Alexandra Stamoulakatou

Magnetic resonance imaging in the evaluation of iron overload in patients with beta thalassaemia and sickle cell disease

Magnetic resonance imaging (MRI) appears to be useful for monitoring iron overload in thalassaemia. We studied 106 patients with beta‐thalassaemia: 80 with thalassaemia major (TM) and 26 with thalassaemia intermedia (TI). Thirty‐five patients with sickle cell disease (SCD) were also evaluated. Serum ferritin, liver and myocardial T2‐relaxation time and liver iron concentration (LIC) were measured. LIC values, based on biopsies from 29 patients, showed a close inverse correlation with the respective liver T2‐values, along with a strong positive correlation with ferritin levels in all patients. Heart T2‐values correlated with left ventricular ejection fraction in TM and SCD, but not in TI patients. Both liver and heart T2‐values were significantly lower in TM patients than those of TI, and SCD patients. Ferritin levels showed a strong correlation with liver T2‐values in all three groups of patients. Similarly, a negative correlation was found between serum ferritin levels and heart T2‐values in TM, but not in TI and SCD patients. Heart and liver T2‐values showed a significant correlation only in TM patients. These results suggest that the MRI technique (T2 relaxation time) used in our study, is a reliable, safe and non‐invasive method for the assessment of the deposition of iron in the liver; results for the heart become reliable only when there is heavy iron deposition.

Phenotypic and molecular diversity of haemoglobin H disease: a Greek experience

Haemoglobin H (Hb H) disease is the severest form of α‐thalassaemia compatible with post‐natal life and occurs when α‐thalassaemia mutations interact to reduce α‐globin synthesis to levels approximately equivalent to the output of a single α‐globin gene. Hb H disease has variable clinical expression, mainly related to underlying genotypes. The spectrum of α‐thalassaemia determinants in Greece appears greater than in any other population studied and, in 75 Greek Hb H disease patients, we found 12 α‐thalassaemia mutations interacting to produce 15 Hb H disease genotypes. Evaluation of haematological, biochemical and clinical findings, and correlation with genotypes, defined genetic predictors of disease severity and factors involved in disease progression. In accordance with previous reports, patients with non‐deletion α‐thalassaemia mutations had more severe clinical expression. Additionally, we found that all patients with the most severe phenotypes had α‐thalassaemic globin variants. Phenotypic severity was not simply related to the degree of α‐globin deficiency: high Hb H levels were found to exacerbate anaemia by negatively influencing tissue oxygenation, and both Hb H and α‐thalassaemic haemoglobin variants appear to reduce red cell survival within the bone marrow and circulation. Together with the long‐term follow‐up in many patients, this report provides comprehensive information for management of Hb H disease and appropriate family counselling.

A novel immunological assay for hepcidin quantification in human serum.

Background Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. Methods and Findings An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L). Conclusions We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.

Arterial elastorrhexis in β‐thalassaemia intermedia, sickle cell thalassaemia and hereditary spherocytosis

Abstract: Arterial and stromal elastorrhexis, an elastic tissue disorder, was recently described in β‐thalassaemia major. Histopathological material from 10 patients with thalassaemia intermedia, 14 with sickle cell thalassaemia and 18 with hereditary spherocytosis was examined in order to investigate the specificity of the arteriopathy. Histological re‐examination was made in a total of 42 spleens with parasplenic lymph nodes in 14 cases, 26 surgical liver biopsies and 16 gallbladders with associated regional lymph nodes. Arteriopathy, qualitatively similar to that seen in β‐thalassaemia major, was found in up to 90% of extrasplenic muscular arteries. Elastorrhexis lesions were also found in intrasplenic arteries and in stromal elastic tissue of spleens and parasplenic lymph nodes, in the absence of tissue iron overload. The arteriopathy appears in the first decade of life even in spleens of normal weight, and seems unrelated to the severity of permanent anaemia. It is suggested that patients suffering from hereditary chronic haemolytic diseases are subject to an elastic tissue disorder which is similar to hereditary pseudoxanthoma elasticum, the earliest and most frequent manifestation of which is arterial elastorrhexis of muscular extrasplenic arteries.

Severe anemia and neutropenia associated with hyperzincemia and hypercalprotectinemia.

Calprotectin, also known as the S100A8/A9 or MRP8/14 complex, is a major calcium-binding protein in the cytosol of neutrophils, monocytes, and keratinocytes. It differs from other S100 proteins in its zinc-binding capacity. The authors describe a 4-year-old girl with severe anemia, neutropenia, inflammation, and severe growth failure. Bone marrow examination showed moderate dyserythropoiesis. No hemolysis, iron deficiency, hemoglobinopathies, immunologic diseases, or autoantibodies were detected. Serum levels of copper and ceruloplasmin were within the normal range, although the serum zinc concentration was markedly increased (310 μg/dL). Urinary zinc excretion and erythrocyte zinc concentrations were within the normal range. Family studies showed normal zinc and copper plasma levels. The patients plasma calprotectin concentration showed a 6,000-fold increase (2,900 mg/L) compared with normal values. The calprotectin concentration is known to be elevated in many inflammatory conditions but is generally below 10 mg/L and thus far below the levels reported in this patient. The authors describe this case as an inborn error of zinc metabolism caused by dysregulation of calprotectin metabolism, which mainly presented with the features of microcytic anemia and inflammation.

Rapid and accurate quantitation of Hb Bart's and Hb H using weak cation exchange high performance liquid chromatography: correlation with the alpha-thalassemia genotype.

The Bio-Rad Variant Hemoglobin testing system is an automated high performance liquid chromatography analyzer marketed with a beta-thalassemia short program to quantify Hbs F and A2, and assist in detecting Hbs A, S, D, C, and E. Although the two hemoglobins present in Hb H disease, Hb Barts and Hb H, are separated by the system, they are not quantitated. In this study we modified the beta-thalassemia short program in order to facilitate quantitation of Hb Barts and Hb H. Blood samples from 60 patients with Hb H disease, with various underlying genotypes, were studied. Analyses were performed on the day of blood collection or on hemolysates stored at -80 degrees C in cyanide or carbomonoxy forms. The mean sum of Hb Barts and Hb H levels in all patients was found to be 12% (range 1.8-35%). Patients with nondeletional mutations (or association of alpha(0) deletion and nondeletional mutations) had notably higher Hb Barts and Hb H levels when compared to patients with deletional genotypes.

Erythroid marrow activity and hemoglobin H levels in hemoglobin H disease

Purpose: To determine serum immunoreactive erythropoietin (Epo) and soluble transferrin receptors (sTfR) levels in patients with hemoglobin H (HbH) disease and the correlation with HbH levels and α-globin genotype. Patients and Methods: Twenty patients with HbH disease were studied. Methods applied included cation-exchange high pressure liquid chromatography for HbH levels, chemoluminescence for Epo concentration, immunoassay for sTfR concentration, and DNA analysis for a-globin genotype characterization. Results: Serum Epo and sTfR levels were significantly elevated (46.6 + 26.8 IU/1 and 5.6 + 1.8 mg/1, respectively) in patients with HbH disease compared to controls (9.2 + 3.3 IU/1 and 1.8 + 0.7 mg/1. respectively). Epo and sTfR levels correlated positively with HbH concentration (r = 0.93 and 0.80, respectively). The highest Epo and sTfR values were observed in three patients with the highest HbH levels who all had nondeletion α-thalassemia mutations. Conclusion: Epo and sTfR levels are increased in patients with HbH disease; this increase is directly related to the HbH concentration that usually reflects the degree of globin polypeptide imbalance. The correlation of Epo. sTfR, and reticulocyte production index in these patients indicates that anemia in HbH disease mainly is caused by ineffective erythropoiesis and a mild degree of peripheral hemolysis.

A Rare Thalassemic Syndrome Caused by Interaction of Hb Adana [α59(E8)Gly→Asp] with an α+-Thalassemia Deletion: Clinical Aspects in Two Cases

Hb Adana is a highly unstable and rare α-globin hemoglobin (Hb) variant, to date described in only three families, in interaction with other α-thalassemia (α-thal) deletions. We describe the clinical and hematological findings in two cases from independent families of Albanian origin, who have an interaction of the codon 59 (Gly→Asp) α2-globin gene variant in trans to a 3.7 kb α+-thal deletion (αcodon 59α/−α). We report their presenting symptoms and laboratory findings as well as complications and differences in their clinical management. Both cases can be characterized as thalassemia intermedia and illustrate the problems associated with selecting the most appropriate options for patient management, especially in cases with rare underlying genotypes.

Interaction of an α+-Thalassemia Deletion with Either a Highly Unstable α-Globin Variant (α2, Codon 59, GGC→GAC) or a Nondeletional α-Thalassemia Mutation (AATAA→AATAAG): Comparison of Phenotypes Illustrating “Dominant” α-Thalassemia

Thalassemia syndromes and unstable hemoglobins traditionally represent two phenotypically separate disorders of hemoglobin synthesis. Highly unstable hemoglobin variants, however, often have phenotypic characteristics associated with both ineffective erythropoiesis (thalassemias) and peripheral hemolysis (unstable hemoglobins). Many highly unstable β chain variants cause a dominant thalassemia-like phenotype, in which simple heterozygotes for such mutations have a clinical expression similar to thalassemia intermedia. The phenotypic expression of highly unstable α-globin variants is usually less severe, due mainly to a gene dosage effect, and they are often only characterized on interaction with other α-thalassemia mutations, whence they are classified as nondeletional α-thalassemia determinants. This study reports the clinical and hematological findings in five cases with rare α-thalassemia genotypes: a single patient with the thalassemic α2-globin gene codon 59 Gly→Asp hemoglobin variant in trans to an ...

The interaction of α° thalassaemia with Hb Icaria: three unusual cases of haemoglobinopathy H

The clinical, haematological, biosynthetic and molecular data of three Greek haemoglobin H (HbH) disease patients with a distinctive clinical phenotype are described. During infancy all three patients had unusually severe clinical manifestations for HbH disease, with anaemia necessitating blood transfusions, signs of bone changes, growth impairment, and splenomegaly. Molecular analysis identified a rare α‐thalassaemia genotype (− −Med/α Icα) . Splenectomy resulted in marked amelioration of the clinical signs; post splenectomy all three patients preserve adequate haemoglobin levels (9–10 g/dl) with growth restored to normal. Despite the initial severe clinical phenotype in these patients, our experience indicates that splenectomy modifies the clinical course to that of mild thalassaemia intermedia. This observation should be considered carefully when giving genetic counselling to families carrying the rare Hb Icaria mutation and an α° thalassaemia mutation.