Liver Iron Concentration Research Articles

NIGELLA SATIVA: ROLE IN IRON OVERLOAD

Background: In thalassemia patients, iron is overloaded because of multiple blood transfusions. This overloaded iron can damage the organs badly which is a major cause of death in these patients. Iron overload in thalassemia major can only be treated by removing the excessive iron through different chelating agents. Commercially available chelating agents are costly and associated with multiple adverse effects. Effectiveness of naturally present chelators is under study. This study was conducted to explore the chelating effect of one of commonly used herb Nigella sativa. Methods: This experimental, randomized controlled trial was conducted in Zoology Department, Government College University, Lahore in its multidisciplinary laboratory. A total of 36 male albino mice were divided into three groups, 12 in each group. Iron was overloaded in groups 2 and 3 by intravenous injections of iron dextran (0.1 ml/g body weight) for 15 days on daily basis. After 15 days, iron dextran injection was discontinued, and mice were allowed to feed on Nigella sativa (200 mg/Kg body weight) for further 15 days. Blood sampling was done at baseline, 15, and 30 days to analyse iron concentration in serum, heart, kidney, and liver. Data was analysed using Student’s t-test. Results: The artificially administered iron in mice increased the iron levels in serum, liver, heart and kidneys to a significant level while administration of N. sativa significantly decreased these levels. Conclusione propose clinical trials of Nigella sativa in thalassemia major patients as an adjunct therapy to explore its efficacy and safety. Pak J Physiol 2021;17(2):20–3

Determination of Non-Invasive Biomarkers for the Assessment of Fibrosis, Steatosis and Hepatic Iron Overload by MR Image Analysis. A Pilot Study

The reference diagnostic test of fibrosis, steatosis, and hepatic iron overload is liver biopsy, a clear invasive procedure. The main objective of this work was to propose HSA, or human serum albumin, as a biomarker for the assessment of fibrosis and to study non-invasive biomarkers for the assessment of steatosis and hepatic iron overload by means of an MR image acquisition protocol. It was performed on a set of eight subjects to determine fibrosis, steatosis, and hepatic iron overload with four different MRI sequences. We calibrated longitudinal relaxation times (T1 [ms]) with seven human serum albumin (HSA [%]) phantoms, and we studied the relationship between them as this protein is synthesized by the liver, and its concentration decreases in advanced fibrosis. Steatosis was calculated by means of the fat fraction (FF [%]) between fat and water liver signals in “fat-only images” (the subtraction of in-phase [IP] images and out-of-phase [OOP] images) and in “water-only images” (the addition of IP and OOP images). Liver iron concentration (LIC [µmol/g]) was obtained by the transverse relaxation time (T2* [ms]) using Gandon’s method with multiple echo times (TE) in T2-weighted IP and OOP images. The preliminary results showed that there is an inverse relationship (r = −0.9662) between the T1 relaxation times (ms) and HSA concentrations (%). Steatosis was determined with FF > 6.4% and when the liver signal was greater than the paravertebral muscles signal, and thus, the liver appeared hyperintense in fat-only images. Hepatic iron overload was detected with LIC > 36 µmol/g, and in these cases, the liver signal was smaller than the paravertebral muscles signal, and thus, the liver behaved as hypointense in IP images.

Measurement of Liver Iron Concentration in a Population of Non-Transfusion Dependent Thalassemia Patients Using a Trained Artificial Neural Network to Analyse Magnetic Resonance Images

Background: Measurements of liver iron concentration (LIC) by magnetic resonance imaging (MRI) have become established and validated in several research intensive centers. However, challenges remain with translating these measurement methodologies for routine clinical use. Current methods involve many steps presenting multiple opportunities for human error and can lead to systematic severe measurement error. Artificial neural networks (ANNs) can be trained to replace human input into image processing and may have a role in providing a simple, robust, and low cost solution for LIC measurement.Aims: The aim of the study wasto determine the limits of agreement between measurements of LIC by a reference standard spin-density-projection-assisted (SDPA) R2-MRI method (FerriScan) and a method using trained ANNs to analyse image data. MR image datasets (400) previously acquired from Siemens scanners were pre-processed and then, together with their corresponding SDPA R2-MRI LIC values, were filtered through publically available ANNs. The ANNs had been pre-trained on large image sets (ImageNet). Relevant features were extracted and then used to train a machine learning (gradient boosting) algorithm to predict LIC values from previously unseen MR image datasets.Methods: Thalassemia patients (N=100) referred by the National Institute of Haematology and Blood Transfusion, Hanoi, Vietnam for routine LIC measurement by MRI were prospectively recruited with informed consent. Patients were randomised to be scanned in either a Philips Ingenia or a Siemens Avanto 1.5T scanner. Image data were acquired using the FerriScan protocol. Images were then processed (1) using a quality controlled core laboratory data analysis service using the SDPA R2-MRI method and (2) by a non-radiologically-trained technician using the trained artificial neural network to generate LIC values. The technician was required to trace the outline of the torso and then click a mouse button. The technician using the ANN was blinded from the SDPA R2-MRI results and the analysts generating the SDPA R2-MRI results were blinded from the ANN results. Reported data were analysed using the statistical methods of Bland and Altman.Results: A plot of the ANN-predicted LIC values against the SDPA R2-MRI LIC values (Figure) shows the data scattered about the line of equivalence indicating that the ANN method is generating results that correlate with the validated SDPA R2-MRI reference standard. The geometric mean ratio of ANN LIC to SDPA R2-MRI LIC was 0.93 (95% CI 0.91 -0.96) indicating a small but statistically significant systematic underestimation of LIC by the ANN. The geometric mean ratios of the two LIC measurements were significantly different for the two scanners (0.98 for Philips and 0.90 for Siemens, p = 0.006) indicating that the bias of the ANN method against the reference standard is not universal but is dependent on scanner. Bland Altman analysis indicates that 95% of pairs of measurements are predicted to have ratios between 1.23 and 0.69 indicating a modest random variability between the ANN method and the reference standard that is better than that between biopsy and the reference standard. The performance of the ANN method in predicting SDPA R2-MRI LIC values above the clinically relevant thresholds of 7 and 15 mg Fe/g dw is characterized in Table 1 showing positive predictive values (PPVs) and negative predictive values (NPVs) together with their 95% CIs.Conclusion: The data indicate that the ANN method of measurement of LIC has potential to be used as a fast and robust method of analysing MR image data to generate LIC values. Further testing is required on a wider range of scanner types and with more patients with lower LICs. The performance of the ANN will also likely be improved through training on a larger number of datasets. [Display omitted] DisclosuresSt Pierre:Resonance Health Ltd: Consultancy, Equity Ownership, Speakers Bureau. Cooper:Resonance Health Ltd: Consultancy. Boulos:Resonance Health Ltd: Employment. House:Resonance Health Ltd: Employment. Pang:Resonance Health Ltd: Employment. Bangma:Resonance Health Ltd: Employment. Taher:Novartis Pharmaceuticals: Honoraria, Research Funding; Celgene: Research Funding.

Surrogate Thresholds for Iron Chelation in Patients with Hematological Malignancies

Background: The reference standard for evaluating the magnitude of body iron overload is measurement of the liver iron concentration (LIC). Until recently, the conventional clinical method for measuring LIC was through analysis of biopsy specimens considered the “gold standard” of LIC measurement. To counter the shortcomings of liver biopsy, several noninvasive techniques have been applied to estimate LIC. The spin-density projection-assisted (SDPA) R2-MRI (FerriScan) technique has been recently validated to estimate LIC. There are reports in the literature that ferritin levels in patients with hematological malignancies (HM) may not correlate sufficiently with LIC. We conducted R2-MRI Ferriscan sequencing in consecutive patients with HM followed in our center to better evaluate the diagnostic value of ferritin levels and packed red blood cell (pRBC) transfusions for iron overload.Methods: HM patients with a history of transfusion requirement were evaluated for inclusion in the study within the Liver Iron Cutino Network (Licnet) project. Patients were excluded if they had contraindications to MRI assessment (heart pacemaker, metallic foreign body or devices, and claustrophobia). Demographics and HM disease data were collected, together with ferritin levels and transfusion requirement. R2 LIC measures by FerriScan were defined as indicating normal/marginally high iron load for values 0 to <3, mild liver iron overload for values 3 to < 7, moderate between 7 and 15 and severe if >15. Correlations and the generation of receiver operating characteristic (ROC) curves were performed to test the diagnostic power of ferritin levels and volume of transfused blood (ml/kg in the preceeding 12 months) to indicate moderate-severe iron overload.Results: 28 patients (20 males) of mean age 62, SD 17 years with HM (myelodysplastic syndromes N=14; acute myeloid leukemia N=7, of which 5 post-transplant; acute lymphoblastic leukemia post-transplant N=2; aplastic anemia N=2; non Hodgkin lymphoma N=1; chronic lymphocytic leukemia N=1; erythroblastopenia N=1) were evaluated. Median baseline ferritin levels were 1617 (interquartile range 1185-2360). Mean R2 values were 10.9, SD 10.1 mg/kg. Three patients had normal or marginally high LIC, 12 patients had mild overload, 6 patients moderate and 7 patients severe overload. Both ferritin levels and amount of blood volume transfused in the preceding 12 months correlated well with R2 measures (Spearman rho=0.478, p=0.10 and r=0.509, p=0.006, respectively). However, neither ferritin levels nor transfusions correlated with R2 MRI in patients with normal-mild iron overload while the correlations were confirmed and highly significant in patients with moderate-severe iron overload (r=0.692, p=0.009 and r= 0.754, p=0.003, respectively). ROC curves denied the diagnostic power of ferritin levels and confirmed transfusions to indicate moderate to severe liver iron overload (Figure 1), indicating that 37 ml/kg/year (about 14 units of pRBC) has a sensitivity of 77% and a specificity of 80% to predict moderate-severe iron overloadConclusions: We confirm that, though ferritin levels do generally correlate with LIC, they are not to be considered an appropriate surrogate marker for iron overload in HM. R2 MRI sequencing is valuable to diagnose the state of LIC. The results of this study indicate that as soon as an HM patient begins to receive regular transfusions, since the probability of developing moderate-severe iron overload is high, it is warranted not to await high ferritin levels, nor a large number of pRBC units, to initiate iron chelation. [Display omitted] DisclosuresOliva:Celgene: Consultancy; La Jolla: Consultancy; Amgen Inc.: Consultancy; Novartis: Consultancy; Janssen: Consultancy.

Serum Ferritin Underestimates Liver Iron Concentration in Patients with Homozygous Alpha-0 Thalassemia

Background: Homozygous α0-thalassemia (Hemoglobin Bart's hydrops fetalis, HBHF) results from deletion of all α-globin genes. Similar to patients with transfusion-dependent beta-thalassemia (TDT-β), survivors of HBHF require life-long transfusions which over time will inevitably lead to significant tissue iron overload (IO). We have recently reported that the underlying disease process in HBHF is different from that of TDT-β (Amid A. et al. Blood, 2016). Unlike TDT-β, hemolysis is the major pathophysiologic process in HBHF and chronically transfused HBHF patients continue to have significant erythropoietic activity and tissue hypoxia (with increased serum erythropoietin) due to the indegenous nonfunctional hemoglobin-H. In addition, almost all survivors of HBHF would have commenced their transfusion therapy in-utero, potentially resulting in earlier IO compared to their TDT-β counterparts. Considering these differences, we hypothesized that the pattern of IO in patients with HBHF would likely differ from those with TDT-β.Methods: Following research ethics board approval, we retrospectively collected longitudinal data on transfusion and chelation history, serum ferritin, and liver iron concentration (LIC) measured by MRI or biopsy, in 7 HBHF (5 males; mean age: 7.3 years) and 14 TDT-β (8 males; mean age: 11.1 years) patients. None of the HBHF or TDT-β patients were splenectomized. Corresponding pairs of serum ferritin and LIC assessment (defined as measurements of not more than two weeks apart with no transfusions in between) were used to calculate ferritin-to-LIC ratios. All patients were treated with the same transfusion protocol to maintain pre-transfusion hemoglobin of >90 g/L and LIC of <5 mg/g of dry liver weight, until 2014. Since April 2014, five patients with HBHF have been commenced on a more aggressive transfusion strategy to decrease the nonfunctional HbH, suppress erythropoiesis, increase tissue oxygenation, and reduce hemolysis. We used linear mixed models to assess the associations between serum ferritin, LIC, and ferritin-to-LIC ratio with age, disease type (HBHF vs. TDT-β), chelation type, and method of LIC assessment. Correlations within a subject and between different ages were effectively accounted for in this model.Results: At 12 month of age, chelation-naïve HBHF patients had significantly higher serum ferritin level reflecting earlier IO compared to chelation-naïve TDT-β patients [1242.4 μg/dL (SD 419.4) vs. 418.3 μg/dL (SD 113.3), p<0.001]. On longitudinal assessments in both chelated HBHF and TDT-β patients, ferritin was significantly correlated with LIC (ρ = 0.88, p <0.001 and ρ = 0.46, p <0.001, respectively). Serum ferritin was significantly lower in HBHF vs. TDT- β [849 μg/dL (SD 573) vs. 2036 μg/dL (SD 1495), p<0.001] but there was no difference in LIC between the two groups [9.68 mg/g (SD 5.73) vs. 8.63 mg/g (SD 7.99), p=0.48]. Consequently, HBHF patients had significantly lower ferritin-to-LIC ratios [86.8 (SD 33.2) vs. 308.5 (SD 182.4), p<0.0001]. In addition, in the HBHF patients, serum ferritin-to-LIC ratio was corrolated with reticulocyte count (ρ = 0.41, p <0.042). In two mixed model analyses that included age, disease type, and method of LIC assessement or chelation type as variable of interset, disease type remained a highly signifiant predictor of serum ferritin-to-LIC ratio (p<0.0001). However, after the implementation of aggressive transfusion strategy (and suppression of erythropoeitic activity and improvement of tissue oxygenation) in HBHF patients, ferritin-to-LIC ratios were significantly higher [304.2 (SD 121.5) vs. 86.87 (SD 33.2), p=0.0003], becoming similar to ratios in TDT-β patients (p=0.88).Conclusion: Patient with HBHF are at-risk of significant IO early in life which poses potential challenges with iron chelation at an early age. Furthermore, in these patients, ferritin significantly underestimates LIC. This finding has important implications for clinical care and surveillance of IO in these patients, especially in areas with limited resources where non-invasive assessement of LIC (MRI) may not be widely available. The lower ferritin-to-LIC ratio observed in HBHF patients and its relation to erythropoietic activity indirectly suggest that in addition to transfusional iron loading, increased gastrointestinal absorption of iron due to suppressed hepcidin may also contribute to siderosis in these patients. DisclosuresNo relevant conflicts of interest to declare.

Effect of Liver and Heart Iron Overload Detected By MRI T2 * on HLA Matched HSCT in Child Patients with β-Thalassemia Major

BackgroundMost children with β-thalassemia major have iron overload which may have negative effect on hematopoietic stem cell transplantation. At present, the serum ferritin is used as an indicator of iron overload .However,since the distribution of iron in the organs of the body is not consistent, the serum ferritin can not exactly reflect the iron overload of important organs such as the liver and the heart .There are many international studies showing that MRI T2 * can accurately measure the iron load of the liver and the heart .In order to further clarify the effect of liver and heart iron overload on HLA matched HSCT among child patients with thalassemia and to provide the basis for clinical evaluation of patients with priority to transplantation,we compared liver and heart iron overload detected by MRI T2 * and serum ferritin with related events of transplantation.Patients and method81 child patients underwent liver and heart MRI T2 * tests.They were over 3 years old from April 2014 to December 2016 ,staying in our department ;they had had HLA matched allogeneic hematopoietic stem cell transplantation, and with β-thalassemia major, they were qualified to participate MRI detection .According to the test results,we calculated the liver and cardiac iron content, which is considered as the level of liver and heart iron overload. Next, it was compared with Serum Ferritin(SF),time of hematopoietic reconstruction,mortality rate, implantation rate and the morbidity of transplantation related complications such as graft versus host disease(GVHD),infection rate,autoimmune hemolysis,pancytopenia, hepatic veno-occlusive disease(VOD)and septicemia.ResultsSF and the liver T2 * were negative correlation (r=- 0.305, P= 0.305), and SF was correlated with the liver iron concent (r= 0.228, P= 0.228).SF was not correlated with cardiac iron content and heart T2 *.Liver iron content was correlated with the time of hemoglobin implantation (r= 0.229, P=0.043), cardiac iron content was correlated with mortality rate (r=0.266, P=0.017) ,SF and implantation rate were negative correlation(r=-0.289, P=0.009),SF was correlated with the morbidity of septicemia(r=0.251, P=0.024),SF was correlated with the morbidity of pancytopenia(r=0.276, P=0.013).SummaryIron overload may have negative effect on HLA matched allogeneic hematopoietic stem cell transplantation among child patients with β-thalassemia major, it is necessary to evaluat SF, liver and heart iron overload before HSCT. DisclosuresNo relevant conflicts of interest to declare.

Optimizing the Management of Iron Overload in Patients with Hemoglobin Disorders in Centers with Limited Access to Magnetic Resonance Imaging

Background:Transfusion therapy for severe chronic anemias is a major cause of iron overload. Patients with hemoglobin disorders are often transfused, either chronically or occasionally, and if iron overload is suspected, it is currently recommended they are subjected to so-called “iron studies” and determination of liver iron content (LIC) and heart T2* by magnetic resonance imaging (MRI). It remains common practice to request both laboratory and imaging tests, even for patients already undergoing iron chelation, despite the unreliability of laboratory markers such as ferritin in the presence of concurrent inflammatory conditions, which do not affect results from MRI scans. Since MRI is not widely available in most underdeveloped and developing countries, we aimed to assess the contribution of laboratory tests to the management of patients with hemoglobin disorders with iron overload by analyzing a cohort of patients who managed to undergo MRI during a period of limited access to MRI scans in our center.Methods: In this single-center, retrospective analysis, data were collected from digital MRI, laboratory information system, and electronic chart databases, upon approval of the local ethics board. Since access to MRI was limited during the period of time studied, physicians used the following criteria to indicate MRI scans to define clinical suspicion of iron overload: patient already undergoing chelation therapy based on previous laboratory tests and clinical assessment, current or previous chronic transfusion therapy, or elevated ferritin with transferrin saturation (TSAT) above 45%.Results and Discussion: From April 2014 to September 2016, 27 patients (17 with sickle cell disease - SCD, 10 with beta thalassemia - BT), median age 35 years (15-62), 9 male (33%), were evaluated at 34 different occasions with MRI and conventional iron studies. Patients with BT had significantly higher levels of serum iron and TSAT than patients with SCD (BT median 266ug/dL, range 93-328 and 0.94, range 0.41-1.00; SCD median 157ug/dL, range 58-264, and 0.64, range 0.21-1.00, P=0.004 and P=0.0138), which reflects a higher percentage of BT patients on chronic transfusions (8/11 vs. 6/17) and the higher transfusion volume for BT in comparison to what is needed for exchange transfusions in SCD. Four patients had normal MRI scans: 1 with SCD not in transfusion, 1 with SCD that had been treated with iron chelation, and 2 BT major in chronic transfusion and optimal response to iron chelation (ferritin <500ng/mL). Only one SCD and one BT patient were diagnosed with cardiac iron overload, with a T2* of 7.5ms and 2.95ms, respectively, in patients with extreme hyperferritinemia (11,567 and 7,090ng/mL) and low compliance to iron chelation. MRI liver iron concentration correlated positively with serum ferritin and total iron binding capacity (TIBC) measurements (r=0.67, 95% CI 0.41-0.83, P <0.0001, and r=-0.49, 95% CI -0.72 to -0.17, P <0.004, respectively) but no serum biomarkers correlated with myocardial iron concentrations. Receiver operating characteristic (ROC) curve analysis showed that ferritin levels performed best in our population as predictor of any increase of LIC above 2mg/g (area under curve 0.9753, P=0.0003), with a cutoff value of 731.6ng/mL with a sensitivity of 96.3% and specificity of 100% to discriminate for the presence of any increase in hepatic iron. In addition, a ferritin cutoff value of 1649ng/mL yielded 76.2% sensitivity and 91.7% specificity to detect a LIC above 5mg/g, comprising 17 (50%) of the MRI scans. Only one patient in this group had less than moderate liver iron overload, but still with an abnormal LIC of 3.5mg/g.Conclusions: Our data suggest that conventional iron studies cannot predict cardiac siderosis, but earlier evaluation with MRI may be warranted in the presence of clinical risk factors such as history of chronic transfusion along with low compliance to iron chelation, independently from laboratory tests. In settings with limited access to MRI, ferritin levels may be useful in pre-screening for hepatic iron overload, so postponing probable negative scans for patients undergoing iron chelation with optimal response or probable positive scans for those with certain levels of hyperferritinemia (e.g.>1649mg/dL) could be appropriate criteria to manage referrals to MRI scans and help prioritize patients with undetermined indication of iron chelation. DisclosuresFertrin:Alexion Pharmaceuticals: Consultancy.

Pituitary Iron: Effect on Pituitary Volume and Function in Transfusion Dependent Thalassemia

The association of iron toxicity with vital organ dysfunction has been extensively investigated in transfusion-dependent-thalassemia (TDT). However, pituitary iron load and its effect on function have been infrequently addressed. Growth delay, hypogonadism and infertility resulting from iron-induced pituitary toxicity are still prevalent, impacting clinical care and patients' quality of life. Early recognition and timely intervention can potentially prevent irreversible damage and restore partial function.AimsWe aimed to determine pituitary iron load and anterior pituitary volume by MRI in various age groups of TDT and examine their association with systemic and organ-specific iron measures. Additionally, we attempted to stratify the occurrence of abnormal anterior pituitary function by these MRI findings. Identifying such risk thresholds could assist in establishing timing and effectiveness of treatment intervention.MRI (1.5T) measurements of anterior pituitary iron (R2) and volume (Vol) were compared to normal controls (Noetzli 2011) with resultant Z-scores. Mean annual ferritin was calculated and cardiac and liver iron concentration (LIC) were determined by MRI-T2* and superconducting quantum interference device (SQUID) biosusceptometry, respectively. Pituitary hormones (LH and FSH) and growth hormone (Z scores for IGF-1 and IGF-BP3) were determined.ResultsFifty-three TDT patients (51% females) age 25±8 years, (range 10-45 years) were enrolled. Thirty-three (62%) had a ZR2 value >2 (mean 5.4) indicating iron accumulation above 97.5th percentile. Thirty-two patients (60%) had a Z-score for anterior-volume (ZVOL) of less than -2 (mean -2.8) indicating gland shrinkage below the 2.5th percent. In 23 patients (43%) both ZR2 and ZVOL exceeded these thresholds. ZR2 inversely correlated with ZVOL; r =-0.32, p<0.02. Pituitary iron increased with age and differed between patients <18 yr (n=14) and those 18-45 yr (n=39) with a mean ZR2 at 0.9±0.6 and 4.3±0.5, respectively (p<0.001). Anterior pituitary volume declined in older patients, lagging after the iron accumulation (Figure).LH/FSH values and ZR2 showed inverse correlation: r = -0.27 (p<0.03) and -0.3 (p< 0.006) respectively. ZVOL and LH/FSH demonstrated a positive correlation: r = 0.40, p<0.05 and 0.37, p<0.003, respectively. Z(IGF-BP3) but not Z(IGF-1) had an inverse correlation with ZR2: r = -0.5 (p<0.001).ZR2 > 4 was found to be a significant predictor for gland shrinkage; Zvol < -2 was present in 17/33 patients with a ZR2>4 compared to 3/20 with a ZR2<4 (p<0.009). Receiver operator (ROC) analysis confirmed it with an AUC of 0.68. ZR2 >4 was also predictive of presence of clinically important cardiac (T2* <20ms, AUC=0.92, p<0.001) and hepatic iron (LIC >10mg/gd.w., AUC= 0.74, p<0.005). Mean ferritin significantly correlated with ZR2 (p<0.001) but not with the gland's volume.ConclusionsAnterior pituitary iron assessed by MRI, increases with age and correlates with total body iron burden in TDT, suggesting cumulative iron toxicity. It results in a decrease in gland size and impaired gonadotropin and growth hormone secretion. Pituitary iron exceeding ZR2 >4, appears to be a significant predictor for change in gland size, that also is associated with cardiac and liver iron-overload. Pituitary MRI and the concomitant liver and cardiac iron quantification can identify patients at high-risk for further pituitary damage, suggesting that early intervention can potentially prevent additional decline in hormonal secretion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cardiorenal Syndrome in Thalassemia Patients

Background: Cardiorenal syndrome (CRS) is a serious condition with high morbidity and mortality. Secondary CRS (type 5 CRS) is characterized by the coexistence of cardiac abnormality and renal dysfunction occurring secondarily to a systemic condition. In thalassemia, a common hematologic disease in Thailand, there is limited information about the prevalence of secondary CRS. Therefore, this study aimed to investigate the prevalence and associated risk factors of secondary CRS in thalassemia patients.Method: In this cross-sectional cohort study, 53 adult thalassemia patients in out-patient clinic of Chiang Mai University Hospital since October 2016 to June 2017 were included. A criterion for diagnosis of secondary CRS is based on a system proposed by Ronco and McCullough. Cardiac abnormalities, assessed by clinical presentation of heart failure, chest X-ray, electrocardiogram, and echocardiogram, included acute or chronic heart failure by using definition from 2016 ESC guideline or structural cardiac abnormalities defined by LV remodeling and dysfunction, diastolic dysfunction, chronic abnormalities in cardiac function or cardiomyopathy. Renal dysfunction is defined by acute kidney injury or chronic kidney according to 2012 KDIGO guidelines. Clinical and laboratory (serum NT-proBNP, spot urine protein, spot urine albumin, spot urine electrolytes, 24-hours urine protein, and 24-hour urine electrolytes) data for 2 consecutive visits, at least three months apart, were assessed. We analyzed correlation between patients with and without cardiorenal syndrome by Chi-square for categorical data, T-test and non-parametric test of 2 independent samples for continuous data. We used binary logistic regression model for multivariate analysis.Result: Of 53 thalassemia patients [42 (79.2%) transfusion dependent thalassemia and 11 (20.8%) non-transfusion dependent thalassemia], 16 (30.2%) has secondary CRS. Cardiac abnormalities was detected in 23 patients (43.4%), included acute or chronic heart failure (N=4, 7.5%), or structural cardiac abnormalities (N=23, 43.4%). Thirty-one patients (58.5%) had renal abnormalities. Compared with the non-CRS group, the CRS group had a significant increase in serum NT-proBNP level [median (range) = 101.4 (79.2-545.1) VS. 64.8 (16.7-281.2) ng/ml, p=0.05], 24-hours urine magnesium level [mean (95%CI) = 5.98 (3.91-8.04) VS. 4.42 (3.01-5.83) mEq/24 hr., p=0.042], 24-hours urine protein level [median (range) = 249 (132-1610) VS. 161 (77-324) mg/24 hr., p=0.003], and 24-hours urine albumin level [median (range) = 45 (3-1303) VS. 5 (2-184) mg/24 hr., p=0.006], but significance lower in mean arterial pressure (MAP) [mean (95%CI) = 72.7(67.5-77.8) VS. 79.5(69.8-89.2) mmHg, p=0.041]. In univariate analysis, CRS was associated with pulmonary hypertension (OR 2.33, p=0.019), presence of extramedullary hematopoiesis (EMH) (OR 3.79, p=0.03). Multivariate analysis confirmed the association of cardiorenal syndrome with presence of EMH (OR 6.55, p=0.04), lower MAP (OR 0.869, p=0.012), elevated serum NT-proBNP (OR =1.013, p=0.012), and elevated 24-hour urine magnesium (OR 2.17, p=0.004). There was no correlation of CRS with type of thalassemia, serum ferritin, liver iron concentration, cardiac T2* and type of iron chelating agents.Conclusion: This study has demonstrated that CRS is common in thalassemia patients. Patients with the presence of EMH, low MAP, increased 24-hour urine magnesium level or an elevated serum NT-proBNP level are at high risk for developing CRS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Systematic Review of the Effect of Iron Chelation Therapy on Survival in Patients with Sickle Cell Disease and β-Thalassemia

Background: Patients with sickle cell disease (SCD) and beta (β)-thalassemia often require regular blood transfusions; however, each unit of blood contains approximately 200 mg of iron, which can rapidly lead to iron overload (IO). This can result in iron deposition in the heart, liver, and endocrine organs, ultimately leading to organ failure, including cardiac disease, liver fibrosis, and metabolic disorders. The introduction of iron chelation therapy (ICT) with deferoxamine (DFO) and more recently deferiprone (DFP) and deferasirox (DFX) has had a significant impact on IO, demonstrating reductions in serum ferritin level and liver iron content. Although ICT has become the standard of care over the past few decades, there are limited data on the effects of these agents on overall survival. The objective of this systematic review is to evaluate the impact of ICT on survival in patients with transfusion-dependent SCD or β-thalassemia.Methods: This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols(PRISMA-P) guidelines. Electronic databases, including Embase, PubMed, Cochrane Library, and the World Health Organization (WHO) Clinical Trials Search Portal, along with American Society of Hematology annual meeting abstracts from 2010-2015 were searched to identify English language articles that were relevant to the topic. Search terms included “sickle cell anemia” or “SCA,” “sickle cell disease” or “SCD,” “beta thalassemia” or “thalassemia,” “iron chelation,” “iron overload,” “survival,” “death,” “study,” and “trial.” Abstracts identified by the literature were independently reviewed and only articles with unanimous support were included; when the eligibility of an abstract was disputed, a full copy of the article was retrieved for review. References were excluded for lack of evidence of survival analysis, comparison between regimens, or if trials had fewer than 25 patients. Details of the included studies were collected for categorization. Although a meta-analysis of the data was initially planned, there were too few studies with similar study parameters to complete the analysis.Results: For SCD, the literature search yielded 203 articles of which 6 were deemed relevant (Figure 1). Of these, 3 were excluded and 3 (published between 2001 and 2014) were included. Reasons for exclusion included small patient number, no comparator, and being a theoretical paper. Only 1 of the 3 was a randomized clinical trial (RCT). For β-thalassemia, theliterature analyses yielded 398 articles of which 40 were deemed relevant (Figure 2). After reviewing the articles, 22 were excluded while 18 (published between 1991 and 2015) were included. Reasons for exclusion included small patient number, no comparator, no survival data, no chelation data, or a focus on hematopoietic stem cell or bone marrow transplantation. Only 3 of the 18 were RCTs; the remainder were retrospective and/or longitudinal studies. Most data on effectiveness of ICT were from longitudinal studies that suggest treatment outcomes have improved significantly over time mainly due to the introduction of new and improved iron chelation agents, including oral treatments and combination therapy. Effectiveness was also investigated in studies evaluating treatment compliance that suggest survival is improved in patients compliant to ICT. Compliance has been enhanced with the introduction of oral agents such as DFP and DFX compared with DFO, which requires daily subcutaneous injections.Conclusions: This systematic review identified few RCTs of ICT reporting survival in SCD and β-thalassemia. Despite limited data, the available literature suggests that ICT improves overall survival in SCD and β-thalassemia. Survival may also have been affected by factors such as hydroxyurea usage and the number of blood transfusions. Newer oral treatments have improved compliance, shown to be an important factor in survival, although there have not been any prospective long-term RCTs adequately powered to compare the efficacy among available iron chelation agents in either SCD or β-thalassemia. [Display omitted] DisclosuresBallas:Novartis: Honoraria, Speakers Bureau. Zeidan:Takeda: Speakers Bureau; AbbVie, Otsuka, Pfizer, Gilead, Celgene, Ariad, Incyte: Consultancy, Honoraria; Otsuka: Consultancy.

Transient Reduction of Transferrin Saturation Contributes to Hepcidin Inhibition Independently of Erfe in Mice Treated with Erythropoietin

IntroductionErythropoiesis is the major consumer of iron for hemoglobin synthesis. Erythroid cells release erythroferrone (ERFE), an erythropoietin (EPO) target gene, to signal their iron needs to the liver through hepcidin inhibition (Kautz et al., 2014). Since hepcidin degrades the iron exporter ferroportin, thus reducing body iron concentration, its inhibition by ERFE increases iron availability for erythroid cells. Hepcidin is mainly controlled by the BMP-SMAD-pathway. We demonstrated that ERFE inhibits hepcidin only when the BMP-SMAD signaling is attenuated (Nai et al., 2016). However, how the BMP-SMAD pathway is reduced in a physiologic context of increased erythropoiesis remains unknown. The major source of iron for erythroid cells is the circulating diferric-transferrin (holo-TF) that is uptaken by TFR1, highly expressed by erythroid cells. Since holo-TF, measured as transferrin saturation, is involved in hepcidin activation by the BMP-SMAD pathway, we hypothesized that increase iron uptake by EPO-stimulated erythropoiesis reduces transferrin saturation, thus inhibiting hepcidin earlier than and independently of ERFE.MethodsC57BL/6 wild type and Erfe KO male mice (8-9 week-old) were challenged with a single EPO injection (200 U/mouse) and sacrificed at early time points (3-15 hrs) post-treatment. To analyze erythroid differentiation and Tfr1 levels at short time points after EPO injection (3 and 6 hrs post-treatment), Ter119+ cells were separated from bone marrow (BM) and spleen according to Forward Scatter (FSC) value and CD44 expression, as described(Liu et al., 2013). Tfr1 was measured by flow cytometry in isolated population and expressed as MFI. Expression of BM and spleen Erfe, GypA and Tfr1 was evaluated by qRT-PCR. Hamp, Id1 and Atoh8 were measured from total liver of EPO- or saline-treated mice. Serum iron, transferrin saturation (TS) and liver iron content were evaluated by standard methods (Pagani et al., 2011).ResultsWild type mice were analyzed at different time point (3, 6, 9 and 15 hrs) after EPO injection. EPO-treated mice upregulate Erfe mRNA both in BM and spleen, starting from 3 hrs post-injection. Hepcidin and the BMP-SMAD target genes Id1 and Atoh8 are significantly decreased at 9 hrs post-EPO. Although Id1 and Atoh8 start to increase 15 hrs post-EPO, hepcidin remains low due to Erfe inhibition. EPO treatment transiently (at 6 and 9 hrs) reduces both serum iron and TS that are increased thereafter (15 hrs) due to hepcidin inhibition. Since EPO stimulates Tfr1 expression in erythroid cells, we measured Tfr1mRNA and protein levels in BM and spleen. Tfr1 mRNA is slightly increased in the spleen and shows a trend towards increase in the BM at 6 hrs post-EPO, whereas it is comparable to untreated mice at 9 and 15 hrs post-EPO. However, flow cytometry analysis shows that, despite the number of erythroid precursors expressing Tfr1 is comparable in untreated and EPO treated mice, membrane Tfr1 is strongly increased at 3 and 6 hrs post-EPO, indicating that increased erythroid uptake accounts for serum iron/TS reduction shortly after EPO treatment. To demonstrate that increased Tfr1 expression by EPO in erythroid cells downregulates the liver BMP-SMAD pathway and hepcidin through a transient reduction in circulating iron/TS and independently of Erfe, we analyzed EPO-injected Erfe KO mice. Both in wild type and Erfe KO animals Id1 is strongly inhibited at 6-9 hrs post EPO. Interestingly, Erfe KO animals show a trend towards a reduction in hepcidin at 9 hrs post-EPO, and a significant decrease at 15 hrs, with low serum iron and TS.The mild but significant effect observed in Erfe KO mice suggests that downregulation of the BMP-SMAD pathway due to transient reduction of TS contributes to hepcidin inhibition in an erythroid-dependent, but Erfe-independent, manner.ConclusionsEPO stimulates erythroid expression of membrane Tfr1 at short time point after treatment, causing a transient decreased in transferrin saturation and a downregulation of the hepatic BMP-SMAD pathway that contributes to hepcidin inhibition independently of Erfe. DisclosuresFerrari:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Camaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

Iron Overload Decreases Engraftment of Mice Receiving Allogeneic or Syngeneic Bone Marrow Transplant

Background:Transfusional iron overload is a poor prognostic factor in patients undergoing hematopoietic stem cell transplantation (HSCT). In multiple clinical studies, elevated serum ferritin correlated with inferior transplant outcomes; however, ferritin is an imperfect marker of iron burden and may not correlate with other markers, such as liver iron content. These studies are also confounded because increased transfusional need often reflects more severe disease, an independent predictor of worse outcome. Thus, it remains unclear whether the correlation between iron status and transplant outcome is due to transfusional iron overload alone, or is more reflective of other comorbidities. We developed a mouse model of parenteral iron overload followed by high dose chemotherapy and allogeneic or syngeneic HSCT to study, in isolation, the effects of iron overload on engraftment and survival after transplant.Methods:Cohorts of female C57BL/6 (H-2Kb) recipients received 0, 5, 25, or 50mg iron dextran, in divided doses over 2 weeks. Mice were then conditioned with busulfan (20mg/kg daily x 5 days) and cyclophosphamide (100mg/kg daily for the last 3 days). Two days later, mice received 30 x 106 bone marrow cells from female BALB/c (H-2Kd) donors (allogeneic HSCT) or 5 x 106 bone marrow cells from female UBC-GFP (H-2Kb) donors (syngeneic HSCT). Mice received enrofloxacin (10mg/kg daily x 5 days) after transplant. Engraftment was evaluated by flow cytometry as the proportion of donor cells in blood or bone marrow. Iron-related parameters were measured at the time of euthanasia (Day +28 post-transplant). Statistical analysis was performed with GraphPad Prism.Results:Our model of iron overload using iron dextran at escalating doses showed a dose-dependent increase in markers of iron load, such as total liver iron (R2=0.86) and ferritin (R2=0.84)(Fig. 1A-B; both P<0.0001 for linear trend). After allogeneic HSCT, there was a statistically significant decrease in overall survival in all groups receiving iron dextran (range: 47-63%), as compared to the saline control (90%)(Fig 1C; P<0.05); this effect was not dose-dependent. Data from 3 pooled experiments (N=30 mice per group) showed an engraftment rate of 59% in the saline group, and 47%, 42%, and 21% in the 5mg, 25mg, and 50mg iron dextran groups, respectively, suggesting a dose-dependent effect of iron overload on engraftment (Fig. 1D). In the syngeneic HSCT model, iron overload prior to transplant similarly resulted in delayed RBC and platelet engraftment and decreased rates of overall engraftment (Fig. 1E,F,G).Conclusions:Iron overload had a dose-dependent negative effect on overall engraftment after allogeneic and syngeneic HSCT. For allogeneic HSCT, there was also a decrease in overall survival in all the groups receiving iron dextran. For syngeneic HSCT, in which graft rejection cannot occur, iron overload still negatively affected post-transplant engraftment, suggesting that this effect is not due solely to potential immunomodulatory effects of excess iron on graft rejection. The mechanism(s) underlying the effects of iron overload on survival and engraftment rates in allogeneic and syngeneic HSCT, along with the potential benefit of chelation therapy, remain to be determined.Figure 1: Effect of iron overload on survival and engraftment. Mean ± S.D. for (A) total liver iron and (B) ferritin for allogeneic HSCT. Kaplan-Meier survival estimates for (C) mortality and (D) percent of mice not engrafting >50% of nucleated cells for allogeneic HSCT (N=10 mice per group per replicate; 30 mice total per group). Mean ± S.D. for (E) percent red cell engraftment over time and (F) percent platelet engraftment of time in syngeneic HSCT. Kaplan-Meier survival estimates for (F) percent of mice not engrafting >50% of red blood cells (N=10 mice per group per replicate; 20 mice total per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as compared to the saline control or as specified. Arrow denotes day of transplant. [Display omitted] DisclosuresSpitalnik:New Health Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Pancreatic Iron Loading in Emoglobinopathies

Background.The MIOT (Myocardial Iron Overload in Thalassemia) project is a multicentric study aimed to validate the T2* Magnetic Resonance Imaging (MRI) technique as non invasive approach for the cardiac and hepatic iron overload assessment in patients with hemoglobinopathies and to correlate the T2* values with other clinical-instrumental parameters. More recently, the E-MIOT (Extension-MIOT) project has been approved, with the aim of adding the assessment of the pancreatic ironMethods. We report the baseline MRI findings at the end of the first year of recruitment in the E-MIOT study, outlying the differences among different emoglobinopathies.Results. First, we selected all transfusion-dependent (TD) patients: 7 with sickle-cell disease-SCD or thalasso-drepanocytosis (42.9% F, 32.47 ± 17.93 years), 16 with thalassemia intermedia-TI (56.3% F, 36.66 ± 13.72 years), and 232 with thalassemia major-TM (55.6% F, 36.95 ± 9.83 years). Sex, mean age, serum ferritin levels, MRI liver iron concentration (LIC) values, and global heart T2* values were comparable among the three groups of patients. Pancreatic T2* values were significantly lower in TM patients versus both SCD and TI patients (see Figure).Second, we focused our analysis on all TI patients, divided in two subgroups: transfusion dependent and no-TD. Global pancreas T2* values and the number of patients with pancreatic iron (T2*<26 ms) were comparable between the two groups (see Table).Conclusion. SCD and TI TD patients have lower pancreatic iron loading than chronically-transfused TM patients. Much of this disparity can be explained by the larger transfusional burdens and durations observed in TM patients (years of regular transfusions: 34.18 ± 10.75 in TM, 22.27 ± 18.53 in SCD and 28.33 ± 15.30 in TI, P=0.023). However, TI-TD and TI-noTD patients had comparable pancreatic T2* values. So, innate differences in iron handling and elimination among different diseases also could contribute to the differences in pancreatic iron loading. [Display omitted] DisclosuresPepe:Chiesi Farmaceutici and ApoPharma Inc.: Other: Alessia Pepe is the PI of the MIOT project, that receives no profit support from Chiesi Farmaceutici S.p.A. and ApoPharma Inc.

The Macrophage Nuclear Receptor Coactivator 4 Counteracts Iron-Deficiency Anemia

The Nuclear Receptor Coactivator 4 (NCOA4) protein has been recently recognized as a novel player in iron metabolism contributing to regulation of cell and systemic iron homeostasis. NCOA4 is a cargo receptor that promotes selectively autophagy of the iron storage ferritin in conditions of iron deficiency, facilitating iron recovery from cellular stores. Inactivation of NCOA4 in cells increases ferritin aggregates [Mancias JD et al., Nature 2014; Dowdle WE et al., Nat Cell Biol 2014; Mancias JD et al., Elife 2015] and C57Bl/6 Ncoa4-ko mice show ferritin and iron accumulation in several organs, in particular spleen [Dowdle WE et al., Nat Cell Biol 2014; Bellelli R et al., Cell Rep 2016]. In these animals, iron retention in tissue ferritin reduces iron availability for erythropoiesis causing mild microcytic anaemia in iron-balanced (IB) conditions. In addition, compared to wt animals, Ncoa4-ko mice fed an iron-deficient (ID) diet develop a more severe anemia [Bellelli R et al., Cell Rep 2016].Given the important impact of mouse strain on iron metabolism, we evaluated the ability of Ncoa4-ko mice on a different background to respond to variation of dietary iron content. To this aim wt and null mice on Sv129/J background were fed an IB or an ID diet for 6 months. The ID diet induced almost no changes in hemoglobin levels of wt mice (IB=14.0±1.9 g/dL; ID=13.3±2.9 g/dL), while Ncoa4-ko developed severe microcytic anemia (Hb: IB=13.5±2.0 g/dL, ID=6.7±2.9 g/dL, p<0.05; MCV: IB=47.8±1.3 fL, ID=36.5±4.8 fL, p<0.05). These results are consistent with wt mice on Sv129/J strain being able to counteract ID by using the large reserves of iron present in their tissues. Notwithstanding such high iron deposits, Sv129/J Ncoa4 -ko fail this compensation. To better evaluate the bone marrow (BM)-derived cells (erythroid precursors, BM and spleen macrophages) contribution to the inability of Ncoa4-ko mice to respond to variation of dietary iron in comparison to other tissues (eg. liver and duodenum), we performed BM transplantation experiments in Ncoa4-ko using Sv129/J background. Lethally irradiated Ncoa4-ko mice were transplanted with wt or Ncoa4-ko BM, generating chimeric null animals with wt (Ncoa4-kowt-BM) or Ncoa4-ko BM cellsas control (Ncoa4-koko-BM). Previously published results obtained in vitro and in vivo in zebrafish suggest an essential role for NCOA4 in erythroid cells development and hemoglobinization [Mancias JD et al., Elife 2015; Ryu MS et al., JCI 2017]. However, we observed that Ncoa4-ko BM cells were able to reconstitute an almost normal erythropoiesis in lethally irradiated mice, indicating that in vivo the loss of Ncoa4 has a limited, if any, effect on the erythroid lineage differentiation. In addition, Ncoa4-ko mice with wt bone marrow have normal hematological parameters, suggesting that the mild microcytosis observed in Ncoa4-ko animals in basal conditions is due to a defect of BM-derived cells. Theoretically this effect may be due to cells belonging either to the erythroid or to the macrophage lineage; based on our findings we suggest a prevalent role for macrophages. In line with this hypothesis, Ncoa4-koko-BM mice fed an ID diet for 3 months developed a more severe anemia than Ncoa4-kowt-BM (Hb= 10.7±0.6 g/dL vs 11.7±1.0 g/dL; p=0.06), accompanied by comparable liver (LIC: Ncoa4-koko-BM=174.8±50.4 μg/g; Ncoa4-kowt-BM=157.1±49.5 μg/g) but higher spleen iron concentration (SIC: Ncoa4-koko-BM=719.0±221.3 μg/g; Ncoa4-kowt-BM=176.1±74.6 μg/g; p<0.05), consistent with decreased iron recycling by spleen macrophages of Ncoa4-koko-BM animals.Over all, our results suggest that NCOA4-mediated ferritinophagy plays a central role in macrophages in counteracting dietary ID, promoting iron release and ensuring efficient erythropoiesis. DisclosuresCamaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

Iron Binding to Specific Lobes of Transferrin Differentially Affects Erythropoiesis and Hepcidin Signaling

Introduction: Transferrin (Tf) plays a role in iron homeostasis as both an iron cargo molecule and as a signaling molecule. Tf is comprised of two homologous lobes (N and C), each of which can bind one iron molecule. As such Tf circulates as four forms: diferric, monoferric C, monoferric N and apo. Upon iron binding, clefts in each lobe close resulting in a conformational shift in the structure of the molecule and altering its affinity for the transferrin receptors. The most abundant molecules in the circulation at normal transferrin saturations are the monoferric forms. The relative abundance of circulating monoferric N in healthy individuals is greater than C, and greater with increased iron status. The N and C lobes differ in several physical properties, including binding affinities for iron. Whether these differences have functional consequences has been a subject of controversy. In vitro data suggest that iron is released initially from the C-lobe during delivery from diferric Tf to the erythron. These observations led to the proposal that iron occupancy of the N lobe might serve as an indicator of erythroid iron sufficiency, and as a signal to regulate dietary iron absorption. We thus hypothesized that iron homeostasis would be influenced by the presence of iron on the transferrin C-lobe vs the N-lobe.Methods: To test this hypothesis, we generated mice producing exclusively monoferric N or C transferrins by mutating two essential tyrosines in the iron binding site of each of the lobes to phenylalanine (Y114 and Y207 in the N-blocked (N-bl) mouse and Y448 and Y537 in the C-blocked (C-bl) mouse). Heterozygous mice were crossed and offspring analyzed for hematological parameters, tissue iron concentrations, and expression by RT-PCR of selected genes relevant to iron homeostasis.Results: The N-bl and C-bl Tf mutant mice each demonstrate hepatic iron loading; however, N-bl mice demonstrated higher non-heme liver iron concentrations than C-bl Tf mutant mice (1542 +/- 89.48 vs 1089 +/- 110.6 ug Fe/g dry weight, respectively; p <0.0001) compared to wild-type (261.8 +/- 22.17 ug Fe/g dry weight; p < 0.0001) at 2 months of age. Liver hepcidin (Hamp1) expression was decreased in both Tf mutants and was significantly lower in the N-bl mutant mice compared to wild-type (1.18 +/- 0.15). Bmp6 expression was not different across strains. Bone marrow Erfe expression was increased ~4-fold in each strain relative to wild-type (p < 0.05). Both strains demonstrate a mild microcytic anemia with hemoglobin concentrations of 12.11 +/- 0.17 and 12.87 +/- 0.24 and MCV values of 42.42 +/- 0.15 and 40.24 +/- 0.13 for N-bl and C-bl mice, respectively, compared to wild type hemoglobin concentrations of 14.98 +/- 0.11 and MCV values of 53.16 +/- 0.11 (p < 0.01 across all categories). Red blood counts are significantly higher in C-bl mutant mice (9.98 +/- 0.12) than in N-bl mutant mice (9.16 +/- 0.09; p <0.001) or controls (8.91 +/- 0.06; p < 0.0001). In contrast, serum erythropoietin (Epo) is significantly higher in N-bl (503 +/- 50.18; p < 0.0001) compared to C-bl (273 +/- 29.5) and wild-type mice (221.6 +/- 10.61; p < 0.0001), resulting in a RBC:Epo ratio of 0.019 +/- 0.003 for N-bl mice, 0.04 +/- 0.003 for C-bl mice and 0.04 +/- 0.001 for wild-type mice (p <0.0001 for N-bl compared to C-bl and wt).Conclusions: N-bl and C-bl Tf mutant mice demonstrate a hemochromatosis phenotype consistent with decreased hepcidin signaling; however, severity differs. Both mouse models demonstrate iron restrictive erythropoiesis; however, erythrocyte count is suppressed relative to erythropoietin levels only in the N-bl mutant mice. The N and C lobes of transferrin thus demonstrate functionally distinct properties. We speculate that iron bound to the N-lobe of transferrin augments the signaling properties of transferrin which regulate hepcidin expression and erythropoiesis. DisclosuresRivella:Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fleming:Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Hyperuricemia and Musculoskeletal Complications in Thalassemia Patients

Background: Thalassemia patients has high cell turnover rate due to its chronic hemolysis and ineffective erythropoiesis, therefore hyperuricemia is anticipated. However, gouty arthritis is not common presentation in this population compared to other diseases which has high cell proliferation and turnover rate. The data about prevalence of hyperuricemia and gouty arthritis in thalassemia patients are also limited. The aim of this study was to identify the prevalence of hyperuricemia , gouty arthritis and associated risk factors in thalassemia patients. We also demonstrated musculoskeletal complications in thalassemia patients.Methods: This was a cross sectional study in thalassemia patients, age 15 years or older at the Adult Hematology Clinic of the Chiang Mai University Hospital. All patients had serum uric acid and 24 hours urine collection test. Hyperuricemia was defined as serum uric acid more than 6.8 mg/dl. The musculoskeletal investigations were plain film, bone mineral density, MRI spine. The data of history taking, physical examination, blood tests, MRI cardiac T2*, MRI liver for liver iron concentration and echocardiogram were also collected to identify associated risk factors. Categorical variables were compared using the Chi-squared test or Fisher exact test as appropriate. Continuous variables were compared by t-test or Wilcoxon rank-sum test (Mann-Whitney test). Level of significance was defined as p-value < 0.05.Results: Fifty-eight thalassemia patients were enrolled. The median age was 23.5 (16-41) years. There were 41 female patients (70.7%). Most of patients were beta thalassemia/hemoglobin E disease (63.8%), homozygous beta thalassemia (29.3%) and hemoglobin H disease (6.9%) respectively. Most of them were transfusion dependent (79.3%) and splenectomized patients (60.3%). Hyperuricemia was found in 29 cases (52.7%) with 7 patients who had gouty arthritis (12.1%). Thirty-two patients (55.2%) had hyperexcretion of uric acid (24 hour urine uric excretion > 600 mg/day). The hyperuricemia was significantly identified in patients with intact spleen more than post splenectomy (34% vs.17.6%, OR=14.49, 95%CI: 3.4-62.5, p<0.01). The hyperuricemia patients had significantly lower mean platelet count (325,000 ± 253,000 vs. 602,000 ± 218,000 per cu.mm., 95%CI: 0.98- 0.99, p=0.01), higher mean serum creatinine level (0.62 ± 0.18 vs. 0.49 ± 0.11 mg/dl, 95%CI: 1.89- 2.43, p=0.03) and lower fraction excretion of uric acid (8.2 ± 2.9% vs. 10.86 ± 3.9%, 95%CI: 0.33- 0.82, p<0.01) than thalassemia patients who had normal uric acid level. The association risk factors for hyperuricemia patients who developed gouty arthritis were older age (mean age 42 ± 11.3 vs. 29.8 ± 8.4 years, 95%CI: 34.38- 25.37, p=0.046), lower cardiac T2* (median 36.4 ± 8.6 vs. 40 ± 13.6 msec, IQR: 12.5, p=0.031) and lower eGFR (mean 110.95 ± 2.89 vs. 128.49 ± 11.03 ml/min/1.73m2, 95%CI: 84.90- 137.00, p=0.046) compared to hyperuricemia without gouty arthritis complication patients. In term of musculoskeletal complications, we identified osteopenia in 56.9%, osteoporosis in 13.8%, chronic joint pain in 13.8%, bone fracture in 6.9% and extramedullary hematopoiesis in 38.6% of our thalassemia patients.Conclusion: Hyperuricemia was found approximately half of thalassemia patients but gouty arthritis was occurred only in ten percent which may be explained by hyperexcretion of uric acid. The significant risk factor were older age, lower eGFR and lower cardiac T2* for gouty arthritis in hyperuricemia patients. Musculoskeletal complications were common in thalassemia patients with more than half of patients had osteopenia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FC 075POST-MORTEM HEPATIC AND BONE MARROW IRON CONTENT IN HEMODIALYSIS PATIENTS: A PROSPECTIVE COHORT STUDY

Abstract Background and Aims Studies using T2 MRI liver scans among Hemodialysis (HD) patients raised concern about the presence of iron overload in this population, regularly treated with intravenous (IV) iron. Histological evidence of tissue iron overload is scarce, since the majority of studies were performed in pre-erythropoiesis- stimulating agents (ESA’s) era, when blood transfusions were common. Primary objective: to quantify iron in the liver and bone marrow by biochemical and histological analysis, in adult CKD stage 5-HD. Secondary objectives: To explore association of clinical, laboratorial parameters, IV iron therapy and iron stores. Method After approval of local Ethical committee and informed consent from families, liver biopsy and bone marrow aspirate were obtained in the first 24h post-mortem from 21 chronic HD patients with anemia or under anemia treatment who died in Hospital Fernando Fonseca. Exclusion criteria: blood transfusion in the previous 2 weeks, acute or chronic liver disease, HIV infection, known hematologic or oncologic disease. Clinical, laboratorial and anemia therapy data were retrieved from hospital registry and outpatient HD centers. Biochemical liver iron content (LIC) was quantified by atomic absorption spectrophotometry. Histological semi-quantitative grading of iron storage was made in the liver and bone marrow using Scheuer’s and Gale’s criteria of grading Perls’ stain, respectively. Results Of 21 patients included, 10 (47,6%) were male, median (IQR) age 76.0 (67.5-85.5) years old, 18 (85.7%) white, 3 (14.3%) black, dialysis vintage was 47.0 (12.5-104.0) months. Charlson Comorbidity index was 10.0 (7.5-11.0), 7 (33%) patients had diabetes, and 11 (52.4%) used an arteriovenous fistula as vascular access. The cause of death was infection (n=9, 42.9%), cardiovascular (N=6, 28.6%), HD withdrawal (n=2, 9.5%) and unknown =3 (14.3%). Median (IQR) hemoglobin was 9.8 (8.5-11.4) g/dl and 11 (52.3%) patients had hemoglobin &amp;lt;10 g/dl. Ferritin was 494.0 (136.0-850.5) ng/ml and TSAT 19.9 % (13.3-26.0). 19 (90.5%) patients were receiving IV iron therapy. Median (IQR) IV iron administered in the previous 6 and 12 months before death was 800 (300-1250) mg and 1500 (650-2175) mg, respectively. All patients were on ESA therapy, median (IQR) dose 5000 (3000-9000) UI/week and erythropoietin resistance index was 9.6 (4.2-16.6). Median (IQR) liver iron content determined by atomic absorption was 42.5 (22.9-69.7) µmol/g. 9 patients (42.9%) had normal LIC (&amp;lt;36 μmol/g), while the remainder had mild to moderate overload. Median (IQR) Scheuer grade was 2 (1-3) and 13 (62%) of liver biopsies had increased (Scheuer grade &amp;gt; 1) iron deposition at histology. Median (IQR) grade of Perls staining in the bone marrow was 3 (3-4) and 9 (45%) had increased (Gale’s grade &amp;gt;3) iron content in the bone marrow. Iron semi-quantitative scores in liver and bone marrow had strong positive correlation (r=0.71, p&amp;lt;0.001). There was a strong positive correlation between LIC and ferritin (r=0.86, p &amp;lt; 0.001) and also TSAT (r=0.56, n=16, p=0.02). Hemoglobin was negatively associated with LIC (r= -0.46, p=0.04), and with iron content in the bone marrow (p=0.04). LIC did not associate with ESA dose, C-reactive protein, dialysis vintage or other clinical parameters. There was no statistically significant association between the dose of IV iron administered in the previous 6 and 12 months with LIC, ferritin,TSAT or iron scores in bone marrow and liver. Conclusion In these HD patients, there was biochemical and histological evidence of iron accumulation in liver and bone marrow. Ferritin and TSAT showed strong correlation with iron deposits, but none was found with the dose of IV iron administered. In this study, anemia severity was associated with higher degree of iron storage both in the liver and bone marrow, suggesting a multilevel blocking mechanism of iron’s utilization.

Comparison of Inline R2* MRI versus FerriScan for liver iron quantification in patients on chelation therapy for iron overload: preliminary results.

MRI quantification of liver iron concentration (LIC) using R2 or R2* relaxometry requires offline post-processing causing reporting delays, administrative overhead, and added costs. A prototype 3D multi-gradient-echo pulse sequence, with inline post-processing, allows immediate calculation of LIC from an R2* map (inline R2*-LIC) without offline processing. We compared inline R2*-LIC to FerriScan and offline R2* calibration methods. Forty patients (25 women, 15 men; age 18-82 years), prospectively underwent FerriScan and the prototype sequence, which produces two R2* maps, with and without fat modeling, as well as an inline R2*-LIC map derived from the R2* map with fat modeling, with informed consent. For each map, the following contours were drawn: ROIs, whole-axial-liver contour, and an exact copy of contour utilized by FerriScan. LIC values from the FerriScan report and those calculated using an alternative R2 calibration were the reference standards. Results were compared using Pearson and interclass correlation coefficients (PCC, ICC), linear regression, Bland-Altman analysis, and estimation of area under the receiver operator curve (ROC-AUC). Inline R2*-LIC demonstrated good agreement with the reference standards. Compared to FerriScan, inline R2*-LIC with whole-axial-liver contour, ROIs, and FerriScan contour demonstrated PCC of 94.8%, 94.8%, and 92%; ICC 93%, 92.7%, and 90.2%; regression slopes 1.004, 0.974, and 1.031; mean bias 5.54%, 10.91%, and 0.36%; and ROC-AUC estimates 0.903, 0.906, and 0.890 respectively. Agreement was maintained when adjusted for sex, age, diagnosis, liver fat content, and fat-water swap. Inline R2*-LIC provides robust and comparable quantification of LIC compared to FerriScan, without the need for offline post-processing. • In patients being treated for iron overload with chelation therapy, liver iron concentration (LIC) is regularly assessed in order to monitor and adjust therapy. • Magnetic resonance imaging (MRI) is commonly used to quantify LIC. Several R2 and R2* methods are available, all of which require offline post-processing. • A novel R2* MRI method allows for immediate calculation of LIC and provides comparable quantification of LIC to the FerriScan and recently published alternative R2* methods.

Serum or plasma ferritin concentration as an index of iron deficiency and overload.

Serum or plasma ferritin concentration as an index of iron deficiency and overload.

Comparative study on iron content detection by energy spectral CT and MRI in MDS patients.

e19032 Background: Myelodysplastic syndrome(MDS) patients may have iron overload due to long-term RBC transfusion or combined with abnormal iron metabolism.what are the differences of iron content detection by magnetic resonance imaging (MRI) and dual energy spectrum computed tomography (DECT)? What are the advantages and disadvantages of the two methods in the state of high and low iron deposition? Can the two methods complement each other? The purpose of this study was to compare the difference of liver/cardiac iron content detection in MDS patients by DECT and MRI under different adjust serum ferritin (ASF) levels. Methods: Liver and cardiac iron content were detected by DECT and MRI. Patients divided into different subgroups according to ASF. Compared the detection rate between DECT and MRI group, and correlation between liver/cardiac iron content detected by DECT/MRI and ASF in each subgroups. Results: The detection rate of iron overload(IO) in DECT group was lower than that of MRI group with ASF &lt; 1000ng/ml subgroup,DECT and MRI had similar detection rates of moderate to severe IO with 1000≤ ASF &lt; 5000ng/ml subgroup, detection rate of severe IO in MRI was lower than that of DECT with 5000ng/ml ≤ ASF subgroup. In patients underwent DECT and MRI examination at the same time, ASF was significantly correlated with hepatic VIC but not with liver iron concentration (LIC), and LIC correlated with ASF after removed these data of ASF &gt; 5000 mg/L. LIC expression are not significantly different among 1000≤ ASF &lt; 5000ng/ml and 5000 ng/ml ≤ASF subgroup. LIC and liver virtual iron content (VIC) were all significant correlation with ASF when ASF &lt; 2500ng/ml, liver VIC was still correlation with ASF but LIC was not when 2500ng/ml≤ ASF. Neither cardiac VIC nor myocardial iron content (MIC) correlation with ASF in these subgroups. Conclusions: This study showed that MRI and DECT can be used complementary to each other. In high iron content, such as ASF ≥5000ng/ml, DECT detection is more reliable. In patients with low iron content, such as SF &lt; 1000 ng/ml, MRI detection is more reliable. According to ASF, the appropriate detection method can be selected to evaluate the iron content more accurately.