Giovanna Graziadei

Elevated liver iron concentration is a marker of increased morbidity in patients with β thalassemia intermedia

Background Patients with β thalassemia intermedia can have substantial iron overload, irrespectively of their transfusion status, secondary to increased intestinal iron absorption. This study evaluates whether iron overload in patients with β thalassemia intermedia is associated with morbidity. Design and Methods This was a cross-sectional study of 168 patients with β thalassemia intermedia treated at two centers in Lebanon and Italy. Data on demographics, splenectomy status, transfusion status, and presence of co-morbidities were retrieved. Laboratory values of serum ferritin, fetal and total hemoglobin levels, as well as platelet and nucleated red blood cell counts were also obtained. Iron burden was determined directly by measuring liver iron concentration using magnetic resonance imaging. Patients were subdivided according to transfusion and splenectomy status into groups with phenotypes of different severity. Results The mean age of the patients was 35.2±12.6 years and 42.9% of them were male. The mean liver iron concentration was 8.4±6.7 mg Fe/g dry weight. On multivariate logistic regression analysis, after adjusting for age, gender, splenectomy status, transfusion status, and laboratory indices, an increase in 1 mg Fe/g dry weight liver iron concentration was independently and significantly associated with higher odds of thrombosis, pulmonary hypertension, hypothyroidism, osteoporosis, and hypogonadism. A liver iron concentration of at least 7 and at least 6 mg Fe/g dry weight were the best thresholds for discriminating the presence and absence of vascular and endocrine/bone morbidities, respectively (area under the receiver-operating characteristic curve: 0.72, P<0.001). Elevated liver iron concentration was associated with an increased rate of morbidity in patients with phenotypes of all severity, with a steeper increase in the rate of vascular morbidity being attributed to aging, and an earlier appearance of endocrine and bone disease. Conclusions Elevated liver iron concentration in patients with β thalassemia intermedia is a marker of increased vascular, endocrine, and bone disease.

Heparin induced thrombocytopenia: Pathogenetic, clinical, diagnostic and therapeutic aspects

Heparin induced thrombocytopenia (HIT) in addition to bleeding complications are the most serious and dangerous side effects of heparin treatment. HIT remains the most common antibody-mediated, drug-induced thrombocytopenic disorder and a leading cause of morbidity and mortality. Two types of HIT are described: Type I is a transitory, slight and asymptomatic reduction of platelet count occurring during 1-2 days of therapy. HIT type II, which has an immunologic origin, is characterized by a thrombocytopenia that generally onset after the fifth day of therapy. Despite thrombocytopenia, haemorrhagic complications are very rare and HIT type II is characterized by thromboembolic complications consisting in venous and arterial thrombosis. The aim of this paper is to review new aspects of epidemiology, pathophysiology, clinical features, diagnosis and therapy of HIT type II. There is increasing evidence that platelet factor 4 (PF4) displaced from endothelial cells, heparan sulphate or directly from the platelets, binds to heparin molecule to form an immunogenic complex. The anti-heparin/PF4 IgG immune-complexes activates platelets through binding with the Fcgamma RIIa (CD32) receptor inducing endothelial lesions with thrombocytopenia and thrombosis. Cytokines are generated during this process and inflammation could play an additional role in the pathogenesis of thromboembolic manifestations. The onset of HIT type II is independent from dosage, schedule, and route of administration of heparin. A platelet count must be carried out prior to heparin therapy. Starting from the fourth day, platelet count must be carried out daily or every two days for at least 20 days of any heparin therapy regardless of the route of the drug administration. Patients undergoing orthopaedic or cardiac surgery are at higher risk for HIT type II. The diagnosis of HIT type II should be formulated on basis of clinical criteria and confirmed by in vitro demonstration of heparin-dependent antibodies detected by functional and antigen methods. However, the introduction of sensitive ELISA tests to measure anti-heparin/PF4 antibodies has showed the immuno-conversion in an higher number of patients treated with heparin such as the incidence of anti-heparin/PF4 exceeds the incidence of the disease. If HIT type II is likely, heparin must be immediately discontinued, even in absence of certain diagnosis of HIT type II, and an alternative anticoagulant therapy must be started followed by oral dicumaroids, preferably after resolution of thrombocytopenia. Further studies are required in order to elucidate the pathogenetic mechanism of thrombosis and its relation with inflammation; on the other hand large clinical trials are needed to confirm the best therapeutic strategies for HIT Type II.

Intrathoracic Masses Due to Extramedullary Hematopoiesis

Extramedullary hematopoiesis often occurs in hemoglobinopathies, hemolytic anemias, leukemias, lymphomas, and myeloproliferative disorders. Liver, spleen, and lymph nodes are frequently involved. However, extramedullary hematopoiesis may also develop in other sites such as thymus, kidney, retroperitoneum, and paravertebral areas of the thorax. Extramedullary hematopoietic masses are often microscopic and asymptomatic, but sometimes they lead to tumor-like masses. We describe massive intrathoracic extramedullary hematopoiesis in a 41-year-old man with compound heterozygosis for beta-thalassemia and sickle cell anemia and functional asplenia. We also describe a 39-year-old man with beta-thalassemia intermedia, who was initially diagnosed as having tumor masses, but was later proved, by magnetic resonance imaging, to have extramedullary erythropoietic tissue. These observations provide further support to include extramedullary hematopoiesis among the differential diagnosis of tumor-like masses in patients with hematologic diseases.

Genetic variability of TMPRSS6 and its association with iron deficiency anaemia.

Transmembrane Protease, Serine 6 (TMPRSS6) has an important role in iron homeostasis and its mutations, performed in TMPRSS6‐deficient mice, have been recently associated with iron‐refractory iron deficiency anaemia (IRIDA). Several variants of TMPRSS6 have been already identified; however the role of polymorphisms and TMPRSS6 haplotypes, causing iron deficiency anaemia, have not yet been investigated. This study sequenced the TMPRSS6 gene in 16 subjects with IRIDA phenotype and identified 27 DNA polymorphisms. Eight single nucleotide polymorphisms and four haplotypes were significantly associated with iron‐refractory anaemia (P < 0·001). Our preliminary results suggest a possible association between specific haplotypes of TMPRSS6 and IRIDA.

Recommendations regarding splenectomy in hereditary hemolytic anemias.

Hereditary hemolytic anemias are a group of disorders with a variety of causes, including red cell membrane defects, red blood cell enzyme disorders, congenital dyserythropoietic anemias, thalassemia syndromes and hemoglobinopathies. As damaged red blood cells passing through the red pulp of the spleen are removed by splenic macrophages, splenectomy is one possible therapeutic approach to the management of severely affected patients. However, except for hereditary spherocytosis for which the effectiveness of splenectomy has been well documented, the efficacy of splenectomy in other anemias within this group has yet to be determined and there are concerns regarding short- and long-term infectious and thrombotic complications. In light of the priorities identified by the European Hematology Association Roadmap we generated specific recommendations for each disorder, except thalassemia syndromes for which there are other, recent guidelines. Our recommendations are intended to enable clinicians to achieve better informed decisions on disease management by splenectomy, on the type of splenectomy and the possible consequences. As no randomized clinical trials, case control or cohort studies regarding splenectomy in these disorders were found in the literature, recommendations for each disease were based on expert opinion and were subsequently critically revised and modified by the Splenectomy in Rare Anemias Study Group, which includes hematologists caring for both adults and children.

Porphyrias at a glance: diagnosis and treatment

Porphyrias are a group of eight rare inherited metabolic disorders of heme biosynthesis pathway. Porphyrias are still underdiagnosed, although examinations of urine and plasma are first-line tests for detecting excess of porphyrins or heme precursors in suspected patients. Diagnosis, particularly for the acute forms, is essential to avoid precipitating factors and the use of triggering drugs. Mutation screening of family members is recommended to identify presymptomatic carriers and to prevent acute attacks. The therapeutic approach should be appropriate regarding specific forms of porphyria and treatment should be started promptly.

Beta-thalassaemia intermedia: evaluation of endocrine and bone complications.

Objective. Data about endocrine and bone disease in nontransfusion-dependent thalassaemia (NTDT) is scanty. The aim of our study was to evaluate these complications in β-TI adult patients. Methods. We studied retrospectively 70 β-TI patients with mean followup of 20 years. Data recorded included age, gender, haemoglobin and ferritin levels, biochemical and endocrine tests, liver iron concentration (LIC) from T2*, transfusion regimen, iron chelation, hydroxyurea, splenectomy, and bone mineralization by dual X-ray absorptiometry. Results. Thirty-seven (53%) males and 33 (47%) females were studied, with mean age 41 ± 12 years, mean haemoglobin 9.2 ± 1.5 g/dL, median ferritin 537 (range 14–4893), and mean LIC 7.6 ± 6.4 mg Fe/g dw. Thirty-three patients (47%) had been transfused, occasionally (24/33; 73%) or regularly (9/33; 27%); 37/70 (53%) had never been transfused; 34/70 patients had been splenectomized (49%); 39 (56%) were on chelation therapy; and 11 (16%) were on hydroxyurea. Endocrinopathies were found in 15 patients (21%): 10 hypothyroidism, 3 hypogonadism, 2 impaired glucose tolerance (IGT), and one diabetes. Bone disease was observed in 53/70 (76%) patients, osteoporosis in 26/53 (49%), and osteopenia in 27/53 (51%). Discussion and Conclusions. Bone disease was found in most patients in our study, while endocrinopathies were highly uncommon, especially hypogonadism. We speculate that low iron burden may protect against endocrinopathy development.

X-chromosomal inactivation directly influences the phenotypic manifestation of X-linked protoporphyria

X‐linked protoporphyria (XLP), a rare erythropoietic porphyria, results from terminal exon gain‐of‐function mutations in the ALAS2 gene causing increased ALAS2 activity and markedly increased erythrocyte protoporphyrin levels. Patients present with severe cutaneous photosensitivity and may develop liver dysfunction. XLP was originally reported as X‐linked dominant with 100% penetrance in males and females. We characterized 11 heterozygous females from six unrelated XLP families and show markedly varying phenotypic and biochemical heterogeneity, reflecting the degree of X‐chromsomal inactivation of the mutant gene. ALAS2 sequencing identified the specific mutation and confirmed heterozygosity among the females. Clinical history, plasma and erythrocyte protoporphyrin levels were determined. Methylation assays of the androgen receptor and zinc‐finger MYM type 3 short tandem repeat polymorphisms estimated each heterozygotes X‐chromosomal inactivation pattern. Heterozygotes with equal or increased skewing, favoring expression of the wild‐type allele had no clinical symptoms and only slightly increased erythrocyte protoporphyrin concentrations and/or frequency of protoporphyrin‐containing peripheral blood fluorocytes. When the wild‐type allele was preferentially inactivated, heterozygous females manifested the disease phenotype and had both higher erythrocyte protoporphyrin levels and circulating fluorocytes. These findings confirm that the previous dominant classification of XLP is inappropriate and genetically misleading, as the disorder is more appropriately designated XLP.

Growth differentiation factor 15 expression and regulation during erythroid differentiation in non-transfusion dependent thalassemia

In the last two decades the research interest in biochemical risk markers has exploded. Among the new biomarkers examined, GDF15, a cytokine member of the transforming growth factor-β (TGFβ) superfamily, is emerging as a biomarker of potential utility in a variety of diseases (cardiovascular disease, diabetes, chronic kidney disease, acute respiratory distress syndromes, rheumatoid arthritis, and many types of cancer) [1,2]. Under normal conditions, GDF15 is expressed at low levels in all organs (serum levels: 200–1150 pg/mL). Although the exact biological functions of GDF15 are still poorly understood, it has been shown to be involved in regulating inflammatory and apoptotic pathways and its expression is upregulated in many different pathological conditions, including inflammation, cancer, cardiovascular disease, pulmonary disease and renal disease [3]. Furthermore, GDF15 concentrations are strongly elevated in disorders hallmarked by increased ineffective erythropoiesis, such as β thalassemia syndromes (up to 100,000 pg/mL), congenital dyserythropoietic anemias (10,000 pg/mL) and myelodysplastic syndromes (5000 pg/mL) [4]. Here, we focused on thalassemia and ineffective erythropoiesis as possible regulator for GDF15 levels. Non-transfusion dependent thalassemia (NTDT) syndromes are characterized by ineffective erythropoiesis and iron overload; GDF15 has been proposed as a link between these two conditions. There is evidence that ineffective erythropoiesis inhibits expression of hepcidin, a hepatic peptide hormone that regulates iron release into the blood stream from duodenal enterocytes, hepatocytes and macrophages [5]. The molecular mechanisms involved in hepcidin suppression in response to increased erythropoietic activity remain unclear, although it was hypothesized that some soluble proteins secreted fromdeveloping erythroblastsmay play a role; GDF15was suggested as one of such proteins. The mechanisms by which GDF15 synthesis is regulated during erythroid differentiation are not known; it has been proposed that GDF15 itself might be responsive to intracellular iron levels [6]. Moreover, correlation between GDF15 levels and the degree of ineffective erythropoiesis has been never explored. In the current study, we evaluated GDF15 expression and regulation during normal and thalassemic erythroid differentiation in vitro andwe investigated the relationship between GDF15 levels and the degree of ineffective erythropoiesis. CD34 cells obtained, after informed consent, from peripheral blood of healthy volunteers (control group) and NTDT patients were cultured for 14 days with a medium stimulating erythroid differentiation [7]. GDF15 expression was evaluated at mRNA levels by real-time PCR (2^-dCt) at different time points (day 0: erythroid progenitors, day 7: proerythroblasts, and day 14: mature erythroblasts) and at protein levels by Western-blot analysis at day 14 of culture. GDF15 secretion

Development of interactive algorithm for clinical management of acute events related to sickle cell disease in emergency department

Sickle cell disease (SCD ORPHA232; OMIM 603903) is a rare hereditary red cell disorder, which global distribution is changed in the last decade due to immigration-fluxes from endemic areas to Western-countries. One of the main clinical manifestations of SCD are the acute painful vaso-occlusive crisis, which cause frequent accesses of SCD patients to the emergency departments (EDs). This has generated the requirement of feasible tools for emergency givers. In the context of the scientific-Italian-Society for the study of Thalassemias and Hemoglobinopathies (SITE), we developed an algorithm with interactive windows to guide physicians in managing SCD patients in EDs.