Maciej Garbowski

Consequences and management of iron overload in sickle cell disease

The aims of this review are to highlight the mechanisms and consequences of iron distribution that are most relevant to transfused sickle cell disease (SCD) patients and to address the particular challenges in the monitoring and treatment of iron overload. In contrast to many inherited anemias, in SCD, iron overload does not occur without blood transfusion. The rate of iron loading in SCD depends on the blood transfusion regime: with simple hypertransfusion regimes, rates approximate to thalassemia major, but iron loading can be minimal with automated erythrocyte apheresis. The consequences of transfusional iron overload largely reflect the distribution of storage iron. In SCD, a lower proportion of transfused iron distributes extrahepatically and occurs later than in thalassemia major, so complications of iron overload to the heart and endocrine system are less common. We discuss the mechanisms by which these differences may be mediated. Treatment with iron chelation and monitoring of transfusional iron overload in SCD aim principally at controlling liver iron, thereby reducing the risk of cirrhosis and hepatocellular carcinoma. Monitoring of liver iron concentration pretreatment and in response to chelation can be estimated using serum ferritin, but noninvasive measurement of liver iron concentration using validated and widely available MRI techniques reduces the risk of under- or overtreatment. The optimal use of chelation regimes to achieve these goals is described.

The Pathophysiology of Transfusional Iron Overload

The pathophysiologic consequences of transfusional iron overload (TIO) as well as the benefits of iron chelation therapy are best described in thalassemia major, although TIO is increasingly seen in other clinical settings. These consequences broadly reflect the levels and distribution of excess storage iron in the heart, endocrine tissues, and liver. TIO also increases the risk of infection, due to increased availability of labile iron to microorganisms. The authors suggest that extrahepatic iron distribution, and hence toxicity, is influenced by balance between generation of nontransferrin-bound iron from red cell catabolism and the utilization of transferrin iron by the erythron.

Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients

In transfusional iron overload, extra‐hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non‐transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond‐Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin‐iron utilization (DBA).

Right ventricular volumes and function in thalassemia major patients in the absence of myocardial iron overload

AimWe aimed to define reference ranges for right ventricular (RV) volumes, ejection fraction (EF) in thalassemia major patients (TM) without myocardial iron overload.Methods and resultsRV volumes, EF and mass were measured in 80 TM patients who had no myocardial iron overload (myocardial T2* > 20 ms by cardiovascular magnetic resonance). All patients were receiving deferoxamine chelation and none had evidence of pulmonary hypertension or other cardiovascular comorbidity. Forty age and sex matched healthy non-anemic volunteers acted as controls. The mean RV EF was higher in TM patients than controls (males 66.2 ± 4.1% vs 61.6 ± 6%, p = 0.0009; females 66.3 ± 5.1% vs 62.6 ± 6.4%, p = 0.017), which yielded a raised lower threshold of normality for RV EF in TM patients (males 58.0% vs 50.0% and females 56.4% vs 50.1%). RV end-diastolic volume index was higher in male TM patients (mean 98.1 ± 17.3 mL vs 88.4 ± 11.2 mL/m2, p = 0.027), with a higher upper limit (132 vs 110 mL/m2) but this difference was of borderline significance for females (mean 86.5 ± 13.6 mL vs 80.3 ± 12.8 mL/m2, p = 0.09, with upper limit of 113 vs 105 mL/m2). The cardiac index was raised in TM patients (males 4.8 ± 1.0 L/min vs 3.4 ± 0.7 L/min, p < 0.0001; females 4.5 ± 0.8 L/min vs 3.2 ± 0.8 L/min, p < 0.0001). No differences in RV mass index were identified.ConclusionThe normal ranges for functional RV parameters in TM patients with no evidence of myocardial iron overload differ from healthy non-anemic controls. The new reference RV ranges are important for determining the functional effects of myocardial iron overload in TM patients.

Synergistic intracellular iron chelation combinations: mechanisms and conditions for optimizing iron mobilization

Iron chelators are increasingly combined clinically but the optimal conditions for cellular iron mobilization and mechanisms of interaction are unclear. Speciation plots for iron(III) binding of paired combinations of the licensed iron chelators desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) suggest conditions under which chelators can combine as ‘shuttle’ and ‘sink’ molecules but this approach does not consider their relative access and interaction with cellular iron pools. To address this issue, a sensitive ferrozine‐based detection system for intracellular iron removal from the human hepatocyte cell line (HuH‐7) was developed. Antagonism, synergism or additivity with paired chelator combinations was distinguished using mathematical isobologram analysis over clinically relevant chelator concentrations. All combinations showed synergistic iron mobilization at 8 h with clinically achievable concentrations of sink and shuttle chelators. Greatest synergism was achieved by combining DFP with DFX, where about 60% of mobilized iron was attributable to synergistic interaction. These findings predict that the DFX dose required for a half‐maximum effect can be reduced by 3·8‐fold when only 1 μmol/l DFP is added. Mechanisms for the synergy are suggested by consideration of the iron‐chelate speciation plots together with the size, charge and lipid solubilities for each chelator. Hydroxypyridinones with low lipid solubilities but otherwise similar properties to DFP were used to interrogate the mechanistic interactions of chelator pairs. These studies confirm that synergistic cellular iron mobilization requires one chelator to have the physicochemical properties to enter cells, chelate intracellular iron and subsequently donate iron to a second ‘sink’ chelator.

Eltrombopag: a powerful chelator of cellular or extracellular iron(III) alone or combined with a second chelator

Eltrombopag (ELT) is a thrombopoietin receptor agonist reported to decrease labile iron in leukemia cells. Here we examine the previously undescribed iron(III)-coordinating and cellular iron-mobilizing properties of ELT. We find a high binding constant for iron(III) (log β2=35). Clinically achievable concentrations (1 µM) progressively mobilized cellular iron from hepatocyte, cardiomyocyte, and pancreatic cell lines, rapidly decreasing intracellular reactive oxygen species (ROS) and also restoring insulin secretion in pancreatic cells. Decrements in cellular ferritin paralleled total cellular iron removal, particularly in hepatocytes. Iron mobilization from cardiomyocytes exceeded that obtained with deferiprone, desferrioxamine, or deferasirox at similar iron-binding equivalents. When combined with these chelators, ELT enhanced cellular iron mobilization more than additive (synergistic) with deferasirox. Iron-binding speciation plots are consistent with ELT donating iron to deferasirox at clinically relevant concentrations. ELT scavenges iron citrate species faster than deferasirox, but rapidly donates the chelated iron to deferasirox, consistent with a shuttling mechanism. Shuttling is also suggested by enhanced cellular iron mobilization by ELT when combined with the otherwise ineffective extracellular hydroxypyridinone chelator, CP40. We conclude that ELT is a powerful iron chelator that decreases cellular iron and further enhances iron mobilization when combined with clinically available chelators.

Clinical and methodological factors affecting non-transferrin-bound iron values using a novel fluorescent bead assay

Nontransferrin-bound iron (NTBI) is a heterogeneously speciated plasma iron, typically detectable when transferrin saturation (TfSat) exceeds 75%. Here, we examine factors affecting NTBI levels by a recently discovered direct chelator-based (CP851) fluorescent bead-linked flow-cytometric assay (bead-NTBI), compared with the established indirect nitrilotriacetate (NTA) assay in 122 iron-overloaded patients, including 64 on recent iron chelation therapy and 13 healthy volunteers. Both methods correlated (r = 0.57, P < 0.0001) but with low agreement, attributable to 2 major factors: (1) the NTA method, unlike the bead method, is highly dependent on TfSat, with NTBI under-estimation at low TfSat and over-estimation once Tf is saturated, (2) the bead method detects <3-fold higher values than the NTA assay in patients on recent deferiprone-containing chelation due to greater detection of chelate complexes but lower values for patients on deferasirox. The optimal timing of sample collection relative to chelation dosing requires further study. Patients with splenectomy, high-storage iron, and increased erythropoiesis had greater discrepancy between assays, consistent with differential access by both methods to the NTBI pools associated with these clinical variables. The bead-NTBI assay has advantages over the NTA assay, being less dependent on TfSat, hence of less tendency for false-negative or false-positive values at low and high TfSat, respectively.

Residual erythropoiesis protects against myocardial hemosiderosis in transfusion-dependent thalassemia by lowering labile plasma iron via transient generation of apotransferrin

Cardiosiderosis is a leading cause of mortality in transfusion-dependent thalassemias. Plasma non-transferrin-bound iron and its redox-active component, labile plasma iron, are key sources of iron loading in cardiosiderosis. Risk factors were identified in 73 patients with or without cardiosiderosis. Soluble transferrin receptor-1 levels were significantly lower in patients with cardiosiderosis (odds ratio 21). This risk increased when transfusion-iron loading rates exceeded the erythroid transferrin uptake rate (derived from soluble transferrin receptor-1) by >0.21 mg/kg/day (odds ratio 48). Labile plasma iron was >3-fold higher when this uptake rate threshold was exceeded, but non-transferrin-bound iron and transferrin saturation were comparable. The risk of cardiosiderosis was decreased in patients with low liver iron, ferritin and labile plasma iron, or high bilirubin, reticulocyte counts or hepcidin. We hypothesized that high erythroid transferrin uptake rate decreases cardiosiderosis through increased erythroid re-generation of apotransferrin. To test this, iron uptake and intracellular reactive oxygen species were examined in HL-1 cardiomyocytes under conditions modeling transferrin effects on non-transferrin-bound iron speciation with ferric citrate. Intracellular iron and reactive oxygen species increased with ferric citrate concentrations especially when iron-to-citrate ratios exceeded 1:100, i.e. conditions favoring kinetically labile monoferric rather than oligomer species. Excess iron-binding equivalents of apotransferrin inhibited iron uptake and decreased both intracellular reactive oxygen species and labile plasma iron under conditions favoring monoferric species. In conclusion, high transferrin iron utilization, relative to the transfusion-iron load rate, decreases the risk of cardiosiderosis. A putative mechanism is the transient re-generation of apotransferrin by an active erythron, rapidly binding labile plasma iron-detectable ferric monocitrate species.

Superior Vena Cava Occlusion by Cardiovascular Magnetic Resonance

A 30-year-old man was referred for cardiac and hepatic iron quantification by cardiovascular magnetic resonance. He was known to have β-thalassemia major and was heavily transfusion dependent, requiring intensive intravenous chelation therapy. However, after nonadherence to anticoagulation and parenteral therapy, his Portacath had thrombosed, necessitating removal. Cardiovascular magnetic …

Interaction of Transfusion and Iron Chelation in Thalassemias

The relationship between blood transfusion intensity, chelatable iron pools, and extrahepatic iron distribution is described in thalassemia. Risk factors for cardiosiderosis are discussed with particular reference to the balance of transfusional iron loading rate and transferrin-iron utilization rate as marked by plasma levels of soluble transferrin receptors. Low transfusion regimens increase residual erythropoiesis allowing for apotransferrin-dependent clearance of non-transferrin-bound iron species otherwise destined for myocardium. The impact of transfusion rates on chelation dosing required for iron balance is also shown.