Post-transfusional iron overload in the haemoglobinopathies

In this report, we review the recent advances in evaluation and treatment of transfusional iron overload (IO). Results of the French thalassaemia registry are described. According to the disease, thalassaemia major or sickle cell anaemia, mechanisms and toxicity of iron overload, knowledge about IO long-term outcome and chelation treatment results, respective value of IO markers, differ. The recent tools evaluating organ specific IO and the diversification of iron chelator agents make possible to individualize chelation therapy in clinical practice. The severity of IO and the level of transfusional iron intake, the preferential localization of IO (heart/liver) as well as the tolerance and adherence profiles of the patient can now be taken into account. Introduction of cardiac magnetic resonance imaging for the quantification of myocardial iron and use of oral chelators have already been reported as decreasing the cardiac mortality rate related to IO in thalassaemia major patients. Long-term observation of patients under oral chelators will show if morbidity is also improving via a more continuous control of toxic iron and/or a better accessibility to cellular iron pools.     

广西地区104例泰国缺失型alpha-地中海贫血1 的研究

目的：对广西地区的泰国缺失型琢-地中海贫血1 的血液学、基因型、临床表现及民族分布进行分析。方法：对门诊病人进行 血常规、血红蛋白电泳及- 地中海贫血基因分析,收集已确诊为泰国缺失型琢- 地中海贫血1 患者的检测数据及临床资料,并用 SPSS 统计软件对以上数据进行统计分析。结果：共检出104 例泰国缺失型alpha- 地中海贫血1,其中71 例基因型为--THAI /alpha-alpha,17 例 基因型为--THAI/-alpha3.7,14 例基因型为--THAI/alpha CS alpha ,2 例基因型为--THAI/alpha QS alpha。统计学分析：--THAI/alpha alpha杂合子和--SEA/alpha-alpha杂合子的血常规 结果比较没有统计学意义,P>0.05;泰国型Hb H病(--THAI/-alpha3.7、--THAI/alpha-CSalpha、--THAI/alpha-CSalpha)和东南亚型Hb H病（--SEA/-alpha-alpha3.7、--SEA/alpha-CS-alpha)的血常 规结果比较有统计学意义,P<0.01;民族分布上,有67 例为壮族,34 例为汉族,其他民族3 例。结论：泰国缺失型- 地中海贫血1 在 广西有一定的发生率,在壮族人群多见;和东南亚缺失型Hb H病相比,泰国缺失型Hb H病出现临床症状的时间更早、贫血更严 重。     

Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera

Despite its importance in iron-overload diseases, little is known about the composition of plasma non-transferrin-bound iron (NTBI). Using 30-kDa ultrafiltration, plasma from thalassemic patients consisted of both filterable and non-filterable NTBI, the filterable fraction representing less than 10% NTBI. Low filterability could result from protein binding or NTBI species exceeding 30 kDa. The properties of iron citrate and its interaction with albumin were therefore investigated, as these represent likely NTBI species. Iron permeated 5- or 12-kDa ultrafiltration units completely when complexes were freshly prepared and citrate exceeded iron by tenfold, whereas with 30-kDa ultrafiltration units, permeation approached 100% at all molar ratios. A g = 4.3 electron paramagnetic resonance signal, characteristic of mononuclear iron, was detectable only with iron-to-citrate ratios above 1:100. The ability of both desferrioxamine and 1,2-dimethyl-3-hydroxypyridin-4-one to chelate iron in iron citrate complexes also increased with increasing ratios of citrate to iron. Incremental molar excesses of citrate thus favour the progressive appearance of chelatable lower molecular weight iron oligomers, dimers and ultimately monomers. Filtration of iron citrate in the presence of albumin showed substantial binding to albumin across a wide range of iron-to-citrate ratios and also increased accessibility of iron to chelators, reflecting a shift towards smaller oligomeric species. However, in vitro experiments using immunodepletion or absorption of albumin to Cibacron blue–Sepharose indicate that iron is only loosely bound in iron citrate–albumin complexes and that NTBI is unlikely to be albumin-bound to any significant extent in thalassemic sera.     

Organ-specific crystalline structures of ferritin cores inβ-thalassemia/hemoglobin E

Summary The cores of ferritins isolated from different organs of human subjects with-thalassemia/hemoglobin E (-thal/HbE) disease have different size distributions and crystallinities depending on the source organ. These patients have not been treated by hypertransfusion regimen or iron chelation therapy.-Thal/HbE spleens and livers yield ferritin cores which are less crystalline than those isolated from normal spleens and livers, reflecting the more rapid deposition of iron in the diseased state. Ferritins isolated from the hearts and pancreases of-thal/HbE subjects were found to have larger, more crystalline cores than those from the-thal/HbE livers and spleens, possibly as a consequence of the role of the heart and pancreas as long-term iron deposition sites in this iron overload pathology.     

Lipid fluidity at different regions in LDL and HDL of beta-thalassemia/Hb E patients

Atherosclerosis-related vascular complications in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients may result from iron induced oxidation of lipoproteins. To identify the specific site of oxidative damage, changes in lipid fluidity at different regions in LDL and HDL particle were investigated using two fluorescence probes and two ESR spin probes. The magnitude of increased lipid fluidity in thalassemic lipoproteins was dependent on the location of the probes. In hydrophobic region, the rotational correlation times for 16-doxyl stearic acid and DPH anisotropy were markedly changed in LDL and HDL of the patients. In the surface region, there was only a slight change in the order parameter (S) for 5-doxyl stearic acid and TMA-DPH anisotropy. Lipid fluidity at the core of LDL and HDL showed good correlation with oxidative stress markers, the ratio of CL/CO, and the level of alpha-tocopherol, suggesting that hydrophobic region of thalassemic lipoprotein was a target site for oxidative damage.     

Absence of JAK2 V617F mutation in thalassemia intermedia patients

JAK2 is a cytoplasmic tyrosine kinase that has a vital role in signal transduction from several hemopoietic growth factor receptors. The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia Vera, essential thrombocythemia, and idiopathic Myelofibrosis but has not been previously described in Thalassemia patients. We studied 36 Lebanese patients diagnosed with thalassemia intermedia and assessed the presence or absence of the JAK2 V617F mutation using JAK2 activating mutation assay (In VivoScribe Technologies) and Polymerase Chain Reaction (PCR). None of the thalassemia intermedia patients were positive for this mutation. To our knowledge, this study is the first to determine the status of JAK2 V617F mutation in thalassemia intermedia patients and expands the international published literature on JAK2. The latter’s V617F mutation does not seem to play a role in this hematologically important clinical entity.     

A flow cytometry approach for quantitative analysis of cellular phosphatidylserine distribution and shedding

Phospholipids are asymmetrically distributed across the membrane of all cells, including red blood cells (RBCs). Phosphatidylserine (PS) is mainly localized in the cytoplasmic membrane leaflet, but during RBC ageing it flip-flops to the external leaflet—a process that is increased in certain pathological conditions (e.g., β-thalassemia). PS externalization in RBCs mediates their phagocytosis by macrophages and removal from the circulation. PS is usually measured by flow cytometry and is reported as the percentage of cells with external PS. In the current study, we developed a novel two-step flow cytometry procedure to quantitatively measure not only the external PS but also the intracellular and shed PS. In this method, PS is first bound to fluorescent annexin V, and then the residual nonbound annexin is quantified by binding to PS exposed on apoptotic cells. Using this method, we measured 1.1 ± 0.2 and 0.12 ± 0.04 μmol inner and external PS, respectively, per 10⁷ normal RBCs. Thalassemic RBCs demonstrated increased PS externalization (1.7-fold) and shedding (11-fold) that was accompanied by lower intracellular PS (31%). These results suggest that quantitative flow cytometry of PS could have a diagnostic value in evaluating the pathology of RBCs in hemolytic anemias associated with increased PS externalization and shortening of the RBC life span.     

Yunnanese(~Aγδβ)~0-地贫缺失桥片段的克隆及缺失连接区的序列测定

为研究导致Ｙｕｎｎａｎｅｓｅ（Ａγδβ）０－地贫缺失事件的分子机制,并从３′并入序列中搜寻增强子类序列,使用ＥＭＢＬ３为载体构建了一例缺失杂合子的基因组文库,筛选到来源于异常染色体１１并包含Ｇγ珠蛋白基因区６．７ｋｂ序列以及１１．５ｋｂ３′并入序列（即缺失桥片段）的克隆．此１１．５ｋｂ序列在正常染色体中位于β珠蛋白基因约下游６６～７８ｋｂ区域．详细分析了这一区域的限制性内切酶图谱．分析了围绕缺失连接区的ＤＮＡ序列,精确定位缺失的５′端点发生在Ａγ珠蛋白基因上游－１１６～－１１７碱基之间．确定缺失的３′端点处于一个Ｌ１序列内,位于β珠蛋白基因下游～６６ｋｂ,距离Ｃｈｉｎｅｓｅ（Ａγδβ）０－地贫缺失３′端点上游～１２．２ｋｂ的一个ＥｃｏＲⅠ位点上游４１３ｂｐ．围绕５′和３′端点的序列之间无明显同源性,说明这一缺失代表了体内的非同源重组事件．这一重组事件可能由Ｌ１序列介导．缺失３′端点下游序列的克隆分离也为进一步从中搜寻加强子类序列奠定了基础     

Inborn defects in the antioxidant systems of human red blood cells

Red blood cells (RBCs) contain large amounts of iron and operate in highly oxygenated tissues. As a result, these cells encounter a continuous oxidative stress. Protective mechanisms against oxidation include prevention of formation of reactive oxygen species (ROS), scavenging of various forms of ROS, and repair of oxidized cellular contents. In general, a partial defect in any of these systems can harm RBCs and promote senescence, but is without chronic hemolytic complaints. In this review we summarize the often rare inborn defects that interfere with the various protective mechanisms present in RBCs. NADPH is the main source of reduction equivalents in RBCs, used by most of the protective systems. When NADPH becomes limiting, red cells are prone to being damaged. In many of the severe RBC enzyme deficiencies, a lack of protective enzyme activity is frustrating erythropoiesis or is not restricted to RBCs. Common hereditary RBC disorders, such as thalassemia, sickle-cell trait, and unstable hemoglobins, give rise to increased oxidative stress caused by free heme and iron generated from hemoglobin. The beneficial effect of thalassemia minor, sickle-cell trait, and glucose-6-phosphate dehydrogenase deficiency on survival of malaria infection may well be due to the shared feature of enhanced oxidative stress. This may inhibit parasite growth, enhance uptake of infected RBCs by spleen macrophages, and/or cause less cytoadherence of the infected cells to capillary endothelium.     

Primaquine-induced superoxde production by β-thalassemic red blood cells

Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, β-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of β-thalassemic cells to oxidative stress.