3rd Pan-European Conference - Abstracts by Thalassaemia International Federation (TIF)

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3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias 24-26 October 2012, Limassol â&#x20AC;&#x201C; Cyprus

THALASSEMIA REPORTS ISSN 2039-4357 - eISSN 2039-4365 Editor-in-Chief Aurelio Maggio, Italy

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Editorial Board Yesim Aydinok, Turkey Paola Baiardi, Italy Erol Baysal, United Arab Emirates Patrizio Bina, Italy Farid Boulad, USA Marcello Capra, Italy Adriana Ceci, Italy Ping Chen, USA Yiu-fai Cheung, Hong Kong Prosanti K. Chowdhury, India Mehmet Akif Curuk, Turkey Vito Di Marco, Italy Giorgio Derchi, Italy Vincenzo De Sanctis, Italy Amal El-Beshlawy, Egypt Aldo Filosa, Italy Graham Foster, UK Suthat Fucharoen, Thailand Renzo Galanello, Italy Shau-Yin Ha, Hong Kong Mehran Karimi, Iran Antonis Kattamis, Greece Marina Kleanthous, Cyprus Li Chi Kong, Hong Kong Philippe Leboulch, USA Dimitris Loukopoulos, Greece Meng-Yao Lu, Taiwan Luigi Mancuso, Italy Nicoletta Masera, Italy Massimo Midiri, Italy David G. Nathan, USA Ching-Tien Peng, Taiwan Alessia Pepe, Italy Debbie Rund, Israel Nicos Skordis, Cyprus George Stamatoyannopoulos, USA Vip Viprakasit, Thailand J Malcolm Walker, UK

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3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

TABLE OF CONTENTS

Posters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Index of authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Scientific program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias 24-26 October 2012, Limassol - Cyprus

SCIENTIFIC PROGRAM

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The important role that Cyprus played in the science of medicine during Antiquity must, to a large extent, be due to the richness of her vegetation and the abundance of her minerals. For centuries, its plants as well as many of its minerals were used for the preparation of medicaments, and the local tradition was ancient and famed. The paper will present the most important of the surviving evidence, culled from ancient texts, and brought to light by excavation. Within a Cypriot perspective, it will deal with the following main themes: doctors, surgical instruments, vegetal and mineral pharmaceutical substances, gods associated with medicine, ex-votos of medical nature, prophylactic amulets, and some new evidence from the palaeopathological study of skeletal remains.

metabolic disorders may be caused by regional (cell or organ) rather than systemic iron accumulation. The regional iron accumulation results from mutations in genes or from humoral factors that affect particular components of cell iron metabolism leading to intracellular maldistribution of the metal. Iron accumulation of iatrogenic nature has recently been identified in the spleen, liver and pancreas of iv-iron supplemented CKD patients with chronic anemia. The present review focuses on nonphysiological (i.e. anomalous) forms of iron in plasma and cells that have been implicated in tissue iron accumulation and multi-organ iron overload and toxicity. Although those pathological forms have not been fully defined in chemical and toxicological terms, they have already been perceived as a major source of iron toxicity in systemic iron overload (SIO) disorders. In this review we explore the possibility that those pathological forms of iron might be of clinical value in the diagnosis of impending IO and in the monitoring of treatment efficacy

ARCHAEOLOGICAL MEDICAL FINDINGS IN CYPRUS Demetrios Michaelides Professor of Classical Archaeology, Director, Archaeological Research Unit, University of Cyprus, Cyprus

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GENETIC/METABOLIC EFFECT OF IRON METABOLISM AND RARE ANEMIAS Clara Camaschella Vita-Salute University and San Raffaele Scientific Institute, Milan, Italy

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THE MOLECULAR AND CELLULAR BASIS OF IRON TOXICITY IN IRON OVERLOAD (IO) DISORDERS. DIAGNOSTIC AND THERAPEUTIC APPROACHES Zvi Ioav Cabantchik,1 Yan Sung Sohn,1 William Breuer,1 Breno Pannia Espósito2 1Institute of Life Sciences, Hebrew University of Jerusalem, Safra Campus at Givat Ram, Jerusalem, Israel; 2Instituto de Química, Universidade de São Paulo, São Paulo, Brasil

Abnormal iron accumulation in human tissues and oxidative damage are emerging issues in the medical field. The most commonly recognized type of pathological accumulation has been associated with the general appearance of plasma non-transferrin bound iron (NTBI) and particularly with a labile iron component that can infiltrate cells in an unregulated manner. A major consequence of excess iron accumulation is a rise in cellular labile iron (LCI) that can promote the formation of reactive oxygenspecies (ROS) from physiological oxygenintermediates (ROI), overriding the cellular antioxidant machineries and causing oxidative damage. The major diseases caused by excess iron accumulation are those that result from tissue exposure to systemic rises in plasma NTBI, as in transfusional siderosis (sickle cell anemia, thalassemia major and some forms of myelodysplasia) or primary hemochromatosis and thalassemia intermedia. However, it is increasingly recognized that various neurological, hematological and more recently chronic diabetes and

Advances in iron metabolism have allowed a novel classification of iron disorders and to identify previously unknown diseases. These disorders include genetic iron overload (hemochromatosis) and inherited iron-related anemias, in some cases accompanied by iron overload. Rare inherited anemias may affect the hepcidin pathway, iron absorption, transport, utilization and recycling. Among the genetic iron-related anemias the most common form is likely the iron-refractory iron-deficiency anemia (IRIDA), due to mutations of the hepcidin inhibitor TMPRSS6 encoding the serine protease matriptase-2. IRIDA is characterized by hepcidin up-regulation, decrease iron absorption and macrophage recycling and by microcytic-hypochromic anemia, unresponsive to oral iron. High serum hepcidin levels may suggest the diagnosis, which requires demonstrating the causal TMPRSS6 mutations by gene sequencing. Other rare microcytic hypochromic anemias associated with defects of iron transport-uptake are the rare hypotransferrinemia, and DMT1 and STEAP3 mutations. The degree of anemia is variable and accompanied by secondary iron overload even in the absence of blood transfusions. This is due to the iron-deficient or expanded erythropoiesis that inhibits hepcidin transcription, increases iron absorption, through the erythroid regulator, as in untransfused β-thalassemia. Sideroblastic anemias are due to decreased mitochondrial

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The accurate evaluation of iron overload is necessary to plan and monitor iron chelation. Beside serum ferritin, which indirectly measures body iron overload, several non invasive techniques have been recently developed to assess organ iron. Magnetic biosusceptometry, by the superconducting quantum interference device (SQUID), is a method used to measure liver iron. The method has been validated but has a limited availability (only two devices across the world) and cannot measure iron in the heart. The introduction of the magnetic resonance imaging (MRI) for the assessment of tissue iron in the early 2000, completely changed our understanding of iron overload and its management. MRI has proven effective in detecting and accurately quantifying iron in the heart (the major letal iron-targeted organ), liver and other organs. MRI is widely available, robust and reproducible in the clinical relevant range. One important advantage of cardiac MRI is its ability to recognize preclinical cardiac iron deposition, allowing early effective treatment. Also iron chelation substantially changed in the last 15 years. Beside deferoxamine, available since 1970 but difficult to be taken by the patients, two oral iron chelators have been approved for clinical use: Deferiprone and Deferasirox. The oral chelators have significantly improved iron chelation therapy, mainly because of greater chelation options (as monotherapy and combinations) and their specific organ targeting. The possibility of accurately assessing organ iron overload and tailoring iron chelation to the needs of each single patients, has substantially changed the prognosis of thalassemia major leading to reduced morbility, increased survival and improved quality of life.

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IRON LOAD Filippo CassarĂ , Aurelio Maggio U.O.C. Ematologia II, A.O.C. Villa Sofia, Palermo, Italy

IRON MONITORING AND TREATMENT Renzo Galanello Pediatric Clinic 2 and Thalassemia Unit, Asl8, University of Cagliari, Italy

iron utilization for heme or sulfur cluster synthesis. Their diagnosis requires demonstrating ringed sideroblasts by Perlâ&#x20AC;&#x2122;s staining of the bone marrow smears. The commonest X-linked form is due to delta-amino-levulinic-synthase-2-acid (ALAS2) mutations. The recessive, more severe form, affects SLC25A38, which encodes a potential mitochondrial importer of glycine, an amino acid essential for ALA synthesis and thus results in heme deficiency. Two disorders affect iron/sulfur cluster biogenesis: deficiency of the ATP-binding cassette B7 (ABCB7) causes X-linked sideroblastic anemia/ataxia, likely impairing the activity of ferrochelatase, which is an iron/sulfur-cluster-dependent enzyme. A recessive form affects GLRX5, a protein involved in the iron/sulfur cluster biogenesis. Aceruloplasminemia is a rare recessive syndrome characterized by anemia, diabetes, retinal degeneration, ataxia and other neurological symptoms, low serum iron but high serum ferritin, due to decreased iron recycling from macrophages and other cells. The study of these rare conditions has greatly contributed to our understanding of iron transport, utilization and recycling. Their distinction is clinically essential in order to plan the best treatment.

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Recent research addressed the main role of hepcidin in the regulation of iron metabolism. However, while this mechanism could be relevant in causing iron load in Thalassemia Intermedia and Sickle-Cell Anemia, its role in Thalassemia Major (TM) is marginal. This is mainly due to the high impact of transfusional requirement into the severe increase of body iron. Moreover, the damage of iron load may be worsened by infections, as HCV hepatitis, or liver and endocrinological damage. One of the most relevant associations was found between splenectomy and increase of risk for mortality due,probably, to more severe iron load. These issues suggest as morbidity and mortality of this group of patients they do not depend only by our ability in controlling heart damage but even in preventing or treating particular infections and complications. This finding is supported by the impairment of survival curves in patients with complications different from heart damage. However, because, during recent years different direct and indirect methods to detect iron overload in patients affected by secondary hemochromatosis have been implemented, our ability to maintain under control iron load is significantly improved. Anyway, the future in iron load management remains to be able to have an iron load map of our body for targeting chelation and other medical treatment according to the single organ damage.

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CARDIAC ARRHYTHMIA THE EMERGING COMPLICATION IN THALASSAEMIA John Malcolm Walker Consultant Cardiologist, University College and The Heart Hospital, London Clinical Director Hatter Cardiovascular Institute UCL, London, UK Since iron overloaded hearts are susceptible to arrhythmia, it is not surprising that palpitations are a relatively frequent complaint in clinic, as they are in any general cardiology service. Historically in non-transfused TM patients, not only were tachycardias frequently seen, but there was also a high incidence of heart block, a situation which has altered radically with the advent of effective chelation strategies. Arrhythmia, presenting as palpitation, are occasionally due to life-threatening ventricular arrhythmia (VT), which in our experience is universally associated with grossly iron overloaded hearts with established ventricular impairment, although VT may be the first clue that the heart is seriously iron loaded. Atrial fibrillation is much more common, but also often occurs in patients who have developed significant iron overload. AF has previously been associated with the risk of impending cardiac failure.

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

APPROACHES TO MANAGEMENT OF β-THALASSEMIA INTERMEDIA Joseph E. Maakaron, Ali T. Taher Department of Internal Medicine, Division of Hematology and Oncology, American University of Beirut Medical Center, Beirut, Lebanon

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IRON OVERLOAD IN NON-TRANSFUSION-DEPENDENT THALASSEMIA Khaled M. Musallam Iron overload due to increased intestinal iron absorption remains a concern in patients with non-transfusiondependent thalassemia (NTDT). A dynamic regulation between ineffective erythropiesis and iron metabolism in these disorders has been recently elucidated. Although the rate of iron loading in NTDT is slower than that observed in regularly transfused patients, the process is cumulative and patients may reach considerably high liver iron concentration levels. The clinical consequences of iron overload in patients with NTDT are various and include hepatic disease, endocrinopathy, bone disease, and vascular outcomes; while cardiac siderosis is less frequently observed. Although serum ferritin levels correlate with LIC in NTDT, they underestimate iron load when compared with transfusion-dependent patients with equivalent LIC. Therefore, direct measurement of LIC is recommended to identify patients at risk and guide iron chelation decisions.

sion-dependent thalassemia (NTDT), transfusion requirements are absent or episodic. Non-transfusion-dependent thalassemia includes β-thalassemia intermedia, HbE/βthalassemia, and α-thalassemia intermedia, also known as HbH disease. β-thalassemia intermedia results from the inheritance of one or two mild β-thalassemia alleles or the co-inheritance, with two severe alleles, of genetic modifiers (i.e. α-thalassemia or genetic determinants increasing fetal hemoglobin) which reduce the imbalance of globin chain synthesis. Recently several genes, including HBS1L-MYB and BCL11A, have been identified that modulate HbF production alleviating the clinical phenotype. In HbE/β-thalassemia the degree of clinical variability depends on the severity of the β-thalassemia allele, but many other genetic factors are involved. The severity of HbH disease is related to the type of molecular defect, being non deletional forms more severe than the deletional forms. However, other not defined mechanisms related to altered proteolytic activity, apoptosis, red cell membrane integrity and the α globin stabilizing protein (AMSP), may play a role. Although extensive studies have been performed questions remain regarding the mechanisms of globin gene regulation and the reasons for extensive phenotypic diversity.

Certainly an iron overloaded heart with some impairment of ventricular function may be precipitated into acute decompensation by the development of AF. This situation constitutes a true clinical emergency and deserves immediate treatment. In our experience the appearance of AF is now changing. Increasingly it is being seen in older patients with little or no current cardiac iron content and with good cardiac function. The factors associated with this clinical demographic change are discussed in this presentation. The problem facing the clinician is differentiating between sinister arrhythmias and the more common benign palpitations experienced by a young population, often anxious, who have first-hand experience of the outcomes of some of their less fortunate peers. Having a quantitative estimate of cardiac iron content, by T2*, will help make this distinction more accurate, especially in those patients with normal ventricular function by conventional assessment. The approach to making an accurate diagnosis of cardiac arrhythmia and their optimal management, including the place of newer interventional technologies will be discussed.

RECENT ADVANCES IN MOLECULAR UNDERSTANDING OF NON TRANSFUSION DEPENDENT THALASSEMIA Renzo Galanello Pediatric Clinic 2 and Thalassemia Unit, ASL8, University of Cagliari, Italy Thalassemias are a group of inherited autosomal recessive hematologic disorders that occur because of defects in the α and β-globin genes of adult hemoglobin (Hb). An imbalance in the synthesis of one or more of the globin chains can result in a wide spectrum of phenotypes, depending on the type and amount of globin synthesized and additional genetic modifiers. In patients with thalassemia intermedia, a condition known as non-transfu-

Thalassemia intermedia is a genetically diverse group of diseases that is the result of an imbalance in the production of the α and β chains with ensuing chronic hemolysis, ineffective erythropoiesis, and iron overload. Resulting complications include bone changes, hypercoagulability, and end-organ damage due to iron overload. This decade has witnessed major breakthroughs in the management of thalassemia. In this article, we examine these novelties in therapy including iron chelation therapy, stem cell transplant, and gene therapy.Iron chelation therapy has been revolutionized with the advent of deferasirox, a once-daily oral iron chelator, that has been shown to be safe and efficacious.Gene therapy was also at the core of this revolution with the discovery of novel gene elements and viral vectors allowing for better control and improved outcomes. OVERVIEW OF PREVENTION APPROACHES Marina Kleanthous Director of the Thalassaemia Research Unit, Institute of Neurology and Genetics (CING), Nicosia, Cyprus Prenatal diagnosis for thalassaemias as well as for other genetic diseases is based mainly on CVS biopsy at the 11th week of gestation and DNA analysis of the fetus for the presence of parental mutations. A number of DNAbased tests, such as allele-specific oligonucleotide probes (ASO) and amplification refractory mutation system

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tors. A number of genetic modifiers for β-thalassaemia and sickle cell disease have been identified, such as BCL11A, KLF1, Myb and others. With genome-wide association studies (GWAS) performed in groups of patients, it is expected that more genetic modifiers and molecular mechanisms that lead to specific phenotypes will be identified. With the knowledge of the genomic background of the patients, a more personalized treatment could be provided while it is also expected to affect prevention approaches and genetic counseling. The use of new technologies such as Next Generation Sequencing (NGS) enables the application of genomic-based approaches to the clinical practice possible. The innovation of reprogramming somatic cells to induced pluripotent stem (iPS) cells provides a possible new approach to treat β-thalassaemia and other genetic diseases. iPS can be made from chorionic villi sampling (CVS) specimens, which are acquired for prenatal diagnosis and can be corrected by gene therapy, differentiated into haematopoietic cells and returned to the fetus. Currently prenatal diagnosis and selective abortion of the affected fetus is the main option of couples at risk. The potential use of iPS for treating thalassaemia raises the possibility of providing a new option following prenatal diagnosis. The treatment in the perinatal periods has the advantage of requiring much fewer cells than adult treatment and of preventing organ damage.

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(ARMS) for point mutations and gap-PCR for specific deletions, can be used to determine the genotype of the fetus. Direct sequencing, multiplex ligation-dependent probe amplification (MLPA) for the detection of deletions, and real-time PCR and melting curve analysis for the detection of point mutation are also used in prenatal diagnosis. Another approach used in prenatal diagnosis for thalassaemias is preimplantation genetic diagnosis (PGD). PGD involves many stages: counselling, in vitro fertilization, the biopsy and analysis of genetic material representing each embryo, embryo transfer, and if implantation occurs, follow-up of pregnancy and baby (or babies) born. Genetic analysis can be performed on the oocyte/zygote biopsies. For PGD applied to monogenic diseases, methods are almost exclusively based on PCR. Inherent difficulties associated with single-cell analysis include total PCR failure, allelic drop-out (ADO, when one of the alleles fails to amplify to detectable levels), and the likelihood of sample contamination. Direct mutation identification methods such as RT-PCR are currently used in PGD. The parallel analysis of polymorphic microsatellite markers alongside the disease-specific assay will monitor contamination in each individual sample and increase the accuracy of the genotyping result by monitoring the presence of ADO, which might otherwise lead to misdiagnosis. PGD for thalassaemia and HLA typing is currently used in order to identify embryos that are both, HLA-matched to an affected sibling and free of the disease. The discovery of cell-free fetal DNA in maternal plasma has opened up new avenues for noninvasive prenatal diagnosis (NIPD). Currently, non-invasive prenatal diagnosis is routinely applied by several centers for the determination of fetal sex in pregnancies at risk for an X-linked disorder and for the fetal Rhesus D (RHD) status in pregnancies at risk for hemolytic disease of the newborn. These approaches are based on the detection of fetal genetic loci absent from the maternal genome. However, the detection of more subtle fetal genetic traits, such as point mutations involved in Mendelian disorders (thalassaemia, cystic fibrosis), is considerably more complex and difficult to perform because they involve fetal genetic changes that differ only slightly from the maternal genome. Approaches permitting the reliable detection of single-gene mutations or single-nucleotide polymorphisms (SNPs) using cell-free fetal DNA in maternal plasma are still in development. Methods for NIPD require both high sensitivity and high specificity. Over the last few years, a number of different strategies have been investigated to meet the challenges for the non-invasive detection of β-thalassemia using maternal plasma. With the advent of single-molecule technologies and high-throughput sequencing, many challenges have been encountered. High-throughput sequencing technology for the development of a NIPD assay for β-thalassaemia based on the detection of paternally inherited fetal alleles using SNPs was used. The analytical power of this method, namely sensitivity, specificity, accuracy and precision should pave the way for the clinical use of NIPD. Thalassaemia as well as other single-gene disorders present clinical variability or phenotypic diversity that is mainly due to allelic heterogeneity, other genetic modifiers and environmental fac-

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DIABETES AND THALASSAEMIA Maria Barnard, Ploutarchos Tzoulis, Romilla Jones, Emma Prescott, Farrukh Shah The Whittington Hospital NHS Trust, Whittington Health, Magdala Avenue, London, UK

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Diabetes is a significant complication of β-thalassaemia major. The aetiology includes iron overload causing β-cell destruction, autoimmunity, insulin resistance secondary to liver disease and development of type 1 or 2 diabetes. There are specific issues for patients with diabetes and thalassaemia which will be discussed here. Impaired carbohydrate metabolism must be detected early, to allow intensification of iron chelation. As life expectancy in thalassaemia rises, diabetic complications are seen. Optimising blood glucose and cardiovascular risk factor control is essential. Insulin remains critical for severely symptomatic patients. With milder hyperglycaemia, oral antidiabetic drugs are increasingly used. At Whittington Hospital, we wanted to address these issues. In 2005, we developed a unique Joint Diabetes Thalassaemia Clinic, where patients are reviewed jointly by specialist teams, including Consultant Diabetologist and Haematologist. The Joint Clinic aims to optimise diabetes, endocrine and thalassaemia care, while supporting patient self-management. A retrospective audit of the Joint Clinic (2005-09), showed improvement in glycaemic control, (Fructosamine falling from 344 umol/L to 319 umol/L). We compared our cohort to the National Diabetes Audit for England (2007-08). Patients attending the Joint Clinic achieved better glycaemic control (target reached: 73% Joint Clinic vs. 63% Nationally), blood pressure control (target reached: 58% Joint Clinic vs. 30% Nationally) and cholesterol control

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

THE CONTRIBUTION OF THE LABORATORY, AND MORE SPECIFICALLY OF MOLECULAR BIOLOGY, IN THE DIAGNOSIS, MONITORING AND TREATMENT OF CHRONIC VIRAL HEPATITIS Petros Karayiannis Section of Hepatology and Gastroenterology, Imperial College, London, UK

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Bone disease represents a prominent cause of morbidity in patients with thalassaemia and other haemoglobin disorders. The delay in sexual maturation, the presence of diabetes and hypothyroidism, the parathyroid gland dysfunction, the haemolytic anaemia, the progressive marrow expansion, the iron toxicity on osteoblasts, the iron chelators, and the deficiency of growth hormone or insulin growth factors have been identified as major causes of osteoporosis in thalassaemia. Adequate hormonal replacement, effective iron chelation, improvement of hemoglobin levels, calcium and vitamin D administration, physical activity, and smoking cessation are the main to-date measures for the management of the disease. During the last decade, novel pathogenetic data suggest that the reduced osteoblastic activity, which is believed to be the basic mechanism of bone loss in thalassemia, is accompanied by a comparable or even greater increase in bone resorption. Therefore, potent inhibitors of osteoclast activation, such as the aminobisphosphonates, arise as key drugs for the management of osteoporosis in thalassaemia patients and other haemoglobin disorders.

BONE DISEASE IN HAEMOGLOBIN DISORDERS Ersi Voskaridou,1 Evangelos Terpos2 1Thalassaemia Reference Center, Laikon General Hospital, Athens, Greece; 2Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece

essary transfusions. Within blood services, the introduction of more advanced serological and molecular techniques for microbiological testing coupled with stricter criteria for selection of donors has greatly reduced the risks of transfusion transmitted infection. Additional steps during processing of blood and components including leuco-depletion and viral inactivation where feasible further improve safety. Hospital blood transfusion practice must focus on ensuring safe and appropriate use including use of clinical guidelines supported by education and training with regular audit of practice. Robust systems are essential for accurate patient identification throughout the transfusion process from taking the initial blood sample, laboratory testing and transfer of blood to clinical areas to the final bedside check prior to transfusion to minimize errors. The reporting and monitoring of all adverse events in relation to blood transfusion via national haemovigilance schemes has highlighted key areas for action resulting in improved transfusion safety.

(target reached: 81% Joint Clinic vs. 78% Nationally). 22.7% of our patients had â&#x2030;Ľ1 microvascular complication. A significant proportion had endocrinopathies (86% hypogonadism, 23% hypoparathyroidism, 18% hypothyroidism). Managing diabetes is one of the greatest challenges a person with thalassaemia can face. Training people to self-manage their diabetes and providing support from specialist teams working together are critical. The unique partnership working of our Joint Diabetes Thalassaemia Clinic allows these very complex patients to be managed effectively.

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Infection with the hepatitis B and C viruses (HBV, HCV) is responsible for a significant burden of morbidity and mortality worldwide. Progression of infection to long-term chronicity is a major risk of development of cirrhosis, hepatocellular carcinoma and liver failure. Antiviral treatment offers the only effective means of interrupting disease progression by suppressing viral replication as in the case of HBV or eliminating the virus as in the case of HCV. Diagnosis of acute infection, monitoring of the chronic phase and evaluating response to antiviral treatments is reliant on the clinical laboratory through the use of serological and molecular tests. The decision to initiate antiviral treatment is very much dependent on these tests, as well as on biochemical and histological criteria. Real time polymerase chain reaction (PCR) has proved invaluable in monitoring viral load in patients during routine follow-up or in those receiving antiviral agents, before, during and after cessation of treatment. What is more, as these tests have become more and more sensitive, they have permitted the definition of viral levels which dictate when to prematurely stop therapy (stopping rules in non-responders) or allow response guided therapy to be instigated for the best benefit to the patient. In the latter case and in relation to HCV treatment, the duration of therapy can be shortened depending on the rapidity of HCV RNA negativity. Similarly, treatment duration is dependent on the genotype of the virus which is determined by a line probe assay employing PCR. Thus genotype 2 and 3 infected patients are treated for 6 months with the standard of care, which involves the combination of pegylated interferon and ribavirin,

BLOOD TRANSFUSION - SAFETY, OPTIMISATION, NEW ADVANCES Shubha Allard Consultant Haematologist, Barts Health NHS Trust and NHS Blood and Transplant, London, UK Regular blood transfusions are a mainstay of therapy for many patients with haemoglobinopathy. It is clearly important to focus on the key current safety issues involved firstly in the provision of a safe blood component and secondly in ensuring safety at all stages of the clinical transfusion process. The World Health Organisation (WHO) promotes an integrated approach for blood safety that has four key elements including establishment of a nationally-coordinated blood transfusion service, collection of blood from exclusively voluntary donors from low-risk populations, testing of all blood for compatibility and transfusion-transmissible infection and appropriate use of blood to reduce unnec-

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GUIDELINES FOR ADULTS Paul Telfer Sickle Cell Disease is a rare condition in many regions of Europe and general clinicians responsible for local services to patients with SCD may have relatively little experience and knowledge of the condition. Inadequate and inappropriate management may deprive patients of treatments and support shown to be beneficial, and may result in sub-optimal outcomes in the short and long-term. A recent report of fatal outcomes in patients with haemoglobin disorders in the UK (National Confidential Enquiry into Patient Outcome and Death, 2008) has highlighted a number of cases where mortality could have been avoided by adherence to standard guidelines or protocols. Local, national, and international guidelines need to take into account differences in health care systems, accessibility and availability of resources. Obviously guidelines for resource-poor, high prevalence countries in subSaharan Africa cannot be the same as those for developed countries. Pan European guidelines may be difficult to implement in individual countries because of the variability in health care systems and differing demographics, health care priorities, and funding arrangements. Local guidelines need to be specifically tailored to the local services and local patient group, and would normally be an adaptation of a national guideline. The main purposes of guidelines include: Informing and assisting health care workers in delivering accepted standards of care, facilitating management of quality of clinical services (e.g. through use in formulating clinical audit standards and peer review of services). They may also form part of a framework for commissioning of health care services, and guide patients in understanding what management they should be receiving. Developing guidelines usually starts with identification of a need, and then an act of commissioning from a regulatory or professional body, and often also from a recognised patient/user group. In the UK, the Thalassaemia and Sickle Cell Societies were involved in developing national guidelines from the outset, and in initiating the process of guideline development. The development of guidelines requires appointment of a chairperson/lead and an expert panel, which should include a range of expert health care workers (not just specialist doctors: Nurses, psychologists, laboratory scientists etc all need to contribute if the guidelines are to represent the multi-disciplinary nature of care). User representatives must be included on the panel. Competing interests of the panel members must be formally declared and considered carefully, as these may influence the objectivity of the final document. The process includes

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EXTERNAL RELATIONS MANAGER, EUPATI WP4 PROJECT MANAGER, DRUG, INFORMATION AND ACCESS (DIA) Matthew May Pharmaceutical medicines development is a highly regulated, costly, long and complex process that is largely unknown to the lay public. Benefits and risks of existing and new treatment alternatives are difficult to understand for patients. In an era of growing demand and emphasis on both quality and sustainability of healthcare, it is critical to address this major gap in public perception and knowledge. Well informed patients and carers have a key role to play in the implementation of patient-centred clinical research strategies and approval processes, access to treatments and treatment optimisation approaches. As demonstrated in the FP7-funded PatientPartner Project, patients are becoming more interested in actively engaging in the many processes involved in the development of new treatments - from participation in clinical trials to advising on protocol and informed consent and ethical review - to the overall medicines development process, marketing authorisation and healthcare policy. However, patients and the lay public are largely unaware of the complex nature of biomedical translational research, personalised and predictive medicine and their potential supportive roles in the research process in order to achieve therapeutic breakthroughs. They would benefit from understanding more about, and thus contributing to, the multiple facets of evidencebased new medicines, drug safety and benefit/risk assessments, health technology assessments, and strategies to enhance patient-centred chronic disease management. The European Patients’ Academy on Therapeutic Innovation (EUPATI), a consortium project led by the European Patients’ Forum, will significantly improve the availability of patient-centric information on medicines R&D, as well as educated patient experts that have the capacity and capability to contribute to medical research. Launched in February 2012 and running for five years, the European Patients’ Academy will develop and disseminate accessible, well-structured and user-friendly information and education resources on therapeutic inno-

vation, build competencies among well informed patients and patient advocates, and create the leading public library on patient information in the seven most common languages. This way, patients will be empowered to join as competent partners of academia, authorities, ethics committees and industry. This presentation will outline the needs of patients in better understanding medical research, and outline how the European Patients’ Academy will address these needs in an independent, collaborative, quality-controlled joint effort.

whilst genotype 1 and 4 patients receive 12 months of treatment. With the introduction of the protease inhibitors, more profound drops in viral load have permitted the shortening of treatment to 6 months even for genotype 1 and 4 patients. In the case of HBV, viral load measurements during treatment allow early detection of viral breakthrough which is attributable to the development of resistance mutations in the polymerase region of the viral genome. Amino-acid substitutions have been identified through nucleotide sequencing studies which confer resistance to the nucleos(t)ide analogues used for long-term suppression of viral replication. Such mutations have been detected with increasing frequency with length of time with low genetic barrier drugs such as lamivudine and adefovir. On the other hand, similar drugs with a high genetic barrier such as entecavir and tenofovir have proven potent inhibitors of viral replication in the absence of resistance mutations.

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3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

LABORATORY DIAGNOSIS OF THE RARE ANAEMIAS: EXTERNAL QUALITY ASSESSMENT BENEFITS PATIENT CARE Barbara De la Salle,1 Andrea Mosca,2 Renata Paleari,2 Vasileios Rapanakis,1 Keith Hyde1 1UK NEQAS for General Haematology, Watford, UK; 2University of Milan, Milan, Italy Since its introduction in the 1960s, external quality assessment has developed to become an essential component of the quality management system of the diagnostic laboratory. External quality assessment provides a long term, retrospective view of laboratory performance, demonstrating the competence of the laboratory to others. The ENERCA project (the European Network for Rare and Congenital Anaemias) has established a list of core laboratory tests that are used in the diagnosis of rare and congenital anaemias, which has been used as the basis for questionnaires to laboratories, to establish the use and quality assurance of diagnostic testing in the congenital and rare anaemias, and to European EQA providers for services in this key area. In general, the provision of EQA for rare and congenital anaemias is widely variable with little provision for the very rare disorders. For the more common congenital anaemias, such as the haemoglobinopathies and thalassaemias, provision is better but there is variation in aspects of the scheme design, especially the frequency of distribution. Where laboratories did not take part in EQA for individual tests, or there was no EQA available, a desire to participate was expressed in 66% (102/154) of cases. The provision of external quality assessment (EQA) services for rare disorders is a challenge. For many of these conditions, the number of patients in any one member state is very small with only a few laboratories providing diagnostic testing. In these cases, the development of pan-European or cross-border EQA may be the only means by which standardisation of methods and results can be achieved. An EQA survey of 243 laboratories for performance in Hb A2 quantification showed encouraging results in that there was a clear differentiation in the results from a β-Thalassaemia carrier and an individual with no evidence of Thalassaemia; however, a bias was observed between different methods of measurement.

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New challenges and priorities are given in the EU Health programme 2007-2013. The objectives of the programme are to improve citizens’ health security, to promote health to improve prosperity and solidarity, and to generate and disseminate health knowledge. If challenges and priorities have been defined globally for rare diseases by the European Commission, persons involved in rare anaemias have taken the opportunity to contribute to the empowerment of patients with rare anaemias. One of the ENERCA partners objectives was the mapping of existing centres that take care of patients with rare anaemias in Europe. Another goal was to obtain a directory of facilities available per centre for patients with rare anaemias. We thought that with those results it could realistically help to define a consensus regarding the criteria to be recognised as a centre of expertise for haemoglobinopathies and very rare anaemias. In various European countries, a questionnaire was distributed via ENERCA partners to colleagues or national and local scientific societies. It is certain that all centres have probably not been informed of this initiative. For France and United Kingdom, there is already recognition of expertise centres in the field of haemoglobinopathies and that is why the questionnaire was not distributed. The main sections of the questionnaire were a general overview of the centre and its activity i.e. centre of experse or general centre, type of patients followed, number of patients followed annually, if a laboratory, average number of samples tested annually, the expertise covered in the centre i.e. diagnosis and prevention, follow-up/case management such as allocated services and staff for management of acute and chronic events, criteria (Proof) of expertise i.e. availability of specialised services, specific treatments, patients services, decision supports and registries, a link with research and finally existence of publications, grant, teaching and training activities. Ninety centres answered the questionnaire. For rare and very rare aneamias, availability of specialized equipments and treatments are satisfactory. But our results show that for laboratories as well as for clinical centres necessary tools to give a diagnosis, to follow and manage the patients are not always available. If it exists, the dedicated and specialized teams are not always implemented. It is obvious that with a rare disease, few patients and sometimes geographic isolation, it is very complex to provide all the expected services. Our results show also that, even in centres of expertise, a registry is not always implemented. Nevertheless, collection of a core data set will support the continuous improvement of clinical care.

EXPERT SERVICES FOR RARE ANAEMIAS ACCROSS EUROPE Béatrice Gulbis Hôpital Erasme, Université Libre de Bruxelles, Belgium, European Network for Rare Congenital Anaemias

Decision supports for the health workers and for the patients are also missing in several centres. In view to improve those points, difficulties to create and collect data should be investigated. Accessibility to grants is not frequent even in centres of expertise. A proposal should be that more grants and funds could be dedicated to rare diseases. Finally, in view to share tools and all aspects in the management of patients with rare or very rare anaemias as well as to improve the knowledge of these diseases at all levels, networking should be encouraged. General recommendations, recommendations for laboratory diagnosis of rare and very rare anaemias or for genetic counselling were proposed by the ENERCA partners. They are presented, will edited in the WhiteBook for the creation of a European Reference Centre for Rare Anaemias and will be available on the ENERCA website (http://www.enerca.org).

deciding on the scope of care are to be covered. This is followed by a literature review, with grading of evidence, then drafting, review of draft, putting the draft out for comments from a range of non-panel experts, redraft and approval of final draft by commissioning/professional organization.

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Hereditary spherocytosis (HS) is the most common congenital red cell membrane defect in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The main clinical features are haemolytic anaemia from compensated to severe, jaundice, splenomegaly and cholelythiasis. The molecular defect is highly heterogeneous, involving the genes encoding for spectrin, ankyrin, band 3 and protein 4.2. The deficiency of any of these proteins, results in a loss of surface area and leads to spheroidal, osmotically fragile cells. The typical laboratory hallmark of HS, although not specific, is the presence of spherocytes on peripheral blood smear, detectable in about 97% of patients; however, spherocytes may be very few in several patients. The laboratory diagnosis of HS therefore commonly relies on indirect tests that exploit the surface area-to-volume ratio, typically reduced in spherocytes i.e. NaCl osmotic fragility tests, Glycerol Lysis and Acidified Glycerol Lysis tests, and Pink test. These methods miss a variable portion of HS cases, particularly the mildest ones, and do not differentiate HS from secondary spherocytosis. The direct flow cytometric EMA-binding test have been recently proposed, showing an high sensitivity and specificity. In atypical cases, the diagnostic workout may be more complex requiring SDS-PAGE analysis of RBC membrane proteins or ekacytometry. SDS-PAGE analysis is also required in differential diagnosis of CDAII. During the course of the ENERCA project a specific survey on laboratory tests used in the diagnosis of RBC membrane disorders was circulated with the final aim to define a consensus among laboratories. Beside red cell morphology and reticulocytes, osmotic fragility tests, performed singularly or in combination, are used in about 50% of centres. EMA-binding test seems to be the most commonly used test performed in 60% of centres. SDSPAGE analysis, ektacytometry, and molecular analysis are performed only in selected atypical cases. There is a wide heterogeneity of opinions about the method with the best specificity and sensitivity; different combinations of tests are used to reach a final diagnosis.

cyte enzyme defect. In some populations, more than 20 percent of people may be affected by this enzyme deficiency. In common polymorphic forms, such as G6PD A– or G6PD Mediterranean, hemolysis occurs only during the stress imposed by infection or administration of oxidative drugs, and in some individuals upon ingestion of fava beans. Patients with uncommon, functionally very severe, genetic variants of G6PD experience chronic hemolysis, a disorder designated hereditary nonspherocytic hemolytic anemia (HNSHA). Hereditary nonspherocytic hemolytic anemia also occurs as a consequence of other enzyme deficiencies, the most common of which is pyruvate kinase deficiency. Deficiencies of glucosephosphate isomerase, triosephosphate isomerase, and pyrimidine 5′-nucleotidase (P5N) deficiency are included among the relatively rare causes of HNSHA. In the case of some deficiencies, notably those of glutathione synthetase, triosephosphate isomerase, aldolase, and phosphoglycerate kinase, the defect is expressed throughout the body, and neurologic and other defects may be a prominent part of the clinical syndrome. There are no exact and verified figures regarding the occurrence of red blood cell enzyme disorders, other than the number of cases reported in literature. Basically, this is due to the lack of a certified registry. Also, like in disorders of the red cell membrane, some enzyme disorders will be difficult to identify because they are either very rare or clinically mild. At the same time, enzyme disorders may well be underdiagnosed due to lack of awareness and the fact that only a limited number of laboratories in the EU are capable of performing the complete panel of tests for detection of enzyme disorders of the red blood cell.

CURRENT DIAGNOSTIC APPROACH AND SCREENING METHODS FOR HEREDITARY SPHEROCYTOSIS Paola Bianchi Hematology and Transplant Unit, Pathophysiology of Anaemia Unit, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

DIAGNOSIS AND EPIDEMIOLOGY OF RED BLOOD CELL ENZYME DISORDERS Richard van Wijk Associate professor, Laboratory For Red Blood Cell Research, Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, The Netherlands Erythrocyte enzyme deficiencies may lead to hemolytic anemia; expression of the defect in other cell lines may lead to pathologic changes such as myopathy and neuromuscular abnormalities. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common erythro-

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ETHNICITY AND HAEMOGLOBINOPATHIES Simon Dyson Director of the Unit for the Social Study of Thalassaemia and Sickle Cell, Scientific Advisor of the Sickle Cell Society, London, and member of the University Hospital of Leicester of the Sickle Society and Thalassaemia Advisory Committee, UK

The UK was unique in Europe when it collected ethnic data for the first time in its 1991 Census. This enabled researchers to deduce likely sickle cell/thalassaemia carriers in UK population centres by comparing numbers allocating themselves into the bureaucratic categories of ethnic groups and what was known about likely carrier rates among such groups. Later, on the basis of local prevalence surveys, such ethnic figures were used by medical researchers to advocate for relevant screening/counselling services. Notions that screening for sickle cell/thalassaemia could be targeted by race were conceptually flawed, and adaptations of the UK census categories to capture Mediterranean, Middle Eastern and Mixed groups who would have been missed by the category White were proposed. These suggestions were tested in a randomized controlled trial of candidate ethnic/family origins screening questions with mixed results: improving the coverage of groups likely to be at risk but reinforcing mistaken beliefs in racial groups by both health professionals and clients. Some White British clients with sickle cell or thalassaemia

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

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GENETIC COUNSELING Adonis Ioannides Course Director, St George’s, University of London Medical Programme at the University of Nicosia, Cyprus

structural haemoglobin variants and the thalassaemia syndroms. They are the most common clinically serious single gene disorders in the world, and their homogygous or genetic compound states result in clinically significant phenotypes of variable severity. It is estimated that over 300,000 affected children are born each year, most with sickle cell disease while about 70,000 are born with βthalassaemia major. Most of these children are born in countries with limited resources and they do not receive the treatment they need, dying in childhood. The high frequency of the haemoglobinopathies, the expensive treatement scheme, as well as the avoidance of fatalities from untreated patients, were among the main reasons that many countries had developed prevention programmes for thalassaemia. The prevention programmes mainly include, screening for thalassaemia traits and abnormal haemoglobins, genetic councelling of the couples at risk which subsequently, will undergo prenatal diagnosis to avoid the birth of an affected child. The genetic services for haemoglobinopathies require close collaboration between several specialities,most notably haematology and molecular genetics. Most of the thalassaemia heterozygotes which are sympton-free present haematological and biochemical characteristics which are useful for their identification. Haematological methods contribute the basis of identification of carrier of the thalassaemias. Red cell indices and morphology, HbA2 quantification and Hb fraction separation are all used. Moreless, no one technic by itself is sufficient to prove the carrier status. Various haematological, biochemical and molecular methods must be implemented in order to get a carrier diagnosis. The results obtained from these methods must be evaluated and a flow chart will be presented illustrating the strategy and the diagnostic critiria which are necessary for the carrier identification. In Cyprus where the frequency of β-thalassaemia carriers is among the highest in the world (1 in 7 carriers), a national thalassaemia control programme was launched in 1978 and implemented succesfully ever since. There is no uniformity on a global basis for screening of Hb disorders and it is therefore logical that different countries in order to meet their own needs adopt methodologies adjusted to their needs and to their budgets.

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trait reportedly reacted in racist ways to their identification as haemoglobinopathy carriers. Classic anthropological evidence emphasizes that new ecologies promoting malaria were produced by the social practices of human agriculture, and were responsible for the spread of genes associated with sickle cell/thalassaemia. This undermines what was previously held to be core aspect of a sociological definition of ethnicity: that peoples could be defined objectively by ancestry and territorial affiliation (to which other, subjective, aspects of the concept ethnicity, such as customs and culture might then be attached). Original screening thresholds in England proposed that groups be defined as at-risk if the indigenous population of a country had a sickle cell/thalassaemia carrier rate of higher than 1 in 1000. Recent data from newborn screening in England suggest that even newborns allocated to the UK Census category White British may have carrier rates around 1 in 500, making White British people a high risk group for carrying genes associated with sickle cell/thalassaemia in terms of original screening thresholds. In conclusion, the example of the relation between ethnicity and haemoglobinopathies in the UK underscores the need for people living with sickle cell and thalassaemia to be accorded appropriate health services in Northern Europe. This is because, at heart, sickle cell and thalassaemia are health issues, not ethnic minority issues. Meanwhile, ethnicity as an analytical concept is itself in decline in sociological analysis, partly because its limitations have been exposed in addressing the issues of sickle cell and thalassaemia.

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The β-thalassaemias are very important genetic disorders of haemoglobin synthesis and are amongst the commonest monogenic disorders. In view of the severity of β-thalassaemia major, a number of screening programmes have been developed aimed at reducing the number of individuals born with the condition. Genetic counsellingplays a vital role in this process supporting the successful implementation of screening and delineating available options to at risk individuals. This review assesses the contribution of genetic counsellingat each stage of this process in the context of new diagnostic techniques and therapeutic options and discusses some of the more challenging aspects such as genotype/phenotype correlation and coinheritance of other genetic conditions or genetic modifiers. SCREENING METHODOLOGY OF HAEMOGLOBIN DISORDERS A. Kyrri, E. Kalogerou, D. Loizidou, X. Makariou, X. Ioannou, L. Kythreotis, G. Xatjilambi Thalassaemia Centre, Nicosia, Cyprus The inherited disorders of haemoglobin, called the haemoglobinopathies, are a large heterogeneous group of autosomal recessive disorders, which include both the

ADVANCES IN MOLECULAR DIAGNOSTICS FOR HAEMOGLOBINOPATHIES Joanne Traeger-Synodinos Assistant Professor, Laboratory of Medical Genetics, Athens University Medical School, St. Sophia’s Children’ Hospital, Athens, Greece More than 280 mutations causing β-thalassaemia have been described, the majority of which are nucleotide variations, and more than 100 α-thalassaemia mutations have been reported, most of which involve deletions from within the α-gene cluster. In addition more than 1150 mutations causing structural variants have been characterized, of which about 6 are clinically relevant (HbS, HbC, HbE, HbD Punjab, HbO Arab and Hb Lepore.). The majority of these mutations can be identified by PCR-based techniques, and many methods have been developed. Many of

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primers. Many NGS systems are now commercially available and the field is continuously advancing at a tremendous pace so technologies are rapidly improving. NGS is applied for research and clinical purposes, including: Identification of the molecular basis of rare, and so far undefined, Mendelian diseases; Targeted genetic testing for diseases caused by many candidate genes. For example retinitis pigmentosa (35 potential known genes); Investigation of cancer genetics by comparing the cancer genome to the germ-line genome in cancer patients; The study of complex diseases and traits and population genetics; Population wide (neonatal) screening and also for noninvasive prenatal diagnosis. For use in a clinical setting, NGS requires improvements with sequence quality, quality control, standardization of protocols, bioinformatics data, and management of the enormous amount of data per analysis. Additionally, ethical aspects need to be considered by patients, professional and the public in the light of the huge amount of information potentially generated for each genome analysed, at least until the prognostic significance of individual genotypes and genotype-interactions are fully understood. Assuming costs do fall, with respect to the haemoglobinopathies, NGS may potentially have a role by facilitating the simultaneous sequencing of all globin genes in a single test, along with any other genes which have been (will have been) confirmed to modify the clinical expression of patients with a haemoglobin disorder. With the rapid progress in NGS technologies perhaps NGS-based genetic testing may become a reality for some laboratories within the next few years.

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the traditional mutation detection methods are targeted to detect known mutations. For detecting nucleotide variations (point mutations) they include Amplification Refractory Mutation System (ARMS) PCR or restriction endonuclease analysis of PCR products (RE-PCR). For detecting common α- or δβ-thalassaemia deletions with known breakpoints, several gap-PCR protocols have been described. As a means to reduce the effort and cost when using targeted assays, scanning methods such as Denaturing Gradient Gel Electrophoresis (DGGE) have proved useful to identify or exclude regions of the genes containing the candidate mutation(s). DGGE is reliable and relatively inexpensive, although it is technically demanding and requires specialized equipment. Also it is imperative to subsequently characterize any nucleotide variation indicated, using either targeted direct mutation assays or automatic sequencing (see below). Overall, especially when addressing a relatively limited number of mutations, the aforementioned methods are generally suitable for the reliable and accurate identification of carrier mutations and prenatal diagnosis in laboratories with limited resources, since they are simple, relatively low cost. However, with a trend of globalization and a broadening of mutation spectrums in each geographical region, more generic methods for detecting nucleotide variations (point mutations) are becoming appropriate. Amongst all methods available, Sanger sequencing using an automated DNA sequencing instrument is probably the most comprehensive, capable of detecting any nucleotide variation within a selected region. However, it is relatively costly, which precludes its use by laboratories with limited budgets. Newer techniques for detecting nucleotide variations include real-time PCR (RT-PCR), mainly used for genotyping nucleotide variations, and High resolution Melting Curve Analysis (HRMA), a scanning method, analogous to DGGE, but more automated. For deletions and/or duplications newer methods include Multiplex Ligationdependent Probe Amplification (MLPA), and finally a non-PCR method using genotyping microarrays (array comparative genome hybridization, aCGH). The principles of application, advantages and pitfalls of these methods, along with experience to date, will be discussed. Despite the promise of new technological developments, to date no single method yet fulfils the ideal criteria of a universal, rapid, reliable, automated and inexpensive method. It is hoped that this may change with the application of the relatively recent new method of Next Generation Sequencing (NGS). NGS is millions of times more efficient than Sanger sequencing, allowing simultaneous sequencing of huge amounts of DNA in a single analytical procedure. For example, an entire genome can now be sequenced within a few days, at a cost that is fast approaching as little as 1000$ US. NGS can be applied to analyse the entire genome of an individual. Alternatively it can be more targeted by pre-selecting for only the protein-coding regions of the genome (otherwise known as exome sequencing) or even only for a more limited selection of candidate genes likely underlying a clinical phenotype. The latter two approaches involve the capture of target sequences in the randomly fragmented DNA sample by molecular probes or ‘baits’ in microarray formats, or bead capture in solution or microfluidics and PCR

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EUROPEAN INTEGRATED RARE DISEASES STRATEGY - EURORDIS’ ACHIEVEMENTS AND PERSPECTIVES John Dart European Organisation for Rare Diseases (EURORDIS) Thirty years ago, the patients were left with manyuncertainties, no networks and no access.The rarity of the disease made it difficult to identify specialists, little information could be found. Most of the patients were engaged in a longstanding process of obtaining the right diagnosis and treatments when these were available. Significant inequalitywas the only standard amongst European countries. In this context, over the last decades, the patients, their parents and relativeshave reached out to other families and established patient organisations for their own diseases in their own country and even beyond. People Living With Rare Diseases have shaped the concept of rare diseases as a public health policy issue: very heterogeneous diseases linked together around the specificity of rarity. Commonalities are: low prevalence, chronic, severe and often life threatening disease,limited scientific and medical knowledge base, lack of investments, winding road to diagnosis and treatment,social exclusion. Gradually, National Alliances of Rare Diseases were created to promote their rights at the national level. In March 1997, the European Organisation for Rare Diseases (EURORDIS) was established to build a strong panEuropean community and critical mass to voice patients’ common needs and expectations, the European level being

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

discrimination. We believe that what can be best performed at EU level is to be performed at EU level (e.g. orphan drug designation and marketing authorisation, clinical added value of orphan drugs, European reference networks, registries, standard of care) and what is best performed at national level is to be performed at national level (e.g. pricing andreimbursement of medicinal products, designation of centres of expertise, provision of care, information). We are confident in the future and shall pursue our efforts in improving the life of rare disease with an integrated, comprehensive and long term strategy… to achieve more success by 2020!

A PATIENT CENTRED APPROACH TO CARE PLANNING FOR PATIENTS WITH CHRONIC GENETIC DISEASES Alastair Kent Director, Genetic Alliance, UK and Chair, European Platform for Patients’ Organizations, Science and Industry (EPPOSI)

This essay proposes seven pre-requisites for the creation of effective programmes of care and support for patients living with the consequences of chronic genetic diseases. It then goes on to discuss the role of patient organisations and other stakeholders in bringing about the development and implementation of these.

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the most relevant to develop meaningful solutions. EU public health challenge: Rare Diseases have indeed a strong European added value due to the scarcity of each disease, widespread medical expertise across European countries as well as patients scattered all over Europe. As of today, Rare Diseases are high on the European public health political agenda:A vast majority of rare diseases still remain untreated, delays in diagnosis can vary from several months to even 30 years (The Voice of 12000 Patients), huge discrepancies in access to treatments exist both within and between Member States, even to marketed orphan drugs. However, compared to thirty years ago, the political landscape has changed a lot with efforts being made in providing incentives to enhance research in the field of rare diseases, offer better provision of careas well as a common European approach to speed up access to orphan drugs. EURORDIS and its members have been greatly involved in shaping the European political and regulatory environment for rare diseases. EURORDIS played a frontline advocacy role in the adoption of important EU Regulations and Directives for rare diseases patients: – Orphan Medicinal Products in 1999; – Medicinal Products for Paediatric Use in 2006; – Advanced Therapy Medicinal Products in 2007; – Patients’ Right in Cross Border Health Care in 2011. The organisation was also highly involved in contributing to founding political texts: – the European Commission Communication in 2008; – the Council Recommendation on action in the field of rare in 2009. EURORDIS’ representatives are involved in major committees at the European Medicines Agency (EMA) and at the European Commission, in the EU Committee of Experts on Rare Diseases (EUCERD). A success at a critical time. EURORDIS is building a unique, integrated, comprehensive and long term strategy to address Rare Diseases patients’ needs in Europe. The active role of patients’ representatives is now recognised as a major contribution to innovation and as catalysing cooperation and sustainable development. They have served in bringing together all stakeholders from researchers, industry, regulators and policy decision makers. European Member States are in the process of adopting national plans for rare diseases integrated within a coherent European policy framework. EURORDIS as well as National Alliances of rare diseases are actively involved in this process. EURORDIS is currently promoting rare diseases in European community programmes for the period 2014-2020 both in the fields of public health and research. EURORDIS’s advocacy priorities are to foster the creation and the development of national Centres of Expertise for rare diseases as well as European Reference Networks of centres of Expertise to facilitate exchange of information, access to proper diagnosis and available treatments. We are promoting the dissemination of best guidelines for diagnosis and care in Europe, the harmonisation of rare diseases registries, the expansion of European research and international research on rare diseases, the continued development of orphan medicinal products and their access to the patients in all European countries in a timely manner, without any

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THE DEEP PROJECT Evi Missouri-Khetab,1 Soteroula Christou2 11st Class Medical Officer, Coordinator of Education; 21st Class Medical Officer, Ministry of Health, In charge of the Thalassaemia Department, Makarios III Hospital, Nicosia, Medical and Public Health Services, Cyprus β-thalassaemia major is one of the most severe forms of anaemia. The recommended treatment consists in regular blood transfusions combined with chelating therapy to remove harmful iron accumulation in the body. The first chelating agent approved for clinical use was deferoxamine that, despite its potentially satisfactory therapeutic effects, is not usable by many patients due to its toxicity. Moreover, its subcutaneous route of administration leads to non compliance in most patients. On August 1999, an oral iron chelating agent, deferiprone, was authorised in Europe, and is currently indicated for the treatment of iron overload in patients with thalassaemia major when deferoxamine is contraindicated or inadequate. Despite a wide experience of deferiprone in thalassaemia patients, limited data are available on its use in children aged 2-10 years old and the need for additional data in this range of age is stated in the 2009 PDCO (the Paediatric Committee at the European Medicinal Agency) priority list. In addition, the anticipated benefit in controlling cardiac iron overload in all paediatric population as well as in other chronic transfusion dependent anaemias, lead the PDCO to expand the request for additional data to the cited patients populations. The DEEP Project has been developed with the specific intent to integrate the existing information on

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BONE MARROW TRANSPLANTATION Lawrence Faulkner Paediatrician, Advisory Board Coordinator, Cure2children Foundation, Italy

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The role of HbF inducers in ameliorating the pathophysiology and severity associated with hemoglobinopathies like sickle cell disease and thalassemia is well demonstrated. So many studies have focused on explain the pathways that involved in the re-activation of γ-globin gene expression. The HbF inducers are mainly includes chemotherapeutic agents (e.g. 5-Azacytidine and hydroxyurea), short-chain fatty acid derivatives (e.g. butyrates, valporate, sodium phenylbutyrate) and other HbF inducers like stem cell factor and synthetic gene-targeted transcription factors. These HbF inducers have significant effects on improve clinical manifestations of hemoglobinopathies and decreasing transfusion dependency. But regarding some limitations of these agents, more studies for the investigation of new agents with more safety and effectiveness is needed.

INDUCTION OF HbF Mehran Karimi, Nader Cohan Hematology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz-Iran

derived vectors, we expressed human anti-α-globin short hairpin RNAs, off a potent PolIII promoter (H1); of the 4 different shRNAs tested, α-globin mRNA reduction varied from 6.3 to 54% of the control CD34+ cells. Similarly, vectors developed for the mouse α-globin, resulted in a significant reduction (range 15-28% of the control) of α-globin in erythroid colonies of Lin- cells. To assay vector performance in vivo, we investigated the hematological parameters in thal3+/- mice transpalnted with FV-transduced Lin- cells, transduced with anti-αglobin shRNA. Despite low chimerism and low vector copy numbers (<0.5 per cell), we observed a 10% reduction in red cell distribution width, a marker for distorted erythropoiesis. We finally developed a combination FV vector expressing β-globin off a HS40 enhancer and antiα-globin shRNA and tested its performance in human CD34+ cells from a thalassemic patient. Globin chain imbalance was ameliorated from a β/α ratio of 0.12 to the level of 0.5, clearly indicating a therapeutic benefit.. Overall, shRNA control of α-globin excess is a feasible target but requires improvements since the RNAi effect is tough to predict and should ideally be combined with controllable elements.

deferiprone use in children aged 2-10 years old, thus covering the lack of knowledge and providing a valid support to the use of the drug in this age group. According to the recent scientific advancements and the opinion expressed by the PDCO, the project has been expanded to include the whole paediatric population and other chronic transfusion dependent anaemias. The aim of this application is to provide data on pharmacokinetics and comparative efficacy/safety of deferiprone, in order to produce an approved Paediatric Investigational Plan to be used for regulatory purposes. The trial is multicentric, open label and non-inferiority active- controlled. There are 12 participating centres from 9 different countries, including industry (Italy, UK, The Netherlands, Tunisia, Greece, Cyprus, Egypt and Canada). At the end of the proposed set of studies, deferiprone, will be available at efficacious dosages in children <18 years as first line treatment. In addition, a three year safety study will evaluate all deferiprone uses in the clinical setting.

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Even though severe thalassemia is a preventable disease, over 100,000 new cases are born yearly, particularly in the Middle East and South-East Asia. Most of these children may not reach adulthood because long-term appropriate supportive care is either inaccessible or unaffordable. Bone marrow transplantation (BMT) remains the only available definitive cure and success rates can be very high in appropriately selected patients, i.e. low-risk younger children with a matched family donor. In these circumstances BMT may be justified medically, ethically as well as financially, in fact, the cost of low-risk BMT is equivalent to that of a few years of non-curative supportive. This manuscript will briefly review the current status of bone marrow transplantation for thalassemia major with particular emphasis on a global prospective and present the experience of the Cure2Children Foundation supporting sustainable and scalable start up BMT programs in low-resource settings. The initial twelve consecutive patients managed in two start up BMT units in Pakistan (Children’s Hospital of the Pakistan Institute of Medical Sciences, Islamabad) and India (South East Asia Institute for Thalassemia, Jaipur) were included in this analysis. These initial six patients per each institution where purposely chosen as the focus of this report because they represent the steepest phase of the learning curve. The median age at transplant was 3.9 years, range 0.9 to 6.0, liver was no greater than 2 cm from costal margin, and all received matched related BMT. A structured on-site focused training program as well as ongoing intensive on-line cooperation was provided by the Cure2Children team of professionals.

DECREASE OF α-CHAINS IN β-THALASSEMIA M. Papadaki,1 G. Vassilopoulos1,2 1BRFAA, Department of Genetics and Gene Therapy, Athens, Greece; 2Division of Hematology, University of Thessaly Medical School, Larisa, Greece In the pathophysiology of β-thalassemia, globin chain imbalance plays a central role in predicting red blood cell (RBC) life span and disease severity. Strategies to improve globin chain imbalance are therefore a legitimate target in the management of this incurable genetic disorder. Classical gene addition with the available retroviral vectors can alter one of the two variables while combined reduction of α-chains could provide a more potent therapeutic effect. We developed foamy virus (FV) vectors for the production of β-globin and vectors targeting the αglobin transcript using the shRNA technology. Using FV-

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3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias 24-26 October 2012, Limassol - Cyprus

POSTERS SAFE BLOOD TRANSFUSION FOR CHILDREN WITH HAEMOGLOBINOPATHIES: ARE WE DELIVERING? A.M.J. Hughes, R. Jayatunga Sandwell and West Birmingham NHS Hospitals NHS Trust, UK

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Paediatric blood transfusions have a greater incidence of adverse outcomes compared to adults. In 2005 following a baseline audit, we designed an admission template incorporating the British Committee for Standards in Haematology (BCSH) guidelines on paediatric blood transfusion for our secondary care Trust. In 2010 the National Comparative Audit (NCA) of the use of Red Cells in Neonates and Children made new recommendations for safe transfusion. All children with β-Thalassaemia Major (BTM) and 10-20% of those with Sickle Cell Disease (SCD) require regular blood transfusions. This is a disruption to their lives and the onus is on healthcare professionals to deliver transfusions smoothly and safely. We have conducted an audit comparing current practice with guidelines and the NCA recommendations, with the aim of improving the quality of care delivered to these children. A retrospective case-notes audit was conducted on all Haemoglopinopathy patients who received a red cell transfusion during one year (11/08/2010 to 10/08/2011). Admission Templates used for these transfusions were analysed. The results were compared with previous audits done in 2005 and 2007. 31 transfusions were analysed; 74% were due to BTM and 26% for SCD. Positive results were demonstrated for documentation of patient details (100%), indication for transfusion (90%), nursing evaluation (90%), patient measurements (100%), observations (100%) and date/time (94%). When compared with previous audits, we demonstrated improvement in the documentation of patient details (88% 2005, 100% 2011), indication (66% 2007, 90% 2011) and observations (96% 2007, 100% 2011) and decline documenting the benefits of transfusion (90% 2007, 48% 2011). Admission Template to be redesigned to include the prescription for blood transfusion on the reverse side, enabling all information relating to the transfusion episode to be documented together. Profile of blood transfusions is to be raised with appointment of Transfusion Nurse Champions and regular updates to doctors and nurses emphasising importance of documentation and distribution of leaflets etc. While some outstanding results were noted, there remains the need for improvement. During close monitoring of transfusion practice for six years and there have been no adverse events. We remain committed to improving upon the smooth delivery of safe blood transfusions to children with Haemoglobinopathies.

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The S-303 Treatment System for RBCs uses S-303 to crosslink nucleic acids and prevents replication of contaminating pathogens and leukocytes. S-303 treated RBCs have met the FDA criteria for 24-h recovery and clinical evaluations in patients with Thalassemia major are planned to assess safety and efficacy after repeat transfusions. RBCs were prepared from CPD WB collections (450 mL) held at 4°C or controlled ambient temperature prior to separation. On D (Day) 1 WB units were leukocyte-filtered, separated into platelet poor plasma and RBC suspended in SAGM. Paired ABO matched RBCs were combined and split for each replicate into Control (C) stored at 4°C and Test (T) processed with the S-303 system (0.2 mM S-303 and 20mM GSH). For in vitro studies, T and C RBCs were stored at 4°C for 35D. For pathogen inactivation (PI) studies, RBCs (ambient WB hold) were spiked with pathogens (S. aureus, Y. enterocolitica, E. coli, S. marcescens, Ad5, CA-HIV, BV, BVDV; n=4) then processed. RBCs were incubated for 3 hours at ambient temperature before sampling for residual viable organisms on rich agar plates (bacteria) or cell culture (viruses). The Hb content of T was 55.7±4.3g (n=12), with a process loss of 2.5±1.6%. Plasma protein was reduced 15-fold [54 mg/dL (T); 800 mg/dL(C)]. D35 hemolysis was lower in Test (0.19%) versus Control (0.28%; P<0.05). Some attributes were statistically different between T and C (Hct, pH, K+, lactate, ATP); however, all results were within reported physiological ranges for RBCs for transfusion. Viruses were inactivated to or below the limit of detection (≥4.4 to >5.9) and inactivation of all bacterial species was greater than 4 log. S-303 treated RBCs met current EU and US guidelines for leukocyte-reduced RBCs in additive solution. PI efficacy was demonstrated across a range of bacteria and viruses.

S-303 TREATMENT SYSTEM FOR PATHOGEN INACTIVATION OF RED BLOOD CELL COMPONENTS (RBCS) Anna Erickson, Debbie Hanson, Betsy Donnelly, Mary Ann Schott, Daniel Arnold, Tracey Diaz, Kent Dupuis, Lynette Sawyer, Nina Mufti Cerus Corporation, Concord, CA, USA

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Most patients suffering from β-thalassemia are dependent on regular blood transfusions which cause hemosidrosis in vital organs like liver and heart leading to organ damage and early death. Increased survival in patients with βthalassemia has allowed for several morbidities like renal dysfunction to manifest. This involvement suggests the need for a reliable and non invasive method to assess the degree of kidney iron overload. The present study was conducted to evaluate the relation between serum ferritin levels, liver and heart MRI T2* relaxation times in thalassemic patients, and their kidney MRI T2* relaxation times as a step to evaluate the feasibility of using MRI T2* to assess the degree of kidney iron overload. This was a prospective study of 120 regularly transfused thalassemic patients (mean age=25.9±9, 60 women and 60 men) suffering from major and intermediate thalassemia from an adult thalassemia clinic located in Tehran, Iran. Cardiac, hepatic and renal MRT T2* were performed. Serum ferritin levels were also measured. Our results indicated a moderate correlation between kidney MRI T2* relaxation time and serum ferritin (r=-0.446, P<0.001). Kidney MRI T2* relaxation time was weakly correlated with liver MRI T2* relaxation time (r=0.388, P<0.001) and cardiac MRI T2* relaxation time (r=0.338, P=0.023) respectively. Moderate correlation between kidney MRI T2* relaxation time and serum ferritin, and its weak correlation with liver and heart T2* relaxation times indicate that relying on these tests to predict the exact condition of kidney iron overload is not a feasible approach. This suggests the need for performing the kidney MRI T2* as a noninvasive approach for evaluating renal iron overload in thalassemic patients. Further studies to investigate the relation between kidney MRI T2* relaxation times and renal function in thalassemic patients is suggested.

T2-STAR (T2*) MAGNETIC RESONANCE IMAGING FOR ASSESSMENT OF KIDNEY IRON OVERLOAD IN THALASSEMIC PATIENTS Mozhgan Hashemieh, Azita Azarkeivan, Shahram Akhlaghpoor, Afshan Shirkavand, Kourosh Sheibani Assistant professor of pediatric hematology/oncology, Imam Hossein Medical Center, Shahid Beheshti University of Medical Science, Tehran, Iran

uate the compliance of couples planning marriage; iii. to determine frequency of β-thalassemia trait among couples; iv. ultimately, to prevent β-thalassemia major. All couples applying for marriage were asked to do CBC on an electronic cell counter. Those with MCV<79 fL were asked to do HbA2 and values >3.5% were diagnosed as B-Thal Trait. If one of the couples is carrier and the other is free, they were given thalassemia-free certificate to proceed in marriage. If both were carriers, they were advised to cancel the marriage. If refused, they were referred to Legitimate Court Qadi Qudah (QQ) for a final decision. There are around 14,273 marriage applications submitted annually to religious courts in the West Bank. From 2001-2009, a total of 140,482 subjects were screened for thalassemia. Of them, 8148 had low MCV and 1904 of them were found carriers for β-thalassemia and none of them had MCV>78.8. In 220 cases, both partners were carriers and their marriages were cancelled either voluntarily or by QQ. Before year 2000, the incidence of thalassemia in West Bank was 29-33 cases while during 2003-2009 it was 5-7 cases. Most of thalassemia patients born after May 2000 resulted from old marriages but 14 out of 80 cases were the result of new marriages. Of which, 4 marriages proceeded abroad and the other 10 cases were the result of misdiagnosis. As the incidence of thalassemia declined sharply in Palestine after year 2000, this study indicates that premarital testing is a very useful tool for detecting carriers and an effective way of controlling β-thalassemia major.

DIAGNOSTIC AND MONITORING TECHNIQUES

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EXPLORING POPULATION AWARENESS AND ATTITUDE TOWARDS HAEMOGLOBINOPATHIES PREMARITAL SCREENING PROGRAM IN DUBAI Kareema AL Arrayed, Rekha Bajoria, Ratna Chatterjee, Khawla Belhoul, Jameel Abu Laban Latifa Hospital, Dubai Health Authority, Unite Arab Emirates

EPIDEMIOLOGY AND PREVENTION PREMARITAL TESTING FOR β-THALASSEMIA TRAIT: PALESTINE EXPERIENCE Khalid Younis Clinical Hematology, Al-Quds University, Palestine Palestine was the first Arab Moslem country to apply the mandatory premarital testing for β-thalassemia in May 2000. The main objectives are: i. to asses the effects of the program on incidence of of β-thalassemia; ii. to eval-

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This study was of a retrospective and a prospective parts. The retrospective part was to study a large sample of HEHPLC tests. All tests were performed as part of premarital screening. Analysis of HE-HPLC reports helped in identifying affected individuals, who were attempted to include in a subsequent population survey. The final group of participants of 84 individuals were interviewed over the phone using a questionnaire exploring knowledge and attitudes towards Haemoglobinopathies, this was the prospective part of the study. Of the 2443 random samples of HPLC reports, 341 (14.0%) were identified with abnormal haemoglobin genotypes. The most commonly identified Haemoglobino-pathies were α-thalassaemia trait (8.1%), β-thalassaemia trait (3.5%), Sickle cell trait (2.2%) and Sickle cell anaemia (0.2%). The sample included 1156 (47.3%) UAE nationals, of whom 237 (20.5%) were affected by one of the Haemoglobinopathies. Within this population, most commonly identified Haemoglobinopathies were α-thalassaemia trait (12.1%), β-thalassaemia (4.4%), Sickle cell trait (3.6%) and Sickle cell anaemia (0.3%). The study showed that β-thalassaemia incidence in UAE is 4% and the survey showed that there was general lack of knowledge on screening procedures, genetic disorders,

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

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The prevention programme of inherited haemoglobin disorders, arranged by Centro Studi Microcitemia Roma (CSMR) since 1975, entails two different types of carrier screening on a voluntary basis: an universal screening offered to secondary school students and a screening offered to young adults. These screenings, carried out in the Italian region called Latium, include providing information to the population, detection of healthy carriers and offering genetic counselling for couples at-risk for βThalassaemia Major (BTM) or Sickle Cell Disease (SCD). The principal aim is the early identification of carriers to reduce the incidence of Thalassaemia major and other severe haemoglobin related disorders. In 36 years of scholastic screening (from 1975 until 2011), 1,466,100 students have been examined and 26,786 (1.8%) carriers of non-α thalassemias have been identified. In the extra-scholastic screening, 388,690 adult subjects (including the carriers’ relatives) have been examined and a total of 38,457 (9.9%) carriers of non-α thalassemias have been detected. New reported cases of thalassemia has been progressively reduced and since 1993 thalassemic patients are no longer born in Latium from autochthonous unaware (not informed) couples at risk. The results demonstrate the many benefits offered from this prevention strategy: through the precocious identification of healthy carriers it allows the prospective identification of at-risk couples, offering them all medical information and options available to prevent the birth of affected children; it has a positive impact on the population, with a good adhesion by both native and immigrant people. In contrast to other screening programmes, it does not involve huge resources and is relatively inexpensive. It is therefore fundamental to continue the prevention programmes in order to reach the complete population that is epidemiologically changing, due to unceasing migratory fluxes from countries in which haemoglobin disorders are common.

A multiethnic society requires to be ready to offer prevention, diagnostics and care to carriers of haemoglobin disorders and to affected people. In the 70’s, the endemic incidence of β-thalassaemia in Latium stimulated the urgent need of a prevention and health education program. After 35 years of application, prevention is totally prospective in autochthons: couples at risk are diagnosed and informed before their first pregnancy. Latium (Central Italy) is one of the Italian region with the highest incidence of immigration. The CSMR has started to monitor the changes in at risk population from endemic areas adjusting the existing screening strategies and offering the same preventive opportunities as for the natives. The health education program for immigrants consists in: – Interaction with multiethnic associations to promote information. – Screening extension to the foreign students. – Genetic counselling for at risk couples, unaware of their carrier condition. – Use of specialized technology for the detection of new molecular spectrum. In 18 years, between 1994 and 2011, a total number of 16,624 subjects from ethnic minorities has been examined at CSMR: 4,639 were carriers of haemoglobin disorders. The most represented groups were from East Europe, followed by Africa, Asia, America. During this screening we observed the increase in number of foreign affected subjects: haemoglobinopaty disease cases are 180 (4% of the screen positives). They include SCD, βThal Maior, Thalassaemia Intermedia and HbH disease, caused often by molecular defects uncommon in Italian population. Until now prevention for immigrants has been mostly retrospective: since 1980 to 2011, 74 immigrant couples at risk were identified and counselled, 45 were retrospective, 29 were prospective. As a consequence of the epidemiological changes, the new challenge is to offer equal access to prevention and medical care among immigrants, in order to reach the same positive results achieved in Latium population.

CARRIER SCREENING FOR INHERITED HAEMOGLOBIN DISORDERS AMONG SECONDARY SCHOOL STUDENTS AND YOUNG ADULTS IN LATIUM, ITALY Antonio Amato,1 Maria Pia Cappabianca,1 Maria Lerone,1 Alessia Colosimo,2 Paola Grisanti,1 Donatella Ponzini,1 Paola Di Biagio,1 Maria Perri,1 Debora Gianni,1 Silvana Rinaldi,1 Roberta Piscitelli1 1ANMI Onlus, Centro Studi Microcitemie, Rome, Italy; 2Department of Comparative Biomedical Sciences, University of Teramo, Italy

EPIDEMIOLOGY AND PREVENTION OF THALASSAEMIA AND HAEMOGLOBINOPATHIES AMONG LATIUM IMMIGRANTS: BUILDING ON KNOWLEDGE AND EXPERIENCE AT ANMI ONLUS - CENTRO STUDI MICROCITEMIE OF ROME (CSMR) Antonio Amato, Paola Grisanti, Maria Lerone, Donatella Ponzini, Paola Di Biagio, Maria Pia Cappabianca, Enrica Foglietta, Fabrizio Mastropietro, Loredana Masi ANMI Onlus, Centro Studi Microcitemie, Rome, Italy

and clinical outcomes. Overall, participants’ attitude towards premarital screening was very positive. Alarmingly though, most participants did not care about the outcome of their tests. This clearly indicates the need for improvement in education.

FAMILY PLANNING PRACTICE AMONG FAMILIES WITH CHILDREN AFFECTED BY β-THALASSEMIA MAJOR IN SOUTHERN IRAN Mehran Karimi, Sezaneh Haghpanah, Sheyda Johari, Shirin Parand, Mohammad Reza Bordbar Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Prevention of the birth of children with β-thalassemia

Thalassemia Reports 2012; 2 (s2) | 15 |

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PREVALENCE OF HAEMOGLOBIN DISORDER IN KUWAITI NATIONALS ATTENDING PREMARITAL SCREENING CENTERS IN KUWAIT Reem Gh Al-Fulaij, R. Chatterjee, R. Bajoria MSc in Haemoglobinopathy, UCL, London, UK

Haemoglobin disorders are the most common clinically serious single gene disorders in the world. This study aims to estimate the prevalence of haemoglobinopathy in adult Kuwaiti population screened as part of Kuwait’s premarital screening program and disclosing the effect of consanguinity on the prevalence of these disorders. Retrospective review of couples attending premarital screening centers from 1 January 2010 to 31 December 2010. Blood samples obtained to check for β-thalassaemia and sickle cell. Couples found to be carriers of hereditary blood disorders were offered genetic counseling. Total of 12,138 prospective couples were tested, age 18-62 years median age 24. Out of 12,138 couples screened, 78 couples were found to have at least one partner carrying some kind of haemoglobinopathy. One partner of 46 couples was found to have β-thalassaemia trait, one of the partners of 23 couples having sickle cell trait, while one partner from 2 couples had sickle cell disease. Both partners from 7 couples were found to have sickle cell trait. 24 couples were declared high-risk. β-thalassaemia trait and sickle cell trait were the commonest type of haemoglobinopathy detected (3.7%). This study and further studies in the same field will help in designing proper awareness programs and help policy makers to modify the current Kuwaiti law to a more firm law that would help prevent the marriage of high risk couple.

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NHS SICKLE CELL AND THALASSAEMIA SCREENING PROGAMME Radoslav Latinovic, Elizabeth Dormandy, Allison Streetly NHS Sickle Cell and Thalassaemia Screening Programme, King’s College London, UK

with known outcomes 2/3 opted to terminate. Gestation as well as condition diagnosed being important factors in termination decision making. 17 out 21 β pregnancies affected by β-Thalassaemia Major opted to terminate. Nearly 690,000 babies were screened, identifying 1 in 2000 screen positive babes for a significant haemoglobinopathy and 1 in 70 babies born in England is a sickle cell carrier, ranging from 1/27 in London to 1/200 in South West. Around 20 f-only babies (likely to be β-thalassaemia major) are born each year in England. Data collected by the screening programme is used to inform policy, e.g. newborn carrier rates used to designate antenatal prevalence, and identify areas for quality improvement.

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major (β-TM) is one of the most important health issues. We investigated family planning practice and related factors among families with affected children. A total of 587 parents of children with β-TM (429 mothers and 158 fathers) were randomly selected from the parents of patients with β-TM who were registered in referral thalassemia clinic in southern Iran. Required information including demographic variables, socio-economic status, and family planning practice were collected. The correlation of family planning practice with related factors was evaluated. Approximately 96% of the parents were practicing contraception at the time of the study. However, only 12.8% of the families whose first child was thalassemic had decided to have no more children. The most frequent contraceptive methods were tubal legation (37.5%) followed by oral contraceptive pill (31.5%). Higher educational level of mothers and higher economic status of the families were found to be related with the lower number of children with β-TM (P=0.001). We determined a high percentage of using safe contraception in at-risk couples. It seems that education programs have been effective on family planning practice. Further attention should be taken into account for increasing knowledge of these at- risk couples with more focus on fathers and parents with low socio-economic status. In Iran due to cultural factors many of these at-risk couples insisted on planned pregnancy to achieve a desired family size, so implementation of a well organized prenatal diagnostic system seems necessary.

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NHS Antenatal and Newborn Sickle Cell and Thalassaemia Screening Programme is fully implemented in England. Newborn screening is universal and offered to all babies while the antenatal screening algorithm depends on prevalence. Aims of the screening programme are to: – Support people to make informed choice during pregnancy and before conception. – Avoid deaths and reduce morbidity. – Promote greater understanding and awareness of the disorders. Screening programme routinely collects data from antenatal, PND and newborn laboratories in England. We have collected a wealth of data that provides a good insight into the sickle cell and thalassaemia screening in England. Newborn data on screen positive results and carriers are available by ethnicity and regional level, while antenatal data (mothers’ haemoglobinopathy status and pregnancy risk) are available by maternity units. In 2010/11 over 700,000 antenatal screening tests were reported to the screening programme with 37% pregnant women being tested before 10 weeks. 17,000 women were identified as screen positive, 9000 fathers were tested and 940 high risk pregnancies were reported in the year. There were 420 PNDs with 95 affected foetal results and of those

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EXPLORING SUSTAINABLE FOLLOW-UP STRATEGIES FOR THALASSEMIA SCREENING, CONTROL AND MANAGEMENT: LESSONS FROM BANGALORE, INDIA Shalini Gambhir, Nutan Dighe, Ankit Mathur, Kamala Eshwar, Shankar AG, Govind Govind, Malashri Biradar, Latha Jagannathan Bangalore Medical Services Trust, Rotary Bangalore TTK Blood Bank, Bangalore, India β-Thalassemia prevalence in India is 1%-3% and higher amongst certain ethnic communities. The prohibitive costs per patient at $1800 p.a., vulnerability to transfu-

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

demiological data is not available for Germany and, consequently, the German NBS programme does not comprise SCD. Between 1 February 2012 and 4 July 2012, 14,797 consecutive babies that were born in Berlin between 1 September 2011 and 4 April 2012, were screened for SCD by means of high-performance liquid chromatography (HPLC) done from dried blood spots. As required by the local ethics committee, samples were pseudonymised prior to the study. Results indicative of SCD, sickle cell trait (SCT), β-thalassaemia or any haemoglobin variant other than haemoglobin S (Hb S) were confirmed by capillary electrophoresis. The haemoglobin patterns of seven newborns were consistent with SCD and 46 babies were identified as Hb S carriers. In another 16 newborns, variant haemoglobins were detected. Although it is possible that these results overestimate the prevalence of SCD in Berlin due to a random error, they suggest that the incidence of SCD is higher than 1.5/10,000 births (P=0.0078), which is a threshold that is widely accepted to define a population at high risk. It is unquestionable that further data is required. Nevertheless, these preliminary results provide evidence that screening for SCD could be a reasonable measure in Berlin.

HAEMOGLOBINOPATHY SCREENING AND PREVENTION IN NORTHERN GREECE. A 10 YEAR SURVEY S. Theodoridou, M. Alemayehou, N. Prappas, Th. Daglis, V. Aletra, O. Karakasidou, E. Boutou, A. Balassopoulou, K. Sinopoulou, V. Delaki, E. Avramidou, V. Alwadi, A. Agapidou, A. Manitsa, A. Loutradi, E. Voskaridou Greece is a Mediterranean country with a high frequency of thalassaemia and haemoglobinopathies. Since 1974 a nation wide program for Thalassaemia prevention has been implemented in our country. The aim was to increase awareness of the public concerning these hereditary anaemias, to screen couples, to genetic counsel and perform prenatal diagnosis. Natives and immigrants are screened free of charge. We report the results of the prevention program in Northern Greece, over a 10year period (2002-2011). A total of 25.520 subjects have been screened. Among them a total of 2602 couples were screened and counselled in our Thalassaemia Prevention Unit. In 259 cases (9.9%), both partners carried an abnormal haemoglobin gene. 248 out of 278 pregnancies, (5 twins pregnancies) were at risk of giving birth to a sick offspring carrying either the homozygous or doubly heterozygous forms of the mutations under discussion. Thirty-two pregnancies involved couples who were heterozygous for mutations that did not cause severe clinical disease, and were exempted from prenatal diagnosis. These gene interactions were β-thal/α-thal, HbΕ/HbΕ, HbΕ-Saskatoon/HbS, HbΕ-Saskatoon/β-thal, HbΟΑrab/HbΟ-Αrab, HbD-Punjab/α-thal, HbD-Punjab/βthal. Prenatal diagnosis was carried out by chorionic CVS (n=218), by amniotic fluid sampling (n=21) and by foetal blood sampling (n=4), In three cases a transvaginal procedure was performed. There has been one obstetric complication out of 248 procedures. Sixty-three (26%) foetuses were found to be homozygotes or double heterozy-

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sion-related complications and the loss of quality of life for those affected, makes it imperative to implement an effective Thalassemia prevention program urgently. Negligible awareness about Thalassemia and its causes explains why, in India, only 5%-10% of Thalassemia patients receive optimal care and many die undiagnosed and untreated before age five. Rotary Bangalore-TTK Blood Bank, Bangalore Medical Services Trust (BMST) has been supporting Thalassemia patients with free blood transfusions since 1984. BMST initiated a Thalassemia Control program in 2009 in the State of Karnataka, South India using the Cascade Screening and Testing model. The program has the support of Government and private transfusion service providers, funding partners and Thalassemia Society, Bangalore. Significant members of the Thalassemia patient’s families are recruited to act as Family Nodal persons to motivate their relatives to participate. Trained counselors then educate and provide pre-test counseling to the family members. Those giving informed consent are tested using Variant Hemogram and Molecular Analysis and the carriers and their families are offered Post-test, genetic counseling. Long-term follow-up of the carriers for future services such as pre-marital counseling, partner screening and pre-natal diagnosis is again facilitated using the Family Nodal persons. Our strategy of using Family Nodal persons has been very effective with 91% of people contacted willing to participate, carrier detection around 40% and genetic counseling availed by 97% of families with carriers. Making the Family Nodal persons responsible for long-term follow-up within their families is working well. For culturally-sensitive, family-oriented populations like India the BMST model of using significant members of the family to help implement a Thalassemia control program could prove effective and sustainable in the long term.

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NEWBORN SCREENING FOR SICKLE CELL DISEASE IN BERLIN – AN ANONYMISED STUDY OF 12000 NEWBORNS Stephan Lobitz,1,4 Claudia Frömmel,2 Oliver Blankenstein,3 Rekha Bajoria,4 Ratna Chatterjee4 1Department of Paediatric Oncology/Haematology/BMT; 2Department of Laboratory Medicine; 3Berlin Brandenburg Neonatal ScreeningLaboratory, Charité, Universitätsmedizin Berlin, Germany; 4University College London, Institute for Women’s Health, London, UK Sickle cell disease (SCD) is an autosomal recessively inherited haemoglobinopathy associated with severe complications that may cause significant morbidity or even mortality before the disorder is diagnosed. As the diagnosis can be made very easily from a liquid blood sample (e.g. cord blood) or dried blood spots, many countries have decided to include SCD testing in their newborn screening (NBS) programmes. Among the indigenous German population, SCD is absent. However, as the number of immigrants from countries at high risk for haemoglobinopathies increases, the prevalence of SCD rises steadily. Nevertheless, robust epi-

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Posters

Thalassemia major (TM) is an autosomal recessive disorder where the parents are asymptomatic β-thalassemia carriers (bTT). On screening 2000 voluntary blood donors from north India we have found the prevalence of bTT to be 3.5%. On analyzing the caste structure we found majority occurring in the khatri-Arora and Jat Sikh castes. Though >300 mutations are described worldwide, few mutations occur in an ethnic background. We analyzed the spectrum of mutations in 1500 TM alleles by PCR-ARMS and β globin gene sequencing from genomic DNA from the peripheral blood. Prenatal diagnosis was offered to 570 pregnancies has been carried out in the last 14 years on DNA from chorionic villous biopsy/amniotic fluid. The pregnancy outcomes and molecular data of these cases were analyzed. The common five Indian mutations were found in 86.8% alleles; commonest being IVS 1,5 (G-C) in 29.4%, followed by Fr 8/9 (+G) in 21%, 619 bp deletion in 17%, IVS 1,1 (G-T) in 8.5% and Fr 41/42 (-TTCT) in 10.8%. Codon 16 (-C) was seen in 4% and the prevalence of uncommon b++ mutations -88 (CT) and Cap+1 (A-C) was 2.6 and 2% respectively. Rare mutations constituted 4.5% alleles and many were identified by β globin gene sequencing. Only 0.1% alleles remained uncharacterized. Some of the mutations could be identified to belong to certain castes or one third of the families had migrated from Pakistan before the partition between India and Pakistan. This finding is based on the fact that most arranged marriages occur within the caste. Results of the prenatal diagnosis of 570 pregnancies showed that in 25.7% cases the fetuses were normal, 49.8% cases were bTT, 23.2% were TM and in 1% cases we were unable to distinguish between bTT and TM. Seven cases had twin pregnancies and all except one case showed similar normal/ bTT/ TM status. This application has helped to prevent the birth of 132 children with thalassemia major in the region and alleviated the anxiety of 430 pregnancies to have children who were either normal or carriers.

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HEMOGLOBIN E AND ITS VARIATIONS IN NORTHERN GREECE. EPIDEMIOLOGICAL SURVEY S. Theodoridou, M. Alemayehou, E. Vlachaki, A. Agapidou, Th. Daglis, V. Aletra, O. Karakasidou, E. Boutou, A. Balassopoulou, V. Delaki, K. Sinopoulou, E. Avramidou, V. Alwadi, E. Voskaridou Greece is a Mediterranean country with a high frequency of thalassaemia and haemoglobinopathies. Hemoglobin E is an inherited variant that results from a substitution of lysine for glutamic acid in the 26th position of the beta (β) globin chain. This nucleotide abnormality also results in mild thalassaemia due to decreased β globin chain production. Haemoglobin E has the highest frequency among people of Southeast Asian heritage. There are other encountered haemoglobin patterns that involve haemoglobin E (Hb E-Saskatoon). Hemoglobin E-Saskatoon (β22Glu>Lys) seems to be an innocuous variant and there are few data available for the association with other haemoglobinopathies. The homozygous state for hemoglobin E (E/E) has a mild degree of anaemia with slightly reduced red-cell survival. The heterozygous state for haemoglobin E is asymptomatic. The aim of the study was to investigate the incidence of Hb E in Northern Greece. A total of 88,003 subjects were screened for heamoglobinopathy in our prevention unit from 1986 since 2011 at the regions of Central and Western Macedonia in Northern Greece with a population of around 2.5 million. The carrier identification was carried out by a standard scheme which included CBC and red cell indices, a Cation Exchange HPLC, electrophoretic techniques, sickling test and measurement of serum ferritin levels. We found 16 heterozygous carriers of Hb E immigrants from Asia, 2 homozygotes for Hb E also fom Asia, and one compound heterozygote for α-thalassaemia and Hb E of Greek origin. Hb E-Saskatoon was found in 6 subjects in heterozygous form, 1 with S- E-Saskatoon and 1 with E-Saskatoon/β ΜΑ.They all had a Greek origin. Identification of Hb E is made by haemoglobin electrophoresis, ion exchange high pressure liquid chromatography (HPLC) as well as DNA studies. It is important to distinguish Hb E disorders because of marked difference in clinical course because of the severe clinical course when they combine with β-thalassaemia. Hb ESaskatoon though when combined with thalassaemia has a mild clinical course. So DNA-based diagnosis of Hb E disorders is optimal and necessary before genetic counseling is undertaken.

SPECTRUM OF GLOBIN GENE MUTATIONS IN NORTH INDIAN PATIENTS WITH THALASSEMIA MAJOR: APPLICATION IN PRENATAL DIAGNOSIS Reena Das,1 Jasbir Kaur,1 Sanjeev Chabra,1 Jasmina Ahluwalia,1 Amita Trehan,2 Subhash Saha,3 Rashmi Bagga,3 Deepak Bansal,2 Inusha Panigrahi,2 R.K. Marwaha,2 G. Garewal1 Departments of 1Hematology, 2Pediatrics and Obstetrics and 3Gynaecology, PGIMER, Chandigarh, India

gotes for clinically significant mutations. These couples were informed of the danger of having a sick child but, as to the termination or continuation of the pregnancy, it was left to the couples to decide for themselves. Nevertheless, all the couples preferred termination of the pregnancies. Our conclusion is that, wide spread population screening for thalassaemia and the implementation of prenatal diagnosis have both decreased the incidence of children born with Thalassaemia Major and Sickle Cell disease. This Thalassemia screening program has proved successful in our case as it has in many other countries that have implemented it.

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3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

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Increased expression of fetal hemoglobin (HbF) may ameliorate the clinical course of haemoglobinopathies like sickle cell disease (SCD) and β-thalassemia. Hydroxyurea (HU) is capable of stimulating HbF production during adult life. Clinical response to HU treatment in patients with haemoglobinopathies is variable, therefore, the presence of non-responders and the potential toxicity of the drug to prolonged treatment have suggested the need to develop an in vitro test to predict the patient’s response to HU before starting the therapy in vivo. In this study we evaluated whether the response of patients with SCD (βs/βs, βs/βthal) and thalassemia intermedia (βthal/βthal) to the treatment with HU in vivo, corresponds to that of primary erythroid cultures derived from the same patients treated with the drug. Liquid primary erythroid cultures. This system reproduce in vitro the erythropoiesis that occurs in the bone marrow. It allows to have cultured cells producing hemoglobin to which it is possible to add a drug for studying the response. QRTPCR assay. To quantify the increase of gamma-globin mRNA. We developed primary erythroid cultures from peripheral blood haematopoietic stem cells of 45 patients (26 βs/βthal, 4 βs/βs and 15 βthal/βthal). The cells were treated with HU and the fold-increase of gamma-globin mRNA was evaluated by QRT-PCR. The same patients were then treated in vivo and the increase of HbF (g/dl) was compared with the increase of gamma-globin mRNA in HU treated cultured erythroid progenitors. The results show that there is a correspondence between the response of erythroid progenitors in terms of gamma-globin mRNA fold-increase and the response in terms of HbF fold-increase observed in vivo. These data indicate that the quantization of gamma-globin mRNA in erythroid cultures from SCD and thalassemia intermedia patients can be considered a parameter predictive of the response to HU treatment in vivo.

The clinical expression of β-thalassaemia, the phenotype, cannot always be fully explained even though the genotype comprising of the β- and α-globin gene loci mutations as well as the Xmn1 polymorphism has already been determined for all thalassaemia patients in Cyprus. Six BCL11A polymorphisms were determined for all the patients in the study based on the evidence that BCL11A gene plays an important role in modulating HbF levels, in an attempt to gather information about the frequency of these variants and to analyse their impact on the patient phenotype. A total of 131 thalassaemic patients aged between 2 to 69 years old, all regularly followed up at the Larnaca Thalasaemia clinic were included in this study 66 patients were males and 65 were females. Clinical data including haematological parameters, frequency of transfusions, volume of blood transfused cardiacT2*, liver T2* and DEXA tests for osteoporosis were collected from patients’ records. For six of those polymorphisms it was possible, using the online tool NEBcutter V2.0 to identify suitable restriction enzymes that allowed us to easily distinguish between the alternative forms of each polymorphic site. The four SNPs in question are Rs11886868 (C/C genotype), Rs4671393 (A/A genotype), Rs7557939 (G/G genotype) and Rs766432 (C/C genotype).The frequency of these SNPs in the group of homozygous β-thalassaemia patients under investigation is 11.4%, 7.6%, 12.2% and 9.9% respectively and for Xmn1 polymorphism at 10.6%. The determination of six BCL11A polymorphisms in this group of patients was informative about the clinical expression of the disease as some of these polymorphisms act as important ameliorating factors of the β-thalassaemia phenotype.

QUANTIFICATION OF GAMMA-GLOBIN MRNA FOLD-INCREASE IN PRIMARY ERYTHROID CULTURES: A PARAMETER TO PREDICT THE RESPONSE OF SICKLE CELL AND β-TALASSEMIA PATIENTS TO HYDROXYUREA TREATMENT Alice Pecoraro, Paolo Rigano, Antonio Troia, Roberta Calzolari, Santina Acuto, Barbara Spina, Elena Baiamonte, Rosalia Di Stefano, Disma Renda, Aurelio Maggio, Rosalba Di Marzo U.O.C. Ematologia II, A.O.C. Villa Sofia, Palermo, Italy

EXISTENCE AND FREQUENCY OF MOLECULAR CHARACTERISTICS FAVOURING FOETAL HAEMOGLOBIN PRODUCTION IN CYPRIOT THALASSAEMIC PATIENTS Maria Sitarou, Marina Kleanthous, Marios Phylactides, Lederer W. Carsten, Marilena Epitropou, Myria Zachariou, Miranda Petrou, Eleni Karitzie, Rekha Bajoria, Ratna Chatterjee Thalassaemia Clinic, Larnaca General Hospital, The Cyprus Institute of Neurology and Genetics, Course Directors of Msc in Haemoglobinopathy, Cyprus

GENE REGULATION AND THERAPY

HBF INDUCTION Mehran Karimi Professor of Pediatric Hematology, Oncology, Hematology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran Hemoglobin F is the major Hb of the fetus and the newborn infant. It consists of two α-globin changes and two γ-globin changes (α2 γ2). Adult production of Hb F is regulated by multiple genetic loci including the Xmn1-HBG2 site in the β-globin locus, the HMIP locus at chromosome 6q23, the bcl11A locus on chromosome 2. Increased γ-globin to bind excess α-globin ameliorates the imbalance in glubin chain synthesis, decreased ineffective erythropoiesis and reduced anemia. So, experimental and clinical data revealed that increased HbF level can ameliorate the sever-

Thalassemia Reports 2012; 2 (s2) | 19 |

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IRON

DEFERASIROX (EXJADE®) DOSE ESCALATION ACHIEVES GREATER REDUCTIONS IN IRON OVERLOAD IN NON-TRANSFUSION-DEPENDENT THALASSEMIA PATIENTS: 1-YEAR RESULTS FROM THE THALASSA STUDY Ali T Taher,1 John B. Porter,2 Vip Viprakasit,3 Antonis Kattamis,4 Suporn Chuncharunee,5 Pranee Sutcharitchan,6 Noppadol Siritanaratkul,7 Renzo Galanello,8 Zeynep Karakas,9 Tomasz Lawniczek,10 Dany Habr,11 Jacqueline Ros,10 Yiyun Zhang,11 M. Domenica Cappellini12 1American University of Beirut, Beirut, Lebanon; 2University College London, London, UK; 3Department of Pediatrics and Internal Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; 4First Department of Pediatrics, University of Athens, Athens, Greece; 5Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 6Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 7Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; 8Ospedale Regional Microcitemie, Cagliari, Italy; 9Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey; 10Novartis Pharma AG, Basel, Switzerland; 11Novartis Pharmaceuticals, East Hanover, NJ, USA; 12Università di Milano, Ca’ Granda Foundation IRCCS, Milan, Italy

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RAP-536 REDUCES INEFFECTIVE ERYTHROPOIESIS AND CORRECTS ANEMIA IN β-THALASSEMIA Rajasekhar NVS Suragani, Robert Li, R. Scott Pearsall, Matthew L. Sherman, Ravi Kumar Acceleron Pharma Inc, Cambridge, MA, USA

Furthermore, RAP-536 treatment reduced splenomegaly (-38%, P>0.05). Analysis of blood smears from RAP-536 treated mice revealed marked improvement in RBC morphology with decreased bilirubin content indicative of decreased hemolysis. Together, these data demonstrates that RAP-536 promotes erythroid differentiation, reduces ineffective erythropoiesis and improves anemia in β-thalassemic mice. Thus ACE-536 represents a potential novel therapy for β-thalassemia patients. Phase I clinical trial of ACE-536 in healthy volunteer is ongoing.

ity of sickle cell disease and β-thalassemia. The HbF inducers can be grouped in several classes based on their chemical structures and mechanisms of action including Hypomethylating agents (like 5-azacytidine and decitibine), Histone deacetylase inhibitors (like sodium butyrate), hydroxyurea, Stem cell factor and erythropoietin. In this present study, we evaluate the effects of these HBF induction drugs in β-thalassemia. The experimental and clinical observations support the idea that agents enhancing HbF synthesis represent a rational approach for the treatment of sickle cell anemia and β-thalassemia and it is expected to be critical for developing countries with low economic status. Therefore, better agents that can induce higher levels of HbF are clearly needed. Hydroxyurea seems to have a good role in the treatment of thalassemia especially thalassemia intermedia, but resistant to this drug is observed in some cases. However, it is important to note that therapy of hemoglobinopathies based on the administration of agents stimulating HbF synthesis cannot be curative and needs a chronic administration.

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β-thalassemia is characterized by the deficiency of globin β-chains resulting in accumulation of unpaired α-globin and apoptosis of erythroid precursors. Ineffective erythropoiesis due to blocking erythroid differentiation and hemolysis of mature RBCs leads to severe anemia in βthalassemia patients. ACE-536 is a modified Activin Type IIB receptor fusion protein that modulates the availability of TGFβ ligands and promotes RBC formation in an erythropoietin (EPO) independent mechanism. While EPO treatment increases erythroid progenitors, ACE-536 promotes maturation of differentiating erythroblasts. In this study, we investigated if RAP-536 (murine ortholog of ACE-536) reduces ineffective erythropoiesis and corrects anemia in a mouse model of β-thalassemia. Threemonth old β-thalassemia intermedia (Hbbth1/th1) mice (N=7/group) were treated subcutaneously twice per week with TBS vehicle (VEH) or RAP-536 (1 mg/kg) for two months. Cell blood counts and bilirubin levels were measured during the study. Bone marrow and splenic erythroid differentiation analysis was carried by using antiCD71 and anti-Ter119 antibodies and flow-cytometric analysis. Prior to dosing β-thalassemic mice were severely anemic with significantly decreased RBC (-31.6% P>0.001), hematocrit (-34.8% P>0.001) and hemoglobin (-35.0% P>0.001) compared to wild type littermates. Following two months of treatment with RAP-536, β-thalassemic mice displayed significant increases in RBC (+32%, P>0.01), hemoglobin (+10.73%, P>0.05), hematocrit (+17.7%, P>0.01) and decreased reticulocytes (33.4%, P>0.05) compared to VEH treatment. Differentiation analysis of bone marrow and spleen revealed significant decreases in basophilic and increases in poly- and ortho-chromatophilic erythroblasts.

| 20 | Thalassemia Reports 2012; 2 (s2)

The 1-year THALASSA study demonstrated the efficacy and safety of deferasirox in iron-overloaded non-transfusion-dependent thalassemia (NTDT) patients. Here we report the effects of dose adjustments on iron burden. NTDT patients aged ≥10 years with liver iron concentration (LIC) ≥5 mg Fe/g dw and serum ferritin (SF) >300 ng/mL were randomized in a 2:1:2:1 ratio to starting dose 5 mg/kg/day deferasirox/placebo or 10 mg/kg/day deferasirox/placebo. Dose could be doubled at 24 weeks in patients with insufficient response (LIC >7 mg Fe/g dw and reduction <15% compared with baseline) provided there were no safety or tolerability issues. Changes in LIC, SF and adverse event (AE) profile were assessed for patients with or without dose escalation over 1 year. One hundred and sixty-six patients (β-thalassemia intermedia [n=95], HbE/β-thalassemia [n=49], α-thalassemia [n=22]) were randomized to 5 mg/kg/day (n=55)/placebo (n=28) and 10 mg/kg/day (n=55)/placebo (n=28). 81 patients (48.8%) received dose escalations. Mean±SD actual deferasirox doses in patients with or without escalation in the starting deferasirox 5 and 10 mg/kg/day

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

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CONSISTENCY OF LIVER IRON REDUCTION WITH DEFERASIROX (EXJADE®) ACROSS SUBGROUPS OF NON-TRANSFUSION-DEPENDENT THALASSEMIA PATIENTS FROM THE 1-YEAR THALASSA STUDY Ali T Taher,1 John B. Porter,2 Vip Viprakasit,3 Antonis Kattamis,4 Suporn Chuncharunee,5 Pranee Sutcharitchan,6 Noppadol Siritanaratkul,7 Renzo Galanello,8 Zeynep Karakas,9 Tomasz Lawniczek,10 Dany Habr,11 Jacqueline Ros,10 Yiyun Zhang,11 M. Domenica Cappellini12 1American University of Beirut, Beirut, Lebanon; 2University College London, London, UK; 3Department of Pediatrics and Internal Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; 4First Department of Pediatrics, University of Athens, Athens, Greece; 5Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 6Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 7Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; 8Ospedale Regional Microcitemie, Cagliari, Italy; 9Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey; 10Novartis Pharma AG, Basel, Switzerland; 11Novartis Pharmaceuticals, East Hanover, NJ, USA; 12Università di Milano, Ca’ Granda Foundation IRCCS, Milan, Italy

ficient response (LIC >7 mg Fe/g dw and reduction <15% compared with baseline). Primary efficacy endpoint was absolute change from baseline in LIC at Week 52 (lastobservation-carried-forward); analyzed here by subgroups based on an ANCOVA model with treatment as factor and baseline LIC as covariate. One hundred and sixty-six patients were randomized with β-thalassemia intermedia (n=95), HbE/β-thalassemia (n=49) or α-thalassemia (n=22). Mean±SD actual deferasirox doses were 5.7±1.4 and 11.5±2.9 mg/kg/day in the 5 and 10 mg/kg/day starting deferasirox dose cohorts, respectively. Overall, at Week 52, least-squares mean LIC significantly decreased from baseline by –1.95 and –3.80 mg Fe/g dw in the deferasirox 5 and 10 mg/kg/day groups, and increased by 0.38 mg Fe/g dw in the placebo. In all subgroups analyzed, there was a trend for greater reduction in LIC in patients treated with deferasirox compared with placebo. A dose-response effect was identified in several subgroups e.g. β-thalassemia intermedia patients (–1.18 [n=28], –4.53 [n=29] and 0.73 [n=32]mg Fe/g dw in the deferasirox 5, 10 mg/kg/day and placebo groups, respectively). No relevant differences were observed in the deferasirox adverse event profile or laboratory evaluations across subgroups. In general, LIC reduction across subgroups was consistent with the primary efficacy analysis with a similar safety profile; the study was not powered to determine differences between subgroup.

cohorts, were 6.8±1.0 and 14.1±2.0 mg/kg/day and 4.6±0.7 and 9.3±1.4 mg/kg/day, respectively. Patients receiving deferasirox 10 mg/kg/day without escalation achieved mean LIC reduction of –3.57 mg Fe/g dw, patients escalated from 10 to 20 mg/kg/day achieved –4.02 mg Fe/g dw. LIC reduction for patients receiving deferasirox 5 mg/kg/day with or without escalation to 10 mg/kg/day was –1.82 mg Fe/g dw and –1.88 mg Fe/g dw, respectively. Mean SF decreased by –128 ng/mL and –308 ng/mL in patients with escalation for starting deferasirox 5 and 10 mg/kg/day cohorts, respectively and by –114 ng/mL and –150 ng/mL in patients without escalation. In the placebo, starting deferasirox 5 and 10 mg/kg/day cohorts, overall AE rates were 80.4, 76.4 and 78.2%, respectively with no relevant differences in patients receiving dose escalation. Larger reductions in LIC and SF were achieved with starting deferasirox dose 10 mg/kg/day, with greatest reductions in patients escalated to 20 mg/kg/day. Dose escalation had no clinically relevant effect on safety.

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USE OF SERUM FERRITIN MEASUREMENTS TO ESTIMATE LIVER IRON CONCENTRATION IN NON-TRANSFUSION-DEPENDENT THALASSEMIA PATIENTS: ANALYSIS FROM THE 1-YEAR THALASSA STUDY Ali T Taher,1 John B. Porter,2 Vip Viprakasit,3 Antonis Kattamis,4 Suporn Chuncharunee,5 Pranee Sutcharitchan,6 Noppadol Siritanaratkul,7 Renzo Galanello,8 Zeynep Karakas,9 Tomasz Lawniczek,10 Dany Habr,11 Jacqueline Ros,10 Yiyun Zhang,11 M. Domenica Cappellini12 1American University of Beirut, Beirut, Lebanon; 2University College London, London, UK; 3Department of Pediatrics and Internal Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; 4First Department of Pediatrics, University of Athens, Athens, Greece; 5Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 6Chulalongkorn University and King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 7Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; 8Ospedale Regional Microcitemie, Cagliari, Italy; 9Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey; 10Novartis Pharma AG, Basel, Switzerland; 11Novartis Pharmaceuticals, East Hanover, NJ, USA; 12Università di Milano, Ca’ Granda Foundation IRCCS, Milan, Italy

The 1-year THALASSA study enrolled a diverse group of NTDT patients with various syndromes, iron burdens and baseline characteristics. The consistency of deferasirox in the reduction of liver iron concentration (LIC) in patient subgroups (age, gender, race, baseline LIC/baseline serum ferritin [SF], splenectomy and underlying NTDT syndrome) was therefore assessed. Ironoverloaded NTDT patients (LIC ≥5 mg Fe/g dw and SF >300 ng/mL) aged ≥10 years were randomized in a 2:1:2:1 ratio: i) 5 mg/kg/day deferasirox, ii) 5 mg/kg/day matching placebo, iii) 10 mg/kg/day deferasirox, iv) 10 mg/kg/day matching placebo, and treated for 52 weeks. Dose could be doubled at Week 24 in patients with insuf-

THALASSA assessed efficacy and safety of deferasirox in iron-overloaded non-transfusion-dependent thalassemia (NTDT) patients. The study provided a large dataset to allow exploration of relationships between liver iron concentration (LIC) and serum ferritin in NTDT patients. THALASSA enrolled iron-overloaded NTDT

Thalassemia Reports 2012; 2 (s2) | 21 |

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atric patients aged 2–<6 (n=68), 6–<12 (n=114) and 12–<16 (n=85) years entered the extension (141 male, 126 female; 92.9% thalassemics; 12.4% had history of hepatitis B and/or C). Median deferasirox exposure was 101.3 weeks (range 55.6–159.9) with mean ±SD deferasirox dose 25.7 ±5.8 mg/kg/day (range 13.4–40.0 mg/kg/day). Median SF decreased from 3222 to 2431 ng/mL (–528 ng/mL; P<0.0001; last observation carried forward). 257/267 (96.3%) patients completed the extension; main reasons (>2 patients) for discontinuation were unsatisfactory therapeutic effect (n=4, 1.5%) and consent withdrawal (n=3; 1.1%). 45/267 (16.9%) patients had two consecutive alanine aminotransferase (ALT) values >5xULN. Importantly, 40/45 patients had high baseline ALT and in 30/45 patients ALT or aspartate aminotransferase was >2.5xULN at baseline, 13/45 had history of hepatitis B and/or C. 6/267 (2.2%) patients had two consecutive serum creatinine values >33% above baseline and >ULN; they had normal values at baseline. Stature, growth and weight assessments indicated positive growth velocity. Mean ±SD growth velocity at end-of-study was 5.9 ±43.3 cm/year. Deferasirox for up to 3 years in pediatric patients was effective in decreasing SF. High liver enzymes were reported at baseline and during the study; renal safety was consistent with previous reports. No adverse effects were observed on pediatric growth/development. THE COMPARISON OF THE EFFECTS OF DESFERRIOXAMINE AND DEFERASIROX COMBINATION THERAPY WITH DESFERRIOXAMINE MONOTHERAPY ON LIVER IRON OVERLOAD OF TRANSFUSION- DEPENDENT THALASSAEMIA PATIENTS Dilek Yazman, Burhan Nalbantoglu, Mevhibe B. Hocaoglu, Ratna Chatterjee, Rekha Bajoria Haematologist, Thalassaemia Centre State Hospital, Nicosia, Cyprus; Department of Psychology, Eastern Mediterrranean University, Institute for Women’s Health, University College London, London, UK

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patients (LIC ≥5 mg Fe/g dw and serum ferritin >300 ng/mL) aged ≥10 years. Data from all screened patients with available serum ferritin (measured centrally) and LIC [measured using validated R2 magnetic resonance imaging (MRI) and read centrally] were used. To determine whether serum ferritin can effectively predict LIC ≥5 mg Fe/g dw for therapy initiation, ≥7 mg Fe/g dw for dose escalation and <3 mg Fe/g dw for therapy interruption; a Receiver Operating Characteristic (ROC) analysis, Positive Predictive Values (PPV) and Negative Predictive Values (NPV) were generated. LIC and serum ferritin were determined at screening in 282 patients with β-thalassemia intermedia, HbE/β-thalassemia or α-thalassemia. Serum ferritin >800 ng/mL was highly predictive of LIC ≥5 mg Fe/g dw (PPV 91.7%). A relatively low NPV (53.6%) for serum ferritin ≤800 ng/mL, indicates that patients with such serum ferritin levels should undergo MRI to detect iron overload requiring chelation. Serum ferritin >2000 ng/mL was highly predictive of LIC ≥7 mg Fe/g dw (PPV 92.9%) and serum ferritin <300 ng/mL was highly predictive of LIC <3 mg Fe/g dw (NPV 80.0%). Serum ferritin could be useful in some NTDT patients to guide iron chelation in the absence of MRI, although MRI-confirmed LIC is desirable. Serum ferritin >800 ng/mL was highly predictive of LIC ≥5 mg Fe/g dw, and serum ferritin >2000 ng/mL was highly predictive of LIC ≥7 mg Fe/g dw; levels at which deferasirox was initiated and dose increases were allowed in THALASSA, respectively. Serum ferritin <300 ng/mL appears adequate to interrupt therapy.

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TREATMENT OF IRON-OVERLOADED PEDIATRIC PATIENTS WITH DEFERASIROX (EXJADE®): A 3-YEAR FOLLOW UP FROM THE EPIC STUDY Amal El-Beshlawy,1 Mohsen El-Alfy,2 Lee Lee Chan,3 Yongrong Lai,4 Kai-Hsin Lin,5 Yesim Aydinok,6 Renzo Galanello,7 Ali El-Ali,8 Nicolas Martin,8 M. Domenica Cappellini9 1Cairo University, Cairo, Egypt; 2Ain Shams University, Cairo, Egypt; 3University Malaya Medical Centre, Kuala Lumpur, Malaysia; 4The First Hospital Affiliated Guang Xi Medical University, Nanning, China; 5National Taiwan University Hospital, Taipei, Taiwan; 6Ege University Hospital, Izmir, Turkey; 7Ospedale Regional Microcitemie, Cagliari, Italy; 8Novartis Pharma AG, Basel, Switzerland; 9Università di Milano, Ca’ Granda Foundation IRCCS, Milan, Italy

Assessment of long-term efficacy and safety of deferasirox (Exjade®) in iron-overloaded pediatric patients is required, since chelation therapy is often lifelong. Transfusion-dependent pediatric patients aged ≥2–<16 years at enrollment with serum ferritin (SF) ≥1000 ng/mL or <1000 ng/mL but >20 transfusions or >100 mL/kg RBC transfused, and LIC >2 mg Fe/g dw confirmed by R2-MRI were included in EPIC. Deferasirox starting dose was 10–30 mg/kg/day, adjusted by 5–10 mg/kg/day based on 3-monthly SF trends and safety. Patients completing 1 year could continue in the extension. SF, adverse events (AEs) and growth/development were monitored. Two hundred and sixty-seven pedi-

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Different combination chelation options are now available with new oral chelator, deferasirox (DFX). Combining desferrioxamine (DFO) and DFX may enhance their efficacy and tolerability and reduce their toxic effects. Here the aim is to compare the efficacy of DFX and DFO combination therapy with regard to DFO monotherapy on liver iron load in transfusion-dependent thalassaemia patients. Ten patients (CTG) were assigned to DFO (40-50 mg/kg/day 3-5 times/week) and DFX (3035 mg/kg/day 7 days) combined treatment and compared to ten patients (MTG) on DFO monotherapy (35-60 mg/kg/day 3-5 times/week). The duration of the study was 18 months. To evaluate the efficacy of therapy, MRI T2* and ferritin levels of both groups were monitored. Transaminases, urea and creatinine were monitored to assess the safety of treatments. After 18 months of therapy there was not a significant difference in the changes after baseline in serum ferritin levels for CTG (142.75±292.91 µg/L) and MTG (-92.67±179.4 µg/L). The mean change in MRI T2* of liver scores for CTG was -2.18±1.02 ms and for MTG was -0.05±0.49 ms. The

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

VITAMIN D DEFICIENCY AND LIVER IRON CONCENTRATION IN TRANSFUSION DEPENDENTHEMOGLOBINOPATHIES IN BRITISH COLUMBIA Hatoon M. Ezzat,1,2,3 John Wu,4 Heather McCartney,4 Heather A. Leitch1,3 1Hematology, St. Paul’s Hospital and the University of British Columbia, Vancouver, BC, Canada; 2BC Adult Provincial Hemoglobinopathies Program, 3BC Provincial Home Hemosiderosis Program, 4BC Children Provincial Hemoglobinopathies Program

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CARDIAC IRON OVERLOAD IN TRANSFUSION-DEPENDENT ANEMIAS: PREVALENCE DATA FROM THE CORDELIA TRIAL COMPARING DEFERASIROX [EXJADE®] WITH DEFEROXAMINE FOR THE REMOVAL OF CARDIAC IRON Dudley J. Pennell,1 John B. Porter,2 Antonio Piga,3 Mohsen El-Alfy,4 Amal El-Beshlawy,5 Yurdanur Kilinç,6 Vip Viprakasit,7 Akif Yesilipek,8 Tomasz Lawniczek,9 Dany Habr,10 Marianne Weisskopf,9 Yiyun Zhang,10 Yesim Aydinok11 1Royal Brompton Hospital, London, UK; 2University College London, London, UK; 3Universitá di Torino, Turin, Italy; 4Ain Shams University, Cairo, Egypt; 5Kasr El Eni University, Cairo, Egypt; 6Cukurova University Medical Faculty, Adana, Turkey; 7Siriraj Hospital, Mahidol University, Bangkok, Thailand; 8Akdeniz University, Antalya, Turkey; 9Novartis Pharma AG, Basel, Switzerland; 10Novartis Pharmaceuticals, East Hanover, NJ, USA; 11Ege University Hospital, Izmir, Turkey

T2*10–≤20 ms. By region, 20.6% of patients in the West, 12.4% in the Middle East and 20.5% in the Far East had T2*<10 ms; 25.3%, 16.5%, and 20.5% had T2*10–≤20 ms, respectively. Mean liver iron concentration (LIC) was 25.9±17.0 mgFe/g dw (West 19.4±14.7 mgFe/g dw, Middle East 25.4±16.3 mgFe/g dw, Far East 35.1±16.9 mgFe/g dw). 64.6% of patients had LIC≥15 mgFe/g dw (West 51.2%, Middle East 64.4%; Far East 82.0%). Median serum ferritin was 3739 ng/mL. 12.1% of patients had LVEF below LLN. The proportion of patients with LVEF below LLN was higher in low T2* categories (23.0, 15.1 and 8.1% in T2*<10, 10–≤20 and >20ms, respectively). Assessment of the prevalence of cardiac siderosis by geographic region in this large cohort of transfusiondependent anemia patients showed that prevalence was lowest in Middle-Eastern patients, but was similar overall to previous reports. Observed differences between geographical regions may reflect transfusion/chelation practices and genetic differences.

mean change in MRI T2* of heart scores for CTG was 6.90±3.74 ms and for MTG was -5.89±4.26 ms. The mean change in ECHO EF% scores for CTG was 0.30±2.60% and for MTG was -2.05±2.84%. The difference in the mean AST levels for CTG was -2.10±10.43 U/L and for MTG was -2.45±5.66 U/L. The difference in the changes after baseline in ALT levels for CTG was 3.24±5.76 U/L and for MTG was -1.05±2.31 U/L. The difference in the changes after baseline in BUN levels for CTG was -1.22±1.42 mg/dL and for MTG was 0.64±1.17 mg/dL. The difference in the changes after baseline in creatinine levels for CTG was -0.11±0.6 mg/dL and for MTG was -0.01±0.03 mg/dL. The changes were all non-significant. Improvement shown in both groups were not found statistically significant however combination group showed better increase of MRI T2*liver scores. Treatments were well tolerated and no significant side effects were observed in both groups.

Enrollment is complete for the multicenter, open-label, randomized, Phase II, 1 year trial evaluating deferasirox versus deferoxamine for the treatment of cardiac siderosis (CORDELIA [NCT00600938]). Patients aged ≥10 years with β-thalassemia major, Diamond-Blackfan anemia, sideroblastic anemia or Low/Int-1 risk myelodysplastic syndromes and history of ≥50 transfusions were screened. Prevalence of cardiac siderosis was assessed by geographic region in this large cohort of patients with transfusion-dependent anemias. Data are summarized by T2* categories and reported by geographic regions (West: Canada, Cyprus, Italy, Turkey, UK; Middle East: Egypt, UAE, Lebanon; Far East: Taiwan, Thailand, China). Left ventricular ejection fraction (LVEF) below the lower limit of normal (LLN) was identified using Westwood criteria (males <59%; females <63%). Nine hundred and twenty-three patients (258 West, 462 Middle East, 203 Far East) were screened (99.1% β-thalassemia major, mean age 19.2±7.8 years). 98.4% had received previous chelation therapy. 16.8% had T2*<10 ms; 20.1% of patients had

Patients with thalassemia major and other chronically transfused hemoglobinopathies are at increased risk of iron overload. Vitamin D deficiency is a common manifestation of these patients. The first step in vitamin D metabolism, known as vitamin D hydroxylation, occurs in the liver. It is not clear whether liver iron overload may affect this step. This study addresses the association between the degree of liver iron overload and vitamin D levels in these patients. Patients with transfusion dependent hemoglobinopathies attending the hemoglobinopathy programs in British Columbia, Canada were identified. Patients included were those who had liver iron concentration (LIC) by MRI (T2* or R2*) and endocrinology assessment including 25 hydroxy vitamin D3 levels between 2009 and 2011. Thirty patients with transfusion dependent hemoglobinopathies were identified. Patients were 6-51 years old. 60% were from Asian descent while only 3.33% were Caucasian. All patients were receiving iron chelation therapy: 60% were receiving desferrioxamine, 30% were on deferasirox, 10% were on a combination. 86.7% patients were receiving vitamin D and calcium supplementation. The mean serum ferritin level was 1182.8 mcg/L while LIC mean was 5.13 mg/g dry weight (DW). Vitamin D deficiency/insufficiency was present in 63.3%. There was a significant association between LIC >5 mg/gDW and vitamin D level <60 nmol/L (P=0.027), with 36.7% patients having moderate iron overload (LIC ≥5 mg/gDW), 8 of whom had a vitamin D level <60 nmol/L, indicating vitamin D insufficiency. We hypothe-

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Haemoglobinopathies are very common in Albania. There are two groups of patients treated at our clinic: the first consists of β-Thalassaemia Major and the second assembles patients with Sickle Cell Disease and Thalassaemia- Sickle Cell Disease. Aim of our study is to evaluate the role of Deferasirox in treatment of our patients. It is a descriptive study, a transversal one, where are studied 107 patients during a two year-period. The Ferritin level is evaluated at the beginning, after the first year of treatment, and after the second year of treatment. The decrease of Ferritin level was very significant (P<0,01). Comparing the mean level of Ferritin in every age-group, it is seen that in the category of children below 14 years old, the mean level of ferritin at the end of study is lower compare to the beginning (P=0,05). Using ANOVA test, it is seen that using the dose of Deferasirox 20 mg/kgP/day, it is not seen significant decrease of ferritin level (P=0,9). At doses over 20 mg/kgP/day, it is seen an important decrease of ferritin level at the end of second year of treatment compare to the level of beginning (P=0,036). The correlation of dose of Deferasirox and hepatic function (ALT) results as a weak one, nonsignificant r=0.1 dhe P=0.2). At the end of second year of treatment, the number of patients with the value under 1000 ng/mL is 25, compare to the initial phase (12) χ2 for trend=10.249 P<0.01. Treatment with oral Iron chelator Deferasirox demonstrates very remarkable role in this category of patients, making their life more healthy and satisfied.

IRON OVERLOAD AND ITS TREATMENT AT HAEMOGLOBINOPATHIC PATIENTS IN ALBANIA Manika Kreka, Eleni Nastas, Gentiana Qirjako, Bledi Kreka, Irena Qendro, Anila Godo, Xhensila Prendushi University Hospital Centee “Mother Teresa”, Tirana, Albania

included. Patients were on chelation treatment with either deferasirox (18 patients), or deferiprone, desferioxamine or both (21 patients). Eight were splenectomised, six received aspirin as prophylactic treatment, seven patients had a liver biopsy. No haemostatic cover was used and no untoward bleeding was reported. One patient had experienced a peptic ulcer bleeding. Four cases with chronic viral hepatitis were included. 66% (26) of the patients showed reduced LTG with adrenaline and 20% (8) showed reduced LTG to two or more of the agonists used. Thirty-four patients were tested with PFA-100 and was prolonged in 73% (25) of them. 43.5% (17 patients) had both a defect in platelet aggregation and a prolonged PFA-100 test, while 10.2% (4) had abnormal LTG and normal PFA-100 time. 20.5% (8) patients had normal aggregation and abnormal PFA-100 test, while 10.2% (4) had both normal aggregation and PFA-100 time. Three patients with chronic hepatitis had reduced LTG and one was found normal. The patients that had undergone liver biopsies had abnormal LTGs and 4 had prolonged PFA100. Among the patients that had undergone a splenectomy, we found five of them to have abnormal LTG and prolonged PFA-100, while two of them had normal tests. The patient with GI hemorrhage had abnormal LTG and PFA-100. We found no correlation with either chelation regimen and LTG or PFA-100. Our patients’ results are indicative of a platelet defect similar to disorders of platelet secretion and signal transduction. The clinical manifestations though were few. In literature and clinical practice there is a lack of consensus about the best diagnostic tests concerning platelet function. In thalassaemics the reduced LTG and the prolonged PFA-100 closure time must not be overestimated due to the limited clinical manifestations. The administration of salicylates in splenectomised thalassaemics should not be ignored. Nevertheless, bleeding can occur in patients with platelet defects of the above type.

sized that vitamin D hydroxylation in the liver may be affected by liver iron overload. The results of this study show a significant association between LIC ≥5 mg/gDW and vitamin D levels <60 nmol/L, suggesting that liver iron overload may indeed affect vitamin D metabolism in the liver. Prospective trials are needed to prove this association.

PLATELET FUNCTION AND CHELATION TREATMENT IN THALASSAEMIC PATIENTS S. Theodoridou, M. Economou, A. Teli, E. Vlackaki, A. Karioti, A. Agapidou, S. Vakalopoulou, V. Garypidou, V. Gkorytsa, N. Gompakis, F. Papachristou Platelet function disorders constitute a rare cause of symptomatic bleeding. The aim of this study was the platelet function investigation in thalassaemics and the detection of any relation with the chelation treatment. The platelet function investigation consisted of the aggregation testing in platelet rich plasma with light transmission aggregometry (LTG) by the use of 5 agonists, and the global test of haemostasis by PFA-100. 39 thalassaemic patients (24 males, 15 females, aged 5 to 45 years) were

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RARE ANAEMIAS MOLECULAR ANALYSIS OF RPS19, RPL5 AND RPL11 GENES IN GREEK PATIENTS WITH DIAMOND BLACKFAN ANEMIA Polyxeni Delaporta,1 Christalena Sofocleous,2 Eftichia Steiakaki,3 Sophia Polychronopoulou,4 Marina Economou,5 Lydia Kossiva,6 Stavroula Kostaridou,7 Antonis Kattamis1 1First Department of Pediatrics, University of Athens, Greece; 2Department of Medical Genetics, University of Athens, Greece; 3Department of Pediatric HematologyOncology, University of Crete, Heraklion, Greece; 4Hematology-Oncology Unit, ‘Agia Sofia; Children’s Hospital, Athens, Greece; 5First Department of Pediatrics, University of Thessaloniki, Greece; 6Second Department of Pediatrics, University of Athens, Greece; 7‘Penteli’ Children’s Hospital, Athens, Greece Diamond Blackfan Anemia (DBA) is a rare inherited disease characterized by congenital defective erythropoiesis. The aim of this study was to assess the incidence and genetic basis of DBA in Greece. A questionnaire was sent

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

shown alteration in band 3 glycosylation. SEC23B gene sequencing analysis was performed to highlight the presence of nucleotide variations and their relationship with the clinical presentation. cDNA was prepared from patients’ mRNA from lymphocytes and erythroblasts. None of the SEC23B mutations was found in 80 unrelated Sicilian control. WE identified SEC 23 B mutations in 4 out of 11 patients analysed. In two patients with suspect of CDAII we found two nucleotide variations in flanking splice junctions, not previously described. RNA analysis studies are in progress to clarify whether such nucleotide variations influence the expression of the gene.

ENDOCRINE COMPLICATIONS

GROWTH RETARDATION AMONG MULTI TRANSFUSED THALASSEMIC PATIENTS IN THALASSAMIA CENTER IN BABIL GOVERNORATE Ahmed Shemran AL-Wataify Department of Paediatric, Babil collage of Medicine, Babil University, Babil, Iraq

Growth retardation can occur as complication of thalassemia as early as the 1st or 2nd year of life but these abnormalities more prominent after the 6-8 years of life. This study was carried out to determine: rate of growth retardation among thalassemia patients in Babil thalassemia center. The relationship of growth failure with certain variable including age, number of blood transfusion, serum ferritin, mean hemoglobin level, size of spleen, type of thalassemia and gender of the patients. A prospective study was conducted on 110 patients, (67 male, 43 female) with β-thalassemia, classified as 79 patients with β-thalassemia major, 23 patients β-thalassemia intermediate and 8 patients with sickle cell thalassemia, who were attending the thalassemia center of Babil maternity and children hospital, from period of march to August 2009, their age ranged from one year to 20 years with mean age of 9±2 years, in comparison to One hundred healthy children were taken as control group,their age ranged from 1-15 years, with mean age 7±2.3 years, non of them had history of blood transfusion and they had no family history of hemoglobinopathy. Thalassemic patients developed 45.5% short stature and 40% weight failure, in comparison to control group (4% short stature, 8% weight failure). Growth retardation was affected by age, increased number of blood transfusion, size of spleen, serum ferritin and hemoglobin level,while there is no difference regarding type of thalassaemia or gender. Growth is significantly affected by thalassaemia and different factors play a role and This study emphasized the importance of maintenance of normalized hemoglobin level, good monitoring of growth parameter and iron over load with optimal iron chelation therapy.

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to all the pediatric hematology, hematology and transfusion units in Greece. Informed consent was obtained from all patients and genomic DNA was isolated from peripheral blood lymphocytes. PCR primers were specifically designed to amplify the whole coding region and the flanking intron/exon junctions of RPS19, RPL5 and RPL11 genes. RPS19 was studied by both ECMA and direct sequencing. RPL5 and RPL11 genes were initially studied by ECMA. Seventeen patients (females: 7, mean age: 11.4±11years) were referred for DBA molecular analysis. Congenital anomalies were observed in 66.7% of the patients. In regard to the clinical course, 4 patients were receiving chronic steroid therapy and 8 were on regular transfusions. Spontaneous resolution of the anemia was observed in one patient. One patient died and 3 patients were lost to follow up. Six patients (35.2%) were found to carry mutations on either the RPS19 gene (3 patients) or the RPL5 gene (3 patients). Mutations c.C390G (p.Υ130X) and c.197_198insA (p.Y66X) detected in the RPL5 gene were novel. Although molecular analysis on the RPL11 gene did not disclose any mutations on the six exons of the gene, nevertheless revealed the previously described c.507+39 A>G variant in intron 5 (2 patients) and a novel c.264+49 A>G variant in intron 3 (1 patient). DBA in Greece presents significant heterogeneity both in clinical and genetic profile. The results of this study are comparable with previous reports from other countries.

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CONGENITAL DYSERYTHROPOIETIC ANEMIA TYPE II: MOLECULAR CHARACTERIZATION IN SICILIAN PATIENTS Serena Sclafani,1 Veronica Agrigento,1 Paolo Rigano,2 Aurelio Maggio,2 Giusi Calvaruso,2 Angela Piazza,2 Alice Pecoraro,2 Elena D’Alcamo2 1Azienda Ospedali Riuniti “Villa Sofia Cervello”; 2U.O.C. Ematologia delle Malattie Rare del Sangue e degli Organi Ematopoietici”, Palermo, Italy

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Congenital dyserythropoietic anemia (CDAs) represents a heterogeneous group of rare hereditary disorders. Three major types have been differentiated ; the most frequent form is CDA type II (CDAII). It is a recessively inherited condition and the diagnosis is based on several biochemical tests: Sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS PAGE), EMA binding test, acid serum lysis test, and bone marrow erythroblasts analysis by electron microscopy. These tests are available only in specialized laboratories, then the diagnosis of CDAII is based on the clinical features. Recently it was shown that the vast majority of CDAII patients carry mutations in the SEC 23B gene encoding an essential component of the coat protein complex (COPII)-coated vesicles. The aim of this study was to identify genetic mutations in patients with suspect of CDAII to confirm the diagnosis and characterize the SEC 23 B gene defect in Sicilian patients. We collected blood samples from 11 Sicilian patients with suspect of CDAII and 80 unrelated control. The diagnosis of CDAII was based on the presence of mild to moderate anemia ineffective erythropoiesis and bone marrow erythroblasts morphological abnormalities. Subjects were analyzed with EMA binding tests and SDS PAGE to

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Abnormal glucose metabolism is a frequent complication in Thalassemia major patients (TMp). The main underlying mechanisms are either insulin deficiency due to the direct toxic damage of iron on pancreatic β-cells, and/or long-standing insulin resistance due to iron overload on different tissues. Usually patients are asymptomatic but it may manifest from mild glucose intolerance to overt diabetes which has an established effect on cardiovascular disease. The widely accepted glycohemoglobin test (A1Ctest) is not accurate in TMp because of haemolysis. Unlikely, fructosamine-test, which measures glycated serum protein and remains unchanged for 14-21 days, provides an overall view of glucose levels for the past few weeks and it is recommended for these cases by the American Diabetes Association (ADA). Fifty TMp, 24 males 26 females, mean age 35.8±2.03 undergo a fructosamine test for their glycemic control. They were classified according to the ADA criteria. Results: - 4/50 with Insulin-dependent Diabetes had abnormal fructosamine levels=364±65 - Between TMp with Diabetes type II 8/50: • 2 treated with Sulfonylureas had also abnormal fructosamine levels=311±47,8 • 4 with Biguanides and Incretin had normal fructosamine levels=185,4±22,2 • 2 with Incretin therapies had normal fructosamine levels=164,3±20,8 - 11/50 with Impaired Glucose Tolerance had normal fructosamine levels=165,3±11,5 - 27/50 with Normal Glucose Metabolism had lower normal fructosamine levels=139,7±27,9 Although fructosamine-test reflects mean blood glucose levels over a couple of weeks, evaluated in the context of patient’s clinical findings, may add up useful information in TMp follow-up. Indeed in certain circumstances, such as pregnancy or recent adjustment of treatment plan, it has advantages. It can also monitor the effectiveness of diet, exercise or medication and encourage TMp’s compliance. Additionally our results lend support to the idea that fructosamine-testing in TMp may be used as a target for glycemic control, like A1C-testing in normal population.

- 50 adult TMp (26 females, 24 males, mean age 35.8±2.03, mean BMI=23 were included in the study. Of them 60% were hypogonadic on hormone replacement therapy. - Bone mineral density (BMD) of lumbar spine vertebrae (L2-L4) and left hip was measured contemporaneously, in the same TMp, by dual-emission X-ray absorptiometry (DEXA). According to WHO, Osteopenia was defined as T-score between -1 and -2.5 and Osteoporosis as T-score less than -2.5. - Total body iron load was assessed by ferritin (CMIA), Signa-MRΙ:1.5-Tesla multi-echo-T2* and Liver iron concentration (LIC) Ferriscan. - Statistical analysis was performed with SPSS. Results: - Bone disease was present in the spine of 82% TMp (mean T-score:-2.35) and at the left hip 78% (mean Tscore:-1.7). Coefficient of correlation between lumbar spine T-score and left hip in contemporary measurements: Spearman rho=0.578, P=0.0001. Sign test two-sided P=0.0043. - There was a positive correlation between left hip Tscore and the age of TMp (P=0,052). - TMp with history of fractures had the less BMD in lumbar spine (P=0.1) and left hip (P=0.07) - Hypogonadism was correlated with less BMD mainly at left hip Mann-Whitney P=0.011 and at lumbar spine Mann-Whitney P=0.15 - No significant correlation was found between lumbar spine and left hip T-score and total body iron load as assessed by Ferritin, LIC, MRI T2*L, T2*H. - Male TMp seem to have better BMD than women, but without statistical significance Despite the optimization of transfusional and chelation regimens and adequate hormone replacement, Calcium, vitamine D, bone demineralization in TMp remains a challenge. There is significant correlation between lumbar spine T-score and left hip in contemporary measurements.

FRUCTOSAMINE IN THE MANAGEMENT OF ABNORMAL GLUCOSE METABOLISM IN β-THALASSEMIA MAJOR PATIENTS Christina Pappa, Ioanna Tzoumari, Kallistheni Farmaki Thalassemia Unit, General Hospital of Corinth, Greece

COMPARISON OF LUMBAR SPINE AND FEMORAL DEXA T-SCORE IN CONTEMPORARY MEASUREMENTS OF THALASSAEMIA MAJOR PATIENTS Ioanna Tzoumari, Christina Pappa, Kallistheni Farmaki Thalassemia Unit, General Hospital of Corinth, Greece Osteoporosis and increased risk of fractures represent a prominent cause of morbidity in adult Thalassemia Major patients (TMp) of both sexes. The pathogenesis of bone loss is multifactorial. Patients and methods:

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IMPAIRED GLUCOSE TOLERANCE TEST IN β-THALASSEMIA MAJOR Jasim M. Al-Marzoki, Zainab W. Al-Maaroof, Widad H. Al-Dujaily Department of Pediatrics, Department of Pathology Babylon Maternity and Children Teaching Hospital, Babylon Medical College, Babylon Medical College, Hilla, Iraq β-thalassemia major is a hereditary disorder of hemoglobin synthesis associated with ineffective erythropoiesis and rapid red cell destruction resulting in sever anemia. Treatment consist of multiple blood transfusions, which is complicated by hemochromatosis in multiple organs including endocrine system. Impaired glucose metabolism is one of the above consequence in the pancreatic β cells. We report the relationship and risk factors of impaired glucose tolerance test in patients with β-thalassemia major in Hilla, Babil, Iraq. Ninety-seven patients with β-thalassemia major were enrolled in this study, which was done in Babylon Thalassemic center,

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

LEPTIN LEVELS IN BLOOD TRANSFUSED β-THALASSEMIA PATIENTS C. Levin,1,4 O. Admoni,2 M. Yunes,1 R. Masalha,3 Y. Tennenbaum Rakover,2,4 A. Koren1,4 1Pediatric Hematology Unit, 2Pediatric Endocrinology Unit, 3Endocrine Laboratory, Emek Medical Center, Afula, 4Rappaport Faculty of Medicine, Technion, Haifa, Israel

Patients with β thalassemia major (βTM) are transfusion dependent, and developed iron overload. Transfusions and chelation improved survival Leptin is a polypeptide hormone, the product of the Ob (obesity) gene, thought to play a key role in the regulation of body weight. Leptin is produced by adipocytes and acts in the hypothalamus suppressing food intake and stimulating energy expenditure. Also affects immunity and inflammation. A proliferative effect on hematopoietic cells was observed. Previous studies on leptin in βTM patients showed low leptin levels. Study aims: measure leptin in β TM; investigate the association between leptin, fat status, (BMI) and hematological and iron metabolism. Thirty-three βTM patients, mean age 19 years were evaluated. All patients underwent anthropometric assessment: Fat %; BMI; pubertal status; and serum leptin. The standard deviation (SD) of serum leptin was utilized. βTM patients have low leptin, SD levels -0.87±1.27. Leptin were lower in females, -0.49 vs. 1.14, decrease with age r=-0.581, P<0.01. Lower levels were found in females over age 21, mean SD -1.89vs-0.28 at age 11 to 20 ys (P<0.02). Negative correlation was found between leptin and BMI (r=-0.489, P<0.01), and fat % (r=-0.416 P<0.02). Positive correlation was found with Hb (mean previous 5 yrs.) (r=0.43, P<0.02). No correlation was found between iron metabolism and leptin. The study results show low leptin in β TM. Leptin decrease with age and are lower in females. The negative correlation between BMI and fat status with leptin can be attributed to a toxic effect or loss of endocrine function of the adipose tissue, possibly related to iron toxicity or anemia and enhanced erythropoiesis. The positive correlation of leptin to hemoglobin suggests a relationship between leptin and erythropoiesis. Further studies need to be done in order to confirm these hypotheses.

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DEFERASIROX COMBINED WITH DEFERIPRONE MAY IMPROVE SHORT STATURE AND PUBERTAL MATURATION IN JUVENILE β-THALASSEMIA MAJOR PATIENTS Kallistheni Farmaki, Ioanna Tzoumari, Christina Pappa Thalassemia Unit, General Hospital of Corinth, Greece

TMp. These results indicate that oral combined chelation (Deferasirox-Deferiprone) seemed to be considerably beneficial in juveniles TMp for attaining normal stature, normal sexual maturity, normal iron load and improved cardiac function. It reverses Desferrioxamine-mediated toxicity leading to a short stature. Moreover it is well tolerated and offers the promise of better compliance.

Hilla, Babil, Iraq. There were 56 males (57.73%) and 41 females (42.26%). The mean age at the time of enrollment was 9.36±3.75 year. The demographic characteristics of patients with β-thalassemic major included the age, gender, height, body weight, age at the first blood transfusion, age at the start of iron-chelation therapy, compliance with iron-chelation therapy, previous splenectomy and family history of diabetes. Estimation of serum ferritin concentration, existence of hepatitis B surface antigen, and hepatitis C antibody were done. All 97 patients underwent oral glucose tolerance test (OGTT). Blood samples for glucose were drawn at 0 and 120 min. Impaired glucose tolerance test (IGT) was found in 5 patients (5.15%), and 92 patients (94.84%) had normal glucose tolerance test. Comparing the patients with normal glucose tolerance test to those with impaired glucose tolerance test, significant differences were found in the serum ferritin concentration. Physicians caring for patients with thalassemia major should be particularly alert to the possibility of diabetes. Serum ferritin concentration, a marker for hepatic iron concentration, was found to be a risk factor in this study.

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Short stature and delayed puberty is the most frequent complication in TMp and persisted despite major therapeutic advances. One male and 2 females TMp, mean age 12.3±2,3, previously on combined chelation: Desferrioxamine-Deferiprone, having a short stature, were switched to combined oral: Deferasirox: 25mg/Kg/day and Deferiprone: 100 mg/Kg/day for 3years. Hospital’s Ethics Committee approval and guardians’ written consent were obtained. Growth-charts, puberty staging (Tanner) and hormonal tests were implemented. Indices of iron overload were analyzed. A close clinical and laboratory monitoring to assure safety was realized. Statistical analyses were performed using SPSS. Although at baseline TMp had a decline of height age percentile varying from <2% to <8%, after oral combined chelation they presented an overall increase in standing and sitting height and reached a normal height age percentile. In all TMp mean body mass index increased (21 vs. 15.8). Sexual development progressed in the 2 elder from Tanner stage II to IV and in the younger from I to III. LH and FSH, as peripheral hormone secretion increased. In both females menarche occurred at the age of 11 and 12years old and the male obtained a normal sperm count. Besides, there was a statistically significant decrease of total body iron load in ferritin=103±52 ng/mL, LIC=0.9 mgFe/g/dw, T2*L=36 ms, T2*H=34.1 ms and cardiac function improvement (mean LVEF=66% vs. 61%, FS=38% vs. 34%). No drug-related neutropenia or agranulocytosis or other toxicities known to be associated with the two chelators were observed. Pre and posttreatment creatinine and eGFR remained unchanged in all

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Cardiac damage remains a major cause of mortality among patients with thalassemia major (TM). The detection of a lower cardiac magnetic resonance T2* (CMR-T2*) signal has been suggested as a powerful predictor of the subsequent development of heart failure. Moreover, the lack of worldwide availability of CMR-T2* facilities prevents its widespread use for annual follow-up evaluations of cardiac function in TM patients, warranting the need to assess the utility of other possible procedures. Actually, only 19/55 countries, the majority in Europe, use the validated MRI T2* for the measurement of cardiac iron overload (TIF, personal communication). In this setting, the determination of left ventricular ejection fraction (LVEF) offers an accurate and reproducible method for heart function evaluation. This retrospective survey suggests that a decrease of 7% or more in LVEF over time may represent a strong and accessible predictive tool for evaluating the risk of cardiac death in TM patients. This report describes a retrospective study of 413 patients with TM who were managed from January 31, 2000 to January 31, 2008. The included patients were divided into two cohorts: i) the not alive and ii) the alive groups. A generalized estimating equations (GEE) model was used to detect variations of mean values of LVEF over time between the two groups. A logistic regression model was used to evaluate the risk of death from heart disease. In this analysis, the mean variations in LVEF values were categorized into three levels: i) patients with an increase in mean LVEF of greater than 0%; ii) patients with a reduction in mean ELVEF of greater than 0% but less than 7%; and iii) patients with a reduction in mean ELVEF of greater than or equal to 7%. Among the 188 analyzed patients, 166 were alive. Of the 22 not alive cohort, 21 patients were being treated with deferoxamine (DFO) alone treatment, while 1 received sequential treatment of DFO and deferiprone (DFP). In the alive cohort 84 patients received DFO-alone, 47 DFPalone and 35 DFO-DFP-sequential chelation treatment

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Glycaemic abnormalities have been reported in patients with β-thalassemia major (TM). The aims of this study were to evaluate glycaemic abnormalities, in young adult TM patients using oral glucose tolerance test (OGTT) and 72-h continuous blood glucose monitoring system (CGMS) and to determine whether glycaemic abnormalities are due to insulin deficiency and/or resistance. Fourteen TM patients were investigated by using a standard 75 gm oral glucose tolerance test (OGTT) and 72-h CGM by Medtronic system. Fasting serum insulin and ferritin concentrations were also measured. HOMA-B, HOMA-IR and QUICKI index were calculated using basal glucose and insulin levels. Using OGTT, 2 patients had impaired fasting glucose (IFG) only, 2 had both IFG and IGT (glucose < 11.1 mmol/L) and 1 had diabetes. In contrast, by CGMS 6 patients had (IGT) only, 3 patients hadhad both IFG and IGT, and 4 patients were Diabetics with glucose level ≥11.1 mmol/L.The mean values of HOMA and QUICKI in patients with TM were (1.6±0.8) and (0.36±0.03) respectively.Ferritin concentrations were positively correlated with the fasting BG r=0.69, P<0.01, serum ferritin correlated with the average (r=0.75; P<0.01) and the maximum BG recorded by CGM (r=0.64, P<0.05). Our data suggest that CGMS is more sensitive than OGTT in detecting glycaemic abnormalities in young adult TM patients.It seems that defective βcell function rather than insulin resistance is the most likely explanation for glycaemic abnormalities in these patients.

P.O. Cervello, Palermo, Italy; 2Department for Mathematical and Statistical Sciences ‘S. Vianelli’, University of Palermo, Palermo, Italy; 3U.O. Medicina Trasfusionale Ospedali Riuniti P.O. Cervello, Palermo, Italy; 4Fondazione Franco e Piera Cutino, Palermo, Italy; 5U.O.C. Centro di Prevenzione Diagnosi e Cura della Talassemia, ARNAS Civico, Di Cristina, Benfratelli, Palermo, Italy; 6Pediatria II, Ospedali Riuniti P.O. Villa Sofia, Palermo, Italy; 7Centro Regionale per la Cura delle Microcitemie, A.O. Cardarelli, Napoli, Italy; 8U.O.C. Pediatria A.O. Cardarelli, Napoli, Italy; 9Unità Operativa Dipartimentale Talassemia, P.O. S. Luigi-Curro, ARNAS Garibaldi, Catania, Italy; 10U.O.S. Talassemia, A.O.O.C.R. Sciacca, Italy; 11U.O.S. Centro Microcitemia, A.O. Umberto I, Siracusa, Italy; 12U.O. Talassemia, A.O. S. Eugenio, Roma, Italy; 13Centro Microcitemia A.O. Perrino, Brindisi, Italy; 14Thalassemia International Foundation, Nicosia, Cyprus; 15Centre for Haematology, Barts and The London School of Medicine and Dentistry, Department of Haematology, Royal London Hospital, London, UK; 16The Heart Hospital, University College London Hospital, London, UK

EVALUATION OF GLYCAEMIC ABNORMALITIES IN β-THALASSEMIA MAJOR USING CONTINUOUS GLUCOSE MONITORING SYSTEM AND ORAL GLUCOSE TOLERANCE TEST Mohamed Abdel Daem Mohamed Yassin,1 Ashraf T. Soliman,2 Ahmed Elawwa,2 R. Kamzoul,2 Ratna Chatterjee,3 Rekha Bajoria3 1MSc Student, Department of Haematology, Doha; 2Department of Haematology and bone Marrow Transplant and Paediatric Endocrinology, Hamad Medical Corporation, Al- Amal Hospital, Doha; 3Clinical Senior Lecturer, Consultant and Course Directors, MSc in Haemoglobinopathy, IFWH, UCL

HEART AND VASCULAR ABNORMALITIES SERIAL ECHOCARDIOGRAPHIC LEFT VENTRICULAR EJECTION FRACTION MEASUREMENTS: A TOOL FOR DETECTING THALASSAEMIA MAJOR PATIENTS AT RISK OF CARDIAC DEATH Aurelio Maggio,1 Angela Vitrano,2 Gaetano Lucania,3 Luigi Mancuso,4 Marcello Capra,5 Lorella Pitrolo,6 Luciano Prossomariti,7 Aldo Filosa,8 Vincenzo Caruso,9 Calogera Gerardi,10 Saveria Campisi,11 Paolo Cianciulli,12 Antonella Quarta,13 Michel Angastiniotis,14 Androulla Elefteriou,14 Giuseppina Calvaruso,1 Paolo Rigano,1 Paul Telfer,15 John Malcolm Walker16 1U.O.C. Ematologia II con Talassemia, Ospedali Riuniti

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3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

SCD patients had significantly lower transfusion index (TI), WBC count, hemoglobin and antithrombin III with markedly elevated D-dimer levels versus β-TM (P<0.05). LV mass, AS, and LV end diastolic diameter were higher in SCD and β-TM than controls (P<0.05), whereas, aortic strain and AD were lower in both groups versus controls (P<0.05). Multiple regression analysis showed that WBCs, HbS, LDH were independently related to ErMPs in SCD (P<0.001) while disease duration, TI, hemoglobin, HbF, LDH,, ferritin, D-dimer and antithrombin III were independently related to ErMPs in β-TM (P<0.001). AS was positively correlated to age, TI, WBCs, LDH, ferritin, D-dimer, PMPs and ErMPs levels in both patients’ groups (P<0.05) as well as HbS in SCD (P=0.01), it was negatively correlated to AD, hemoglobin and HbF. Multiple regression analysis showed that PMPs and ErMPs were independently related to aortic stiffness and distensibility. PMPs and ErMPs could be considered potential biomarkers for vascular dysfunction and disease severity and may be implicated in the pathogenesis of coagulation abnormalities encountered in CHA patients.

HEPATOLOGICAL COMPLICATIONS

MARKED IMPACT OF IL28B GENOTYPE IN THE NATURAL CLEARANCE OF HCV-RNA CLERANCE IN PATIENTS WITH HEMOGLOBINOPATHIES Angela Piazza,1 Emanuela Fecarotta,1 Disma Renda,1 Veronica Agrigento, Serena Sclafani,1 Salvatore Madonia,2 Mario Cottone,2 Aurelio Maggio1 Maria Concetta Renda1 1UOC Ematologia per le malattie Rare del Sangue e degli Organi Ematopoietici ; 2UOC Medicina I, AOR ‘‘Villa Sofia-Cervello’’, Palermo, Italy

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regimens. The number of patients with a mean basal LVEF <55% was not statistically different between the two groups. Patients in the not alive cohort had statistically significant higher LIC and serum ferritin levels compared to those of the alive group. The regression coefficent of status × year suggested a statistically significant linear decrease over time of LVEF values among the not alive patients (Coeff. -1.51, 95%; CI -2.31-0.71; P<0.0001) compared with the alive ones. Actually, the alive cohort had increased LVEF values over time, although this result was not statistically significant (Coeff. 0.27, 95% CI -0001; 0.54; P<0.051). The risk of death due to cardiac failure in patients with a mean reduction of LVEF greater or equal to 7% was 4.93, with a 95% confidence interval (CI) of 1.61 to 15.11 (P=0.005). Falls in LVEF of less than 7% were not associated with statistically significant risk for heart disease (OR=1.22, P=0.762). Serum ferritin levels greater or equal than 2500 μg/L were associated with a statistically significant risk of death for heart disease (OR=5.05, P=0.002). This retrospective survey suggests that variations in LVEF values over time, as determined by 2D-echocardiography, may be used for risk assessment of heart failure in patients with TM. A fall of LVEF 7%, over time may be considered a strong predictive tool for the detection of TM patients with increased risk of death due to heart failure. Our observation that serum ferritin levels greater or equal than 2500 μg/L were associated with a statistically significant risk of death due to heart disease confirmed findings previously published. However, because of limitations related to the study design, such findings should be confirmed in a prospective randomized clinical trial.

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MICROPARTICLES: A NEW LINK BETWEEN VASCULAR WALL STIFFNESS AND COAGULATION DISTURBANCES IN CHRONIC HEMOLYTIC ANEMIA Azza Abdel Gawad Tantawy,1 Amira Abdel Moneam Adly,1 Eman Abdel Rahman Ismail,2 Nevine Mamdouh1 1Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt; 2Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Circulating microparticles (MPs) are membrane-derived vesicles released by RBCs, platelets, endothelial cells and monocytes and may contribute to the hypercoagulable state in sickle-cell disease (SCD) and β-thalassaemia major (TM). We aimed to measure the levels of platelet and erythrocyte-derived MPs (PMPs and ErMPs) in young SCD and TM patients in relation to clinicopathological characteristics and aortic elastic properties. Forty SCD and 40 βTM of mean age 10.5 yr were compared with 40 healthy controls. Patients were studied for history of disease and therapy, hematological profile, hemoglobin electrophoresis, D-dimer, antithrombin III and flow cytometric expression of PMPs (CD41b+) and ErMPs (glycophorin A+), echocardiography to assess aortic stiffness (AS) and distensibility (AD) and LV functions. Both MPs were elevated in SCD and TM compared to controls (P<0.001).

Patients with thalassemia major (TM) and sickle/β-thalassemia (S/βThal), have a high risk of hepatitis C virus (HCV) infection. Association studies detected a singlenucleotide-polymorphism (SNP) 3 kilobases (kb) upstream of IL28B gene (19q13,-3kbC>T) associated to HCV-RNA spontaneous clearance and in linkage disequilibrium with the IL28B exon2 variant K70R. Because of the treatment of chronic HCV infection with pegylatedinterferon-α2 and ribavirin (PEG-IFN-α/RBV) could be compromised by severe side effects in TM and S/βThal patients (ribavirin-associated haemolysis), the possibility of predicting the spontaneous C virus clearance is crucial. The aim of our study was to evaluate, in patients with TM and S/βThal, the influence of the two quoted variants in hepatitis C virus clearance. Forty two patients with hemoglobinopathies, not-treated with PEG- IFN-α /RBV, were studied. DNAs were extracted from peripheral blood mononuclear cells. DNA variants were investigated by endonuclease digestion and reverse dot-blot hybridization. PCR primers were designed in our laboratory on the basis of IL28B gene map sequence. According to literature, the genotype IL28B -3kbCC was considered most frequently associated with the spontaneous HCV-RNA clearance, whereas the genotypes -3kbTT and -3kbCT were considered most frequently associated with a persistent infection. Twenty of 42 studied patients (47.6%)

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Hepatitis is the infections of a common cause disease among poly Transfused Patients. Hepatitis C is slowed progression and inducing HCC. This study assessed HCC incidences, the role of iron and possible antitumor activity of chelators in 170 thalassemia patients using deferoxamine (DFO) therapy. They are diagnosed with Hepatitis C due to positive PCR-RNA. They are treated with IFN.The follow up program including tests every 3 Months and PCR-RNA, AFP and liver US every 6 months. Whenever there was suspicion of liver malignancy, Biopsy was performed. From the total of 170 patients 59.4% were male and 40.6% were female. Mean age of thalassemia diagnosis was 2.69±5.403 (1-41) years and mean Age of hepatitis diagnosis was 17.37±7.263 (3-51) years. 92.4% of Patient’s MT, 0.6% SS, 2.9% TI. From them, 119 Patients had positive PCR-RNA. The viral genome was 1a3a. 73.5% of patients had first course of therapy. The frequency of AFP greater than 10 was 5.9%. And the incidence of HCC was 0.6%. In our study of 170 patients with CHC, one TM patients with liver siderosis and fibrosis and late introduction of iron chelation developed HCC. The main risk factor for HCC was HCV infection in TM patients but it was iron activity in TI patients. Iron chelation with DFO appeared to play a Protective role.

INCIDENCE OF HEPATOCELLULAR CARCINOMA IN PATIENTS WITH THALASSEMIA WHO HAD HEPATITIS C Ansari Shahla, Azarkivan Azita, Halagi Farideh Department of hematology and oncology, Ali Asghar Children’s Hospital, Tehran University of Medical Sciences, Tehran, Iran

the consequent preventive measures, have reduced very much the risk of transfusion trasmitted infectious diseases. However, there are still many patients who were infected before the screening of donors; they show chronicity of hepatitis C virus, and for them there is the problem of eradication of viral infection, since the possible evolution towards hepatocarcinoma is emerging as a cause of death of these patients. In our Department of Hematology with Thalassemia about 170 patiens with Thalassemia Major are being treated. The patiens with chronic hepatitis C were 51 in 2001. Of these, 20 became HCV-RNA negative thus achieving SVR after treatment with IFN monotherapy (years 1988-98) or with Peg-IFN only (years 2002-07). So in 2010, there were still 31 patients HCV-RNA positive, among these 23 (74%) with genotype 1b, 2 patiens (6%) with genotype 2a/2c, 2 patiens (6%) with genotype 3a, and 4 patiens (13%) with genotype 4c/4d. After the approval in August 2009 in Italy, by the AIFA, of the use of Ribavirin in patients with thalassemia, we proposed all of them a combined treatment with Peg-IFN and Ribavirin with varying duration of therapy (from 24 to 72 weeks) according to genotype and EVR. The patients who agreed to undergo this treatment were 14, divided as follows: 12 patients (85%) with genotype 1b, 1 patient (7.5%) with genotype 3a, 1 patient (7.5%) with genotype 4c/4d. Only 1 patient was naïve to treatment, the others being relapsers or not responders to previous treatment. Duration of treatment: 2 patients underwent 24 weeks of therapy (one because non-responder, the other for favorable genotype), 1 patient stopped it after 36 weeks for thrombocytopenia, 8 patients underwent the therapy for 48 weeks, 1 patient was treated for 54 weeks and 2 patients had 72 weeks of antiviral therapy. Only 2 patients developed severe neutropenia or agranulocytosis, which required the repeated use of GCSF. All patients increased, as expected, the need of transfusions, requiring adequate iron chelation regimens with careful monitoring of iron overload. 12 out of 14 patients resulted negative to HCV-RNA during treatment, but only 6 (43%) reimaned HCV-RNA negative 6 months after the end of antiviral therapy so with a SVR rate wich is comparable to that of the non-thalassemic population. Our experience demonstrates the feasibility of combined antiviral therapy IFN+ribavirin in patients with transfusion dependent thalassemia. Adverse events were well controlled and the results obtained in terms of SVR are satisfactory.

showed a spontaneous HCV-RNA clearance: 15/20 (75%) had an IL28B genotype -3kbCC, 5/20 (25%) had a genotype -3kbCT. Twenty two of 42 studied patients (52.4%) showed an HCV-RNA persistence: 13/22 (59.1%) showed an IL28B genotype -3kbCT; 4/22 (18.2%) showed a genotype -3kbCC; 4/22 (18.2%) showed a genotype -3kbTT, 1 was homozygous for the 3kb C allele but heterozygous for the K70R. These findings suggest that IL28B genotype seems to play a role in natural clearance of HCV-RNA. Actually, 15 out of 20 (75%) untreated patients who showed a spontaneous HCV-RNA clearance had an IL-28B -3kbCC genotype.

TREATMENT OF CHRONIC VIRAL HEPATITIS IN THALASSAEMIA MAJOR: FEASIBILITY OF THE COMBINED THERAPY IFN+RIBAVIRIN G.B. Ruffo, L. Cuccia, F. Gagliardotto, M.R. Marocco, M. Capra, Z. Borsellino U.O.C. Hematology with Thalassemia, A.R.N.A.S. Civico - Di Cristina Benfratelli, Palermo, Italy The chronic liver damage in patients with thalassemia is the result of a multifactorial process, in which a major role is played by iron accumulation and infection by hepatotropic viruses. Over the past 25 years the discovery of an effective vaccine against hepatitis B virus and identification of new pathogens, particularly hepatitis C virus, with

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SICKLE CELL DISEASE SOLUBLE CD163 LEVELS AS A NOVAL RISK MARKER FOR PULMONARY HYPERTENSION IN YOUNG SICKLE CELL DISEASE PATIENTS Azza Abdel Gawad Tantawy,1 Amira Abdel Moneam Adly,1 Eman Abdel Rahman Ismail2 1Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt; 2Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt Sickle cell disease (SCD) vasculopathy is mainly linked to intravascular hemolysis and free plasma haemoglo-

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

haemolytic transfusion reactions. Only one randomized control trial was identified with a small number of retrospective or prospective cohort or case control studies. Considerable variation was found in the timing and method of administration of prophylactic transfusion regimes amongst studies. Variation was also found in the outcome measures reported and in the nature of control groups used. This limited the statistical analysis and level of evidence that could be gained. This review failed to demonstrate consistent evidence that prophylactic transfusion improves fetal or maternal outcome to a greater degree than that which can be obtained from careful monitoring by specialist obstetric and haematology teams. Prophylactic transfusion should be limited to individual cases where the potential benefits clearly outweigh the risks.

ADHERENCE TO PROPHYLACTIC PENICILLIN IN PATIENTS WITH SICKLE CELL DISEASE - ALDER HEY CHILDREN’S HOSPITALS EXPERIENCE L. Smith, R. Keenan, L. Barton, R. Chatterjee, R. Bajoria Children with sickle cell disease (SCD) have an increased susceptibility to severe bacterial infections. Infections are a major cause of mortality and morbidity among children with SCD. A large multi-centred clinical trial (PROPS) was undertaken and an 84% reduction in the incidence of infection in the group treated with penicillin was identified, resulting in the recommendation that all children with SCD should be commenced on prophylactic penicillin. Non-adherence to medications is common in children and adolescents with chronic illness. Literature highlights an adherence rate to penicillin in children with SCD as low as 19% and no higher than 69%. The objective of this study was to establish the adherence rates to prophylactic penicillin in our cohort of patients with SCD. Three methods were used to assess adherence on 32 patients with SCD. A self-report tool and a health belief questionnaire were used in the routine haematology clinic to identify patients/parents self-report of adherence and their beliefs on SCD and infection. A urine sample was then obtained and assayed to detect the presence of penicillin. Further urine samples were then obtained when the children came to subsequent clinic appointments or attended the hospital for other reasons to try to establish a pattern of adherence. A self report rate of 90% was obtained from parents but this corresponded with a 55% adherence rate as detected by the presence of penicillin in the urine. Parents acknowledged that SCD and infections were serious for their child but very few actually thought that their child was susceptible to infections. Despite the low adherence rate to penicillin only 2 infections were detected during the studies and non of the isolates were sensitive to penicillin. The low adherence rate and lack of infections poses the question what is the ideal rate of adherence in order to prevent infections. It also highlighted the fact that more attention to adherence was needed in our cohort of patients.

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bin,and contributing to serious pulmonary hypertension (PH). Soluble CD163 (sCD163) is a biomarker of monocyte-macrophage activation and is implicated in clearance of free plasma hemoglobin. This study aimed to assess sCD163 levels in SCD in relation to clinicopathological characteristics and as a potential biomarker for PH. Sixty SCD patients aged 9.8-18.9 years were compared to 30 healthy controls. Patients were in steady state and had no clinical cardiovascular disease. They were subjected to: history of sickling crisis, transfusion and hydroxyurea therapy; hematological profile, hemoglobin electrophoresis, serum ferritin and measurement of serum sCD163; and were screened for PH defined as tricuspid regurgitant velocity (TRV) >2.5 m/s using DopplerEchocardiography. The prevalence of PH was 37.5%, 8.6% had severe PH. The mean TRV in SCD was 2.17±0.55 m/s compared to 1.47±0.4 m/sec in controls (P<0.001). sCD163 was elevated in SCD patients compared to controls (P<0.001) with the highest values in patients with PH compared to those without PH (P<0.001). Mean TRV was positively correlated with sCD163,LDH, ferritin (P<0.01) and negatively correlated with Hb (P<0.0001). The strongest predictor of PH was serum ferritin (P<0.0001), and sCD163 levels (P<0.01). sCD163 was higher in SCD without hydroxyurea therapy compared to treated patients and controls (P<0.001). There was significant positive linear relationships between sCD163 and age, leucocytic count, HbS and TRV with negative correlations with hemoglobin and HbF levels (P<0.05). A cutoff value of sCD163 at 1400 ng/mL could be considered predictor for PH with sensitivity 92.3% and specificity of 94.1% (P<0.001). High prevalence of PH was detected in young SCD, with hemolysis, asplenia and iron overload implicated in its pathogenesis. Serum CD163 can be considered a prognostic biomarker for development of PH, which needs further long term studies.

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DOES PROPHYLACTIC BLOOD TRANSFUSION DURING PREGNANCY IN SICKLE CELL DISEASE IMPROVE MATERNAL AND FETAL OUTCOMES – A SYSTEMATIC REVIEW OF THE LITERATURE L.M. Defoe, S. Cook, R. Chatterjee, R. Bajoria South Tees Hospitals NHS Foundation Trust. Dr D Plews, South Tees Hospitals NHS Foundation Trust, Haemoglobinopathy, UCL, London, UK Pregnancy in Sickle Cell Disease (SCD) is associated with an increased risk of maternal and fetal morbidity and mortality. The use of prophylactic blood transfusion during pregnancy has become standard practice in some centres whilst others consider this to be controversial. Blood transfusion is not without risk and transfusion related complications may result in maternal or fetal morbidity or mortality. The objective of this review was to determine the evidence for the use of prophylactic transfusion regimes during pregnancy in SCD. Primary outcomes measures were maternal mortality and live birth rates with secondary outcome measures of stillbirth, premature delivery, low birth weight, caesarean section, painful crisis, urinary tract infection, hepatitis, alloimmunization and delayed

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Stroke and Silent cerebral infarct (SCI) are the most common cause of brain damage in patients with Sickle-CellDisease (SCD). Approximately 10% of patients will have overt stroke. The prevalence of SCI is 21.8% in children (6 to 19 years old) with SCD. We evaluated a population of 168 Sicilian patients with SCD (141 HbS/β-thalassemia genotype, 27 HbS homozyguos) treated in our center, with MRI of the brain. 42/168 patient (25%) had SCI and 7/168 (4,1%) overt stroke. Also we report clinical data of 104 patients with S/β-thalassemia genotype treated with hydroxyurea (HU) during 11-years mean follow-up. A statistically significant reduction was observed in the number of sickle cell crises (86%, 7.8±6.9 per year, P<0.0001), hospitalizations (2.5±2.9 per year vs. 0.3±1.5, P<0.0001), and days in the hospital (22.4±21.9 per year vs. 0.3±1.5, P<0.0001). In 20 patients was performed MRI before the treatment with HU and then it was repeated periodically during HU therapy (mean follow-up, 11 years), aimed at the detection of SCI or, eventually, at determining its progression. Progressive cerebral infarcts occurred in 65% (15 of 23) of HU-treated patients. Notably, 3 overt strokes (1 of 3 relapse strokes) and 12 SCI were observed. In the latter group, 2 out of the 5 patients developed new lesions. In these patients, neurocognitive deficits were observed. Moreover, stroke and SCI were not related to the HU clinical response. A large silent infarct was observed after 7 years of therapy in one excellent responder patient (from 12 to 0 crises/year). Therefore, HU therapy appear not to prevent these complications. Most likely, factors related to chronic inflammation and vascular wall damage are involved, and a new therapeutic approach is needed.

Arabia was conducted between April 2005 and May 2010. The medical records were reviewed to define the perioperative risks and the postoperative complications in relation to the type of transfusion modality selected. The medical record of 75 SCD patients who undergoing surgeries were reviewed. Preoperatively, 25.3% had complete exchange transfusion (CETX), 17.3% had partial exchange transfusion (PETX), 26.7% had simple top up transfusion (STX) and 30.7% had no transfusion (NTX). The postoperative complications were 20% vaso-occlusive crises (VOC), 2.7% acute chest syndrome (ACS), and 16% had fever. There was 33.3% patients with prolonged duration of the hospital stay. There was no significant difference in the outcome of postoperative fever, VOC, ACS, and the length of hospital stay between all types of transfusion modalities. However, The correlation was highly significant between the pre-operative haemoglobin (Hb) level and postoperative fever (P=.001) and VOC (P=.002). Interestingly, SCD patients who received hydroxyurea were observed to have less postoperative complication like fever (P=.015) and vaso-occlusive crises (P=.011), while those who received prophylactic heparin in the postoperative period were found to have a reduced length of hospital stay (P=.005) and vaso-occlusive crises (P=.001). The guidelines for preoperative transfusion in SCD patients was effective in reducing the postoperative morbidity and mortality. However, this guidelines establish the surgical situations where preoperative transfusion is needed and the optimum regimen for different surgical types.

CEREBROVASCULAR EVENTS IN SICKLE CELL DISEASE PATIENTS TREATED WITH HYDROXYUREA Paolo Rigano, Giuseppina Calvaruso, Antonino Giangreco, Gaetano Restivo Pantalone, Disma Renda, Alice Pecoraro, Aurelio Maggio A.O. Ospedali Riuniti “Villa Sofia-Cervello” UOC Ematologia per le Malattie Rare del Sangue e degli Organi Ematopoietici, Ospedali Riuniti “Villa SofiaCervello”, Palermo, Italy

THE OUTCOME OF PREOPERATIVE TRANSFUSION GUIDELINE ON SICKLE CELL DISEASE PATIENTS AT KING FAHD HOSPITAL-JEDDAH (KSA) Sameera M. Refaie Felemban,1,2 Rekha Bajoria,2 Amani H. Alsawaf,1 Ratna Chatterjee,2 Abdulelah I. Qadi1 1King Fahd Hospital, Jeddah, Saudi Arabia; 2Institute For Women’s Health, UCL, London, UK We have developed a local hospital preoperative transfusion guidelines for sickle cell disease (SCD) patients to reduce the perioperative and the postoperative complications. This study was conducted to evaluate the outcome of practice on SCD patients undergoing surgeries in our institution. A retrospective review of 75 SCD patients undergoing surgery at King Fahd Hospital, Jeddah, Saudi

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ADMISSIONS OF SICKE CELL DISEASE CHILDREN IN HOSPITAL DE MATARÓ: THE LAST 6 YEARS Anna Cabot Dalmau, Sonia Amoretti Cubero, Sheila Tubio Gómez, Iris Collazo Vallduriola, Víctor Sanmartín Sánchez, Maria Trabazo Del Castillo Servicio de Pediatría, Hospital de Mataró, Barcelona, Spain Sickle cell disease (SCD) is now a prevalent chronic disease that has appeared in our area in the last decades mostly because of African immigration. Children affected often require hospitalization because of its frequent complications. To evaluate the frequency of hospital admissions and their main diagnoses in children affected of SCD. Retrospective and descriptive analysis of clinical recorded data of 10/17 children with SCD who required hospital admission, collected through the last 6 years. Amidst 17 SCD children under 18 y controlled in our Paediatric Unit, (12 SS and 5 S-β-thalassaemia0) 10 required hospitalizations in the last 6 years (8 SS and 2 Sβ-thalassaemia0). Among 7 patients who did not require hospitalizations 3 were under three at the end of followup ant the other 4 had high HbF levels, media 24%. The rate of admissions was 0.09/patient/year, similar to SCD literature (0-5 times per y). The causes of hospitalization were: 26 vaso-occlusive crisis (VOC) (61.9%), 3 Acute thoracic syndrome (ATS) (7.14%), 6 cases of worsening of basal levels of Hb that needed red cell transfusion (14.3%), 1 splenic infarction complicating an admittance

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Thalassemia hemoglobinopathies,chronic hemolysis and iron overload cause functional and pathological abnormalities in various organ systems. This study aimed to assess prevalence and severity of renal glomerular and tubular dysfunction in young β-thalassemia major (β-TM) and thalassemia intermedia (TI ) patients and identify risk factors and early predictors for renal impairment. Sixtysix β-TM and 26 TI patients, aged 2.5-16 years were compared to 40 healthy controls. Clinical history and examination were done,laboratory investigations included: CBC, liver/ kidney functions, hepatitis B and C markers, serum ferritin and bicarbonate, plasma osmolality; urinary testing for total urinary proteins, microalbumin excretion, N-acetyl-β-D-glucosaminidase (NAG), retinol binding protein (RBP), α-1 microglobulin, bicarbonate and osmolality. Calculation of corrected creatinine clearance according to surface area, % fractional excretion of bicarbonate (% Fr. Ex. Bicarb.) and the ratio of urine/serum osmolality were done. Renal abnormalities among thalassemics included :proteinuria (71%), increased RBP (69.4%) and α-1 microglobulin (54.8%), increased urinary NAG (58.1%), decreased urinary osmolality (58.1%), increased % Fr. Ex. Bicarb. (32.2%), microalbuminuria (29%). Urinary total proteins and microalbuminuria were higher in thalassemics versus controls (P<0.01). NAG, RBP, α-1 microglobulin were elevated in thalassemics versus controls (P<0.01), RBP was higher in TM versus TI (P<0.01). Correlation was positive between NAG, RBP, α-1 microglobulin and serum ferritin (P<0.01),with negative correlation between urine osmolality and ferritin (P< 0.01). Urinary total protein was positively correlated with microalbuminuria, NAG, RBP,α-1 microglobulin (P<0.01). The Z-score analysis for discrimination of patients with renal dysfunction proved superiority of both urine total protein and RBP compared to other studied parameters. Asymptomatic glomerular and tubular

Haemoglobinopathies are the commonest single gene disorders in the world. They manifest as hemolytic anaemia of differing severity. There is relative imbalance between the α and non α chains. They separate from haem and precipitate on the red cell membrane, prompting premature haemolysis, and apoptosis, precipitating anaemia - the hallmark of haemoglobinopathy. This results in generation of free radicals mainly via Fenton reaction. Toxic iron species also catalyze the formation of free radicals, inducing multi-organ damage. This is an end organ disease – if remains untreated. The present study was carried out to assess the improvement in haematological, biochemical, oxidative stress and clinical parameters after introduction of antioxidants. Fifteen subjects suffering from HbE-β-thalassaemia and their normal siblings were tested for common haematological, biochemical and oxidant parameters at initiation. Then they were started on curcuminoid and piperine derivatives at a dose of 250mg twice daily and continued for 6 months. There after the medication was stopped. Blood samples were collected every three months till the 9th month, to test for the same parameters. The results obtained was analyzed statistically and presented. Increased oxidative stress was noted in all patients suffering from haemoglobinopathy, clearly in excess to their normal siblings. Curcuminoid and piperine supplementation of affected subjects, showed improvement of all the parameters of free radical induced injury. Some also exhibited stabilization of haemoglobin levels and marginal reduction of serum ferritin. Three months after withdrawal, most parameters returned to near pre- treatment levels. As Curcumin and piperine derivatives have shown reduction in free radical induced damage in subjects suffering from haemoglobinopathy, it may be combined with existing therapy, to reduce the oxidant induced cellular damage.

BIOMARKERS OF RENAL DYSFUNCTION IN β-THALASSEMIA MAJOR AND INTERMEDIA PATIENTS Azza Abdel Gawad Tantawy, Nagham S. El Beblawy, Amira Abdel Moneam Adly Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

EFFECTS OF TREATMENT WITH CURCUMINOID AND PIPERINE DERIVATIVE ON SUBJECTS SUFFERING FROM HAEMOGLOBINOPATHY Prosanto Chowdhury,1 Rekha Bajoria,1 Ratna Chatterjee,1 Manikanchan Das,2 Nabendu Chaudhuri2 1Institute for Women’s Health, UCL, London, UK; 2KPC Medical College, Kolkata, India

OTHER

dysfunction are prevalent in young asymptomatic TM and TI patients. Regular screening by urine total protein and RBP may be cost-effective for early detection of renal dysfunction in thalassemics.

because a VOC (2.3%), 2 osseous infections (4.76%), one with sepsis, and 5 surgical procedures (11.9%) such as bone drilling of femoral head, circumcision, cholecystectomy, cholecystectomy+splenectomy, and adenoid exeresis+myringotomy. One child with osteomyelitis required surgical drainage. 4 patients (9.5%) required ICU, 1 sepsis and 3 ATS. 19 children (45.2%) received a red cell transfusion. SCD often needs hospitalization because of its frequent acute complications, which are often severe and may require ICU. The SCD cohort of 17 children followed in this small comarcal Hospital showed a rate of admissions similar to what is described, and as usual, most of them were due to VOC, and needed ICU 7,14%. There have been no deaths.

ADHERENCE OF THALASSEMICS TO THE VACCINATION PROGRAM FOR THE FLU AND STREPTOCOCCUS PNEUMONIAE Kalpaka Anastasia, Stefanidou Christine, Vlahou Evangelia, Aggelaki Maria Thalassaemia Unit, “Saint Paul” Hospital, Thessaloniki, Greece Patients with thalassaemia major are relatively susceptible to the flu and infections from streptococcus pneumo-

Thalassemia Reports 2012; 2 (s2) | 33 |

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In thalassaemia, red cells are hypochromic and microcytotic. On the contrary, the lack of vitamin B12 causes megaloblastic anemia. The co-existance of megaloblastic anemia and lack of B12 vitamin may result as a diagnostic problem in patients with thalassaemia from the hematological point of view (volume of red blood cells). The early diagnosis of the co-existance of thalassaemia and megaloblastic anemia. We studied 44 patients with thalassaemia (11 men and 33 women) with an average age 41.21. From them, 35 had thalassaemia major and 9 had thalassaemia intermedia. All the patients had vitamin B12 levels within the normal range, except from only one patient with thalassaemia major. The cause of the lack of vitamin B12 was investigated, if it was nutritic or organic. Nothing pathological was found. The patient underwent a per os treatment and the vitamin B12 levels were restored. Patients with thalassaemia major may present other hemopoetic disorders. The appearance of microcytosis in the CBC may conceal a lack of folic acid or vitamin B12.

B12 VITAMIN LEVELS IN THALASSAEMIA PATIENTS A. Kalpaka, C. Stefanidou, E. Vlahou, M. Papazaharia Thalassaemia Unit, “Saint Paul” Hospital, Thessaloniki, Greece

quently iron overload in β-thalassaemia patients. The aim of this study was to demonstrate the outcome of splenectomy during a long follow up period with emphasis in thromboembolic events (TEE), pulmonary hypertension (PHT) and non- transfusion related infections, to investigate the hypothesis that splenectomy may be a risk factor for these complications. One hundred and sixty-two patients were investigated retrospectively. Patients classified according to splenectomy status as splenectomized (54%) or non-splenectomized and according to phenotype as thalassaemia major (TM) (83 patients) or intermedia (TI) (79 patients). Splenectomies performed between 1952 and 2009. PHT defined as a sustained PASP>36 mm Hg. Clinical characteristics and risk factors were reviewed in groups of patients with or without TEE, PHT and infections. The overall prevalence of TEE was 10.5%. All these events occurred in splenectomized patients (P<0.0001). The risk of TEE was higher in older aged patients (P<0.001), but we did not find any association with phenotype. The overall prevalence of PHT was 17.4%. There was significant association of PHT with splenectomy, TI (P<0.05), and the number of transfusions were less in PHT (P<0.001). The risk was higher in older aged patients (P<0.001). The overall incidence of infections was 0.62 per 100 patient years. There was no association of infection with splenectomy and phenotype. Pneumonia was the most common event of infections. Splenectomy can be associated with an increased risk of TEE, PHT, and severe overwhelming infections in non protected patients. Of particular importance is the need for assessment of the risk in the older patients, and the more vulnerable groups mainly the transfusion independent TI patients, providing appropriate strategies to prevent those consequences.

niae. The purpose of the present study was to evaluate the percentage of the thalassemics monitored and transfused in our thalassaemia unit that complied with the vaccination program that we follow for the influenza virus and streptococcus pneumonia with the pneumococcal polysaccharide vaccines. Forty-four persons were studied. All of them had b- thalassaemia major. From them, there were 11 men and 33 women. The average age was 41.21. Twenty one of them, 47.7%, (6 men and 11 women) had been splenectomized and 23 had not (52.27%). 95.5% of the thalassaemia patients had been vaccinated for the influenza virus and 90.9% for streptococcus pneumoniae. We also noted that all the thalassemics who had undergone a splenectomy (100%), had followed closely the vaccination program and were all vaccinated against both infectious agents. The percentage of the thalassemics vaccinated was very high. The findings of this study underline the important role of the physician in the special approach of a person with thalassaemia, that should be from all aspects.

THE OUTCOME OF SPLENECTOMY IN β-THALASSAEMIA Michael Hadjigavriel,1 Krikor Simamonian,1 Yiota Christou,1 Michael Angastiniotis,2 Ratna Chatterje,3 Rekha Bajoria3 1Thalassaemia unit, Limassol General Hospital, Cyprus; 2Thlassaemia International Federation, Nicosia, Cyprus; 3Institute for Women’s Health, University College Hospital, London, UK Splenectomy had been the most common management strategy for reducing transfusion requirements and subse-

| 34 | Thalassemia Reports 2012; 2 (s2)

HLA-TYPING BY BUCCAL SWAB TO FACILITATE ACCESS TO BONE MARROW TRANSPLANTATION GLOBALLY. THE CURE2CHILDREN FOUNDATION PRELIMINARY EXPERIENCE Mohamed El Missiry,1 Priya Marwah,2 Rajpreet Soni,2 Sadaf Khalid,1 Naila Yaqub,3 Tatheer Zara,3 Sarah Khan,3 Fatima Itrat,3 Mohamed Hamed Hussain,1 Nezih Cereb,4 Lawrence Faulkner1 1Cure2Children Foundation, Florence, Italy; 2South East Asia Institute for Thalassemia, Jaipur, India; 3Children’s Hospital Pakistan Institute of Medical Sciences, Islamabad, Pakistan; 4Histogenetics, Ossining, NY, USA Bone marrow transplantation (BMT) remains the only definitive cure for thalassemia and sickle cell disease (SCD). Finding a compatible family member is an initial crucial step. Compatibility testing has relied on blood samples which are logistically complicated to collect and send to distant labs performing HLA-typing. The Cure2Children-Foundation (C2C) thanks to a special rate for HLA-typing by Histogenetics (HG) for this project, has offered centralized buccal swab DNA-based low-resolution HLA class I A and B typing, directly or through partner centers, in lower-income countries for thalassemia patients and potential donors. After registration

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

EX-VIVO EXPANSION OF CORD BLOOD DERIVED HAEMATOPOIETIC STEM CELLS Alice Pecoraro, Rosalia Di Stefano, Laura Scolaro, Elena Baiamonte, Barbara Spina, Antonio Troia, Rosalba Di Marzo, Germana Fiorentino, Desiderio Gueli Alletti, Aurelio Maggio, Santina Acuto UOC Ematologia per le Malattie Rare del Sangue e degli Organi Ematopoietici, Ospedali Riuniti “Villa Sofia-Cervello”- Palermo, Italy

Gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cell (HSCs), holds the promise to treat β-thalassemia and SCD patients lacking a HLA-compatible donor and it is becoming a therapeutic reality. Several factors, including HSC source, will be important in determining efficient gene transfer and subsequent engraftment. Umbilical cord blood (UCB) HSCs have biological features that favor hematological recovery after transplantation and are more amenable to gene transfer than those of adult; thus the idea to collect UCB cells of the patient at birth, to genetically modify and store them until the young patient presents optimal clinical conditions for the autologous transplantation is very attractive. Since one of the major obstacle lies in the low number of HSCs per CB unit the aim of this study was to expand UCB-HSCs and to characterize them. Ex-vivo expansion of immune-selected CD34+ cells from 10 UCB units. FACS analysis to determine number and percentage of cells populations that express surface markers typical of stemness or differentiation; CFU assay in methylcellulose. The CD34+ cells expansion profiles were similar in all samples: an average of 5 fold-increase at day 4-6 and 10 fold-increase at day 810. The CD34+ percentages were maintained up to 5 days declining on day 8 when the CD34- began to expand. The expansion of CD34+ refers to a more immature HSC subpopulation (CD34+/CD133+/CD38). In CFU assay a similar frequency of colonies from primitive progenitors (CFU-GEMM, BFU, and CFU-GM) was detected at day 0 and at day 4-6 of expansion, while at day 8 these colonies were much less represented than those derived from more mature progenitors (CFU-G and CFU-M). Our results demonstrate the ability to expand 5-10 times HSCs (CD34 +) from UCB which show characteristics of stemness as evidenced by phenotypic and clonogenic analyses.

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CLINICAL AND HAEMATOLOGICAL PHENOTYPE OF THE -101 C>T SUBSTITUTION IN GREEK β-THALASSAEMIA PATIENTS Polyxeni Delaporta,1 Konstantinos Stokidis,1 Joanne Traeger-Synodinos,2 Emmanuel Kanavakis,2 Antonis Kattamis1 1First Department of Pediatrics, University of Athens, Greece; 2Department of Medical Genetics, University of Athens, Greece

yurea (1 in group A). The -101 C>T mutation causes mild reduction of β-globin chain synthesis. Compound heterozygotes with other severe β-thalassaemia gene defect result in thalassemia intermedia of moderate severity but with appreciable complications, which may increase with age. These data are useful for planning therapeutic approach and genetic counseling.

in C2C database, buccal swab collecting kits were labeled and sent from India to worldwide locations accompanied by simple photographic guides. Once collected, samples were sent by airmail to HG which issued typing results to C2C electronically. Families and/or referral centers were then notified. From December 2009 to April 2012 a total of 54 cases were included from ten countries: India (n=36), Pakistan (n=9), Iraq (n=2), and from Afghanistan, Algeria, Morocco, Nigeria, Uganda, UAE and Vietnam (n=1 each). BMT indication was thalassemia (n=50), SCD, acute myeloid leukemia, Diamond-Blackfan anemia, and severe combined immunodeficiency (n=1 each). BM donors were mainly siblings except for three who were first degree relatives. In 18 cases (33%) one or more compatible donors were identified. Duration between collection and results reporting was 21 (9-75) days, and between HG sample delivery and HLA reporting was 4 (2-37) days, median (range). C2C covered BMT costs, completely or partially, in the 18 compatible cases: 4 successfully undergone transplantation, 13 being prepared, including confirming HLA-typing locally, and one opted out. To date, no major discrepancy were identified between HG and local confirmative HLA-typing. HLAtyping using a buccal swab is simple, reliable and globally applicable.

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The β-thalassaemias are due to gene defects causing either decreased (β+or β ++) or absent (β0) β-globin chain synthesis. The C>T substitution at position -101 from the Cap site within the distal CACCC box is considered the most common amongst the β++ (silent) β-thalassaemia mutations in Mediterranean populations. In this study, we evaluated the clinical and haematological parameters in compound heterozygotes for the -101 C>T and a β-thalassaemia gene defect. Patients with β-thalassaemias followed in our unit, were categorized in Group A (β101C>T/β0) or Group B (β-101C>T/β+) based on their genotype. The mean values of the hemoglobin, MCV, serum ferritin and reticulocyte levels were calculated. Clinical and haematological data were collected. Twenty-eight patients (14 females) were included in this report. Hemoglobin values were some lower in Group A (21 patients, mean age 26 years) than Group B (7 patients, mean age 15 years) (9±1.2 vs. 10±1.2 g/dL, P>0.05, respectively). Complications were observed mainly in Group A and involved: iron overload (4), cholelithiasis (5), cholecystectomy (4), thyroid disorders (5), splenectomy (3), thrombosis (1) and portal hypertention (1). The only complications in Group B were splenomegaly (4). Treatment modalities included: occasional transfusions (3 in group A), iron chelation (2 in group A) and hydrox-

Thalassemia Reports 2012; 2 (s2) | 35 |

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Patient with sickle cells disease (SCD) experience serious and potentially life – threatening complication that can affect their health and well being. To assess the prevalence of disease complications among sickle cell disease at our institute. To provide accurate data on Health Related Quality of Life (HRQoL) for patients with SCD and to measure the impact of sickle cell disease, severity, disease complication and outcome through Patient Health Questionnaire (PHQ). This was a cross-sectional study of SCD patients attending Hematology clinic at King Abdul Aziz University Hospital from Jan. 2009 to Dec. 2011. The study participants were patients with SCD age 2 to 48 years who presented for a routine visit to the clinic. A total of 50 tailored questions were customized to measure the HRQoL, the mean outcome for children was measured by the CHQ-Parent. A total of 115 consecutive patients with SCD completed the PHQ. Fifty two (45.2%) were males and sixty three (54.86%) were females. Patient age range from 2 to 48 years with mean age 19.80 old, less than 12 years old were 22 (19%) from 12 to 18 years old 33 (28.6%) above 18 years old 60 (52%) patient. A total of 78 (67.8%) were classified as a severe while 37 (32.2%) were classified as mild. The PHQ is a valid tool to assess HRQL in patients with SCD and could serve as an important adjunct to determine the effect of SCD on life style. Organ damage and bone diseases have a serious negative impact on the quality of life. In spite of severe non benign sickle cell disease among our population, adherent to the treatment and early diagnosis, treatment can improve the outcome and the quality of life of sickle cell patient.

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The International Centre of Bone Marrow TransplantationMIH Foundation, directed by Guido Lucarelli, Rome, Italy, is a pioneer in pediatric bone marrow transplantation (BMT) for hemoglobinopathies as thalassemia and sickle cell disease. We have a specialized team of 21 nurses and 11 doctors achieving survival rates up to 97%. We have families from a wide variety of cultures and who speak thirteen languages including Arabic, Indian and English. Health education for transplantation is continuous, holistic, and includes the intercultural involvement of various healthcare professionals including interpreters. Family education first occurs in the day hospital, which is critical to prepare the patient for transplantation. The transplant process, risks and benefits, life after transplantation and potential risk for complications post-BMT are explained to all families. The nursing staff has to ensure that patient and family support is continuous and culturally appropriate. Respecting family’s traditions and values, while ensuring the safety of the child during transplantation and post-transplant is essential to success. Counseling in our center includes teaching for child and family skill development, access to and use of resources, homecare management, decision making, and self care. During transplant when the children often feel frustrated and stressed, a clown therapy group made up of specialists and nurses provides psychological support to children. Parents and children are included in the care plan and assistance of the child during all stages of the transplant. Our work was efficient and satisfactory and we have received so many thanks from our young patients and their relatives, coming from a diverse variety of countries such as Romania, Pakistan, Palestine and Iraq, which makes us proud and respected while helping us to grow as humane professionals. Success is based on the active role of the all the team members particularly nurses.

PATIENT AND FAMILY EDUCATION IN A MULTI-ETHNIC BONE MARROW TRANSPLANT CENTRE: NURSE’S ROLE Musa Dubali International Centre of Bone Marrow TransplantationMIH Foundation, Rome, Italy

DISEASE COMPLICATIONS OF PATIENT WITH SICKLE CELLS DISEASE AND QUALITY OF LIFE OUTCOMES AT KING ABDUL AZIZ UNIVERSITY HOSPITAL JEDDAH, KINGDOM OF SAUDI ARABIA Soad K. Al Jaouni,1 Taher Halawa,1 Mutasem S. Al Mehayawi,2 Mohammad Al Muhayawi1 1Hematology Department, 2Third Year Medical Student, King Abdul Aziz University Hospital, Faculty of Medicine, Jeddah, Kingdom of Saudi Arabia

QUALITY CARE FOR QUALITY OF LIFE

| 36 | Thalassemia Reports 2012; 2 (s2)

of authors 3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012,Index Limassol - Cyprus

Bajoria R.................................................................16, 31

Admoni O. ....................................................................27 Agapidou A. ....................................................17, 18, 24 Aggelaki M. ..................................................................33 Agrigento V.............................................................25, 29 Ahluwalia J. ..................................................................18 Akhlaghpoor S. .............................................................14 AL Arrayed K...............................................................14 Al Jaouni S.K................................................................36 Al Mehayawi M.S.........................................................36 Al Muhayawi M............................................................36 Al-Dujaily W.H.............................................................26

Barton L. .......................................................................31 Belhoul K......................................................................14 Bianchi P. ........................................................................8 Biradar M......................................................................16 Blankenstein O..............................................................17 Bordbar M.R. ................................................................15 Borsellino Z. .................................................................30 Boutou E. ................................................................17, 18 Breuer W. ........................................................................1 Cabantchik Z.I. ...............................................................1 Cabot Dalmau A. ..........................................................32 Calvaruso G. .................................................................25 Calvaruso G. ...........................................................28, 32

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Al-Fulaij R.G. ...............................................................16

Barnard M.......................................................................4

Adly A.A.M......................................................29, 30, 33

Bansal D........................................................................18

Acuto S. ..................................................................19, 35

Balassopoulou A. ...................................................17, 18

Abu Laban J..................................................................14

Index of authors

Al-Maaroof Z.W. ..........................................................26

Calzolari R. ...................................................................19 Camaschella C. ...............................................................1

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Al-Marzoki J.M. ...........................................................26

AL-Wataify A.S. ...........................................................25

Alemayehou M. ......................................................17, 18

Aletra V...................................................................17, 18 Allard S...........................................................................5

Cappabianca M.P. .........................................................15 Cappellini M.D. ................................................20, 21, 22 Capra M. .......................................................................28 Capra M. .......................................................................30

Alwadi V. ................................................................17, 18

Carsten L.W. .................................................................19

Amato A........................................................................15

Caruso V........................................................................28

Amoretti Cubero S........................................................32

CassarĂ F. ........................................................................2

Angastiniotis M. ...........................................................28

Cereb N.........................................................................34

Angastiniotis M. ...........................................................34

Chabra S........................................................................18

Arnold D. ......................................................................13

Chan L.L. ......................................................................22

Avramidou E...........................................................17, 18

Chatterje R. ...................................................................34

Aydinok Y. ..............................................................22, 23

Chatterjee R..............................14, 17, 19, 22, 28, 32, 33

Azarkeivan A..........................................................14, 30

Chatterjee R. ...........................................................16, 31

Bagga R. .......................................................................18

Chaudhuri N..................................................................33

Baiamonte E. ..........................................................19, 35

Chowdhury P.................................................................33

Bajoria R. ...........................14, 17, 19, 22, 28, 32, 33, 34

Chuncharunee S......................................................20, 21

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Alsawaf A.H. ................................................................32

Campisi S......................................................................28

Thalassemia Reports 2012; 2 (s2) | 37 |

Erickson A. ...................................................................13

Cohan N. .......................................................................12

Eshwar K. .....................................................................16

Collazo Vallduriola I.....................................................32

Espósito B.P. ...................................................................1

Colosimo A...................................................................15

Ezzat H.M. ....................................................................23

Cook S. .........................................................................31

Farideh H. .....................................................................30

Cottone M. ....................................................................29

Farmaki K. ..............................................................26, 27

Cuccia L........................................................................30

Faulkner L...............................................................12, 34

D’Alcamo E. .................................................................25

Fecarotta E. ...................................................................29

Daglis Th. ..............................................................17, 18

Filosa A.........................................................................28

Dart J.............................................................................10

Fiorentino G..................................................................35

Das M............................................................................33

Foglietta E.....................................................................15

Das R. ...........................................................................18

Frömmel C. ...................................................................17

De la Salle B...................................................................7

Gagliardotto F. ..............................................................30

Defoe L.M.....................................................................31

Galanello R...............................................2, 3, 20, 21, 22

Delaki V. .................................................................17, 18

Gambhir S.....................................................................16

Delaporta P..............................................................24, 35

Garewal G.....................................................................18 Garypidou V..................................................................24 Gerardi C. .....................................................................28

Di Stefano R. ..........................................................19, 35

Giangreco A..................................................................32

Diaz T............................................................................13

Gianni D........................................................................15

Dighe N.........................................................................16

Gkorytsa V. ...................................................................24

Donnelly B....................................................................13

Godo A..........................................................................24

Dormandy E..................................................................16

Gompakis N. .................................................................24

Dubali M.......................................................................36

Govind G. .....................................................................16

Dupuis K.......................................................................13

Grisanti P.......................................................................15

Dyson S...........................................................................8

Gueli Alletti D. .............................................................35

Economou M. ...............................................................24

Gulbis B. .........................................................................7

Economou M. ...............................................................24

Habr D. .............................................................20, 21, 23

El Beblawy N.S. ...........................................................33

Hadjigavriel M..............................................................34

El Missiry M.................................................................34

Haghpanah S.................................................................15

El-Alfy M. ..............................................................22, 23

Halawa T. ......................................................................36

El-Ali A. .......................................................................22

Hanson D. .....................................................................13

El-Beshlawy A........................................................22, 23

Hashemieh M................................................................14

Elawwa A......................................................................28

Hocaoglu M.B...............................................................22

Elefteriou A. .................................................................28

Hughes A.M.J. ..............................................................13

Epitropou M..................................................................19

Hussain M.H. ................................................................34

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Di Marzo R. ............................................................19, 35

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Di Biagio P....................................................................15

Cianciulli P....................................................................28

Index of authors

| 38 | Thalassemia Reports 2012; 2 (s2)

Kythreotis L. ...................................................................9

Ioannides A.....................................................................9

Lai Y..............................................................................22

Ioannou X. ......................................................................9

Latinovic R. ..................................................................16

Ismail E.A.R. ..........................................................29, 30

Lawniczek T. ....................................................20, 21, 23

Itrat F.............................................................................34

Leitch H.A. ...................................................................23

Jagannathan L. ..............................................................16

Lerone M. .....................................................................15

Jasbir Kaur ....................................................................18

Levin C. ........................................................................27

Jayatunga R...................................................................13

Li R. ..............................................................................20

Johari S. ........................................................................15

Lobitz S.........................................................................17

Jones R............................................................................4

Loizidou D. .....................................................................9

Kai-Hsin L. ...................................................................22

Loutradi A. ...................................................................17

Kalogerou E. ...................................................................9

Lucania G......................................................................28

Kalpaka A. ....................................................................33

Maakaron J.E. .................................................................3

Kalpaka A. ...................................................................34

Madonia S.....................................................................29

Kamzoul R. ...................................................................28

Maggio A. ..................................2, 19, 25, 28, 29, 32, 35

Kanavakis E. .................................................................35

Makariou X.....................................................................9 Mamdouh N. .................................................................29 Mancuso L. ...................................................................28

Karayiannis P. .................................................................5

Manitsa A. ...................................................................17

Karimi M. .........................................................12, 15, 19

Marocco M.R................................................................30

Karioti A. .....................................................................24

Martin N........................................................................22

Karitzie E. .....................................................................19

Marwah P. .....................................................................34

Kattamis A..................................................20, 21, 24, 35

Marwaha R.K................................................................18

Keenan R. .....................................................................31

Masalha R. ....................................................................27

Kent A...........................................................................11

Masi L...........................................................................15

Khalid S. .......................................................................34

Mastropietro F...............................................................15

Khan S. .........................................................................34

Mathur A.......................................................................16

Kilinรง Y. ........................................................................23

May M. ...........................................................................6

Kleanthous M. ..........................................................3, 19

McCartney H. ...............................................................23

Koren A. ......................................................................27

Michaelides D.................................................................1

Kossiva L. .....................................................................24

Missouri-Khetab E........................................................11

Kostaridou S. ................................................................24

Mosca A..........................................................................7

Kreka B.........................................................................24

Mufti N. ........................................................................13

Kreka M. .......................................................................24

Musallam K.M................................................................3

Kumar R........................................................................20

Nalbantoglu B...............................................................22

Kyrri A. ..........................................................................9

Nastas E. .......................................................................24

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Karakasidou O. .......................................................17, 18

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Karakas Z................................................................20, 21

Hyde K............................................................................7

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

Thalassemia Reports 2012; 2 (s2) | 39 |

Saha S. ..........................................................................18

Panigrahi I.....................................................................18

SanmartĂn SĂĄnchez V. ...................................................32

Papachristou F...............................................................24

Sawyer L.......................................................................13

Papadaki M. ..................................................................12

Schott M.A....................................................................13

Papazaharia M. .............................................................34

Sclafani S................................................................25, 29

Pappa C...................................................................26, 27

Scolaro L.......................................................................35

Parand S. .......................................................................15

Scott Pearsall R.............................................................20

Pecoraro A..................................................19, 25, 32, 35

Shah F. ............................................................................4

Pennell D.J....................................................................23

Shahla A........................................................................30

Perri M. .........................................................................15

Shankar A.G. ................................................................16

Petrou M. ......................................................................19

Sheibani K. ...................................................................14

Phylactides M. ..............................................................19

Sherman M.L. ...............................................................20

Piazza A..................................................................25, 29

Shirkavand A. ...............................................................14

Piga A. ..........................................................................23

Simamonian K. .............................................................34

Piscitelli R.....................................................................15

Sinopoulou K..........................................................17, 18

Pitrolo L. .......................................................................28

Siritanaratkul N.......................................................20, 21 Sitarou M. .....................................................................19 Smith L. ........................................................................31

Porter J.B. .........................................................20, 21, 23

Sofocleous C.................................................................24

Prappas N......................................................................17

Soliman A.T. .................................................................28

Prendushi X. .................................................................24

Soteroula C. ..................................................................11

Prescott E. .......................................................................4

Spina B. ..................................................................19, 35

Prossomariti L...............................................................28

Stefanidou C. ................................................................33

Qadi A.I. .......................................................................32

Stefanidou C. ................................................................34

Qendro I. .......................................................................24

Steiakaki E. ...................................................................24

Qirjako G. .....................................................................24

Stokidis K. ....................................................................35

Quarta A........................................................................28

Streetly A. .....................................................................16

Rapanakis V. ...................................................................7

Sung Sohn Y. ..................................................................1

Refaie Felemban S.M. ..................................................32

Suragani RNVS ............................................................20

Renda D. ...........................................................19, 29, 32

Sutcharitchan P. ......................................................20, 21

Renda M.C....................................................................29

Taher A.T. ...........................................................3, 20, 21

Restivo Pantalone G. ....................................................32

Tantawy A.A.G.................................................29, 30, 33

Rigano P......................................................19, 25, 28, 32

Telfer P. .....................................................................6, 28

Rinaldi S. ......................................................................15

Teli A. ..........................................................................24

Ros J. ......................................................................20, 21

Tennenbaum Rakover Y................................................27

Ruffo G.B. ....................................................................30

Terpos E. .........................................................................5

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Ponzini D. .....................................................................15

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Polychronopoulou S......................................................24

Paleari R..........................................................................7

Scientific Program

| 40 | Thalassemia Reports 2012; 2 (s2)

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias, 24-26 October 2012, Limassol - Cyprus

Theodoridou S. .................................................17, 18, 24

Voskaridou E...................................................................5

Trabazo Del Castillo M. ...............................................32

Walker J.M................................................................2, 28

Traeger-Synodinos J. ................................................9, 35

Weisskopf M. ................................................................23

Trehan A. ......................................................................18 Troia A....................................................................19, 35

Wu J. .............................................................................23

Tubio G贸mez S. ............................................................32

Xatjilambi G. ..................................................................9

Tzoulis P..........................................................................4

Yaqub N. .......................................................................34

Vakalopoulou S.............................................................24 van Wijk R. .....................................................................8 Vassilopoulos G. ...........................................................12

Yassin M.A.D.M. ..........................................................28 Yazman D. ....................................................................22 Yesilipek A. ..................................................................23

Tzoumari I. .............................................................26, 27

Yiota C. .........................................................................34

Vitrano A. .....................................................................28

Younis K. ......................................................................14 Yunes M. .......................................................................27

Vlachaki E. ...................................................................18

Viprakasit V. .....................................................20, 21, 23

Vlackaki E. ...................................................................24

Zara T............................................................................34

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Vlahou E. ......................................................................34

Zachariou M..................................................................19

Vlahou E. ......................................................................33

Zhang Y.............................................................20, 21, 23

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Voskaridou E...........................................................17, 18

Thalassemia Reports 2012; 2 (s2) | 41 |

ly on se lu ia er c m om -c on N PAGEPress Publications, Pavia, Italy | www.pagepress.org |

3rd Pan-European Conference on Haemoglobinopathies and Rare Anaemias 24-26 October 2012, Limassol â&#x20AC;&#x201C; Cyprus