Higher Liver Iron Concentration Research Articles

Quantitative computed tomography assessment of transfusional iron overload

Quantitative computed tomography (QCT) has been proposed for iron quantification for more than 30 years, however there has been little clinical validation. We compared liver attenuation by QCT with magnetic resonance imaging (MRI)-derived estimates of liver iron concentration (LIC) in 37 patients with transfusional siderosis. MRI and QCT measurements were performed as clinically indicated monitoring of LIC and vertebral bone-density respectively, over a 6-year period. Mean time difference between QCT and MRI studies was 14 d, with 25 studies performed on the same day. For liver attenuation outside the normal range, attenuation values rose linearly with LIC (r(2) = 0·94). However, intersubject variability in intrinsic liver attenuation prevented quantitation of LIC <8 mg/g dry weight of liver, and was the dominant source of measurement uncertainty. Calculated QCT and MRI accuracies were equivalent for LIC values approaching 22 mg/g dry weight, with QCT having superior performance at higher LIC's. Although not suitable for monitoring patients with good iron control, QCT may nonetheless represent a viable technique for liver iron quantitation in patients with moderate to severe iron in regions where MRI resources are limited because of its low cost, availability, and high throughput.

Safe and Efficient Chelation by Deferasirox In Thalassemia Major Patients with Low Liver and Cardiac Iron Concentrations

AbstractAbstract 5168Chelation treatment of iron overload from chronic blood (RBC) transfusion is still a challenge to both, patients and medical caretakers. Different treatment regimes have been recommended so far, especially for chronically transfused patients with low or even normal liver iron concentration. We report the results from 16 regularly transfused patients with thalassemia major (TM) who were on iron chelation treatment under normal to mild liver iron concentration (LIC). All patients received deferoxamine (DFO) treatment before they changed to deferasirox (DSX) treatment.16 TM patients (mean age 13.6 y) were treated with DSX (median dose 18 mg/kg/d, range: 7 – 33 mg/kg/d) for 6 to 71 months. Liver iron measurements by biomagnetic susceptometry (BLS) and/or MRI-R2 as well as cardiac MRI-R2* were performed in intervals of 6 to 12 months. The median LIC was 782 μ g/g-liver wet weight (range: 460 μ g – 1122 μ g). Median RBC transfusion rate was 8500 ml/y, equivalent to about 2 erythrocyte concentrates per 3 weeks or a daily iron influx of 16.2 mg/d. For each measurement interval, the ratio of daily iron influx and DSX dose rate was calculated. This represents the equilibrium molar efficacy for iron balance.In all 16 TM patients no severe side effects were observed and creatinine was in the normal range of < 0.9 mg/d throughout the treatment with DSX. From baseline DFO treatment interval to the endpoint of DSX treatment, liver iron decreased by 124 – 4689 μ g/g-liver (conversion factor of 6 for mg/g-dry-wgt), while serum ferritin decreased by -596 to 8283 μ g/l. For all measurement intervals, molar chelation efficacies between 18 % and 56 % were calculated at equilibrium with a median efficacy of 31 % (interquartile range = 16 %). This agrees with molar efficacies of DSX reported earlier, but for relatively higher LIC and chelation doses (Blood 2005; 106(11):#2690 and Blood 2007; 110(11):#2776). The cardiac R2* (median R2* = 38 s-1) was either below the normal threshold of 50 s-1 (T2* > 20 ms) or decreased by about 24 %/y under DSX treatment. In these few patients at low LIC, this was even higher than recently reported. Conclusion:Even in patients with normal to mild LIC iron chelation treatment with DSX is safe, does not result in increased creatinine levels or severe side effects and is as efficient as in patients with higher LIC. Disclosures:No relevant conflicts of interest to declare.

Combination of Two Orally Active Iron Chelating Agents: Efficacy and Safety In a Clinical Setting

AbstractAbstract 2064For patients with transfusion dependent anemias, in particular thalassemia major, as part of a compassionate –use program that was approved by the hospital's IRB, we report the efficacy and safety outcomes of combining therapy with deferasirox (DFX) and deferiprone (DFP). The combination of DFP and deferoxamine (DFO)has been shown to be effective in reducing cardiac iron overload but some patients are unable or unwilling to use DFO. DFP alone has also been reported to be cardioprotective. Based on concerns for their welfare in the presence of excessive cardiac iron load, a reduced left ventricular ejection fraction and either severe allergy or intolerance to DFO, 4 adult patients with thalassemia major have been treated with the combination of DFX and DFP for between 6 and 60 months (mean 18 months). All four patients were initially treated with DFO or DFX but DFP at 75–100 mg/kg/day was initiated because of severe cardiac iron overload. DFX at 15–40 mg/kg/day was added based upon high liver iron concentrations(LIC). Efficacy was evaluated by monthly ferritin levels and semiannual cardiac T2* and LIC estimates (using MRI R2 and R2*). All 4 patients had at least two MRI assessments. Table 1shows the changes in ferritin, cardiac T2*, LVEF and mean LIC. Cardiac T2* improved from 5.8 ±1.5 to 7.0 ± 1.5 ms., (p=0.15). If the T2* is recalculated as cardiac iron concentration then the change was from 4.1± 1.3 to 3.3± 1.1 mg/g dry weight (p=0.09). One patient who has been receiving the treatment for 6 months has shown a 1% per month deterioration in T2*. The patients who have been on the combination for 12, 24 and 60 months, have had reductions of 2.7%, 0.5% and 1.5% per month respectively. LVEF improved overall from the baseline value of 52.8% to 58.9% (p=0.02). Ferritin fell from a mean of 5826 to 5544 ng/L (p=0.86). LIC increased from a mean of 20.7 to 28.1 mg/g dry weight (p=0.36). Table 2 shows the baseline and minimum absolute neutrophil counts and baseline and final ALT (IU/L). No drug-related neutropenia (ANC of <1.5 ×109/L), agranulocytosis or arthralgia were observed. No patients demonstrated significant proteinuria and mean creatinine levels were unchanged. ALT's showed fluctuations that were compatible with the degree of LIC. It is important to note that compliance was extremely variable in that one patient (P3), only took DFX once monthly with many lapses in her DFP treatment as well. The other patients also had significant lapses in their compliance as well as long vacation periods during which they did not receive treatment. These results indicate that the combined use of the two oral chelators (DFP & DFX) prevented further cardiac iron loading with a tendency for its reduction, significant improvement in left ventricular ejection fraction, maintenance of ferritin levels and LIC. It is also possible that the DFP cardioprotective effect may be activated even in the presence of poor compliance with it. The combination was well tolerated and easier to manage. It is more acceptable for life-long chelation and positively influenced patients' quality of life. It seems likely that maximum doses of both medications need to be prescribed for better outcomes and longer periods of follow up are essential. In particular, a prospective randomized study in patients with excessive cardiac iron will be necessary before this combination could be considered standard therapy.Table 2Showing baseline absolute and lowest neutrophil count (ANC ×109/L), baseline and latest ALT (IU/L).IdentifierBaseline ANCLowest ANCBaseline ALTLatest ALTP13.42.29164P24.82.12395P33.82.87269P44.74.513364 Disclosures:Berdoukas:ApoPharma Inc.: Consultancy. Wood:Novartis Inc.: Research Funding; ApoPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees. Coates:Novartis: Research Funding, Speakers Bureau.

Deferiprone Is Associated with Lower Serum Ferritin (SF) Relative to Liver Iron Concentration (LIC) Than Deferoxamine and Deferasirox- Implications for Clinical Practice

AbstractAbstract 4246 IntroductionSerum ferritin (SF) is typically used as a surrogate marker for body iron load in thalassemia major (TM) patients. SF is useful as it can be regularly and inexpensively measured, allowing early trend recognition in chelation management. Various guidelines have recommended target SFs for TM patients, regardless of the prescribed chelator. The emergence of non-invasive LIC determinations has now allowed us to examine whether relationships between SF and LIC vary with chelator. Any differences in this relationship among chelators can have important implications for treatment guidelines. MethodsWe assessed whether the relationship between LIC measured by Ferriscan® (LIC-R2) and ferritin (using most recent SF within 3 months of LIC-R2 and mean SF in the year prior to LIC-R2 (SFmean)) varied according to chelators. TM patients (n=84), mean age 33 (±10) years, who were compliant and continuously on one chelation modality for > 1 year prior to LIC-R2 were retrospectively analysed. Chelation regimes included deferoxamine (DFO) (n=17), deferasirox (DFX) (n=47) or deferiprone (DFP) (n=23). The following patients were excluded: those with rheumatological diseases, HCV-RNA+ hepatitis, steatohepatitis and recent infections to minimize confounders of SF elevation; those on combination DFO and DFP due to their highly variable DFO frequencies; those with LIC >43mg/g dw as their LICs could not be reliably quantified. ResultsPatients had received a median of 10 (1-33), 1.5 (1-4) and 8 (1-19) years of DFO, DFX and DFP respectively prior to LIC-R2. Baseline characteristics were not significantly (sig) different among chelator groups (P>0.25): 51% had splenectomy; SF was determined at a median of 11(0-55) days from LIC-R2. The 3 chelator groups had similar median LICs (P=0.99) but DFP patients had sig lower median SF and SFmean than DFO (P=0.00) and DFX (P<0.01) patients (Table). This was observed across clinically relevant LIC thresholds of <3.2, 3.2–7 and >7 mg/g dw (P<0.05; DFP vs DFO and DFX). For high LICs associated with increased cardiac risk (>15mg/g dw), the median of DFP patients' SFmean was only 2529 (1776-2734) μg/L, which had a tendency to be less than that of DFX patients (median 4563 (1761-6871) μg/L, P=0.09) despite similar median LICs (P= 0.71). These were reflected by a lower median SF/LIC ratio in DFP than DFO (P=0.00) and DFX (P=0.00). Linear regressions of LIC vs SF were then compared between different chelators for SF <4000μg/L and LIC ≤ 20 mg/g dw as the best linear correlations were observed for these values across all 3 chelators. DFP's linear regression line (LIC =(0.0043 ±0.0011) × SF+ (0.74 ± 1.63), R =0.65) was sig different from DFO's (LIC =(0.0025 ±0.0010) × SF + (-0.55 ±2.28), R =0.58, P =0.00) and DFX's (LIC =(0.0022 ±0.0004) × SF +(0.55 ±0.75), R =0.69, P =0.047). DFP had higher predicted LICs at various SF compared to DFO and DFX (table). DFO was not sig different from DFX for the above parameters. ConclusionsOnce patients are stabilized on >1 year of a given chelator, the relationship between LIC and SF shows differences among chelators, especially for SF <4000μg/L and LIC ≤20 mg/g dw. Patients on DFP monotherapy will tend to have higher LICs at any given SF in this range than those on DFO or DFX. About half of our DFP patients had LIC >15mg/g dw despite SFmean <2500μg/L. As SF <4000μg/L mainly reflects reticuloendothelial (RE) rather than hepatocellular iron, these are consistent with a preferential removal of RE compared with hepatocyte iron by DFP, likely due to its rapid inactivation by glucuronidation within the hepatocyte, while DFO and DFX have a relative trophism for hepatocellular iron. These findings suggest that for chelation involving DFP, the SF trend may not reflect the LIC in the same way as with DFO or DFX, emphasizing the value of LIC monitoring in addition to SF.Table 1LIC, ferritin and predicted LIC.DFODFXDFPMedian LIC(mg/g dw)5 (1.2–30.6)4.8 (0.8–36.5)5 (0.5–34.9)Median SF (μg/L)1927 (1378–5182)1713 (312–6085)1142 (133–2897)Median SFmean (μg/L)2147 (950–6063)2006 (773–7290)1240 (230–2734)Median SF/LIC (μgL–1/mgg–1)523 (120–1562)403 (52–1188)181 (56–910)Predicted LIC (95%CI) (mg/g dw) at:SF 1000 μg/L1.9 (0–4.9)2.8 (1.9–3.7)5.1 (3.2–6.9)SF 2000 μg/L4.4 (2.8–5.9)5 (4.2–5.8)9.4 (6.8–12.0)SF 4000 μg/L9.3 (5.1–13.5)9.5 (7.3–11.6)18 (11.1–24.9) Disclosures:Off Label Use: Deferiprone, an oral iron chelator, is an off-label medication in North America. Shah:Swedish Orphan/Apotex: Honoraria; Norvatis: Honoraria, Speakers Bureau. Porter:Norvatis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Visão atual da hemocromatose hereditária

A hemocromatose hereditária (HH) está relacionada a diversos distúrbios do metabolismo do ferro que ocasionam sua sobrecarga tecidual. A HH clássica está associada às mutações do gene HFE (homozigose para C282Y ou duplo heterozigose para C282Y/H63D), sendo encontrada quase exclusivamente em descendentes do norte Europeu. A hemocromatose hereditária, quando não relacionada ao gene HFE, é causada por mutações de outros genes, recentemente identificados, envolvidos no metabolismo do ferro. Hepcedina é o hormônio regulador do ferro que inibe a ferroportina, proteína exportadora de ferro dos enterócitos e dos macrófagos; um defeito na expressão do gene da hepcedina ou na sua função costuma ser a causa da maioria dos tipos de hemocromatose hereditária. Os alvos acometidos pela HH são órgãos e tecidos - fígado, coração, pâncreas, articulações e pele -, sendo a cirrose e o diabetes melito os sinais tardios da doença em pacientes com expressivo aumento da concentração hepática de ferro. Pacientes com diagnóstico estabelecido de hemocromatose hereditária e sobrecarga de ferro devem ser tratados com flebotomia para a obtenção de depleção do ferro do organismo; em seguida, com flebotomia de manutenção. As causas mais frequentes de morte por hemocromatose hereditária são câncer hepático, cirrose, miocardiopatia e diabete; entretanto, pacientes submetidos à depleção do ferro de maneira satisfatória e antes do desenvolvimento da cirrose ou da diabete podem ter sobrevida normal.

Calibration of Improved T2* Method for the Estimation of Liver Iron Concentration in Transfusional Iron Overload.

Abstract 2004Poster Board I-1026 Background and rationaleNon-invasive estimation of tissue iron concentration is being used increasingly to follow responses to chelation therapy. Magnetic resonance (MR) T2* was originally developed to estimate myocardial iron but the first description of the myocardial T2* method also demonstrated a clear relationship between liver T2* and liver iron concentration (LIC) measured by biopsy (Anderson, Eur Heart J 2001). The inclusion of liver analysis at this time was intended mainly to show the relationship of T2* to tissue iron rather than as a standard method for LIC measurement, particularly as the echo times used were too long to measure the high iron levels commonly found in the liver. Since this work, other MR methods such as the R2 technique (St. Pierre, Blood 2004) have been specifically validated to measure LIC. However, if T2* could be validated and calibrated appropriately for LIC, it may be convenient to measure liver and heart iron at the same time using this rapid technique. Since the initial calibration of the T2* method, there have been major improvements in scanner technology and hardware. In the original paper, eight different echo times were acquired (TE 2.2- 20.1ms), each of which required a separate breath-hold with important implications for image registration regarding the measurement of T2*. Much shorter, more closely spaced echo times are now possible in a single breath-hold which makes the calculation of T2* more accurate, especially at higher liver iron concentrations where T2* is short. Methods:A large cohort of patients at UCLH have had liver biopsies as part of iron chelation studies on Deferasirox, while continuing to be monitored by T2* of both heart and liver. Data pairs for LIC calibration curve plots were chosen as nested values by shortest biopsy-to-MR time lapse criterion (±75 days) from all biopsy and hepatic T2* results. Liver biopsy iron was measured in a single central laboratory in Rennes, France (Clinique des Maladies du Foie [Clinic for Hepatic Illnesses], Center Hospitalier Universitaire) on paraffin embedded sections as previously described (Soriano-Cubells MJ, Atomic Spectrosc. 1984). All MR scans were performed on a 1.5T Sonata MR scanner (Siemens, Germany) using a 4-channel anterior phased array coil at Royal Brompton Hospital, London. A transverse slice through the centre of the liver was imaged using a multi-echo single breath-hold gradient echo T2* sequence with a range of echo times (TE 0.93-16.0ms). T2* decay was measured using Thalassaemia tools (Cardiovascular Imaging Solutions, London, UK) from a region of interest (ROI) in an area of homogeneous liver tissue, avoiding blood vessels and other sources of artefact. To account for background noise, a truncation method was used for curve-fitting (He, Magn Reson Med 2008). All T2* measurements were performed in triplicate by 2 independent observers choosing three separate ROIs to analyse. The ROIs were chosen to be as large as possible in three separate areas of the liver (anterior, mid/lateral and posterior). Spearman correlation method was used to estimate the degree of relationship between biopsy LIC and hepatic R2* (1/T2*) values, both of which showed positive skew. Results and conclusions:61 patients had both liver biopsy and liver MR measurements undertaken. However, only 18 had biopsies within 75 days of MR T2* measurement and these are the patients included in the following analysis. There is a linear relationship between biopsy LIC and the reciprocal of T2* (R2*), given by: LIC = 0.03 x R2* + 0.74 (R2=0.96 p<0.0001, slope 95% CI 0.027-0.033, with y intercept CI -0.73 to 2.2). The 95% confidence intervals for the linear regression line were calculated from the SEM and are shown as the broken lines (Figure 1). Staging of fibrosis/cirrhosis provided in biopsy reports was not found to significantly affect regression analysis contrary to previous studies. The inset shows the relationship between T2* and biopsy LIC. The new calibration shows acceptable linearity and reproducibility over an LIC range up to 30mg/g dry weight and gives LIC values approximately 1.94 times higher than the original method reported by Anderson. Comparison of these values with those obtained by SQUID and Ferriscan would also be of interest. [Display omitted] Disclosures:Pennell:Cardiovascular Imaging SOlutions, London, UK: Director.

Microarray analysis of liver gene expression in iron overloaded patients with sickle cell anemia and beta‐thalassemia

Chronic transfusion therapy is used clinically to supply healthy erythrocytes for patients with sickle cell anemia (SCA) or beta-thalassemia major (TM). Despite the benefits of red blood cell transfusions, chronic transfusions lead to iron accumulation in key tissues such as the heart, liver, and endocrine glands. Transfusion-acquired iron overload is recognized as a cause of morbidity and mortality among patients receiving chronic transfusions. At present, there is little understanding of molecular events that occur during transfusional iron loading and the reasons for the large inter-individual variation observed clinically in transfusion-acquired iron accumulation. To address these issues, we examined whether any liver-expressed genes in SCA or TM patients with transfusional iron overload were associated with the degree of iron accumulation. Specifically, we performed microarray analysis on liver biopsy specimens comparing SCA patients with mild or severe iron overload and also compared SCA with TM patients. Fifteen candidate genes were identified with significantly differential expression between the high and low liver iron concentrations. SCA patients and 20 candidate genes were detected between the SCA and TM patient comparison. Subsequent quantitative PCR experiments validated 12 candidate genes; with GSTM1, eIF5a, SULF2, NTS, and HO-1 being particularly good prospects as genes that might affect the degree of iron accumulation. Future work will determine the baseline expression of these genes prior to transfusional iron overload and elucidate the full impact of these genes on the inter-individual variation observed clinically in transfusion-acquired iron accumulation.

Deferasirox (Exjade®) Significantly Improves Cardiac T2* in Heavily Iron-Overloaded Patients with β-Thalassemia Major

Introduction: Iron overload currently remains a major cause of morbidity and mortality in patients (pts) with thalassemia major (TM), despite the availability of iron chelation therapy. Magnetic resonance imaging (MRI) can be used to measure tissue iron levels and identify pts with iron overload. The aim of this study was to monitor cardiac siderosis using MRI T2* in a cohort of Omani pts with TM.Methods: Cardiac MRI T2* was assessed in 19 pts enrolled at a single center in Oman. All pts had transfusional iron overload and had received chelation therapy (16/19 pts DFO + deferiprone; 2/19 DFO; 1/19 deferiprone) prior to enrollment in the ESCALATOR study. After completing a 1-year core phase of the ESCALATOR study, cardiac iron was evaluated as part of the site standard of care. Hence, pts had been receiving once-daily oral deferasirox starting with 20 mg/kg/day with subsequent dose adjustments for 12 months prior to the first cardiac T2* assessment (baseline [BL]). Pts continued on deferasirox throughout the 18-month period of this present evaluation.Results: All 19 pts (11 male; 8 female) completed 18 months of evaluation. Mean age (±SD) was 18 years (±4.5; range 10–29). At BL, pts had a mean cardiac T2* (±SD) of 17.2 (±10.8) ms (table) indicative of cardiac iron loading, (normal >20 ms) and a high median BL serum ferritin (SF) of 5497 ng/mL, signifying a high total body iron burden (r = −0.35). Pts also had a high liver iron concentration (LIC), which was correlated with both BL cardiac T2* and SF levels (r = −0.52 and 0.53, respectively). When the data were analyzed by BL cardiac T2* subgroups (<10 ms [n=6], 10–20 ms [n=7], <20 ms [n=13] and >20 ms [n=6]), all subgroups demonstrated a high BL SF. Mean deferasirox dose (±SD) in all pts was 25.9 (±2.3) mg/kg/day at BL, 32.0 (±4.4) mg/kg/day at 6 months and 37.7 (±5.5) mg/kg/day at 18 months, with dose adjustments carried out as per ESCALATOR study protocol.Overall, deferasirox significantly improved mean cardiac T2* by 3.6 and 4.3 ms at 6 (P=0.007) and 18 months (P=0.02), respectively. Further analysis showed a significant improvement in T2* (P<0.05) at 18 months in all subgroups except those pts with normal BL T2* (>20 ms), who only showed an improvement at 6 months. Moreover, deferasirox significantly reduced SF (P=0.001) and LIC (P=0.01) in the total study population at 18 months. Additional subgroup analyses for the changes at 6 and 18 months relative to BL are shown in the table.Table. Efficacy of deferasirox after 6 and 18 months of therapyAll pts (n=19)<10 ms (n=6)10–20 ms (n=7)<20 ms (n=13)>20 ms (n=6)Mean cardiac T2*, ms (±SD)BL17.2 (10.8)6.3 (2.2)14.9 (3.1)10.9 (5.2)30.8 (5.1)6 months20.8 (13.7)‡6.2 (2.6)19.2 (6.8)13.2 (8.5)37.2 (4.9)‡18 months21.5 (12.8)‡7.8 (3.2)‡21.4 (8.7)‡15.1 (9.6) ‡35.2 (6.0)Median SF, ng/mLBL549763855497549747336 months41286100480348033031‡18 months4235‡593746554674‡1793‡Mean LIC, mg/g dw (±SD)Pre-BL†24.17 (8.96)29.57 (11.21)24.81 (7.69)27.01 (9.38)18.02 (3.44)6 months19.71 (11.42)‡25.23 (10.73)23.34 (11.16)24.22 (10.55)9.93 (5.89)‡18 months17.62 (12.93)‡20.02 (10.85)23.97 (13.96)22.15 (12.28)7.82 (8.50)‡†These values were measured 6 months prior to BL T2* MRI; ‡significantly improved (P<0.05) compared with BLConclusions: Deferasirox therapy significantly improved cardiac T2* in these heavily iron-overloaded pts with TM. Improvement was seen in pts with various degrees of cardiac siderosis, including those pts with BL cardiac T2* <10 ms, indicative of high cardiac iron burden. SF and LIC were also significantly reduced in all pts, indicating that deferasirox reduced both cardiac and total body iron burden in these pts. That deferasirox dose was increased in all pts over the 18 months of the study highlights the importance of dose titration to achieve treatment goals.

Safety and Efficacy of High Dose Intravenous Desferoxamine for Reduction of Iron Overload Due to Chronic Transfusion in Sickle Cell Patients.

Background: Patients with sickle cell disease (SCD) receiving chronic blood transfusions are at risk of developing iron overload and organ toxicity. Chelation therapy with either subcutaneous (SQ) desferoxamine (DFO) or oral deferasirox is effective in preventing and reducing iron overload but poses significant challenges with patient compliance. Intravenous (IV) infusions of high dose DFO (HDD) have been utilized in non compliant patients with heavy iron overload in small case series. We review our experience of high dose IV DFO in a large cohort of SCD patients with significant iron overload who are non compliant with SQ DFO.Methods: The medical records of SCD patients who received HDD in our center between 1993 and 2004 were reviewed. All of them were on chronic transfusion, had significant iron overload defined by serum ferritin > 1500 and/or liver iron concentration (LIC) more than 10 μg/g of liver tissue and were non-compliant with SQ DFO. All patients underwent annual ophthalmologic, hearing, pulmonary and cardiac evaluation. Demographic data, treatment details, serum ferritin levels, liver iron concentration (LIC), liver enzymes, renal function tests, audiogram and other relevant clinical data were collected.Results: There were 27 patients (19 males, 8 females), 19 patients were on transfusion for history of cerebrovascular accident, 5 for abnormal transcranial Doppler flow velocity, 2 for transient ischemic attack and one for recurrent pain crises. All continued to receive packed red blood cell transfusions aimed to keep HbS levels below 30 or 50% during this time. They were treated in-hospital with DFO 15 mg/kg/hr IV for 48 hrs every 2 weeks (20 patients), 3 weeks (4 patients) and 4 weeks (3 patients). The mean age at start of high dose regimen was 14.6 years (range 9–27 years). The mean duration of HDD treatment was 8.9 months (range 3–49 months). Fourteen patients had LIC determined by liver biopsy. Significant reductions in LIC were observed after HD (table I). This was more pronounced in patients who had higher LIC and received at least 6 months of HDD. Histological examination of liver biopsies revealed a decrease in the grade of liver iron storage. Four patients had portal triaditis initially which resolved after starting HDD therapy. Also there was significant improvement in liver enzymes (ALT, AST) after HDD. There was a trend in decreasing ferritin levels after HDD but this did not achieve statistical significance. All patients tolerated HDD without any major reactions. No audiologic or ophthalmologic toxicity or acute or chronic pulmonary complications were observed. Blood urea nitrogen remained normal in all patients after HDD but there was mild increase in serum creatinine. One patient had high serum creatinine (1.2 mg/dL) after two doses HDD. This patient had focal segmental glomeurosclerosis which was most probably the cause for the rise in creatinine. There was no significant increase in serum creatinine in our series when this patient was excluded.Conclusions: In our cohort of SCD patients we observed a significant decrease in liver iron burden with high dose IV DFO. Our patients tolerated the therapy well without any major toxicity. This regimen is safe and may be an option for poorly compliant patients with significant iron overload. In addition, combination of this regimen with oral iron chelators may be of benefit to patients with significant iron overload and organ dysfunction.Table 1: Laboratory characteristics of sickle cell patients before and after high dose IV DFOParameterNo. of PatientsMean (SD) prior to HDDMean (SD)after HDDp Value*Liver iron (μg/g of liver tissue )1416864 (10903)12681 (8298)0.04Liver iron min of 6 months of HDD (μg/g of liver tissue )818677 (8319)9362 (4521)0.01Liver iron >10 mg & minimum 6 months of HDD (μg/g of liver tissue)721181 (7054)10092 (4443)0.01Grade of liver iron storage143.57 (0.9)3.07 (1)0.05Serum Ferritin (ng/mL)273842 (2619)3238 (1780)0.06Serum AST (IU/L)2754.1 (27.2)44.6 (17.6)0.04Serum ALT (IU/L)2739.2 (36)27.5 (14.2)0.01Blood urea nitrogen (mg/dL)278.9 (2.9)9.5 (4.3)0.20Serum Creatinine (mg/dL)+260.50 (0.1)0.55 (0.2)0.07* Changes in mean levels analyzed using two-tailed Paired T Test with significant p value ≤ 0.05.SD – Standard deviation+ See text

Iron Overload in C282Y Heterozygotes: Identification of New Rare HFE Gene Mutants and a Step Strategy for Diagnosis.

Introduction: Homozygozity for the p.Cys282Tyr (C282Y) mutation of the HFE gene is the main genotype associated with the common form of adult hereditary hemochromatosis. C282Y carriers do not usually develop iron overload, unless they have additional risk factors such as liver diseases, a dysmetabolic syndrome or an associated genetic defect. The commonest is the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele. However, a few rare HFE mutations can be found on the 6th chromosome in trans, some of which are of clinical interest to fully understand the disorder.Patients and Methods: We recently investigated four C282Y carrier patients with unusually high iron parameters, including increased levels of serum ferritin (SF), high transferrin saturation (TS) and high iron liver content measured by MRI. They were males, aged 37, 40, 42, 47 at diagnosis. Two brothers (aged 40 and 42) were referred separately. The HFE genotype, including the determination of the C282Y, H63D and S65C mutations was performed using PCR-RFLP. HFE sequencing was undertaken using the previously described SCA method (1). Sequencing of other genes (namely, HAMP, HJV/HFE2, SLC40A1, TFR2) was possibly performed in a last step using the same method.Results: We identified three rare HFE mutant alleles, two of which are undescribed, in the four studied patients. One patient bore a 13 nucleotide-deletion in exon 6 (c.[1022_1034del13], p.His341_Ala345>LeufsX119), which is predicted to lead to an abnormal, elongated protein. The two brothers had a substitution of the last nucleotide of exon 2 (c.[340G>A], p.Glu114Lys) that may modify the splicing of the 2d intron. The third patient, who bore an insertion of a A in exon 4 (c.[794dupA],p.[trp267LeufsX80]), has already been reported (1).Discussion: A vast majority of C282Y carriers will not develop iron overload and can be reassured. However, a careful step by step strategy at the clinical and genetic levels may allow to correctly identify those patients deserving further investigation. First, clinical examination and the assessment of iron parameters (SF and TS) allow identifying C282Y heterozygotes with an abnormal iron status. Once extrinsic factors such as heavy alcohol intake, virus or a dysmetabolic syndrome have been excluded, MRI is very useful to authenticate a high liver iron content. Second, HFE genotype must first exclude the presence of the H63D mutation. Compound heterozygozity for C282Y and H63D, a very widespread condition in our area, is usually associated with mild iron overload. Third, HFE sequencing can be undertaken and may identify new HFE variants as described here. The two novel mutations, a frameshift modifying the composition and the length of the C terminal end of the HFE protein and a substitution located at the last base of an exon, are likely to lead to an impaired function of HFE in association with the C282Y mutant. However, it is noteworthy that three of the four patients were diagnosed relatively late, after the 4th decade, as it is the case for C282Y homozygotes. Three further unrelated patients are currently under investigation in our laboratory for a similar clinical presentation. Finally, it can be noted that in those patients who will not have a HFE gene mutant identified, analysis of other genes implicated in iron overload must be performed to search for digenism or multigenism. None of our investigated patients had an additional gene abnormality.

Liver biopsy results in patients with sickle cell disease on chronic transfusions: Poor correlation with ferritin levels

Chronic transfusions are effective in preventing stroke and other complications of sickle cell disease. The aim of this study was to determine whether serum ferritin levels correlated with liver iron content in sickle cell patients on chronic transfusion. Forty-four liver biopsy specimens from 38 patients with homozygous sickle cell anemia (HbSS) and one patient with sickle thalassemia receiving chronic transfusions were studied. Five patients underwent a second liver biopsy for follow up. Three ferritin measurements were used to calculate a mean for each patient. The association between serum ferritin levels and liver iron quantitation was measured using the Spearman rank correlation, and sensitivity and specificity were determined for selected threshold values of serum ferritin. Serum ferritin levels ranged from 515 to 6076 ng/ml, liver iron concentration ranged from 1.8 to 67.97 mg/g dry weight. The amount of iron per gram liver dry weight was moderately correlated with serum ferritin values (r = 0.46). The correlation of duration of transfusion with serum ferritin (r = 0.40) and with liver iron content (r = 0.41) also indicated moderate correlation. Liver biopsy results led to changes in the management after 29/44 (66%) of the biopsies. Serum ferritin >/=2500 ng/ml predicted high liver iron content (>/=7 mg/g), with a sensitivity of 62.5% and a specificity of 77.8%. We found a poor correlation between serum ferritin levels and liver iron content (LIC). Despite being on chelation therapy, many patients on chronic transfusion had high levels of liver iron. Measurement of LIC is highly recommended in these patients.

Iron preloading aggravates nutritional steatohepatitis in rats by increasing apoptotic cell death

High serum ferritin and liver iron concentrations were found in some patients with NASH, suggesting a role for iron as a co-factor that aggravates liver injury. The aim of this study is to investigate the effects of parenteral iron in a rat model of NASH induced by a methionine choline deficient diet (MCDD). Wistar rats were divided into 1 - Control, 2 - Iron (Fe), 3 - MCDD, 4 - MCDD&Fe groups. Iron dextran 100mg/kg was administered intra-muscularly in groups 2 and 4. All rats were fed MCDD, Groups 1 and 2 were supplied with choline and methionine. Blood and tissue samples were obtained after 4weeks. The iron injection alone did not affect the liver whereas MCDD led to steatohepatitis. Iron worsened steatosis without any obvious effect on accompanying inflammation. It aggravated tissue injury by increasing apoptosis. Liver fibrosis was observed only in 3 out of 10 rats in the MCDD&Fe group. Observation of liver fibrosis only in the MCDD&Fe group suggests that iron induced increase in apoptosis contributes to the development of fibrosis at an earlier time than expected.

Liver iron content assessment by routine and simple magnetic resonance imaging procedure in highly transfused patients

Liver iron content (LIC) assessment by magnetic resonance imaging (MRI) is validated but not standardized. In a single center, we tried to assess the accuracy of a specific, simple MRI procedure adapted to high LIC from a well-established simple and routine procedure known to quantify LIC. In 27 cases of monthly transfused patients, we compared biochemical values of LIC assessed on liver biopsy specimens and results obtained by two signal intensity ratio of gradient echo imaging (R2*) MRI protocols. The first was Gandon's routine procedure previously validated in liver disease and the second, our own method, was an addition of a gradient echo sequence specifically adapted to high LIC encountered in hematology practice. Twenty-seven liver biopsies were performed in 18 adult patients (myelodysplastic syndrome = 5, beta-thalassemia = 13). LIC by biopsy ranged from 1.4 to 54 mg/g liver dry weight (mg/g dw) (median 9.4 mg/g dw). Correlation between LIC by biopsy and by MRI with Gandon's procedure was good (R = 0.80) in patients with LIC falling within the range reported by Gandon. By contrast, a weak correlation was demonstrated (R = 0.52) in patients with high LIC (above 11.2 mg/g dw). With our sequences, the correlation was good both in the entire group of patients (R = 0.83) and in patients with LIC above 11.2 mg/g dw (R = 0.85). Our results suggest that the addition of a specific shorter-gradient echo sequence to a very simple, fast technique produces an accurate estimation of LIC in post-transfusional iron overload.

Hepatocyte iron loading capacity is associated with differentiation and repression of motility in the HepaRG cell line

High liver iron content is a risk factor for developing hepatocellular carcinoma (HCC). However, HCC cells are always iron-poor. Therefore, an association between hepatocyte iron storage capacity and differentiation is suggested. To characterize biological processes involved in iron loading capacity, we used a cDNA microarray to study the differentiation of the human HepaRG cell line, from undifferentiated proliferative cells to hepatocyte differentiated cells. We were able to identify genes modulated along HepaRG differentiation, leading us to propose new genes not previously associated with HCC. Moreover, using Gene Ontology annotations, we demonstrated that HepaRG hepatocyte iron loading capacity occurred both with the repression of genes involved in cell motility, signal transduction, and biosynthesis and with the appearance of genes linked to lipid metabolism and immune response. These results provide new insights in the understanding of the relationship between iron and hepatocyte differentiation during iron-related hepatic diseases.

Liver iron concentration evaluated by two magnetic methods: Magnetic resonance imaging and magnetic susceptometry

Quantification of liver iron concentration (LIC) is crucial in the management of patients suffering from certain pathologies that can produce iron overload, such as Cooley's anemia and hemochromatosis. All of these patients must control the level of iron deposits in their organs to avoid the toxicity of high LIC, which is potentially lethal. This paper describes experimental protocols for LIC measurement using two magnetic techniques: magnetic resonance imaging (MRI) and biomagnetic liver susceptometry (BLS). MRI proton transverse relaxation rate (R2) and image intensity, evaluated pixel by pixel, were used as indicators of iron load in the tissue. LIC measurement by BLS was performed using an AC superconducting susceptometer system. A group of 23 patients with a large range of iron overload (0.9 to 34.5 mgFe/g(dry tissue)) was evaluated with both techniques (MRI x BLS). A significant linear correlation (r = 0.89-0.95) was found between the LIC by MRI and by BLS. These results show the feasibility of using two noninvasive methodologies to evaluate liver iron store in a large concentration range. Both methodologies represent an equivalent precision.

Reproducibility and Sensitivity in Assessing Liver Iron Content (LIC) in Highly Transfused Patients by Routine Magnetic Resonance Imaging(MRI).

Intoduction Due to the poor signal to noise ratio of MRI, there are no strong data pertaining to the validity of MRI techniques in the assessment of LIC in high post-transfusionnal iron overload. Moreover, reproducibility (from center to center) of this technique is not clearly established. We have compared chemically determined high LIC and two different MRI protocols of measuring LIC in patients transfused monthly: the first, to evaluate the exact reproducibility of a simple and widely recognized procedure used in hepatic diseases and the second, to improve sensitivity at higher LIC.Patients and MethodsMRI method: MRI studies were performed using a 1.5 T system (Philips intera, The Netherlands). Transverse images were recorded, visualizing the right liver lobe (L) and posterior vertebral muscles (M) in the same slice. The signal intensity (SI) for hepatic tissue and the posterior vertebral muscle were determined. The L/M SI ratio (SIR) was then calculated. First procedure: Four magnetic resonance gradient echo sequences (T2++/t2+/PD/T1), exactly as previously published in hepatic disease studies (Gandon,Y Lancet 2004), were performed and the same software for LIC determination was used in order to evaluate the center to center reproducibility. Second procedure: EGT0.5 and EGT0 sequences using shorter echo times with the aim of improving accuracy at higher levels of iron overload were performed.Patients and liver biopsy: 21 liver biopsies were performed, after written consent, in 16 patients (MDS=4, b thalassemia=12). In the case of repeated biopsy, an interval of a minimum 12 months was respected between the two biopsies. Results: 21 biopsies, each more than one mg, in 16 adult patients (8 males, 8 females) with a median age of 41 (range 23–73 ). LIC ranged from 2.6 to 54mg/g liver dry weight (mg/gdw), (median 12.6 mg/gdw). Hepatic fibrosis and cirrhosis were histologically found in 4/21 and 4/21 cases, respectively. First procedure: In biopsies with LIC below 375 micromol/g dw (n= 14), a strong correlation was demonstrated between LIC and L/M SIR (r = 0.86), for the best sequence, similar to that previously reported (r =0.91). For the entire group, including cases with LIC > 375 micromole/g dw (n= 21), the correlation was not as good (r=0.72) as the previous group using the same sequence. Second procedure: EGT0.5: 4 cases having low LIC and SIR >1.5 were excluded. Among the 17 remaining cases, linear agreement between LIC and L/M SIR was excellent (r=0.89). EGT0: 16 cases had SIR<1.5 and were analyzed with similar results (r=0.83).Conclusion In mild post-transfusionnal iron overload, our results show: measurement by routine MRI produces an accurate and reproducible estimation of LIC from center to center, with the same level of correlation as previously reported, independent of the machine used. At high liver iron concentration (LIC> 375 micromol/gdw) (33% of our cases), some simple and rapid specific sequences can also provide an accurate measurement of LIC. These results suggest that, whatever the level of iron overload encountered, the LIC determination by simple and rapid routine MRI is more a question of the standardization than of the sensitivity of the method.

Cu,Zn-superoxide dismutase is lower and copper chaperone CCS is higher in erythrocytes of copper-deficient rats and mice.

Discovery of a sensitive blood biochemical marker of copper status would be valuable for assessing marginal copper intakes. Rodent models were used to investigate whether erythrocyte concentrations of copper,zinc-superoxide dismutase (SOD), and the copper metallochaperone for SOD (CCS) were sensitive to dietary copper changes. Several models of copper deficiency were studied in postweanling male Holtzman rats, male Swiss Webster mice offspring, and both rat and mouse dams. Treatment resulted in variable but significantly altered copper status as evaluated by the presence of anemia, and lower liver copper and higher liver iron concentrations in copper-deficient compared with copper-adequate animals. Associated with this copper deficiency were consistent reductions in immunoreactive SOD and robust enhancements in CCS. In most cases, the ratio of CCS:SOD was several-fold higher in red blood cell extracts from copper-deficient compared with copper-adequate rodents. Determination of red cell CCS:SOD may be useful for assessing copper status of humans.

Liver disease in transfusion dependent thalassaemia major

Aims: To study the prevalence and severity of liver diseases of transfusion dependent thalassaemia major patients, and correlate the histological and biochemical changes of iron overload in liver with the...

The hepatologist in the haematologists' camp.

The hepatologist in the haematologists' camp.

Elements in autopsy liver tissue samples from Greenlandic inuit and Danes. II. Iron measured by X-ray Fluorescence Spectrometry

The purpose of this study was to measure the content of iron (Fe) in liver tissue samples from urbanized Greenlandic Inuit using X-ray fluorescence spectrometry, and compare the results with those obtained in liver tissue samples from urbanized Danes. Normal liver tissue samples were obtained at autopsy from 50 Greenlandic Inuit (27 men, 23 women) with a median age of 61 years (range 23-83) and from 72 Danes (42 men, 30 women) with a median age of 62 years (range 15-87). In the entire series, there was no significant difference between liver iron in Inuit compared with Danes. Likewise, there was no significant gender difference concerning liver iron content, either in Inuit or in Danes. The median iron content (with 5-95 percentile) in Inuit was 17.23 mmol/kg dry liver (5.80-91.80) and in Danes 16.51 mmol/kg dry liver (7.83-39.05). However, when stratified according to age, a trend was revealed showing that Inuit men and women < or = 50 years had a lower liver iron content than Danes (p = 0.05 and p = 0.08) whereas Inuit men and women > 50 years had a higher liver iron content than Danes (p = 0.18 and p = 0.02). There was a significant correlation between liver iron content and age in both Inuit men (rs = 0.49, p = 0.01) and in women (rs = 0.64, p = 0.003), but not in Danes. In Inuit, the median hepatic iron index (liver iron content divided by age) was 0.33 in men and 0.32 in women. The median estimated iron content in the whole liver was 6.54 mmol (365 mg) in Inuit men and 5.41 mmol (302 mg) in Inuit women (p = 0.6). There was no correlation between hepatic iron index and age. In Danes, the median hepatic iron index was 0.46 in men and 0.29 in women (p = 0.01). There was a significant inverse correlation between hepatic iron index and age in the two genders and in the entire series (rs = -0.71, p = 0.0001). The results indicate that young and middle-aged urbanized Inuit have slightly smaller iron stores than urbanized Danes, whereas elderly Inuit have higher iron stores than Danes. In Danes, iron stores plateau at 30 to 40 years of age in men and some years after the menopause in women. In Inuit, iron stores continue to increase in old age, probably to due a lifelong dietary intake of haem iron.