients with Period Average Serum Ferritin (SF) > 2145 vs ≤ 2145 ng/mL were 7.1-fold (P < 0.001) or with Absolute Change SF > 1330 vs ≤ 1330 ng/mL increase were 21.5-fold (P < 0.001) more likely to die from any cause. Patients with Period Average Liver Iron Concentration (LIC) > 8 vs ≤ 8 mg/g were 20.2-fold (P < 0.001) or with Absolute Change LIC > 1.4 vs ≤ 1.4 mg/g increase were 27.6-fold (P < 0.001) more likely to die from any cause. Patients with Index (first) cardiac T2* (cT2*) < 27 vs ≥ 27 ms were 8.6-fold (P < 0.001) more likely to die from any cause. Similarly, results at varying thresholds were identified for death from cardiovascular disease. These findings should support decisions on iron chelation therapy by establishing treatment targets, including safe iron levels and clinically meaningful changes over time., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Tissue iron overload is a life-threatening scenario in children with transfusion-dependent β-thalassemia major, miRNAs that are involved in iron hemostasis could serve as therapeutic targets for control of iron overload. We aimed to find out the association between three iron-related miRNAs "miR-let-7d, miR-122, and miR-200b" and excess iron in tissues, in transfusion-dependent β-thalassemia major patients. Circulating miRNA expressions are measured in peripheral blood (PB) samples using qPCR of transfusion-dependent (TDT) β-thalassemia patients (n = 140) and normalized to non-transfusion-dependent (NTDT) β-thalassemia (n = 45). Results revealed that plasma expression levels of miR-let-7d and miR-200b were significantly downregulated in TDT patients; however, miR-122 was upregulated. In terms of tissue iron load, aberrant expression of miRNAs was significantly associated with increased-iron accumulation in hepatic and cardiac tissues. We concluded that circulating miRNAs are strong candidates that associate iron hemostasis in transfusion-dependent β-thalassemia major patients. And by extension, targeting miR-let-7d, miR-122, and miR-200 might serve as novel sensitive, specific and non-invasive predictor biomarkers for cellular damage under condition of tissue iron excess., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hydroxyurea (HU) and thalidomide have been reported to improve clinical and hematological parameters in transfusion-dependent beta thalassemia (TDT). Therefore, we retrospectively analyzed the combination of HU and thalidomide in 140 transplant ineligible TDT, ≥ 10 years old, visiting our thalassemia clinic between October 2014 and November 2019. Responses were defined as maintenance of hemoglobin ≥9gm/dl without transfusion as complete response (CR) and with at least 50% reduction in transfusion burden as partial response (PR). Patients with less than 50% transfusion burden reduction for consecutive 6 months of therapy were defined as non-responders (NR), and treatment was discontinued thereafter. Primary end point was overall response rate (ORR) at last follow-up. At median follow-up of 22.6 (95% CI 16.4-28.7) months, 76 (57.2%) patients achieved CR and 19 (14.3%) achieved PR, accounting to an ORR of 71.5%. Among responders at last follow-up, a significant increase in the post-treatment hemoglobin (0.88±0.37gm/dl, p<0.0001) and drop in serum ferritin (-1490.5ng/ml, p<0.0001) were observed. Median time to CR was 124 (95% CI 75.3-172.6) days. Median longest continuous CR was 791 (95% CI 662.2-919.7) days. Common toxicities observed were sedation (25%), hyperbilirubinemia {(23.57%, grade 3/4 =17 (12.14%)}, and constipation (22.8%). Nearly three-fourth of the patients has responded with majority having CR. Adverse events are a concern; hence, regular close monitoring is a prerequisite.

Curcuminoids, polyphenol compounds in turmeric, possess several pharmacological properties including antioxidant, iron-chelating, and anti-inflammatory activities. Effects of curcuminoids in thalassemia patients have been explored in a limited number of studies using different doses of curcuminoids. The present study aims to evaluate the effects of 24-week curcuminoids supplementation at the dosage of 500 and 1000 mg/day on iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfused β-thalassemia/Hb E patients. In general, both curcuminoids dosages significantly lowered the levels of oxidative stress, hypercoagulability, and inflammatory markers in the patients. In contrast, reductions in iron parameter levels were more remarkable in the 1000 mg/day group. Subgroup analysis revealed that a marker of hypercoagulability was significantly decreased only in patients with baseline ferritin ≤ 1000 ng/ml independently of curcuminoids dosage. Moreover, the alleviation of iron loading parameters was more remarkable in patients with baseline ferritin > 1000 ng/ml who receive 1000 mg/day curcuminoids. On the other hand, the responses of oxidative stress markers were higher with 500 mg/day curcuminoids regardless of baseline ferritin levels. Our study suggests that baseline ferritin levels should be considered in the supplementation of curcuminoids and the appropriate curcuminoids dosage might differ according to the required therapeutic effect. Thai Clinical Trials Registry (TCTR): TCTR20200731003; July 31, 2020 "retrospectively registered".

Thalassemia intermedia is a subgroup of β-thalassemia which originates from mutations in the beta-globin gene. Zinc and copper play important roles in the metabolism. Due to its significant therapeutic effects, curcumin has led many studies to focus on curcumin. In a double-blind clinical trial study, 30 patients with beta-thalassemia intermedia with an age range of 20 to 35 years were randomly selected 1:1 to receive either curcumin or placebo for 3 months. Before and after the intervention period, 5 ml of blood was taken to determine the serum levels of zinc and copper. The laboratory tests were checked at baseline and at the end of the treatment. While the serum levels of zinc and zinc/copper significantly increased, the serum levels of copper decreased after 3 months of curcumin intake. In addition, on the basis of baseline characteristics, a negative correlation was found between zinc and body mass index and positive correlations were identified between copper with triglyceride and high-density lipoprotein. Also, the level of ferritin protein in the curcumin group compared to the placebo group showed a significant decrease after 3 months of curcumin use. Therefore, it could be concluded that curcumin might exert a net protective effect on copper toxicity in thalassemia intermedia patients. The investigation also implicated that curcumin represents an approach to regulating zinc homeostasis and may be useful as a complementary treatment of patients with thalassemia intermedia, especially in patients with zinc deficiency or low serum zinc/copper ratio. Clinical Trial Registration Number: IRCT20190902044668N1.

Oxidative stress is a major mechanism contributing to the progression of β-thalassemia. To assess the effect of vitamin E and N-acetyl cysteine (NAC) as antioxidant agents on total oxidative stress (TOS) status and total antioxidant capacity (TAC) in patients with transfusion-dependent β-thalassemia (TDT). In this open-label randomized controlled trial, from May to August 2019, 78 eligible patients with TDT over the age of 18 were enrolled. All patients were registered at the Thalassemia Clinic of Shiraz University of Medical Sciences in Southern Iran. Patients were randomly allocated to the NAC group (10 mg/kg/day, orally), vitamin E group (10 U/kg/day, orally), and control group. The duration of the study was 3 months. The mean age of the participants was 28.5 ± 5.1 (range: 18-41) years. At the end of the study, TOS significantly decreased only in the vitamin E group (mean difference (MD), 95% confidence interval (CI): 0.27 (0.03-0.50), P = 0.026). TAC significantly decreased in both supplemented groups at the 3rd month of treatment (NAC group: MD (95% CI): 0.11 (0.04-0.18), P = 0.002 and vitamin E group: 0.09 (0.01-0.16), P = 0.022 respectively). Hemoglobin did not significantly change at the end of the study in each group (P > 0.05). Mild transient adverse events occurred in 4 patients of the NAC group and 5 patients of the vitamin E group with no need to discontinue the treatment. Vitamin E can be a safe and effective supplement in improving oxidative stress in patients with TDT. Moreover, it seems that a longer duration of using antioxidant supplements needs to make clinical hematologic improvement in TDT patients.

Patients with beta thalassemia major (TM) are transfusion-dependent (TD) since early childhood and for life. Development of alloantibodies and autoantibodies against red blood cell (RBC) antigens is increasingly recognized as a significant transfusion hazard, especially among heavily transfused patients. The aim of this study is to assess RBC alloimmunization and autoimmunization rates in TD TM patients treated in our Comprehensive Center of Adult Thalassemia, Hemoglobinopathies and Rare Anemias. TD TM patients, regularly transfused every 2-3 weeks, were included in the study. Clinical and RBC transfusion records, including RBC antibodies, since diagnosis in early childhood, were retrieved from patients' files and from the blood bank database. Forty TD TM patients, > 18 years of age, were included in the study. Alloimmunization was demonstrated in 17 (42.5%) patients. Thirty-four alloantibodies were detected, with the most frequent being RH related (12 of 34, 35.3%) followed by those of the Kell system (8 of 34, 23.5%). Age at first transfusion was positively related to the probability of developing alloantibodies (p = 0.02). Splenectomy was found to be correlated with developing alloantibodies (p = 0.016). Logistic regression analysis of the lifelong probability of developing alloantibodies on the age at first transfusion and splenectomy demonstrates a strong positive relationship (p = 0.002). A substantially high rate of alloimmunization was found among adult TD TM patients. Early initiation of RBC transfusions, avoidance of splenectomy and extended Rh and K antigen matching, can reduce the incidence of alloimmunization in TD TM patients.

β-Thalassemia is an inherited single gene disorder related to reduced synthesis of the β-globin chain of hemoglobin. Patients with β-thalassemia present variable clinical severity ranging from asymptomatic trait to severe transfusion-dependent anemia and multiple organs complications. Moreover, multiple immune abnormalities are a major concern in β-thalassemia patients. Aberrant neutrophil effector function plays a pivotal role in infection susceptibility in these patients. In severe and persistent inflammation, immature neutrophils are released from the bone marrow and are functionally different compared with mature ones. Despite some abnormalities reported for thalassemia patient's immune system, few data exist on the characterization of human neutrophils in β-thalassemia. The aim of this study was to investigate the phenotype and function of circulating neutrophil subsets in patients with β-thalassemia major and with β-thalassemia intermedia divided in transfusion-dependent and non-transfusion-dependent. By the use of immunochemical and cytofluorimetric analyses, we observed that patients' CD16+ neutrophils exhibit abnormalities in their phenotype and functions and the abnormalities vary according to the clinical form of the disease and to the neutrophil subset (CD16 bright and CD16 dim ). Abnormalities include altered surface expression of the innate immune receptor CD45, Toll-like receptor 4, and CD32, reduced ability to produce an oxidative burst, and elevated levels of membrane lipid peroxidation, especially in patients with a more severe form of the disease. Overall, our results indicating the occurrence of an immuno-senescent phenotype on circulating neutrophils from thalassemia patients suggest the usefulness of neutrophil feature assessment as a tool for better clinical management of β-thalassemia.

Large deletions in the β-globin gene cluster lead to increased HbF levels by delaying the γ- to β-globin switch process. However, these deletions when inherited as a homozygous condition or when co-inherited with β-thalassemia result in variable clinical phenotypes. Individuals or families with a clinically presenting child, where the parents had HbF levels ≥ 10%, were further screened for the presence of large β-globin cluster deletions. Six deletions in the β-globin gene cluster were screened by GAP-PCR, and the uncharacterized deletions were further analyzed by gene dosage or by multiplex ligation-dependent probe amplification (MLPA). Among 192 individuals suspected for the inheritance of large deletions, 138 were heterozygous for large deletions, 45 were compound heterozygous of a large β-globin cluster deletion and β-thalassemia, and 9 were found to be homozygous for deletions. Among the heterozygotes, the Asian Indian inversion-deletion was found to be the most common deletion (39.9%), followed by the HPFH-3 deletion (30.0%). Other deletions 49.3 kb, δβ-thalassemia (21.2%), and 32.6 kb deletion (4.4%) were also found to be prevalent in our population. Patients compound heterozygous or homozygous for HPFH-3 and 32.6 kb deletions showed a milder clinical presentation, as compared with the patients compound heterozygous or homozygous for the Asian Indian inversion-deletion and 49.3 kb δβ-thalassemia. This comprehensive study highlights the mutation spectrum of large β-globin cluster deletions and the clinical heterogeneity in the patients homozygous or compound heterozygous with β-thalassemia, thus asserting the need for molecular characterization of these deletions.

The phenotype/genotype relationship of patients with transfusion-dependent thalassaemia (TDT) is particularly complex and variable, thus generating different levels of severity and of annual transfusion volume (ATV). In this study, we explored the role and the contribution of several factors potentially involved in determining mean ATV in a cohort of TDT patients which have been followed since long time. We collected data on one-hundred and twenty-seven patients with transfusion-dependent β-thalassaemia followed at Rare Blood Cell Disease Unit, AORN Cardarelli Hospital. Age at first transfusion, genotype, spleen status (splenectomy or not), and mean soluble transferrin receptor (sTfR) were the parameters included in the analysis. At stepwise regression analysis which included all the parameters, only splenectomy and mean sTfR significantly predicted the mean ATV (F = 70.94, P < 0.0001, R 2  = 0.540). Overall, our data may suggest that in patients with TDT, the measurement of sTfR level together with the spleen status could contribute, more accurately than genotype, to provide a basal evaluation of residual erythropoietic activity and mean ATV.

β-thalassemia major is one of the most common hematologic disorders in the world. It causes severe anemia and patients require regular blood transfusions, which causes different complications such as iron overload and alloimmunization. Regulatory T cells (Tregs) have an important role in regulation of immune responses. FoxP3 is the major marker of Tregs and its expression can be influenced by different factors. GDF-15 is another gene that plays a role in iron homeostasis and regulation of immune system in different diseases. The aim of this study was to assess the frequency of Tregs and FoxP3/GDF-15 gene expression in β-thalassemia major patients with and without alloantibody as well as its correlation with different factors such as serum ferritin and folate levels. This study was conducted on 68 β-thalassemia major patients with and without alloantibodies in comparison with 20 healthy individuals with matched age and sex as control group. Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and real-time PCR were performed in order to evaluate serum ferritin and folate levels, frequency of Tregs, and the expression of FoxP3 and GDF-15 genes, respectively. The percentage and absolute count of Tregs were increased in patients compared with controls (P = 0.0003), but there was no difference between responders and non-responders (P > 0.05). The Tregs count correlated positively with serum ferritin. No correlation was observed between target genes and serum ferritin and folate, but there was a positive significant correlation between the expression of FoxP3 and GDF-15 genes, which shows the immunosuppressive role of GDF-15.

Since iron overload is the commonest cause of morbidity and mortality in β thalassemia major (β-TM), it represents one major target in therapeutic management of the disease. The recently discovered erythroid regulator, erythroferrone (ERFE), governed by high levels of erythropoietin, was found to suppress hepcidin expression, thus increasing iron availability for developing erythroid progenitors. We aimed to investigate ERFE levels in Egyptian β-TM patients as an attempt to understand its role in the prediction of iron overload states. Our study included 70 β-TM patients, divided into two subgroups according to the degree of iron overload, and 30 sex and age-matched healthy subjects. ERFE gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and serum hepcidin was measured using enzyme-linked immunosorbent assay (ELISA) technique. Both ERFE gene expression levels and transferrin saturation (TS%) values were able to discriminate among cases with different degrees of iron overload, in contrast to hepcidin. TS% was acknowledged as the best predictor of iron overload (AUC 0.893) in comparison with serum hepcidin and ERFE gene levels (AUC 0.807 and 0.677, respectively), and ERFE gene expression was an independent predictor for the estimated TS%. In conclusion, we suggest that using the ERFE gene expression, combined with serum hepcidin estimation, can substantiate the role of estimated TS% as a promising tool in screening for iron overload in β-TM patients.

Despite the advances in the management of hemoglobinopathies, further insight into disease pathophysiology is necessary to improve our therapeutic approach. Activin-A has emerged as a regulator of erythropoiesis and bone turnover in malignant disorders; however, clinical data in hemoglobinopathies are currently scarce. Thus, we aimed to investigate the role of activin-A among hemoglobinopathy patients and evaluate the rationale of its targeting. Circulating levels of activin-A were measured in patients (n = 227) with beta-thalassemia major (TM) (n = 58), beta-thalassemia intermedia (TI) (n = 43), double heterozygous sickle cell/beta-thalassemia (HbS/beta-thal) (n = 109), or homozygous sickle cell disease (n = 17), and we explored possible correlations with clinical and laboratory data. Seventeen age- and gender-matched, healthy individuals served as controls. Bone marrow density (BMD) was determined using dual-energy X-ray absorptiometry. TM and HbS/beta-thal patients had elevated activin-A compared to controls (p = 0.041 and p = 0.038, respectively). In TM patients, high circulating activin-A showed strong correlations with hemolysis markers, namely reticulocyte count (p = 0.011) and high lactate dehydrogenase (LDH; p = 0.024). Similarly, in HbS/beta-thal patients, activin-A showed positive correlations with indirect bilirubin (p < 0.001), ferritin (p = 0.005), and LDH (p = 0.044). High activin-A correlated with low Z-score of both lumbar spine BMD in TI patients (p < 0.01) and femoral neck BMD in TM patients (p < 0.01). Serum activin-A is elevated in patients with TM and HbS/beta-thal and correlates with markers of hemolysis and low BMD. These data support a role of activin-A in the biology of these disorders and provide further rationale for the broader clinical development of activin-A inhibitors in this setting.

Thalassemia patients have a high cell turnover rate due to chronic hemolysis and ineffective erythropoiesis; therefore, hyperuricemia is anticipated. This study aimed to identify the prevalence of hyperuricemia, gout and nephrolithiasis, conditions associated with serum uric acid (SUA), and urine uric acid excretion (UUA) in thalassemia patients. This was a cross-sectional study in patients aged 15 years or older at Chiang Mai University Hospital. All patients had blood and 24-h urine collection test. We enrolled 112 thalassemia patients in which 67.0% were female, 64.3% had beta thalassemia/Hb E, 76.8% were transfusion dependent, and 59.8% were post splenectomy. The median age was 29 (16-58) years. Mean SUA was 6.7 ± 2.0 mg/dl and hyperuricemia (SUA > 6.8 mg/dl) was found in 47 cases (45.2%). Intact spleen (ORs 4.3, 95%CI 1.55-12.50, p = 0.01) and lower FEuric (ORs 2.08, 95%CI 1.35-3.33, p < 0.01) were associated with hyperuricemia significantly. Seven (6.3%) had gouty arthritis and nine (8%) had microscopic hematuria, one case being confirmed nephrolithiasis. The mean UUA excretion was 981.3 ± 335.0 mg/day and UUA hyperexcretion (> 700 mg/24 h) was found in 83.3%. UUA hyperexcretion patients had renal hyperfiltration 46%, glomerular dysfunction 84%, and tubular dysfunction 7.7%. From our study, hyperuricemia was found in approximately 40% of thalassemia patients but gouty arthritis occurred only in few patients (6%). This may be explained by urinary uric hyperexcretion which is found in over 80%. The significant risk factors for hyperuricemia were intact spleen and lower fraction excretion of uric acid.

Beta-thalassemia can present with a wide spectrum of phenotypes determined by the coinheritance of α-thalassemia, hereditary persistence of fetal hemoglobin, and polymorphic variants in the BCL11A, HMIP, and HBB clusters. The codon 29 (cd29) mutation in the beta gene has been associated with a broad diversity of thalassemia phenotypes, possibly through genetic modifiers determining the genotype-phenotype relationship. In this study, we evaluated the effect of 10 single nucleotide polymorphisms (SNPs) on β-thalassemia severity in a group of 21 Lebanese patients bearing the cd29 mutation. Hematological parameters and clinical characteristics were evaluated according to transfusion dependence. The proportions and absolute concentrations of HbF were found to be higher in non-transfusion-dependent (NTD) patients than in transfusion-dependent (TD) ones. Iron parameters were found to be higher in TD patients. The SNPs that were evaluated included the XmnI-158 polymorphism in the HBG gene and SNPs in the BCL11A and HMIP loci. It was noted that individuals homozygous or heterozygous for the effect allele in the BCL11A and HMIP SNPs had higher HbF levels, lower ferritin concentrations, and lower liver iron content and were less likely to be transfusion dependent. Our results showed that HbF production variants may have an important impact on the severity of β-thalassemia, which might provide a severity prediction tool that can help in the anticipation of patients' phenotypes and therefore in future therapeutic decision making.

Streptococcus pnemoniae is a major cause of morbidity and mortality among splenectomised patients with β-thalassemia major. We have previously shown that a 13-valent pneumococcal conjugate vaccine (PCV13) induces robust early immune responses in such patients, while history of repeated immunisations with the 23-valent polysaccharide pneumococcal vaccine (PPSV23) results in attenuation of the response to PCV13. However, the duration of vaccine-induced protection in splenectomised thalassemic patients and the associated need for booster immunisation remains unclear. In the current study, we enumerate antibody persistence 5 years post-PCV13 and investigate any correlation with early immune response and immunisation history. Pneumococcal serotype (PS)-specific antibodies against 5 vaccine antigens were measured 5 years post-PCV13 in 34 asplenic adults with β-thalassemia. PS-specific antibodies against 5 vaccine serotypes had declined significantly at 5 years post-PCV13 (year 5).Year 5 antibody titres remained above baseline for PS9V, 19A and19F, returned to baseline for PS23F, and dropped below baseline for PS3 (p < 0.001).Year 5 antibodies were positively correlated with day 28 antibody titres, while no correlation was found with early memory B cell response. Previous PPSV23 history was correlated with impaired antibody persistence against serotype 19A. Antibody levels dropped significantly but remained at protective levels 5 years post-PCV13.We propose that asplenic patients with β-thalassemia may benefit from measurement of antipneumococcal antibodies after 5 years post-PCV13 as they may eventually be in need for booster pneumococcal vaccination. Clinical Trials Registration ID: www.clinicaltrials.gov NCT01846923.

Thalassaemia are the most common inherited autosomal recessive single gene disorders characterized by chronic hereditary haemolytic anaemia due to the absence or reduced synthesis of one or more of the globin chains. Haemoglobin E-β thalassaemia is the genotype responsible for approximately one half of all severe beta-thalassaemia worldwide. This study proposes to evaluate the effect of various molecular parameters on the response of hydroxyurea. Hydroxyurea was started at an initial dose of 10 mg/kg of body weight/day on 110 transfusion-dependent HbE-β thalassaemia patients. HbF level was measured by HPLC analysis. β-Thalassaemia mutations, XmnI and five other SNPs, and α-globin gene deletions and triplications were detected by ARMS-PCR, RFLP-PCR and Gap-PCR, respectively. Based on the factors for evaluating hydroxyurea-response, 42 patients were responders as they showed an increment of Hb from a mean baseline value of 6.45 g/dl (± 0.70) to 7.78 g/dl (± 0.72) post-therapy. Based on increase in HbF above the median value (14.72%) post-therapy, 78 patients were found to be responders. All the 78 responders showed mean decrease in transfusion of 74.26% (± 8.32) with a maximum decrease of 98.43%. There was a significant correlation between decrease in transfusion and increase in HbF level for all 78 responders. XmnI polymorphism showed the strongest association (p < 0.0001) with increase in HbF levels and Hb levels. Patients with α-globin gene deletions were better responders. It was concluded that hydroxyurea treatment is effective in transfusion-dependent HbE-β thalassaemia patients and the response is best in patients having both XmnI polymorphism and α-deletion.

Pulmonary arterial hypertension (PAH) is a serious complication in β-thalassemia. The mechanism of PAH development is believed to be through chronic platelet activation and red cell (RBC) membrane abnormality contributing to a hypercoagulable state and thrombosis, which consequently leads to the development of PAH. Extracellular vesicles (EVs) shed from the plasma membrane of platelets and RBCs are found to be associated with thrombotic risk. This study aimed to investigate the involvement of phosphatidylserine (PS)-bearing cells and EVs in accelerating the progression of the hypercoagulable state in transfusion-dependent thalassemia (TDT) patients. Fresh whole blood samples from splenectomized TDT-β-thalassemia/HbE patients (11 with PAH and 14 without PAH) and 15 normal subjects were analyzed for platelet activation by measuring P-selectin expression using flow cytometry and the number of dense granular using an electron microscope. The amounts of PS-bearing RBCs, large RBC-EVs, platelets, and medium EVs were determined by flow cytometry. Platelet activation in PAH patients was not significantly different from other groups; however, the amounts of PS-bearing large RBC-EVs, platelets, and medium platelet-derived EVs were significantly increased in PAH patients as compared to normal subjects, but they were not different from patients without PAH. This could be affected by antiplatelet therapy that reduced the levels of platelet activation and the amount of PS-bearing cells, including EVs, in PAH patients as well as in patients without PAH.

Recently, resveratrol showed induction of γ-globin mRNA synthesis in human erythroid precursors and reducing oxidative stress in red cells of thalassemia patients in many in vitro studies. We aimed to investigate the efficacy and safety of resveratrol, for the first time, in non-transfusion-dependent beta-thalassemia intermedia (B-TI) in Southern Iran. In this double-blind randomized clinical trial, 54 patients with B-TI were investigated during 6 months between October 2016 and March 2017. Patients were randomly allocated into three groups by simple randomization method. Group 1 (hydroxyurea (HU) and placebo, 18 patients), group 2 (resveratrol/piperine and placebo, 16 patients), and group 3(HU and resveratrol/piperine, 20 patients). Primary end point was considered as change in hemoglobin (Hb) levels and need for blood transfusion. Drug safety was considered as a secondary end point. Mean age of the patients was 28.2 ± 5.6 (18-42) years. Response rate was not significantly different among the three groups (P > 0.05). Higher percentages of adverse events were detected in groups 2 (31.3%) and 3 (25%) compared to group 1 (5.6%). However, the difference was not statistically significant (P > 0.05). All reported adverse events were gastrointestinal symptoms. Resveratrol showed a similar efficacy with HU in the small population of non-transfusion B-TI patients during a 6-month follow-up. Complications, mostly gastrointestinal, were observed more frequently in resveratrol groups compared to the HU group. Although it was not statistically significant, more attention should be given to safety and efficacy of resveratrol as an oral HbF-augmenting agent.

To investigate the efficacy and safety of thalidomide in patients with thalassemia intermedia (TI). Patients with a confirmed diagnosis of TI who met the trial criteria and signed consent forms were prescribed oral thalidomide 50 mg qn for 3 months from February 2017. Complete blood counts, Hb analysis, and liver and kidney functions were monitored monthly during treatment and any differences were compared before and after treatment. Patients with Hb increments > 2.0 g/dL were termed main responders (MaR), and those with Hb increments between 1.0 and 2.0 g/dL as minor responders (MiR), otherwise they were termed non-responders. Relevance analysis was performed to explore parameters predicting Hb increments after treatment. Adverse effects during treatment were carefully recorded. The overall response rate (ORR = MaR + MiR) and MaR rates were 78.6 and 50% after 1 month of treatment, respectively, and 85.7 and 71.4% after 3 months treatment. At the end of the treatment period, Hb and HbF increased by 2.5 ± 1.8 g/dL and 2.5 ± 1.6 g/dL, while bilirubin, lactate dehydrogenase, and the nucleated red blood cell count (NRBC) were significantly decreased, while the reticulocyte count significantly increased. Correlation analysis showed that the Hb increments correlated significantly with the ratio of HbF before treatment (r = 0.683, P = 0.007) rather than age, Hb, reticulocyte count, and NRBC before treatment. Adverse events during treatment were mild, and drug reduction or withdrawal from the trial was not required. Thalidomide had rapid and significant effects in patients with TI, and also, it is safe and convenient. But larger scale clinical trials will be required to confirm our conclusions., Trial Registration: NCT02995707, https://www.clinicaltrials.gov/ct2/show/NCT03184844?term=thalidomide+thalassemia&rank=1 .

To study safety, efficacy (hemoglobin and hemoglobin F percentage increment in non-transfusion-dependent patients and decrease in transfusion frequency in transfusion-dependent patients), and determinants of response of decitabine in patients with HbE/β-thalassemia. Thirty patients of HbE/β-thalassemia (age > 18 years) were enrolled. Both transfusion-dependent (TDT) and non-transfusion-dependent (NTDT) patients were included after obtaining informed consent. Participants received 0.2 mg/kg of 5-aza-2'-deoxycytidine (decitabine) subcutaneously on 2 consecutive days a week for at least 12 weeks. Complete hemogram was done every 2 weeks and HPLC at every 4-week interval, until 2 months after last dose of drug for response assessment. Various factors like XMN1 polymorphism, IVS 1-5, alpha deletion, alpha triplication, baseline hemoglobin F, and baseline total hemoglobin were evaluated as determinants of response. Mean therapy period was 20.32 weeks. For NTDT group, peak mean increment in hemoglobin was 0.938 g/dl (p value < .001) and hemoglobin F percentage was 9.62% (p value < .001). Transfusion requirement decreased to 0.25 units compared to 0.96 units per patient per month for TDT patients over a period of last 1 year. Common side effects were respiratory tract infection (grade I/II) in three patients, chest tightness in one patient (grade I), and gastric erosion (grade III) in one patient. Decitabine is safe and efficacious in HbE/β-thalassemia.

The impaired biosynthesis of the β-globin chain in β-thalassemia leads to the accumulation of unpaired alpha globin chains, failure in hemoglobin formation, and iron overload due to frequent blood transfusion. Iron excess causes oxidative stress and massive tissue injuries. Advanced glycation end products (AGEs) are harmful agents, and their production accelerates in oxidative conditions. This study was conducted on 45 patients with major β-thalassemia who received frequent blood transfusions and chelation therapy and were compared to 40 healthy subjects. Metabolic parameters including glycemic and iron indices, hepatic and renal functions tests, oxidative stress markers, and AGEs (carboxymethyl-lysine and pentosidine) levels were measured. All parameters were significantly increased in β-thalassemia compared to the control except for glutathione levels. Blood glucose, iron, serum ferritin, non-transferrin-bound iron (NTBI), MDA, soluble form of low-density lipoprotein receptor, glutathione peroxidase, total reactive oxygen species (ROS), and AGE levels were significantly higher in the β-thalassemia patients. Iron and ferritin showed a significant positive correlation with pentosidine (P < 0.01) but not with carboxymethyl-lysine. The NTBI was markedly increased in the β-thalassemia patients, and its levels correlated significantly with both carboxymethyl-lysine and pentosidine (P < 0.05). Our findings confirm the oxidative status generated by the iron overload in β-thalassemia major patients and highlight the enhanced formation of AGEs, which may play an important role in the pathogenesis of β-thalassemia major.

Bone marrow transplantation (BMT) serves as the only curative treatment for patients with β-thalassemia major; however, hemostatic changes have been observed in these BMT patients. Aggregability of thalassemic red blood cells (RBCs) and increased red blood cell-derived microparticles (RMPs) expressing phosphatidylserine (PS) are thought to participate in thromboembolic events by initially triggering platelet activation. To our knowledge, there has been no report providing quantitation of these circulating PS-expressing RBCs and RMPs in young β-thalassemia patients after BMT. Whole blood from each subject was fluorescently labeled to detect RBC markers (CD235a) and annexin-V together with the known number TruCount™ beads. PS-expressing RBCs, RMPs, and activated platelets were identified by flow cytometry. In our randomized study, we found the decreased levels of three aforementioned factors compared to levels in patients receiving regular blood transfusion (RT). This study showed that BMT in β-thalassemia patients decreases the levels of circulating PS-expressing RBCs, their MPs, and procoagulant platelets when compared to patients who received RT. Normalized levels of these coagulation markers may provide the supportive evidence of the effectiveness of BMT for curing thalassemia.

We retrospectively evaluated the relationship between serum transferrin receptor-1 (sTfR1) and some fundamental events in the life and the management (the age at diagnosis, the age at the first red blood cells transfusion, the age at splenectomy, and the overall need of chelation therapy) of 111 patients with non-transfusion-dependent thalassemia (NTDT) subdivided in four genetic entities: patients with homozygous or compound heterozygous state for β-thalassemia, patients with triplicated α genotype associated with β heterozygosity, patients with deletional HbH, and patients with the combination of a β defect plus a β chain variant. We found that the group with homozygous or compound heterozygous state for β-thalassemia had the highest sTfR1 levels and that the presence of increased sTfR1 levels (>5 times normal) was associated with a complex and severe history of disease requiring splenectomy, occasional red blood cells transfusions, and early start and continuous iron chelation therapy.The complexity in the management of NTDT patients is an emerging issue due to the wide heterogeneity of clinical behavior. Our data indicate that the measurement of sTfR1 levels, a common laboratory test, could contribute to correctly stratify disease history and the iron chelation strategy in NTDT patients.

An improvement in quality of life and survival occurred among thalassemia major (TM) patients: pregnancy in such patients has become a reality. Safe pregnancy and delivery require efforts to ensure the best outcomes. Between 2007 and 2016, 30 TM patients had 37 pregnancies. We analyzed the hematological parameters before, during, and after pregnancies and in 19 patients a cardiovascular magnetic resonance (CMR) T2* was performed. The mean age at first pregnancy was 30 ± 4 years; the current mean age is 35 ± 5 years. Twenty-four patients (80%) had a single pregnancy, five patients (17%) had two pregnancies, and one patient (3%) became pregnant three times. Seventeen pregnancies (46%) were spontaneous, 20 (64%) needed gonadotrophin-induced ovulation and/or reproductive technologies. All pregnancies resulted in live births. Seven were twin pregnancies (19%). The mean gestational hemoglobin was 9.2 ± 0.5 g/dl, lower than pre- and postpregnancy (9.8 ± 1 g/dl, p = ns and 9.6 ± 1 g/dl, p = 0.02, respectively). Median ferritin levels increased progressively (1071, range 409-5724 ng/ml, before pregnancy vs 2231, range 836-6918 ng/ml, after pregnancy, p < 0.0001). CMR before pregnancy showed a normal cardiac T2* (mean 35.34 ± 8.90 ms) and a mean liver iron concentration (LIC) of 3.37 ± 2.11 mg/g dry weight (dw). After pregnancy, the mean cardiac T2* was 31.06 ± 13.26 ms and the mean LIC was significantly increased (9.06 ± 5.75 mg/g dw, p = 0.0001). Pregnancy is possible and safe in thalassemia major. During pregnancy, iron accumulates, especially in the liver; a prompt resumption of chelation after delivery is mandatory.

The presence of elevated numbers of circulating microparticles (MPs) has been hypothesized to be responsible for the occurrence of thromboembolic events (TEEs) in thalassemic patients. Our aim is to evaluate the presence and the thrombotic risk of circulating MPs in thalassemia patients and to determine the difference in MPs between β-thalassemia major (β-TM) and thalassemia intermedia (TI). The percentage of the annexin-labeled MPs, platelet-derived MPs (PMPs), erythrocyte-derived MPs (RMPs), and endothelial-derived MPs (EMPs) was measured by flow cytometry, in 87 thalassemia patients (39 β-TM and 48 TI). By multiple regression analysis, we then assessed the various independent risk factors for the occurrence of TEE. The thalassemic patients who experienced TEE had a significantly higher platelet count, higher percentage of annexin-labeled MPs, and higher percentage of PMPs (p value = 0.014, 0.003, and 0.014, respectively). There was no significant difference between β-TM and TI patients at the level of any of the studied MPs. The predictive risk factors for TEE in thalassemic patients were splenectomy, total and direct bilirubin, the RMPs, and the EMPs (OR = 10.07 (CI = 3.7-27.1), 4.3 (CI = 2.1-8.7), 1.4 (CI = 1.5-6.2), 1.6 (CI = 1.1-2.2), 3.0 (CI = 1.9-4.9), respectively). In conclusion, the elevated numbers of circulating MPs is a risk factor for the TEE in thalassemia patients.

Thromboembolic events including cerebral thrombosis, deep vein thrombosis, and pulmonary embolism are major complications in β-thalassemia. Damaged red blood cells and chronic platelet activation in splenectomized β-thalassemia/HbE patients were associated with increased microparticles (MPs) releases into blood circulation. MPs are small membrane vesicles, which play important roles on coagulation. However, the role of MP in thalassemia is poorly understood. In this study, the effects of splenectomized-MPs on platelet activation and aggregation were investigated. The results showed that isolated MPs from fresh platelet-free plasma of patients and normal subjects directly induce platelet activation, platelet aggregation, and platelet-neutrophil aggregation in a dose-dependent manner. Interestingly, MPs obtained from splenectomized patients are more efficient in induction of platelet activation (P-selectin + ) when compared to MPs from normal subjects (P < 0.05), tenfold lower than pathophysiological level, at 1:0.1 platelet MP ratio. Co-incubation of splenectomized-MPs with either normal-, non-splenectomized- or splenectomized-platelets at 1:10 platelet MP ratio increased platelet activation up to 5.1 ± 2.2, 5.6 ± 3.7, and 9.5 ± 3.0%, respectively, when normalized with individual baseline. These findings suggest that splenectomized patients were proned to be activated by MPs, and splenectomized-MPs could play an important role on chronic platelet activation and aggregation, leading to thrombus formation in β-thalassemia/HbE patients.

Thalassemia is a common genetic disorder in Tunisia. Early iron concentration assessment is a crucial and challenging issue. Most of annual deaths due to iron overload occurred in underdeveloped regions of the world. Limited access to liver and heart MRI monitoring might partially explain these poor prognostic results. Standard software programs are not available in Tunisia. This study is the first to evaluate iron overload in heart and liver using the MRI T2* with excel spreadsheet for post processing. Association of this MRI tool results to serum ferritin level, and echocardiography was also investigated. One hundred Tunisian-transfused thalassemia patients older than 10 years (16.1 ± 5.2) were enrolled in the study. The mean myocardial iron concentration (MIC) was 1.26 ± 1.65 mg/g dw (0.06-8.32). Cardiac T2* (CT2*) was under 20 ms in 30 % of patients and under 10 ms in 21 % of patients. Left ventricular ejection function was significantly lower in patients with CT2* <10 ms. Abnormal liver iron concentration (LIC >3 mg/g dw) was found in 95 % of patients. LIC was over 15 mg/g dw in 25 % of patients. MIC was more correlated than CT2* to LIC and serum ferritin. Among patients with SF <1000 μg/l, 13 % had CT2* <20 ms. Our data showed that 30 % of the Tunisian thalassemia major patients enrolled in this cohort had myocardial iron overload despite being treated by iron chelators. SF could not reliably predict iron overload in all thalassemia patients. MRI T2* using excel spreadsheet for routine follow-up of iron overload might improve the prognosis of thalassemia major patients in developing countries, such as Tunisia, where standard MRI tools are not available or expensive.

Hyperhemolysis syndrome (HHS) is characterized by severe intravascular hemolysis with a decrease in the reticulocyte count, which is triggered and aggravated by transfusion and cannot be explained by standard immunohematological studies. A nationwide study was conducted in order to retrospectively identify thalassemia patients with HHS in Spain in order to assess pre-disposing mechanisms for this syndrome. For this, the expression of adhesion (CD49, CD36) and complement-related molecules (C3a, CD59) and the levels of reticulocyte apoptosis and macrophage activation were measured in 4 thalassemia patients with HHS, 14 patients without HHS, and 10 healthy subjects. Five of the six thalassemia patients had δβ-thalassemia. The patients were not alloimmunized prior to the syndrome, which was developed after the first transfusion in all but one case. Patients with δβ-thalassemia did not respond to corticoids or immunoglobulins; only splenectomy was successful. The expression of CD49 (α4β1 integrin) was far higher in patients who had experienced HHS (85.07 ± 18.46 vs. 46.28 ± 24.31; p < 0.01), and the difference remained significant after correcting by the number of molecules analyzed (Bonferroni p < 0.05). In our population, δβ-thalassemia was the most common hemoglobinopathy in patients with HHS. Furthermore, the risk to develop this syndrome may be associated with an increased expression of α4β1 integrin.

The discovery of fetal DNA (f-DNA) opens the possibility of early non-invasive procedure for detection of paternally inherited mutation of beta-thalassemia. Since 2002, some studies have examined the sensitivity and specificity of this method for detection of paternally inherited mutation of thalassemia in pregnant women at risk of having affected babies. We conducted a systematic review of published articles that evaluated using this method for early detection of paternally inherited mutation in maternal plasma. A sensitive search of multiple databases was done in which nine studies met our inclusion criteria. The sensitivity and specificity was 99 and 99 %, respectively. The current study found that detection of paternally inherited mutation of thalassemia using analysis of cell-free fetal DNA is highly accurate. This method could replace conventional and invasive methods.

During erythropoiesis, iron levels need to be carefully regulated to ensure there is sufficient iron available for hemoglobin synthesis, but that there is no excess to cause damage to the developing erythroblast. Iron influx to the developing erythroblast is controlled by the expression of the transferrin receptor, while iron efflux is regulated by ferroportin (FPN), the sole iron-exporting protein. FPN is encoded through multiple messenger RNAs (mRNAs) some of which contain an iron-responsive element (variant I mRNAs) and some of which do not (variant II mRNAs). This study sought to investigate the expression of the FPN mRNAs in developing erythroblasts from normal controls and β(0)-thalassemia/Hb E patients. While levels of FPN protein were relatively constant, marked reductions of the variant I message were seen in erythroblasts from β(0)-thalassemia/Hb E patients as compared to normal control cells, particularly in late erythropoiesis. Variant II mRNAs were generally increased during erythroid differentiation. No difference was seen in levels of either transferrin or ferritin heavy chain expression. While no difference was observed in labile iron pools under normal culture conditions, erythroblasts from β(0)-thalassemia/Hb E patients showed a significantly reduced expression of total FPN message under high iron conditions as compared to normal control erythroblasts. These results are consisted with dysregulation of iron efflux from the maturing erythroblast in β(0)-thalassemia/Hb E patients, and this dysregulation possibly contributes to ineffective erythropoiesis seen in these patients.

Deferasirox is a novel once-daily, oral iron chelator. The aim of this study was to evaluate the long-term efficacy and tolerability of deferasirox in Taiwanese patients with transfusion-dependent β-thalassemia who have been treated with deferasirox for 7 years. Taiwanese patients aged ≥2 years with transfusion-dependent β-thalassemia whose serum ferritin levels were ≥1000 ng/mL and had started deferasirox treatment since December 2005 at the National Taiwan University Hospital were enrolled. Sixty patients were recruited for analysis, and 11 (18.3 %) patients discontinued deferasirox during the study. In the 42 patients included in the efficacy analysis, the mean serum ferritin levels decreased significantly by 2566 ng/mL after 7 years of treatment (P < 0.001). Forty-one of these patients received a cardiac T2* evaluation after 3 years of deferasirox treatment, and the mean cardiac T2* value increased significantly from 30.6 ± 16.6 to 45.9 ± 22.6 ms after 7 years of deferasirox treatment (P < 0.001). Deferasirox-related adverse events assessed by investigators were reported in 46 (76.7 %) patients. The most common adverse events related to deferasirox were skin rashes (n = 29, 48.3 %), followed by abdominal pain (n = 23, 38.3 %) and diarrhea (n = 16, 26.7 %). Most adverse events were manageable. This study demonstrated that long-term treatment with deferasirox was effective in improving iron overload, including cardiac iron overload, in patients with transfusion-dependent β-thalassemia. Deferasirox was well tolerated; however, the incidences of common adverse events related to deferasirox appeared higher in our Taiwanese patients than other studies.

Hydroxycarbamide (hydroxyurea or HU) has been shown to increase fetal hemoglobin (HbF) in patients with β-thalassemia intermedia (TI). The reported effects of HU in increasing the total hemoglobin (Hb) have been inconsistent. Studies of long-term therapy with HU in pediatric TI are rather uncommon. A retrospective observational study was carried out to evaluate the clinical responses to HU in Egyptian patients with β-TI. One hundred patients; children (n = 82, mean age 9.9 ± 4.1 years) and adults (n = 18) were studied for the mean Hb, HbF%, median serum ferritin, transfusion history, and splenic size before and after HU therapy (mean dose 20.0 ± 4.2 mg/kg/day, range 10-29 mg/kg/day) over a follow-up period 4 to 96 months (mean 35.4 ± 19.2 months). Molecular studies were also done for group of patients (n = 42). The overall response rate to HU was 79 %; 46 % were minor responders (with a reduction in transfusion rate by 50 % or more and/or an increase in their total hemoglobin level by 1-2 g/dl) and 33 % major responders (becoming transfusion-free and/or having an increase in total hemoglobin level by >2 g/dl). Mean hemoglobin increased among responders from 6.9 ± 0.9 g/dl to 8.3 ± 1.4 g/dl (p < 0.001). A significant rise in mean HbF (27.0 vs. 42.5 %; p < 0.011) and a decrease in median serum ferritin (800 vs. 644 ng/ml; p < 0.001) were also observed among responders (n = 45). Transfusions stopped in 44 % of pretreatment frequently transfused responders (n = 11/25). Splenic size decreased in 37 % of patients (n = 30/81). The predominant β-thalassemia mutation was 1-6 (T > C) in 32/42 (76 %) of studied patients; 28/32 were responders. Bivariate analysis showed no predictors of response as regards sex, pediatric and adult age, splenic status, or genotype. Hydroxycarbamide is a good therapeutic modality in the management of pediatric as in adult TI patients. It can minimize the need for blood transfusion, concomitant iron overload, and blood-born viral transmission especially in developing countries like Egypt.

Myocardial siderosis in thalassemia major remains the leading cause of death in developing countries. Once heart failure develops, the outlook is usually poor with precipitous deterioration and death. Cardiovascular magnetic resonance (CMR) can measure cardiac iron deposition directly using the magnetic relaxation time T2*. This allows earlier diagnosis and treatment and helps to reduce mortality from this cardiac affection. This study aims to determine the prevalence of cardiac siderosis in Egyptian patients who are heavily iron loaded and its relation to liver iron concentration, serum ferritin, and left ventricular ejection fraction. Eighty-nine β-thalassemia patients receiving chelation therapy (mean age of 20.8 ± 6.4 years) were recruited in this study. Tissue iron levels were determined by CMR with cardiac T2* and liver R2*. The mean ± standard deviation (range) of cardiac T2* was 28.5 ± 11.7 ms (4.3 to 53.8 ms), the left ventricular ejection fraction (LVEF) was 67.7 ± 4.7 % (55 to 78 %), and the liver iron concentration (LIC) was 26.1 ± 13.4 mg Fe/g dry weight (dw) (1.5 to 56 mg Fe/g dw). The mean serum ferritin was 4,510 ± 2,847 ng/ml (533 to 22,360 ng/ml), and in 83.2 %, the serum ferritin was >2,500 ng/ml. The prevalence of myocardial siderosis (T2* of <20 ms) was 24.7 % (mean age 20.9 ± 7.5 years), with mean T2* of 12.7 ± 4.4 ms, mean LVEF of 68.6 ±5.8 %, mean LIC of 30.9 ± 13 mg Fe/g dw, and median serum ferritin of 4,996 ng/ml. There was no correlation between T2* and age, LVEF, LIC, and serum ferritin (P = 0.65, P = 0.085, P = 0.99, and P = 0.63, respectively). Severe cardiac siderosis (T2* of <10 ms) was present in 7.9 %, with a mean age of 18.4 ± 4.4 years. Although these patients had a mean T2* of 7.8 ± 1.7 ms, the LVEF was 65.1 ± 6.2 %, and only one patient had heart failure (T2* of 4.3 ms and LVEF of 55 %). LIC and serum ferritin results were 29.8 ± 17.0 mg/g and 7,200 ± 6,950 ng/ml, respectively. In this group of severe cardiac siderosis, T2* was also not correlated to age (P = 0.5), LVEF (P = 0.14), LIC (P = 0.97), or serum ferritin (P = 0.82). There was a low prevalence of myocardial siderosis in the Egyptian thalassemia patients in spite of very high serum ferritin and high LIC. T2* is the best test that can identify at-risk patients who can be managed with optimization of their chelation therapy. The possibility of a genetic component for the resistance to cardiac iron loading in our population should be considered.

The severity of thalassemia is currently classified based on clinical manifestations and multiple tests. In the present study, we performed a plasma proteome analysis to identify differentially expressed proteins compared between normal subjects and patients with mild and severe forms of β-thalassemia/hemoglobin E (Hb E). Plasma samples were collected from patients with mild (n = 8) and severe (n = 12) forms as well as healthy normal individuals (n = 12). Clinical chemistry revealed that several parameters, i.e., hematological indices, oxidative stress markers, antioxidant enzymes, and erythropoietic activity, had significant differences among these three groups. After removal of seven major abundant proteins, the plasma proteome profiles were compared using two-dimensional gel electrophoresis. Spot matching, quantitative intensity analysis, and statistics revealed differential levels of 32 and 9 proteins when comparing normal vs. patients and mild vs. severe forms, respectively. These proteins were successfully identified by quadrupole time-of-flight mass spectrometry and/or tandem mass spectrometry. The decreased level of ADP-ribosylation factor guanine nucleotide-exchange factor 2 in β-thalassemia/Hb E patients compared to healthy individuals and the decreased level of endothelin-converting enzyme 2 in severe form compared to the mild form of the disease were validated by Western blot analysis. Our data provide a number of proteins that may lead to better understanding of the pathophysiology of thalassemia or for novel biomarkers which can be used to simply differentiate mild and severe forms of β-thalassemia/Hb E without any need for multiple tests.

We report a new β-thalassaemia allele detected in a young Italian woman, suffering with mild non-haemolytic anaemia (Hb < 10 g/dL) and not showing Hb variant or Heinz bodies. The allele is characterised by duplication of tetranucleotide 'AG/CT' (+1344/+1347) including the invariant dinucleotide 'AG' of IVS-II acceptor splicing site and the first two nucleotides of codon 105. β-Globin complementary DNA (cDNA) sequencing did not reveal any mutation and qualitative analysis of the reverse transcription PCR reaction showed that only the proximal 3' splice site present in the duplicated gene is used giving race to an anomalous messenger RNA (mRNA) present in trace (1.5 %) because, most probably, rapidly degraded. In the anomalous mRNA, the insertion causes a frameshift and synthesis of an abnormal truncated β-chain (139 residues), unable to form Hb variant because of the severe conformational changes. The duplication might have arisen from secondary structures generated by quasi-palindromic sequence 5'-CCCA(C)AG/CT(CC)TGGG-3'. Restriction fragment length polymorphism analysis for the β-globin haplotype and familiar segregation analysis indicated that the mutant β-globin gene was associated with the haplotype V.

Stored red blood cells become deficient in nitric oxide that limits their ability to transfer oxygen to tissues that need it. The aims of this study are to assess the endogenous nitric oxide metabolites (NOx) and arginase I levels in transfusion-dependent β-thalassemic patients; to compare these levels in patients transfused with fresh RBCs with patients transfused with old RBCs, β-thalassemic minor patients, and normal control; and to correlate these levels with some clinical variables. Group I was composed of 23 patients with homozygous β-thalassemia on hypertransfusion regimen. They were adequately transfused with fresh RBC. Group II was composed of 17 patients with homozygous β-thalassemia on hypertransfusion regimen. They were adequately transfused with old RBCs. Group III was composed of 30 patients with homozygous β-thalassemia. They were adequately transfused with fresh RBCs. Group IV was composed of 18 patients with homozygous β-thalassemia. They were adequately transfused with old RBCs. Both group III and group IV were supposed to be on hypertransfusion regimen, but they did not follow the regimen. Group V was composed of 21 patients of β-thalassemia minor. Nineteen apparently healthy individuals (HbAA) served as a control group (group VI). In addition to routine laboratory investigations, plasma levels of NOx and serum levels of arginase I were assessed in all subjects. The mean values of plasma NOx were significantly decreased in groups III and IV compared to the other groups. Also, the levels of NOx were significantly decreased in patients who received old RBCs compared to the other groups. There were high serum levels of arginase I in groups III and IV compared to the other groups. There were significant negative correlations between plasma NOx and some hemolytic biochemical markers in groups III and IV. There were significant positive correlations between serum arginase I and some hemolytic biochemical markers in groups III and IV. Also, there was a significant negative correlation between plasma NOx and serum arginase I levels in groups III and IV. In non-adequately transfused patients with β-thalassemia major, inactivation of NO correlates with hemolytic rate and is associated with the erythrocyte release of cell-free hemoglobin, which consumes NO directly, and the simultaneous release of the arginine-metabolizing enzyme arginase, which limits bioavailability of the NO synthase substrate, arginine, during the process of hemolysis. New treatments aimed at improving arginine and NO bioavailability through arginase inhibition, suppression of hemolytic rate, oral arginine supplementation, predonation testing, and transfusion of fresh RBCs or use of NO donors represent potential therapeutic strategies for this common hemolytic disorder.

The association between iron overload indices and pathology of the heart and liver in transfusion-dependent patients with β thalassemia major (TM) has been extensively studied. Nonetheless, data on endocrine disease remains limited. This was a cross-sectional study of 382 TM patients treated with regular transfusions and desferrioxamine at the Thalassemia Center in Dubai, UAE. Retrieved data included demographics, splenectomy status, steady-state serum ferritin levels, and the presence of endocrinopathies (diabetes mellitus, hypothyroidism, hypoparathyroidism, and hypogonadism). Multivariate logistic regression analyses were used to determine which variables were independently associated with the occurrence of each endocrinopathy. The mean age of patients was 15.4 ± 7.6 years, with an equal sex distribution. The mean serum ferritin level was 2597.2 ± 1976.8 μg/l. The frequencies of specific endocrinopathies were diabetes mellitus (10.5%), hypothyroidism (6.3%), hypoparathyroidism (10.5%), and hypogonadism (25.9%). On multivariate logistic regression analysis, patients with a serum ferritin level >2,500 μg/l, but not >1,000-2,500 μg/l, were 3.53 times (95% CI 1.09-11.40) more likely to have diabetes mellitus, 3.25 times (95% CI 1.07-10.90) more likely to have hypothyroidism, 3.27 times (95% CI 1.27-8.39) more likely to have hypoparathyroidism, and 2.75 times (95% CI 1.38-5.49) more likely to have hypogonadism compared to patients with a serum ferritin level ≤1,000 μg/l. However, splenectomized patients with serum ferritin levels ≤2,500 μg/l had comparably high rates of all endocrinopathies as patients with serum ferritin levels >2,500 μg/l. Endocrinopathy is common in TM patients treated with desferrioxamine therapy, especially in patients with serum ferritin levels >2,500 μg/l or those splenectomized.

In thalassemia intermedia (TI), the increase in bone marrow hemopoietic activity frequently leads to extramedullary erythropoeisis (EMH), but its relationship with the soluble form of transferrin receptor (sTfR) which fully reflects the marrow erythropoietic activity, has not yet been explored. From January 2007 to December 2010, all TI patients attending at our center were prospectively enrolled to undergo sTfR assay and MRI or CT (if claustrophobic) scan evaluation for the presence of paraspinal EMH. A total of 59 patients with TI were studied; EMH involved 23 (39%) patients; overall, the concentration of sTfR varied from 2.6 to 20.6 (mean = 8.7) mg/L, but in splenectomized group and in unsplenectomized group, it varied from 4.2 to 17.8 (mean ± SD = 9.86 ± 3.33) mg/L and from 2.6 to 20.6 (mean ± SD = 7.25 ± 3.9) mg/L, respectively with a statistically significant intergroup difference (p < 0.01). The cutoff point at 8.6 mg/L using the ROC curve showed a sensitivity of 78.3% and a specificity of 72.2%, in predicting EMH but, in unsplenectomized subgroup, they raised to 100% and 90.9%, respectively. These data showed that in TI the level of sTfR could represent a predictive factor of EMH particularly in patients with spleen.

Erythropoiesis in β-thalassemia patients is ineffective, primarily because of death of the erythroid progenitor cells at the polychromatic normoblast stage. While it is known that autophagy plays a critical role during erythropoiesis by removing organelles from erythroid cells during terminal differentiation, its role in erythroid cells whose function is impaired remains to be explored. To investigate this, CD34+ erythroid progenitor cells from normal controls and β-thalassemia/Hb E patients were isolated from peripheral blood and cultured under conditions driving differentiation into an erythroid lineage, and levels of autophagy and apoptosis were analyzed both directly and after biochemical manipulation with L: -asparagine. A significantly higher level of autophagy was seen in β-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts during erythropoiesis. Interestingly, this activation was mediated in part by the presence of high levels of Ca(2+) as modulation of Ca(2+) levels significantly reduced the level of autophagy in these cells. Inhibition of autophagic flux in normal erythroblasts significantly increased apoptosis in normal erythroblasts, but not in thalassemic erythroblasts, although sensitivity to autophagic flux inhibition was restored by reduction of Ca(2+) levels. These results suggest that high levels of autophagy in β-thalassemia/HbE erythroblasts may contribute to the increased levels of apoptosis that lead to ineffective erythropoiesis in β-thalassemia/Hb E erythroblasts.