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Objective Alpha thalassemia major (ATM) is often fatal in utero due to severe hydrops fetalis. Although in utero transfusions (IUTs) are increasingly used to allow fetal survival in ATM, prenatal and postnatal outcomes are not well described. Methods We retrospectively reviewed cases of ATM at our institution treated with consecutive IUT. Clinical records were reviewed for transfusion history, neurodevelopmental outcomes, anatomic abnormalities, survival to hematopoietic cell transplantation, and transfusion independence. A systematic review was performed, and additional reported cases are discussed. Results Three patients who underwent IUT for ATM were identified, and review of the literature revealed 17 reported cases. Of patients who received IUT, reported neurodevelopmental deficits occurred in 29% (4/14) and anatomic abnormalities in 55% (11/20). Four patients eventually underwent successful hematopoietic cell transplantation. Transfusion volumes were less than suggested guidelines for other causes of fetal anemia in 91.7% of the transfusions. Conclusion This series demonstrates the potential for achieving full fetal development with normal neurologic outcomes in those affected by ATM. It provides support for continued patient and provider education about current benefits and risks of active prenatal therapy for fetuses with ATM, as well as continued research to optimize therapeutic strategies such as in utero transplantation. © 2016 John Wiley & Sons, Ltd.

A high tricuspid regurgitant jet velocity (TRV) signifies a risk for or established pulmonary hypertension (PH), which is a serious complication in thalassemia patients. The underlying pathophysiology in thalassemia subgroups and potential biomarkers for early detection and monitoring are not well defined, in particular as they relate to spleen removal. To better understand some of these unresolved aspects, we examined 76 thalassemia patients (35 non-transfused), 25 splenectomized non-thalassemia patients (15 with hereditary spherocytosis), and 12 healthy controls. An elevated TRV (>2.5m/s) was found in 25/76 (33%) of the patients, confined to non-transfused or those with a late start of transfusions, including patients with hemoglobin H-constant spring, a finding not previously described. These non or late-transfused patients (76% splenectomized) had significantly increased platelet activation (sCD40L), high platelet count, endothelial activation (endothelin-1), and hemolysis (LDH, plasma-free Hb), while hypercoagulable and inflammatory markers were not significantly increased. The same markers were increased in the seven patients with confirmed PH on cardiac catheterization, suggesting their possible role for screening patients at risk for PH. A combination of hemolysis and absence of spleen is necessary for developing a high TRV, as neither chronic hemolysis in the non-splenectomized thalassemia patients nor splenectomy without hemolysis, in the non-thalassemia patients, resulted in an increase in TRV.

This study aimed to demonstrate the validity, reliability and responsiveness of a new disease-specific quality of life (QoL) questionnaire for children and adults with thalassaemia major, the Transfusion-dependent QoL questionnaire (TranQol). 106 participants (51 adults and 55 children) were recruited from six North American thalassaemia treatment centres with a mean age of 20·7 years (standard deviation [SD] 9, range 7-51 years). The mean total TranQol score was 71 (SD 17, 32-97) on a scale of 0-100. Patients with co-morbidities had significantly lower scores (63 vs. 75, P = 0·001). TranQol scores showed substantial agreement (P < 0·001) with the Health Utilities Index Mark 3 (all patients, r = 0·65), the Pediatric QoL (children, r = 0·77) and the Short Form (36) physical (adults, r = 0·69) and mental summary scores (r = 0·76). In the subgroup who rated their QoL as better, there was a 4·0 point (SD 9·0) improvement in TranQol scores, from baseline of 67·1-71·1 one week later (P = 0·008). Test-retest reliability was excellent (intra-class correlation coefficient, 0·93). The TranQol was valid, with acceptable correlation for all administered measures and was reliable and responsive to change. The TranQol can be incorporated into future studies of thalassaemia major.

Neurodegeneration with brain iron accumulation (NBIA) describes a heterogeneous family of diseases characterized by high brain iron, particularly in the basal ganglia. The most common manifestation of childhood NBIA is classic pantothenate kinase-associated neurodegeneration (PKAN), a severe, progressive type of autosomal recessive neuroaxonal dystrophy characterized by early onset of symptoms (as opposed to atypical PKAN, with an average age of onset of 14 yrs). There is currently no established therapy for the disease. Intrathecal baclofen has been reported to improve ease of care and dystonia in patients with PKAN. Deferiprone, an iron chelator, has been shown to be safe and tolerable in patients with PKAN as well as effective in reducing brain iron accumulation, as measured by magnetic resonance imaging. This case report highlights the potency of combining intrathecal baclofen and oral deferiprone in a patient with classic PKAN. Although treatment with deferiprone alone was not attempted, this combination therapy seems to be more efficacious than treatment with only intrathecal baclofen.

Painful episodes of vaso-occlusion are the leading cause of hospitalizations and emergency department visits in sickle cell disease, and are associated with increased mortality. Low nitric oxide bioavailability contributes to vasculopathy in sickle cell disease. Since arginine is the obligate substrate for nitric oxide production, and an acute deficiency is associated with pain, we hypothesized that arginine may be a beneficial treatment for pain related to sickle cell disease. Thirty-eight children with sickle cell disease hospitalized for 56 episodes of pain were randomized into this double-blinded placebo-controlled trial. Patients received L-arginine (100 mg/kg tid) or placebo for 5 days or until discharge. A significant reduction in total parenteral opioid use by 54% (1.9 ± 2.0 mg/kg versus 4.1 ± 4.1 mg/kg, P=0.02) and lower pain scores at discharge (1.9 ± 2.4 versus 3.9 ± 2.9, P=0.01) were observed in the treatment arm compared to the placebo one. There was no significant difference in hospital length of stay (4.1 ± 01.8 versus 4.8 ± 2.5 days, P=0.34), although a trend favored the arginine arm, and total opioid use was strongly correlated with the duration of the admission (r=0.86, P0.0001). No drug-related adverse events were observed. Arginine therapy represents a novel intervention for painful vaso-occlusive episodes. A reduction of narcotic use by50% is remarkable. Arginine is a safe and inexpensive intervention with narcotic-sparing effects that may be a beneficial adjunct to standard therapy for sickle cell-related pain in children. A large multi-center trial is warranted in order to confirm these observations.

Iron overload is the primary cause of mortality and morbidity in thalassemia major despite advances in chelation therapy. We performed a pilot clinical trial to evaluate the safety and efficacy of combined therapy with deferasirox (DFX, 20-30 mg/kg daily) and deferoxamine (DFO, 35-50 mg/kg on 3-7 days/week) in 22 patients with persistent iron overload or organ damage. In the 18 subjects completing 12 months of therapy, median liver iron concentration decreased by 31% from 17.4 mg/g (range 3.9-38.2 mg/g) to 12.0 mg/g (range 0.96-26.7 mg/g, P

Alpha thalassemia major (ATM) is often fatal in utero due to severe hydrops fetalis. Although in utero transfusions (IUTs) are increasingly used to allow fetal survival in ATM, prenatal and postnatal outcomes are not well described.We retrospectively reviewed cases of ATM at our institution treated with consecutive IUT. Clinical records were reviewed for transfusion history, neurodevelopmental outcomes, anatomic abnormalities, survival to hematopoietic cell transplantation, and transfusion independence. A systematic review was performed, and additional reported cases are discussed.Three patients who underwent IUT for ATM were identified, and review of the literature revealed 17 reported cases. Of patients who received IUT, reported neurodevelopmental deficits occurred in 29% (4/14) and anatomic abnormalities in 55% (11/20). Four patients eventually underwent successful hematopoietic cell transplantation. Transfusion volumes were less than suggested guidelines for other causes of fetal anemia in 91.7% of the transfusions.This series demonstrates the potential for achieving full fetal development with normal neurologic outcomes in those affected by ATM. It provides support for continued patient and provider education about current benefits and risks of active prenatal therapy for fetuses with ATM, as well as continued research to optimize therapeutic strategies such as in utero transplantation. © 2016 John WileySons, Ltd.

An elevated tricuspid regurgitant jet velocity (TRV) is associated with hemolysis and early mortality in sickle cell disease, yet risk factors, clinical parameters, and mortality associated with this biomarker in thalassemia are poorly defined. This report summarizes the prevalence of an elevated TRV in 325 patients screened by Doppler echocardiography in the Thalassemia Clinical Research Network. A documented TRV was reported in 148 of 325 (46%) of patients. Average age was 25.9 years (range, 5-56 years) and 97% were transfusion-dependent. Mean TRV was 2.3 ± 0.4 m/s (range, 0.2-3.5 m/s). An abnormal TRV ≥ 2.5 m/s was identified in 49 of 148 (33%) of patients with a documented TRV, 5% (8/148), with a TRV ≥ 3.0 m/s, suggesting significant PH risk. Older age was strongly associated with a high TRV; however, 16% of children had a TRV ≥ 2.5 m/s. A history of splenectomy, hepatitis C, smoking, or high white blood cell count was associated with TRV elevation. In summary, an elevated TRV is noted in one-third of transfusion-dependent thalassemia patients with a documented value and develops in both children and adults. Age, splenectomy, hepatitis C, and smoking are significant univariate risk factors, with splenectomy surfacing as the dominant risk factor over time. Mortality was low in this cohort. Prospective longitudinal studies are needed. This study is registered at http://www.clinicaltrials.gov as NCT00661804.

Preserving fertility, preventing early menopause, and predicting reproductive ability have become crucial for many adult thalassemia major females. Luteinizing hormone/follicle-stimulating hormone (LH/FSH) and estradiol, commonly used for assessment of fertility potential in thalassemia, have a poor predictive value. Current reproductive practice uses markers of ovarian reserve testing, which were not yet studied in thalassemia women. We explored the relationship between liver iron concentration (LIC) and fertility status in 26 females (mean 30 years old). Seventeen (65%) of them experienced primary or secondary amenorrhea. Levels of LH/FSH and estradiol were low or undetectable in 48% and 35% of patients, respectively and did not correlate with age, presence of amenorrhea, and LIC. This further addresses the need for utilization of current available methods for assessment of fertility capacity in thalassemia, which will also allow future correlation with pituitary iron measures by MRI as well as early intervention for fertility preservation.

Advances in the management of thalassemia have resulted in increased life expectancy and new challenges. We conducted the first survey of education and employment status of people with thalassemia in North America.A total of 633 patients (349 adults and 284 school age children) enrolled in the Thalassemia Clinical Research Network (TCRN) registry in Canada and the U.S. were included in the data analysis. Predictors considered for analysis were age, gender, race/ethnicity, site of treatment (Canada vs. United States), transfusion and chelation status, serum ferritin, and clinical complications.Seventy percent of adults were employed of which 67% reported working full-time. Sixty percent had a college degree and 14% had achieved some post-college education. Eighty-two percent of school age children were at expected grade level. In a multivariate analysis for adults, Whites (OR = 2.76, 95% CI: 1.50-5.06) were more likely to be employed compared to Asians. Higher education in adults was associated with older age (OR = 1.67, 95% CI: 1.29-2.15), female gender (OR = 2.08, 95% CI: 1.32-3.23) and absence of lung disease (OR = 14.3, 95% CI: 2.04-100). Younger children (OR = 5.7 for 10-year increments, 95% CI: 2.0-16.7) and Canadian patients (OR = 5.6, 95% CI: 1.5-20) were more likely to be at the expected education level. Neither transfusion nor chelation was associated with lower employment or educational achievement.Individuals with thalassemia in North America can achieve higher education; however, full-time employment remains a problem. Transfusion and chelation do not affect employment or education status of this patient population.

Patients with E/beta(0) thalassaemia, the most common haemoglobinopathy in many Asian countries, might benefit from drugs that increase fetal and total haemoglobin and thereby decrease the need for transfusions. The long-term clinical efficacy and safety of such therapy is unknown, limiting its use in countries where resources for safe and regular transfusion are scarce. In this study, 45 patients were treated with hydroxyurea (18-20 mg/kg) for 24+/-9 months, hydroxyurea with sodium phenyl butyrate (n=8) and hydroxyurea with erythropoietin (n=9), each for approximately 6 months, and followed for 3 years from study exit. Hydroxyurea had minimal toxicity, resulted in a mean 1.3 g/dl steady-state increase in haemoglobin in 40% of patients, and a milder response (

Background: Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of rare genetic disorders characterized by progressive degenerative motor symptoms and the accumulation of iron in the basal ganglia. Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of NBIA caused by a mutation in the PANK2 gene leading to a deficiency in pantothenate kinase. Phospholipase A2G6-Associated Neurodegeneration (PLAN) is caused by a mutation in the PLA2G6 gene resulting in impaired phospholipase activity. Current understanding of systemic changes in NBIA disorders is limited, leaving no clear diagnostic biomarkers. Monitoring the systemic changes could identify candidate biomarkers for assessing disease severity and evaluating the efficacy of new therapies. Previous studies of Parkinson's disease (PD) have found a systemic burden of increased oxidative stress and chronic inflammation accompanies the neurological symptoms of the disease. Similarly, abnormal systemic iron regulation associated with brain iron accumulation as well as damage associated with neuromuscular degeneration could lead to increased oxidative stress and a state of chronic inflammation in NBIA. Our initial investigation of a patient with PLAN1, revealed elevated levels of systemic oxidative stress. We investigated a group of PKAN patients as well as continued our investigation of a patient with PLAN to evaluate the possibility of abnormal iron trafficking, increased oxidative stress and chronic inflammation in NBIA. Our aim was to expand our investigation of circulating levels of inflammatory cytokines, oxidative stress markers and iron regulatory and metabolic proteins in NBIA patients to include a group of patients with PKAN. Methods: Plasma samples from 15 PKAN patients were collected at the UCSF Benioff Children's Hospital in Oakland, California. Similarly, a plasma sample from a patient with PLAN was collected in Campbell River, British Columbia. Plasma samples from a matched group of 15 healthy controls were also collected at the University of Victoria. All patients provided informed consent to the study. The pro-inflammatory cytokines IL-6 and TNFα as well as the anti-inflammatory cytokine IL-10 were measured by ELISA. Total levels of the lipid peroxidation product malondialdehyde (MDA) were measured using N-Methyl-Phenyl-Indole (NMPI). Free, acutely generated, MDA not bound to proteins, was measured by removing plasma proteins via a 10KD spin filtration then measuring the MDA content of resulting filtrate using NMPI. Results: The levels of MDA and Free MDA were significantly elevated in PKAN patients at baseline in comparison to controls (p = 0.05, p = 0.03). IL-6 and TNFα were slightly, but not significantly elevated at baseline compared to controls. We previously demonstrated, similar elevations of oxidative stress in our case study of an NBIA patient with PLAN1. Additionally, all three inflammatory cytokines measured for this study expansion in PLAN were higher than average levels observed in the PKAN and control groups (S ee Table 1). Further analysis of systemic biomarkers in NBIA including proteomic analysis of 30 systemic blood proteins, including iron trafficking proteins is ongoing. Conclusions: We expand previous findings of elevated levels of systemic oxidative stress in other neurodegenerative diseases such as PD to include NBIA patients with PKAN and PLAN. We provide novel evidence of elevated levels of Free MDA; representative of an acute oxidative stress burden in NBIA in addition to the previously noted elevation in total MDA levels. We provide preliminary signs that of an accompanying inflammatory burden in NBIA, but a larger sample group may be needed to determine its significance. References M. Minkley, A. Jackson, D. Smith, C. Borchers, E. Vichinsky, R. Nashmi, P.B. Walter and P. M. Macloed. (2017). Neurodegeneration with Brain Iron Accumulation: PLA2G6-Associated Dystonia-Parkinsonism: Clinical and Animal Studies. Presented at the 2017 European Human Genetics Conference, Copenhagen, Denmark . Disclosures Minkley: Apopharma: Research Funding. Neumayr: Apopharma: Research Funding. Walter: Apopharma: Research Funding.

Pulmonary hypertension (PH) commonly develops in thalassaemia syndromes, but is poorly characterized. The goal of this study was to provide a comprehensive description of the cardiopulmonary and biological profile of patients with thalassaemia at risk for PH. A case-control study of thalassaemia patients at high versus low PH-risk was performed. A single cross-sectional measurement for variables reflecting cardiopulmonary status and biological pathophysiology were obtained, including Doppler-echocardiography, 6-minute-walk-test, Borg Dyspnea Score, New York Heart Association functional class, cardiac magnetic resonance imaging (MRI), chest-computerized tomography, pulmonary function testing and laboratory analyses targeting mechanism of coagulation, inflammation, haemolysis, adhesion and the arginine-nitric oxide pathway. Twenty-seven thalassaemia patients were evaluated, 14 with an elevated tricuspid-regurgitant-jet-velocity (TRV) ≥2.5m/s. Patients with increased TRV had a higher frequency of splenectomy, and significantly larger right atrial size, left atrial volume and left septal-wall thickness on echocardiography and/or MRI, with elevated biomarkers of abnormal coagulation, lactate dehydrogenase levels and arginase concentration, and lower arginine-bioavailability compared to low-risk patients. Arginase concentration correlated significantly to several echocardiography/MRI parameters of cardiovascular function in addition to global-arginine-bioavailability and biomarkers of haemolytic rate, including lactate dehydrogenase, haemoglobin and bilirubin. Thalassaemia patients with a TRV ≥2.5m/s have additional echocardiography and cardiac-MRI parameters suggestive of right and left-sided cardiac dysfunction. In addition, low arginine bioavailability may contribute to cardiopulmonary dysfunction in β-thalassaemia.

Pulmonary hypertension is a common but often overlooked complication associated with thalassemia syndromes. There are limited data on the safety and efficacy of selective pulmonary vasodilators in this at-risk population. We, therefore, designed a 12-week, open-label, phase 1/2, pilot-scale, proof-of-principle trial of sildenafil therapy in 10 patients with β-thalassemia and at increased risk of pulmonary hypertension based on an elevated tricuspid regurgitant jet velocity >2.5 m/s on Doppler-echocardiography. Variables compared at baseline and after 12 weeks of sildenafil treatment included Doppler-echocardiographic parameters, 6-minute walked distance, Borg Dyspnea Score, New York Heart Association functional class, pulmonary function, and laboratory parameters. Treatment with sildenafil resulted in a significant decrease in tricuspid regurgitant jet velocity by 13.3% (3.0±0.7 versus 2.6±0.5 m/s, P=0.04), improved left ventricular end systolic/diastolic volume, and a trend towards a improved New York Heart Association functional class. No significant change in 6-minute walked distance was noted. Sildenafil was well tolerated, although minor expected adverse events were commonly reported. The total dose of sildenafil (mg) was strongly correlated with percent change in nitric oxide metabolite concentration in the plasma (ρ=0.80, P=0.01). There were also significant increases in plasma and erythrocyte arginine concentrations. Our study suggests that sildenafil is safe and may improve pulmonary hemodynamics in patients at risk of pulmonary hypertension; however, it was not demonstrated to improve the distance walked in 6 minutes. Clinical trials are needed to identify the best treatment strategy for pulmonary hypertension in patients with β-thalassemia. (clinicaltrials.gov identifier: NCT00872170)

To determine the effects of l-arginine (l-Arg) supplementation on nitric oxide metabolite (NOx) production, oral l-Arg was given to normal controls, sickle cell disease (SCD) patients at steady state and SCD patients hospitalized with a vaso-occlusive crisis (VOC). l-Arg (0·1 g/kg) increased NOx formation by 18·8 ± 68% in normal controls, whereas steady-state SCD patients demonstrated a paradoxical decrease in NOx of −16·7 ± 4% (P = 0·004). In contrast, patients with VOC demonstrated a dramatic increase in NOx production by +77·7 ± 103%, a response that was dose dependent. l-Arg appears to be the rate-limiting step in NOx production during VOC. Oral arginine may therefore benefit SCD patients by inducing an increase in NO production during VOC.

In thalassemia, fetal hemoglobin (HbF) augmentation with hydroxycarbamide (also known as hydroxyurea) is not always successful. The expected parallel effects on red cell (RBC) membrane deformability, cell hydration, and membrane phospholipid organization, all important for extending RBC life span and increasing Hb, have been infrequently examined. We analyzed these characteristics in 15 nontransfused E/beta(0) thalassemia patients treated with HU (mean 10.2 months). Membrane deformability and cell hydration mildly improved in association with increased HbF levels approaching statistical significance (r = 0.51, P = 0.06). All measures improved considerably in splenctomized patients. These findings underscore the disappointing results of hydroxyurea treatment in clinical trials and the importance of examining the effect on RBC characteristics for the development and understanding of HbF-enhancing agents.

Introduction: β-thalassemia major requires a lifetime of transfusion and chelation therapy and despite improved chelation therapies, patients endure organ iron overload and increased hemolysis leading to organ injury and release of damage associated bio-markers. The structure of these markers are recognized by pattern recognition receptors (PRR) of the innate immune system as damage associated molecular patterns (DAMPs), which trigger inflammation and increased reactive oxygen species (ROS). The specificity of this recognition depends on PRRs, such as toll-like receptor 4 (TLR4), to provide the first line of defense against pathogens and DAMPS to initiate inflammation. Therefore, chronic organ iron loading is implicated in the chronic inflammation and morbidity of β-thalassemia major; however, the mechanism of how monocytes respond to iron loading in β-thalassemia remains largely unknown. This study investigated how chronic iron and heme overload affected innate immune cell morphology and expression of TLR4 and ROS by modeling THP-1 cellscompared to β-thalassemia monocytes. Methods: Cultured THP-1 monocytic cells were exposed to increasing concentrations of ferric citrate (0-250 μM) or heme (0-50 μM) for 24 hours or to chronic exposures of 40 μM ferric citrate for up to 3 weeks. Anti-human TLR4 phycoerythrin conjugated antibodies (TLR4-PE) were used to quantify TLR4 expression and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure ROS. Flow cytometry was done using a BD FACSCalibur. Peripheral-blood samples were obtained from 13 consenting β-thalassemia patients as part of the Novartis sponsored study investigating the effects of combined chelation therapy (CICL670AUS42T). Samples were also obtained from ethnically and age matched healthy controls. Peripheral-blood monocytes and THP-1 cells were gated on the parameters of forward and side scatter to stratify cells based on size and granularity, separating cells into two populations: larger more granular (LG) and smaller less granular (SG) cells. Results are reported as the MFI ratios of LG versus SG cells for TLR4-PE or DCFH-DA as a functional measure of the difference in TLR4 or ROS expression between these populations. Results: In vitro, LG THP-1 cells expressed 4-fold more TLR4 and 10-fold more ROS than SG cells (p < 0.001). Treatment of THP-1 cells with increasing concentrations of iron or heme for 1 day or 3 weeks, increased the ROS and TLR4 heterogeneity between LG and SG monocytes. The LG versus SG ratio of ROS production significantly correlated to the concentration of ferric citrate or heme added to the culture media (r = 0.64, p = 0.019 and r = 0.58 and p = 0.048, respectively). The mean ratio for iron but not heme treated THP-1 cells was significantly greater than for untreated cells (p = 0.008 and p = 0.406, respectively). Mean LG versus SG ratios of TLR4-PE MFI were 5.20 ± 1.56 for ferric citrate, 4.11 ± 0.90 for heme, and 3.78 ± 1.41 for untreated cells. We observed a similar TLR4 morphological heterogenic pattern in peripheral-blood monocytes from iron overloaded β-thalassemia patients. β-thalassemia LG monocytes had significantly greater TLR4 MFI than SG monocytes (LG mean = 11.91 ± 1.17, SG mean = 6.56 ± 1.02, p < 0.001). Long-term treatment of THP-1 monocytic cells for 3 weeks with 40 μM ferric citrate also resulted in the same phenomena regarding TLR4. No difference was observed in ROS production for control LG cells and SG cells; however, THP-1 cells treated for 3 weeks with 40uM ferric citrate revealed populations of LG cells that expressed 3 fold more ROS than SG cells. Conclusion: THP-1 monocytic cells and β-thalassemic peripheral-blood monocytes exhibit a morphological heterogeneity where LG cells express more TLR4 and in vitro, produce more ROS than SG cells. As TLR4 is one of the receptors for the initiation of inflammation, LG cells are likely greater contributors to the chronic inflammation experienced by β-thalassemia patients. Thus, we hypothesize that iron overload may give rise to monocytes that are larger and more granular and express more TLR4 and ROS. Disclosures Walter: Novartis: Research Funding. Porter:Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy.

Patients with hemoglobin E (Hb E)-beta 0-thalassemia, one of the most common hemoglobinopathies worldwide, could benefit from drugs that increase fetal and total hemoglobin levels and thereby decrease the need for transfusions. The long-term clinical outcome of such therapy, its hematologic effects, and which patients are likely to benefit from treatment are unknown. Consequently, the use of such drugs for Hb E-beta 0-thalassemia is limited, and countries where resources for safe and regular transfusion are scarce cannot benefit from them. In a multicenter trial of 42 patients treated with hydroxyurea for two years, almost half the patients demonstrated a significant increase in steady-state hemoglobin level. Drug toxicity was minimal. Combined treatment of hydroxyurea with erythropoietin benefited selected patients, but the addition of sodium phenyl butyrate was ineffective. After 5 years of follow-up, a subset of patients remained off transfusions. Hydroxyurea should be considered for a subset of Hb E-beta 0-thalassemia patients.

Introduction The thalassemias are inherited anemias sometimes characterized by severe transfusion dependence that can lead to extra-hepatic cardiac iron overload, causing cardiomyopathy. Despite improved chelation therapies, patients with transfusion-dependent thalassemia still endure cardiomyopathy and chronic inflammation. The innate immune system provides the first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) interacts with CD14 on innate immune cells transducing the signal for bacterial lipopolysaccharide. Another cell surface protein that is not a PRR, but aids phagocytosis and is important in granulocytes function and chemotaxis is the adhesive polysaccharide antigen, CD15. The role that excess iron plays in determining expression level of these innate immune proteins is unknown. Thus, the goal in these studies is to investigate the relationship of cardiac iron overload and its chelation to innate immune cell expression of TLR4 and CD15 in patients with transfusion-dependent thalassemia. Patients and Methods Eighteen patients with transfusion dependent thalassemia (11 – 29 years old) (participating in the Novartis sponsored CICL670AUS24T) were enrolled in a substudy investigating innate immunology (Novartis sponsored CICL670AUS42T). Patients were investigated at baseline, then after 6 months and one year of combined chelation therapy with deferasirox and deferoxamine. Fasting blood samples were obtained after a 72 hr washout with no chelators. Fourteen healthy controls (10 - 35 yrs old) were also enrolled. Changes in LIC (ferritometer), cardiac function (MRI) and myocardial iron (MRI T2*) were monitored. Peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from blood samples using density gradients. Monocytes and granulocytes were further purified using antibody-linked magnetic microbeads. Highly enriched populations of CD14+ monocytes and CD15+ granulocytes were verified by flow cytometry. The expression level of CD15 and TLR4 was determined. Results Previously we found that transfusion-dependent thalassemia patients had 37% higher TLR4+ neutrophils than control patients and a smaller percentage of CD15+ neutrophils. We have also observed a decrease in TLR4 expression during the course of combined chelation therapy on neutrophils but not monocytes, indicating that TLR4 is differentially modulated on neutrophils compared to monocytes. Now we find that these flow cytometry parameters show significant relationships to markers of iron burden. The percentage of TLR4+ monocytes was related to liver iron concentration (r=- 0.49, p = 0.039), ferritin concentration (r=-0.47, p = 0.049), serum iron level (r=0.61, p = 0.008), and total iron binding capacity (TIBC; r=0.51, p = 0.021), while the percentage of CD15 positive neutrophils predicted myocardial iron, as measured by MRI T2* (r= 0.69, p Conclusions This study found evidence that the innate immune system may be modulating iron trafficking not only to the liver but to the heart as well. The negative correlation between LIC and TLR4 expression suggests that severe iron overload may lead to heptocellular damage causing monocyte dysfunction, the pathology of which could be due to altered TLR4 expression. This relationship may also be driving the positive correlation observed between TLR4 expression and TIBC. CD15 seems to play an important role in cardiac health as it is positively correlated to LVEF and MRI T2*. This relationship is further validated by our previous finding that transfusion-dependent thalassemia patients had a smaller percentage of CD15+ neutrophils, which we now show improved during one year of combination chelation therapy. Taken together, this suggests that chelation therapy may enhance cardiac health by increasing the percentage of CD15+ neutrophils. Disclosures: Walter: Novartis: Research Funding. Porter:Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Celgene: Consultancy. Vichinsky:Novartis: Honoraria, Research Funding.

Abstract 2122 Introduction: Pulmonary hypertension (PH) commonly develops in thalassemia syndromes, but is poorly characterized. Since limited data are available we set out to provide a comprehensive description of the cardiopulmonary and biological profile of patients with thalassemia at-risk for PH. Methods: We designed a case-control study of thalassemia patients at-risk for PH, defined by a tricuspid-regurgitant-jet velocity (TRV)≥2.5m/s on Doppler-echocardiography (echo) compared to those with low PH-risk, define by a TRV Results: Twenty-seven thalassemia patients with a mean TRV=2.5±0.6m/s (range:1.0–4.9m/s) were evaluated, 14 with a TRV≥2.5m/s. Mean age was 35±12 years, 70% were male, 74% were splenectomized, and 67% had thalassemia major (TM). Patients with PH-risk (mean TRV=3.1±0.7m/s) had a significantly higher frequency of splenectomy (OR:11.1 [95% CI: 1.11–112.0], p=0.04), higher NYHA functional class, Borg Dyspnea Score after 6MWT, and significantly larger right atrial size, left atrial volume, and left septal-wall thickness on echo and/or MRI, with elevated biomarkers of coagulation abnormalities, lactate dehydrogenase (LDH) levels, arginase concentration (5.7±7 vs. 78.7±108 ng/ml, p=0.003) and arginase activity, and lower arginine-bioavailability compared to low-risk patients (mean TRV=2.1±0.4m/s). Arginase concentration and activity correlated significantly to several echo/MRI parameters of cardiovascular function including cardiac index (r=0.75, p Conclusions: Thalassemia patients with a TRV≥2.5m/s have additional echo and MRI parameters suggestive of PH-risk. Dysregulation of the arginine metabolome and the consequences of excess plasma arginase are associated with cardiopulmonary complications in thalassemia and warrants further study. Disclosures: Taher: Novartis: Research Funding, Speakers Bureau.

Abstract 1023 Introduction. Pulmonary hypertension (PH) is a complication associated with thalassemia syndromes, particularly thalassemia intermedia. There are limited data on the safety and efficacy of selective pulmonary vasodilators in this at-risk population. Methods. We evaluated the safety and efficacy of a 12-week prospective, phase 1/2 pilot scale study of sildenafil (100 mg TID) in ten b-thalassemia patients with elevated TRV on Doppler-echocardiography ≥ 2.5 m/s suggestive of PH risk. Patients were evaluated at baseline and at weeks 2, 4, 8 and 12 of sildenafil therapy, and six minute walk distance (6MWD), biomarkers of hemolysis, coagulation, inflammation and adhesion were assessed. Results. Our study population had an average age of 37±12.3 years, 8/10 were male, and 50% were thalassemia intermedia. Splenectomy prevalence was high (90%), and only 30% of patients were transfused since infancy. The mean pre-transfusion hemoglobin was 10.4±1.5 g/dL. A 12-week open-label trial resulted in a significant decrease in TRV by 13.3% (3.0±0.7 vs. 2.6±0.5m/s, p=0.04, Figure 1), improvements in left ventricular end systolic/diastolic volume (p≤0.02), diffusion capacity for carbon monoxide (DLCO, p=0.003) and a trend towards a reduced Borg Dyspnea Score and improved NYHA Functional class. No significant change in 6MWD was noted, although 6MWD correlated strongly with DLCO (ρ=0.72, p=0.03) suggesting that oxygen diffusion across the alveolar-capillary membrane was an important determinant of exercise capacity. Sildenafil was generally well tolerated, but most patients experienced anticipated transient headaches and visual/color disturbances associated with sildenafil use. One patient withdrew from the study due to worsening dyspnea. No other serious adverse events were reported. A strong direct correlation between total dose of sildenafil (mg) taken and % change in plasma NO metabolite concentration was observed (ρ =0.80, p=0.01). A significant increase in plasma and erythrocyte arginine concentration occurred, without an associated change in plasma arginase activity/concentration, nitric oxide metabolites or vascular endothelial growth factor. However arginase concentration was elevated in this cohort similar to prior reports, and correlated inversely to hemoglobin (ρ=-0.41, p=0.01), and directly to ALT (ρ=0.40, p=0.004), AST (ρ=0.38, p=0.04), left ventricular end systolic volume (ρ=0.77, p=0.001), and end-diastolic volume (ρ=0.79, p=0.001). Conclusion. Our study suggests that sildenafil is safe and may improve cardiopulmonary hemodynamics in patients at risk for PH, however improved exercise capacity as reflected by an improved 6MWD was not observed. The reduction in left ventricular dimensions is promising, and could reflective of either increased inotropy or chronotropy, or decreased systemic afterload. This is also the first report of an influence of sildenafil on diffusion capacity of the lungs in patients with thalassemia and the first description of increased plasma and erythrocyte arginine concentration after sildenafil therapy. Given the association of arginine bioavailability with long-term survival in cardiovascular disease, this is an unexpected effect of sildenafil that warrants further investigation. These data support the need for further clinical trials evaluating the use of sildenafil in thalassemia. Disclosures: Taher: Novartis: Research Funding, Speakers Bureau.

Abstract 1025 Introduction: Thalassemia major patients endure chronic RBC transfusions, high levels of tissue iron, iron chelation and organ injury. Patients with thalassemia also have reduced immune function and are at risk for infection. Infection is in fact the second most common cause of death in thalassemia. The innate immune system provides the first line of defense against infection and it specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) interact with CD14 on innate immune cells transducing the signal for bacterial Lipopolysaccharide (LPS), resulting in cytokine production. The role iron plays in thalassemia in determining expression level of PRRs is unknown. Thus, the goal in these studies is to investigate the relationship of iron overload and its chelation to innate immune cell expression of PRRs in thalassemia. Patients and Methods: Eighteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox and deferoxamine (Novartis sponsored CICL670AUS24T) were enrolled in a substudy investigating innate immunology (Novartis sponsored CICL670AUS42T). Fasting blood samples were obtained i) at baseline after a 72 hr. washout of chelator, and ii) at 6 and 12 months on study. Fourteen healthy controls (10 – 35 yrs old) were also enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and then from these cells monocytes and granulocytes were purified using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes and CD15+ granulocytes were verified by flow cytometry. The expression level of CD14 and CD15 in PBMCs and TLR4 in purified cells were determined and reported as the median fluorescent intensity (MFI). Liver iron concentration (LIC) was determined by biomagnetic susceptibility (“SQUID”, Ferritometer®) in patients with thalassemia; healthy controls were shown to have normal ferritin. Results: In PBMCs from thalassemia patients at baseline, the expression of monocyte CD14 and TLR4 were significantly increased 22% and 6.5% respectively compared to healthy controls (p < 0.05). Granulocytes from patients with thalassemia at baseline were also found to have a 50% higher expression of TLR4 compared to controls. Markers of iron burden, such as LIC and ferritin also significantly correlated with the expression of monocyte TLR4. In longitudinal analysis markers of iron burden, the expression of TLR4 on monocytes and granulocytes all significantly decreased in the follow-up period in thalassemia patients receiving intensive combination chelator therapy (p Conclusions: These studies support the hypothesis that iron burden influences the innate immune response in thalassemia as demonstrated by the increased monocyte expression of both CD14 and TLR4 at baseline, both of which likely contribute to the commonly observed susceptibility to infection. After intensive chelation, the levels of CD14 and TLR4 decreased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. These changes in CD14 and TLR4 may be able to restore proper innate immune function in thalassemia patients. Disclosures: Walter: Novartis: Research Funding. Harmatz:Novartis: Research Funding; FerroKin BioSciences: Research Funding. Porter:Novartis: Consultancy, Research Funding. Vichinsky:ARUP Research Lab: Research Funding; ApoPharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Abstract 2077 Introduction: Pulmonary arterial hypertension (PAH) has been accepted to be a clinical entity associated with hemoglobinopthies, mostly reported in sickle disease and thalassemia intermedia (TI). Anecdotal cases of PAH in other chronic hemolytic disorders have been reported; many of which have been associated with a prior splenectomy. The pathogenesis of PAH in the non sickle-cell RBC hemolytic disorders has not been well defined and the large variability in its frequency and severity are not clear. We aimed to characterize biomarkers in thalassemia and in other RBC disorders and evaluate the specific role of splenectomy (spleen.) in these diseases. Patients and Method: A total of 106 patients were analyzed: Forty two were regularly transfused thalassemia major (TM), 34 had TI (16 with Hb E/beta thalassemia, 11 with beta thalassemia, and 7 with hemoglobin H-Constant Spring (Hb H-CS); 18 with hereditary spherocytosis (HS) or pyruvate kinase (PK) deficiency, 2 with an unstable Hb (Hb Koln), and 10 with a history of splenectomy for non-RBC related reasons (trauma or chronic ITP). PAH was determined by using a Doppler echocardiogram showing a tricuspid regurgitation jet velocity (TRV) of >2.5m/s. Markers of platelet activation (P- selectin, soluble CD40 (sCD40L); abnormal exposure of Phosphatidyl Serine (PS) on RBCs, thrombin generation (thrombin-anti-thrombin, TAT); increased ventricular pressure strain (brain natriuretic peptide (BNP); plasma free Hb, LDH and NOx, and endothelin-1, soluble vascular cell adhesion molecule (sVCAM) were analyzed. Result: Nineteen (56%) of the TI patients (15 with beta-TI and 4 with Hb H-CS) had abnormally elevated TRV (Mean 3.0±0.5). The 2 patients with Hb Koln had increased TRV. There were no PAH cases detected among patients with membrane or RBC enzyme deficiencies, neither was there an increased TRV in patients who had been splenectomized without a RBC disorder. There were also no cases of increased TRV in the transfused TM cohort. Patients with TI, in addition to a prior splenectomy and increased hemolysis had increased platelet activation, abnormal RBC PS exposure and anemia. TI patients also had an increase in ET-1 levels, a potent vasoconstrictor. The 2 patients with Hb Koln had increased thrombin generation in addition to hemolysis and absence of spleen. Conclusions: PAH was not detected in splenectomized patients with only a mild chronic hemolytic disorder or in those without a RBC disorder. In addition to the absence of spleen, a higher rate of hemolysis, resulting in NO scavenging and increase in ET-1 and the involvement of platelet activation or thrombin generation are likely required for the development of dysregulation of the pulmonary vascular function. PAH is rare in TI patients which have not undergone splenectomy; absence of spleen likely enhances the effects of thrombocytosis and platelet activation. Our data indicates that PAH is common in patients with Hb H-CS, mostly splenectomized, and should be screened for. In addition our data shows that blood transfusions reduce the elements of anemia, hemolysis and coagulation activation and prevent the development of PAH in splenectomized and non-splenectomized patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4269 Therapeutic regimens that combine two iron chelators may enhance chelation efficiency by improving access to different tissue iron stores and control of the toxic labile iron pool. The combination of two chelators can reduce toxicity through averting the need for high doses of a single drug, but it is essential to establish the safety such regimens. We therefore explored the combined use of deferasirox (DSX) and deferoxamine (DFO) in patients with transfusion-dependent thalassemia who had failed standard chelation therapy with single drug. Patients were eligible if the liver iron concentration (LIC) >15 mg/g dry liver-weight or if iron-induced end organ injury was present. Subjects were monitored for hepatic and renal toxicity, visual or auditory changes, and the development of new complications from iron overload. The ability of the combined therapy to control systemic iron burden (serum ferritin and LIC) and myocardial iron overload (MRI T2*) was evaluated. We also measured changes in plasma levels of non-transferrin bound iron (NTBI) and labile plasma iron (LPI). Fifteen subjects were enrolled in 3 groups: adults with LIC 15 mg/g (group B), and children 8–18 years with LIC >5 mg/g (group C). The duration of therapy was 52 weeks. DSX (20-30 mg/Kg) was administered daily and DFO (35-50 mg/Kg/infusion) was infused on 3–7 days/week (as 8–12 hour infusion) based upon the degree of iron overload present at baseline. At the initiation of the study, the mean daily dose of DFO was 16, 33, and 17 mg/Kg/day and mean DSX dose was 21, 25 and 22 mg/Kg/day for groups A, B and C, respectively. At the conclusion of the trial, the median LIC declined by 48% from 10.8 mg/g (3.9-34.8 mg/g) to 5.7 mg/g (1.0-24.0 mg/g, p=0.003). The median ferritin fell by 43% from 2030 ng/mL (1000-5230 ng/mL) to 1150 ng/mL (421-5260 ng/mL, p=0.008). Myocardial iron in the 3 subjects who had T2* No significant toxicity or unusual adverse events were observed with combined chelation therapy in this group of high-risk patients with thalassemia. Elevation of serum creatinine or ALT was not observed in any subject. One subject from group B died at 9 weeks from start of trial from sepsis. One subject interrupted DSX therapy because of abdominal pain. In all other cases the treatment was well tolerated and no dose adjustment or suspension of therapy was required on account of toxicity. Protocol-mandated modification of treatment (temporary cessation of DSX or DFO) occurred in three subjects owing to a marked fall in serum ferritin and LIC. These results suggest that simultaneous administration DSX and DFO is well tolerated and has low potential for toxicity. Combined chelation therapy appears to be effective in rapidly reducing systemic iron burden, lowering myocardial iron, and controlling plasma NTBI and LPI in patients at risk of developing end-organ damage. Disclosures: Harmatz: Ferrokin: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Porter:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Vichinsky:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 2016 Poster Board I-1038 BACKGROUND: Pulmonary hypertension (PH) contributes to heart disease, the leading cause of death in thalassemia. PH in thalassemia is multifactorial, including oxidative stress, hemolysis, thrombosis, splenectomy, abnormal arginine-nitric oxide bioavailability, and iron overload. Despite the high mortality rate associated with PH, no intervention studies in thalassemia have been completed. This report summarizes the prevalence of elevated tricuspid regurgitant jet velocity (TRV) as a proxy for PH in patients clinically screened by Doppler echocardiography (echo) in the Thalassemia Clinical Research Network (TCRN). PATIENTS AND METHODS: The TCRN is an NIH-sponsored network of major thalassemia centers in the US, Canada and London, UK. This analysis is part of the Thalassemia Longitudinal Cohort (TLC), which collects longitudinal data from 428 patients at 16 centers. We report results from 303 patients (71%) who were assessed by echo at least once. Predictors considered for analysis were age, gender, splenectomy, hepatitis C, WBC, ferritin, hemoglobin, liver function, creatinine, chronic transfusion status, number of transfusions, chelation status, and hydroxyurea use. Multivariate effects of predictors significant in univariate analysis were modeled using logistic regression for elevated TRV. RESULTS: Measurable TRV jet was detectable in 144/303 (48%) of patients. Average age of the cohort with a detectable TRV was 25.8 years (range 5-56 years); half were female.. 97% of the patients studied were transfusion dependent. Mean TRV was 2.3±0.4 m/s (range 0.2-3.5 m/s). An abnormal TRV ≥ 2.5 m/s was identified in 45/144 (31%) of patients screened, 18% (8/45) of whom had a TRV ≥ 3.0 m/s suggesting significant PH. Older age was strongly associated with PH (r = 0.38, p CONCLUSIONS: Elevated TRV is noted in at least 1/3 of transfusion-dependent thalassemia patients with measurable TR jets. We observed elevated TRV in both children and adults. Less than half of patients screened by echo had a measurable TRV, suggesting that technical diligence is needed to increase the fraction of echos with a measured TRV to better identify patients at risk of PH. Age, splenectomy and history of hepatitis C are significant univariate risk factors in this population, with age remaining an independent risk factor in multiple regression analysis. Ongoing assessment in the TLC and specific prospective longitudinal studies of PH in thalassemia are needed to understand the natural history and pathophysiology in thalassemia in order to determine optimal therapy. Interventional trials for PH in thalassemia are needed. Disclosures: No relevant conflicts of interest to declare.

Abstract 573 BACKGROUND: Vaso-occlusion is the principal factor in the morbidity of sickle cell disease (SCD). Vaso-occlusive painful episodes (VOE) are common, debilitating, and the leading cause of hospitalizations, emergency room visits, and are associated with an increased mortality rate. There is no effective therapy that targets the underlying mechanisms of VOE. Symptomatic relief with analgesics is the only availabel treatment. Nitric oxide (NO) is a potent vasodilator that contributes to a variety of vaso-occlusive events in SCD. We have found that an arginine deficiency and low NO bioavailability occurs during VOE in SCD. Since arginine is the obligate substrate for NO production, and an acute deficiency is associated with VOE, we hypothesized that arginine supplementation may be a safe and beneficial treatment for sickle cell pain. PATIENTS AND METHODS: Hospitalized SCD patients > 3 years diagnosed within 24 hours with VOE and without associated complications were eligible; written informed consent was obtained. A total of 56 patients completed randomization in this double-blinded, placebo controlled trial. A standardized treatment and monitoring program for VOE was followed. Average age was 13.9 ± 4 years (range 3.6-19 years), and 52% were female. Patients received intravenous (IV) or oral arginine (0.1 gram/kg TID, n=28) or placebo (n=28) for 5 days or until discharge from the hospital. Narcotic records for 2 patients (randomized to placebo arm) were incomplete and were not included in the narcotic use analysis. An intention to treat analysis was performed for narcotic use applying an unpaired t-test with Welch's correction to adjust for unequal variance. RESULTS: Age was equally distributed between treatment and placebo groups. 57% of the arginine treatment group and 46% of the placebo group were female. A significant reduction in narcotic use (defined as total morphine use over the course of the hospital stay in mg/kg) by 56% was observed in the treatment arm receiving IV or oral arginine compared to placebo (mean ± SEM: 1.8 ± 0.4 mg/kg; n=28 vs. 4.1 ± 0.8mg/kg; n=26, p=0.01). Average length of hospitalization was 4.5 ± 0.4 days, and there was no significant difference between the 2 groups (4.1 ± 0.3 vs. 4.8 ± 0.5 days, p = 0.27; arginine vs. placebo arm). Four episodes of acute chest syndrome (ACS) developed during the study, three in the treatment arm and one in the placebo arm. There was one patient who experienced clinical deterioration associated with ACS requiring emergent transfusion and a transfer to the pediatric intensive care unit (PICU) in the placebo arm. No clinical deterioration or PICU transfers occurred in the arginine arm. Five in the treatment arm received transfusion vs. four in the placebo arm. No drug-related adverse events were observed. No significant differences were observed between pre and post therapy liver or renal function, or hematological parameters in the arginine treatment group vs. placebo. Two patients admitted for pain management ultimately did not receive IV narcotics. Both had been randomized into the arginine-treatment arm and received arginine therapy per protocol throughout their hospital stay and required only oral narcotics and non-steroidal analgesia. Reduction in narcotic use in the treatment arm remained significant even when these 2 patients were excluded from the analysis (p=0.02). CONCLUSIONS: IV arginine therapy represents a novel nutritional intervention for the treatment of pain in hospitalized patients with SCD. Use of IV arginine should also be considered in the treatment of VOE in the emergency department setting prior to hospitalization, although further investigation is warranted. A reduction of narcotic use by over 50% observed in this study is remarkable, as this is the first successful intervention for sickle cell-related pain that targets the underlying mechanism of vaso-occlusion through a promising NO-based therapy. Arginine is a safe and inexpensive intervention with narcotic-sparing effects that should be considered as an adjunct to standard therapy for VOE requiring hospitalization. Disclosures: Off Label Use: Arginine for treatment of sickle cell vaso-occlusive pain episodes.

Abstract 2021 Poster Board I-1043 Therapeutic regimens that combine two chelators have the potential to improve iron excretion while avoiding toxicities associated with high doses of single agents. This pilot study was designed to explore the safety of the combined treatment with deferasirox (DFX) and deferoxamine (DFO) in individuals with transfusion-dependent thalassemia and iron overload who had failed standard therapy. Subjects underwent baseline evaluation of liver iron, cardiac iron, cardiac function and target organ damage, and were enrolled in 3 groups (n=5 in each group) - Group B: adults with liver iron concentration (LIC) >15 mg/g dry weight, group A: adults with LIC >5 and 5 mg/g dry weight plus iron related organ dysfunction. The duration of therapy was 52 weeks, with DFX (20-30 mg/Kg) given daily and (DFO 35-50 mg/Kg/infusion) given for 3-5 days/week (groups A and C), or 5-7 days/week (group B). Subjects were monitored for hepatic and renal toxicity, visual or auditory changes, and development of new symptoms. Changes in serum ferritin, LIC (ferritometer), cardiac function (MRI) and myocardial iron (MRI T2*) were monitored. We have enrolled 14 subjects (target 15 subjects) with a median follow up of 29 weeks (range 18-52). The mean daily dose of DFO was 16, 33, and 17 mg/Kg/day for groups A, B and C, respectively, at the start of the study. The corresponding mean DFX dose was 21, 25 and 22 mg/Kg/day. At the end of 26 weeks, the mean LIC (mg/g dry wt) in evaluable patients declined from 13.0 (3.9-21.7) to 10.6 (0.60-18.3, p=0.015), and the mean ferritin (ng/mL) fell from 2631 (1000-5230) to 2158 (319-5845, p=n.s.). Cardiac evaluation revealed that mean MRI T2* (msec) improved from 22.7 (6.7-32.6) to 25.5 (10.7-38.1) and the mean LVEF (%) from 63.4 (47.5-68.5) to 64.3 (53.4-72.2), but these changes were not statistically significant. No subject developed evidence of significant myocardial iron (T2* Disclosures: Lal: Novartis: Research Funding. Harmatz:Novartis: Research Funding. Vichinsky:Novartis: Consultancy, Research Funding.

Low bone mineral density (BMD) is commonly seen in regularly transfused thalassemia patients; however, there have been few reports for bone mineral density assessment in transfusion independent thalassemia patients. The present report includes the results of BMD assessments in patients with transfusion independent thalassemia who were referred to the bone density clinic through 2002–2006. BMD was evaluated by dual energy x-ray absorptiometry (DXA, Hologic Delphi A). A convenience sample of 24 patients (Females=15) with transfusion independent thalassemia were measured with a mean age of 22.1 ± 13.8 years. Subjects younger than 10 yrs old (n = 7) underwent scans for Lumbar spine (LS; L1-L4) and whole body (WB), patients ages 10–20 (n = 5) were assessed for LS, WB, and non-dominant hip, and for patients older than 20 (n = 12), LS and hip scans were completed. Z-scores specific for age and gender were generated using Zemel BS et al.(J. Bone Min Res 2004) database. Z-scores less than −2.0 were considered as low bone density. Calcium intake was assessed by a brief food frequency questionnaire. Past medical history, medications, history of fractures, and family history of osteoporosis were obtained by chart review and patient interview. Data is presented as Mean ± SD. T-test was used to assess differences in continuous variables. The mean LS Z-score (n = 24) was −1.5 ± 1.0 and the mean hip Z-score (n = 17) was −0.5 ± 1.1. Mean WB Z-score (n = 10) was −2.0 ± 1.2. There was a significant (p

Determination of red blood cell (RBC) survival in the peripheral blood, to evaluate the efficacy of pharmacological agents that augment fetal hemoglobin (HbF) and total hemoglobin (Hb) in patients with beta-thalassemia, has not been implemented. We report the use of biotin labeling, a nonisotopic, safe and effective method to determine the erythrocyte lifespan, proven to be equivalent to 51Cr. We studied RBC survival in 9 Hemoglobin E-β-thalassemia (ET) patients, 4 thalassemia intermedia (TI) patients and compared RBC survival to normal control. Repeat measures were performed in 3 ET patients while on hydroxyurea (HU) treatment at 15–19 mg/Kg/day. RBC from 30 ml blood were ex-vivo biotinylated under sterile conditions using EZ-Link Sulfo-NHS-Biotin (Pierce), and re-infused to the patient as described before (Franco et al,1998). At set time points, the number of biotinylated cells was determined in small blood samples by flowcytometry using fluorescently labeled strepavidin. All patients tolerated the procedure well without an indication of re-infusion induced infection or anti-biotin antibody related hemolysis. The presence of biotinylated cells in the circulation was followed until only 0.05-0.08% biotinylated cells could be detected. The data were mathematically fitted to 100–100*[1-(1/T)*t]exp(-kt), where t is the time point, T is the extinction time, and k the exponential rate of RBC removal. The control data showed a linear removal rate (k=0), and a T of 100 days (R=0.95), consistent with a normal red cell life span. In the 9 ET and 4 TI patients, a faster disappearance of biotinylated cells was noted, and the number of surviving (biotinylated) cells in the population followed an exponential pattern, consistent with random removal (k=0.033, R= 0.97). Mean survival was 43±11 and 36±15 days, respectively. The 3 patients who received 10–12 months of HU treatment had a mean 0.8 gr/dL increase in their Hb and 20% increase in HbF. However, no difference was found in the RBC survival curves of these patients (k=0.034, R= 0.97), as compared to the non treated patients. One patient who was treated with HU and Erythropoietin (500u/Kg x3/wk) and had a 1.6 gr/dL increase in hemoglobin and RBC survival analysis showed the biotinylated cells to follow a similar survival curve. While these results cannot exclude a longer survival in selected cells, specifically those containing higher HbF, the overall effect of HU treatment has not proven to prolong the lifespan of labeled thalassemia erythrocytes. These results indicate that biotin label provides RBC survival characteristics in thalassemia patients. The method has advantages over 51Cr, including lack of radioactivity, avoiding labeling elution, low expense and recovery of labeled cells after prolonged time in the circulation. Moreover, the method is a valuable tool to measure effect of various treatments on the survival characteristics of the thalassemia erythrocytes. Preliminary findings indicate that despite the effect of HU on an increase in HbF and a mild to moderate increase in total Hb, the overall thalassemic erythrocyte lifespan has not increased, suggesting other mechanisms for these results including erythroid expansion, improved effective erythropoiesis and cell selection.

Abstract 4071 Poster Board III-1006 Introduction Infection is the second most common cause of death in thalassemia. The innate immune system provides a first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) are important for transducing the signal for bacterial Lipopolysaccharide (LPS), resulting not only in cytokine production, but also in the control of extracellular iron levels through production of neutrophil gelatinase associated Lipocalin (NGAL). However, the exact role that NGAL plays and the expression level of PRRs are unknown in thalassemia. Thus, the goal in these studies is to investigate the relationship of iron overload to the innate immune cell expression of PRRs and NGAL in thalassemia. Patients and Methods Fifteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox (an oral iron chelator) and deferoxamine were enrolled (Novartis sponsored CICL670AUS24T). Fasting blood samples were obtained i) at baseline after a 72 hr washout of chelator, and ii) at 6 and 12 months on study. Five healthy controls (13 - 18 yrs old) were also enrolled. Fresh monocytes were isolated using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes were verified by flow cytometry. The expression of TLR4, also examined by flow cytometry is reported as the mean fluorescent intensity (MFI). In patients with thalassemia, liver iron concentration (LIC) was analyzed by biomagnetic susceptibility (“SQUID”, Ferritometer®). The plasma levels of NGAL were analyzed by ELISA. Results At baseline the expression of monocyte TLR4 (mean 18.8 ± 3.5 MFI) was reduced 30% compared to the healthy controls (mean 26.9 ± 7.6 MFI, p Conclusions These preliminary studies support the hypothesis that iron burden has a negative impact on the innate immune response in thalassemia as demonstrated by the decreased expression of TLR4. After intensive chelation, the levels of TLR4 increased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. Finally, the iron transport protein NGAL is significantly elevated in thalassemia possibly acting to prevent essential iron uptake by pathogenic bacteria. Disclosures: Harmatz: Novartis: Research Funding; Apotex : Membership on an entity's Board of Directors or advisory committees; Ferrokin: Membership on an entity's Board of Directors or advisory committees. Vichinsky:Novartis: Consultancy, Research Funding.