Pensri Pootrakul

Clinical manifestation of beta-thalassemia/hemoglobin E disease.

Purpose To review the clinical manifestation and changes in hematologic parameters of patients with &bgr;-thalassemia/hemoglobin (Hb) E. Materials and Methods Retrospective analysis of the clinical manifestation of 378 patients with &bgr;-thalassemia/Hb E attending the hematology clinic at Siriraj Hospital between 1957 and 1982. Results A wide spectrum of clinical phenotypes has been observed. Most patients show clinical symptoms by 10 years of age. The majority of patients survive with or without occasional transfusion. Splenectomy was performed in 26.5% of patients. Patients come to the hospital because of anemia, fever, abdominal mass, and jaundice. Gastrointestinal tract disturbances are the most common presenting symptoms (34.6%), especially abdominal pain (10%) and cholecystitis (5.1%). Respiratory tract infections were found in 21.8% of patients and cardiovascular complications, including congestive heart failure, occurred in 11.9%. Other less common symptoms and complications included bone pain, chronic leg ulcers, paraplegia and hypertension-associated convulsions, and cerebral hemorrhage after multiple blood transfusion. Patients usually die between 20 to 40 years of age (67%), mainly from congestive heart failure and septicemia. Septicemia was often caused by Gram-negative bacteria. Conclusion These clinical features observed in patients with &bgr;-thalassemia/Hb E are probably the results of chronic anemia and iron overload. The study of the life history and clinical courses of patients with &bgr;-thalassemia/Hb E should provide important information for the better management of these patients.

Clinical trial of deferiprone iron chelation therapy in β‐thalassaemia/haemoglobin E patients in Thailand

Summary. Nine patients with either β‐thalassaemia/haemoglobin E (7) or homozygous β‐thalassaemia (2) not requiring regular transfusions were treated with the oral iron chelator, deferiprone 25–50 mg/kg/d for between 17 and 86 weeks (mean 49 weeks). There were significant decreases in serum ferritin (initial mean ± standard deviation 2168 ± 1142, final 418 ± 247 µg/l; t‐test for paired samples, P = 0·005), hepatic iron (initial 20·3 ± 6·26, final 11·7 ± 4·83 mg/g/dry weight; P = < 0·02), red cell membrane iron (initial 76·2 ± 3·64, final 7·2 ± 0·56 mmol/mg protein; P = < 0·0005) and serum non‐transferrin bound iron (initial 9·0 ± 0·56, final 5·9 ± 0·89 µmol/l; P = < 0·0005). There was also a significant rise in serum erythropoietin (initial 240 ± 195·1, final 433·2 ± 269·2 U/l; P = 0·034). The haemoglobin level rose in three patients and transfusion requirements were reduced substantially in four patients. Serum thiobarbituric acid reactive substance (TBARS) also fell in six of eight patients. Patients generally improved clinically, with weight gain observed. Side‐effects were mild and included gastrointestinal symptoms (6) and arthralgia (1), not requiring withdrawal of the drug. One patient died at 17 weeks of therapy as a result of an intercurrent infection. His neutrophil count was normal. We conclude that deferiprone is an effective, well‐tolerated iron chelator for patients with thalassaemia intermedia. Further studies are needed to determine the optimum dose and length of treatment needed to reduce iron burden to a safe level in these patients.

The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai β-thalassemia/hemoglobin E patients and Australian β-thalassemia patients

Mössbauer spectra of 12 beta-thalassemia/hemoglobin E spleen samples from Thai patients who had not received multiple blood transfusions and chelation therapy and seven beta-thalassemia spleen samples from Australian patients who had received multiple blood transfusions and chelation therapy were recorded with sample temperatures of 78 K. Each spectrum was found to consist of a superposition of a relatively intense central doublet characteristic of high-spin Fe(III), a low intensity sextet of peaks due to magnetic hyperfine-field splitting, and occasionally a doublet that could be attributed to heme iron. A significant (P=0.01) difference (Kolmogorov-Smirnov statistic of 0.71) between the distributions of sextet signal intensity as a fraction (Fs) of the total non-heme iron Mössbauer spectral signal for the two groups of patients was detected. The distribution of Fs for the Thai beta-thalassemia/hemoglobin E spleens had a mean value of 0.128 (S.D. 0.035) while that for the Australian beta-thalassemia spleens had a mean of 0.27 (S.D. 0.12). No significant difference between the distributions of non-heme iron concentrations in the tissues for the two groups of patients was detected by atomic absorption spectrometry. This study shows that the Australian beta-thalassemia patients had a higher fraction of their non-heme spleen iron in a goethite-like form than the Thai beta-thalassemia/Hb E patients.

Renal Function in Adult Beta-Thalassemia/Hb E Disease

β-Thalassemia hemoglobin E (β-thal/Hb E) is the commonest form of hemoglobinopathy in Thailand. Shortened red cell life span, rapid iron turnover and tissue deposition of excess iron are major factors responsible for functional and physiological abnormalities found in various forms of thalassemia. Increased deposition of iron had been found in renal parenchyma of thalassemic patients, but no systematic study of the effect of the deposits on renal functions has been available. The purpose of this study is to describe the functional abnormalities of the kidney in patients with β-thal/Hb E and provide evidence that increased oxidative stress might be one of the factors responsible for the damage. Urine and serum samples from 95 patients with β-thal/Hb E were studied comparing with 27 age-matched healthy controls. No difference in the creatinine clearance was observed. β-thal/Hb E patients excreted significantly more urinary protein (0.8 ± 0.5 vs. 0.3±0.1 g/day, p < 0.001). Aminoaciduria was found in 16% of the patients. Analysis of urinary protein by SDS-PAGE electrophoresis and silver staining revealed abnormal pattern of protein with increased small molecular weight (<45 kD) bands. Morning urine analysis showed significant lower urine osmolality (578.3 ± 164.6 vs. 762.4 ± 169.9 mosm/kg, p < 0.001) in patients. Patients excreted more NAG (N-acetyl beta-D-glucosaminidase, 26.3 ± 41.3 vs. 8.4 ± 3.9 U/g Cr, p < 0.0001) and β2-microglobulin, 124.3 ± 167 vs. 71 ± 65.5 µg/g Cr, p = 0.001. Plasma and urine MDA (malonyldialdehyde) levels were both raised (p < 0.0001). Nine patients were selected for renal acidification study. All were found to be normal, but showed poor response to DDAVP challenge (urine osmolality 533 ± 71). This is the first report of renal tubular defects found associated with β-thal/Hb E disease. The mechanism leading to the damage is not known but it might be related to increased oxidative stress secondary to tissue deposition of iron, as indicated by the raised levels of serum and urine MDA. It is not known whether these functional defects would have any long-term effects on the patients. Further studies are warranted and means of prevention of these defects should urgently be sought.

Serum Ferritin Levels in Thalassemias and the Effect of Splenectomy

Iron overload is a constant and the more important complication in thalassemia. Serum ferritin concentration accurately reflects body iron stores. A total of 245 thalassemic patients aged 12-55 years were examined, 71 having Hb H disease and 174 beta-thalassemia/Hb E disease. The patients received minimal or no blood transfusions. 73 patients with beta-thalassemia/Hb E were studied 1-28 years after splenectomy. The serum ferritin levels in both Hb H and beta-thalassemia/Hb E patients were higher than normal. They were higher in beta-thalassemia/Hb E than Hb H disease. Most striking was the significantly higher serum ferritin levels in splenectomized patients with beta-thalassemia/Hb E disease than in the nonsplenectomized ones. The observation is compatible with previous observations that splenectomy in thalassemia is associated with increased iron deposition and increased transferrin iron saturation. The further increase in iron overload after splenectomy in thalassemia should be borne in considering removal of this organ.

Homozygous haemoglobin constant spring: A need for revision of concept

SummaryTwenty-two patients with mild haemolytic anaemia and haemoglobin (Hb) Constant Spring (CS) of around 6% were studied because they were suspected of having homozygous Hb CS. Family studies revealed Hb CS trait in both parents of eight patients, supporting that they were homozygous for Hb CS. The other patients were included because they had clinical and haematological features similar to the diagnosed cases of homozygous Hb CS. Heterozygosity and homozygosity for Hb CS are clearly distinguishable in that the former is asymptomatic but the latter is associated with overt haemolytic anaemia, and the levels of Hb CS in the two conditions of less than 1% and around 6%, respectively, do not overlap. The findings in homozygous Hb CS contracdict prediction. There are four a-structural genes per normal human diploid genome. Hb CS trait is believed to be almost equivalent to a-thalassaemia 2 or a loss of one a-gene because HB CS, an a-variant, is barely or not detectable. Homozygosity for Hb CS has thus been predicted to be equivalent to a-thalassaemia 1 or a loss of two genes. The latter is asymptomatic and associated with microcytic-hypochromic red cells. However, Hb CS homozygosity presents with mild overt haemolytic anaemia and normal sized red cells. Pathogenesis associated with Hb CS inheritance is more complex than originally believed. There is a possibility that the unstable aCS mRNAs precipitate and aggregate leading to pathology of red cells and to the basophilic stippling appearance, so striking in this syndrome.

Association between bone mineral density and erythropoiesis in Thai children and adolescents with thalassemia syndromes

Increased marrow erythropoiesis in patients with thalassemia syndromes results in the expansion of bone marrow cavities and consequently decreases bone tissues, leading to osteoporosis. Whether the soluble transferrin receptor (sTfR), a marker of erythropoietic activity, correlates with the bone mineral density (BMD) in thalassemic patients has not previously been addressed. Forty-six children and adolescents with thalassemia syndromes, who were either not transfused or suboptimally transfused, were studied. BMD was determined by dual-energy X-ray absorptiometry. Blood samples were obtained in order to determine sTfR and hemoglobin. The patients were categorized into four groups: 1, β-thalassemia/hemoglobin E (β-thal/E) with transfusion-dependency (TD) (n = 18); 2, β-thal/E with transfusion-independency (TI) (n = 15); 3, β-thalassemia major (β-major) (n = 6); 4, hemoglobin H (HbH) (n = 7). All patients had normal serum free thyroxine (FT4) and thyroid-stimulating hormone (TSH), and intact parathyroid hormone (PTH), serum calcium (Ca), phosphate (P), and 25-OH-vitamin D levels. The BMD of patients in the β-major and β-thal/E with TD groups were not significantly different. In comparison with the β-major and β-thal/E with TD groups, the β-thal/E with TI and HbH groups had significantly higher BMD of the total body (TB), femoral neck (FN), and lumbar spine (LS), as well as higher levels of hemoglobin. In contrast, the sTfR levels of the β-major, β-thal/E with TI, and HbH groups were significantly lower than those of the β-thal/E with TD group. The BMD of TB, FN, and LS was negatively correlated with the sTfR level, but positively correlated with the hemoglobin level. In conclusion, increased marrow erythropoiesis is one of the major determinants of reduced bone mass in thalassemic patients with either no transfusion or suboptimal transfusion.

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

SummaryThe cores of ferritins isolated from different organs of human subjects withβ-thalassemia/hemoglobin E (β-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.β-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 ofβ-thal/HbE subjects were found to have larger, more crystalline cores than those from theβ-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.

Hemin: A Possible Cause of Oxidative Stress in Blood Circulation of β-Thalassemia/Hemoglobin E Disease

A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of g -thalassemia/hemoglobin E disease ( g -thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 w M in serum of g -thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of g -thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of g -thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum hemin, r =0.778 (p <0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p =0.259) and 0.519 (p =0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of g -thal/Hb E.

The effects of vitamin E on platelet activity in β‐thalassaemia patients

Summary.  A double‐blind, crossover, placebo‐controlled study of the effect of vitamin E on platelet functions was performed on nine splenectomized and 16 non‐splenectomized β‐thalassaemia/haemoglobin E (β‐thalassaemia/HbE) patients. The patients were supplemented with a daily dose of vitamin E (525 IU) for 3 months. The functions of platelets were assessed by adenosine diphosphate (ADP)‐induced platelet aggregation and adenosine triphosphate release. Plasma α‐tocopherol, plasma thiobarbituric reactive substances (TBARs) and serum ferritin levels represented patients’ antioxidant status, lipid peroxidation status and iron status respectively. Before experimentation, all patients had low plasma α‐tocopherol levels. The splenectomized patients showed severe iron overload iron, had higher plasma TBAR levels and their platelets were more reactive to ADP than those of non‐splenectomized patients. Three months of daily vitamin E supplementation resulted in a significant increase in plasma α‐tocopherol levels and reduction in plasma TBAR levels in all patients. Serum ferritin levels of the patients were not altered; however, vitamin E reduced the platelet reactivity of the splenectomized patients towards normal levels. The influence of vitamin E on platelet reactivity may result in delaying hypoxaemia and pulmonary occlusion that commonly occurs in splenectomized β‐thalassaemia/HbE patients.