Non-transferrin bound iron in Thalassemia: differential detection of redox active forms in children and older patients.

Non-transferrin bound iron (NTBI) is commonly detected in patients with systemic iron overload whose serum iron-binding capacity has been surpassed. It has been perceived as an indicator of iron overload, impending organ damage and a chelation target in poly-transfused thalassemia patients. However, NTBI is a heterogeneous entity comprising various iron complexes, including a significant redox-active and readily chelatable fraction, which we have designated as "labile plasma iron" (LPI). We found that LPI levels can be affected by plasma components such as citrate, uric acid, and albumin. However, the inclusion of a mild metal mobilizing agent in the LPI assay (designated here as "eLPI"), at concentrations that do not affect transferrin-bound iron, largely overcomes such effects and provides a measure of the full NTBI content. We analyzed three distinct groups of poly-transfused, iron overloaded thalassemia patients: non-chelated children (3-13 yrs, Gaza, Palestine), chelated adolescents-young adults (13-28 yrs, Israel), and chelated adults (27-61 yrs, Israel) for LPI and eLPI. The eLPI levels in all three groups were roughly commensurate (r(2) = 0.61-0.75) with deferrioxamine-detectable NTBI, i.e., DCI. In older chelated patients, eLPI levels approximated those of LPI, but in poly-transfused unchelated children eLPI was notably higher than LPI, a difference attributed to plasma properties affected by labile iron due to lack of chelation, possibly reflecting age-dependent attrition of plasma components. We propose that the two formats of NTBI measurement presented here are complementary and used together could provide more comprehensive information on the forms of NTBI in patients and their response to chelation.

Increased platelet adhesion under flow conditions is induced by both thalassemic platelets and red blood cells.

Thromboembolic complications are not uncommon in thalassemia. Previous studies suggest increased platelet aggregation and a potential role of pathological changes in the red blood cell (RBC) lipid membrane, induced by oxidative stress. In the present study, platelet adhesion and the effect of thalassemic RBC on platelet adhesion under flow conditions were evaluated, using the Cone and Plate (let) Analyzer(CPA). Twenty-two beta-thalassemia patients and 22 blood type-matched healthy controls were studied. An increased platelet adhesion (% surface coverage, SC), was observed in patients as compared to controls (p < 0.05). When platelet count and haematocrit were normalized by autologous reconstitution, a significant increase in platelet aggregation (average size, AS) was observed (p < 0.05). Increased platelet adhesion (SC and AS), was demonstrated in six patients with a history of thrombosis as compared to 16 patients without any history of thrombosis (p < or = 0.007) and in 17 splenectomized patients as compared to five non-splenectomized patients (p = 0.003). In reconstitution studies, thalassemic RBC mixed with normal platelet-rich plasma significantly increased platelet adhesion compared to normal RBC (SC p < 0.03, AS p < 0.02). Thalassemic platelets reconstituted with normal RBC, had increased aggregation (AS, p < 0.004) in comparison with normal platelets. The results indicate that increased platelet adhesion in beta-thalassemia is induced by both platelets and RBC. Increased platelet adhesion correlated with clinical thrombotic events and thus may suggest a mechanism of thrombosis in thalassemic patients. The potential application of the CPA in identifying thalassemic patients with high risk for thrombosis should be studied prospectively in a larger cohort of patients.

Fermented papaya preparation as redox regulator in blood cells of beta-thalassemic mice and patients.

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. Fermented papaya preparation (FPP) was tested for its antioxidant effects: the scavenging effect was determined spectrofluorometrically in a cell-free system using 2'-7'-dichlorofluorescin-diacetate (DCF). Both spontaneous and H(2)O(2)-induced DCF oxidations were decreased by FPP in a dose-dependent fashion. Using flow cytometry, it was shown that in vitro treatment of blood cells from beta-thalassemic patients with FPP increased the glutathione content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their reactive oxygen species, membrane lipid peroxidation and externalization of phosphatidylserine. These effects result in (a) reduced thalassemic RBC sensitivity to hemolysis and phagocytosis by macrophages; (b) improved PMN ability to generate oxidative burst - an intracellular mechanism of bacteriolysis, and (c) reduced platelet tendency to undergo activation, as reflected by fewer platelets carrying external phosphatidylserine. Oral administration of FPP to beta-thalassemic mice (50 mg/mouse/day for 3 months) and to patients (3 g x 3 times/day for 3 months), reduced all the above mentioned parameters of oxidative stress (p < 0.001 in mice and p < 0.005 in patients). These results suggest that FPP, as a potent antioxidant, might alleviate symptoms associated with oxidative stress in severe forms of thalassemia.

Phenylhydrazine as a partial model for beta-thalassaemia red blood cell hemodynamic properties.

beta-Thalassaemia is a congenital haemoglobinopathy, associated with red blood cells (RBC) anomalies, leading to impairment of their flow-affecting properties, namely, RBC deformability, self-aggregability, and adherence to endothelial cells (EC). Treatment of normal RBC with phenylhydrazine (PHZ) causes selective association of oxidized alpha-globin chains with the membrane skeleton, leading to reduced RBC deformability, characteristic of beta-thalassaemia. PHZ has thus been used to mimic phenotypes of beta-thalassaemia RBC. The present study was undertaken to further elucidate the suitability of PHZ-treated RBC as a model for beta-thalassemic RBC, by comparing the aggregability and adhesiveness of PHZ-treated RBC to those of RBC from thalassaemia intermedia (TI) patients, using image analysis of RBC under flow. In addition, the externalization of phosphatidylserine (PS), a mediator of RBC/EC interaction, was determined. It was found that PHZ caused enhanced RBC adhesiveness to extracellular matrix, similar to TI-RBC. Furthermore, in both conditions, the enhanced adhesiveness was mediated by PS translocated to the RBC surface. In contrast, PHZ treatment completely abolished RBC aggregability, while TI-RBC aggregability was slightly elevated. It is proposed that PHZ-treated RBC resemble beta-thalassaemia RBC in their deformability and adhesiveness, but not in their aggregability, and thus can be used as a limited model for beta-thalassaemia RBC phenotypes.

Growth hormone reserve in adult beta thalassemia patients.

Reduced serum insulin-like growth factor-1 (IGF-1) and hypogonadotrophic hypogonadism are common features of adult beta-thalassemia, and warrant evaluation of the growth hormone (GH)-IGF-1 axis. The aim of this study was to determine GH reserve in beta-thalassemia patients (9 females, 7 males, 15 major, 1 intermedia), age 29.3 +/- 6.9 years, BMI 21.3 +/- 1.9 kg/m2, and in 20 age, sex and BMI-matched healthy controls, using the GH-releasing hormone (GHRH)-arginine test. The associations between peak GH response and hormonal and biochemical indices were evaluated. Using BMI-related cut-off limits for peak GH response in the GHRH-arginine test, 4/16 beta-thalassemia patients had peak GH lower than 11.5 microg/l, the cut-off limit suggested for lean subjects, and were diagnosed as GH deficient (GHD). Using 9 microg/l as the cut-off limit 2/16 patients were GHD. Reduced serum IGF-1 and IGFBP-3 were present in 69% and 19% of the patients, respectively. Peak GH did not correlate with serum IGF-1, TSH, and fT4 levels or gonadal status. Neither peak GH nor IGF-1 correlated with serum ferritin. Our findings suggest that GHD is present in up to a quarter of adult beta-thalassemia patients. The clinical benefits of GH therapy need to be determined. GHD alone does not account for the high prevalence of reduced IGF-1 in adult beta-thalassemia.

Severe infections in thalassaemic patients: prevalence and predisposing factors.

The incidence of infections among patients with thalassaemia and the role of risk factors for infection are uncertain. We studied the occurrence of infections necessitating hospitalisation in 92 homozygous beta-thalassaemia patients who had been followed longitudinally for decades, and investigated the role of potential risk factors for these infections. Pneumonia accounted for 26% of the infections and fever of unknown origin for 14%. Staphylococcus aureus was the major pathogen possibly related to injections associated with intensive chelation with deferoxamine. There was a significant increase in the rate of infection over time, notably after 15 years. Splenectomy correlated with the incidence of infection (P < 0.001) without being confounded by other variables and with highest frequencies of infections present after 10 years. A direct correlation between iron overload and infection was evident only before the initiation of iron-chelating treatment (P < 0.01). Following initiation of deferoxamine, paradoxically, the infection rate increased (P = 0.046). The combination of splenectomy and deferoxamine treatment was associated with the highest adjusted infection rate. Parathyroid dysfunction and glucose-6-phosphate dehydrogenase deficiency were significantly associated with infection (P = 0.02 and P = 0.04 respectively). The infection rate in thalassaemia is affected mainly by the duration of the disease and is increased by splenectomy and, in the long term, by treatment with deferoxamine.

Flow cytometric analysis of the oxidative status of normal and thalassemic red blood cells.

BACKGROUND: The oxidative status of cells has been shown to modulate various cell functions and be involved in physiological and pathological conditions, including hereditary chronic anemias, such as thalassemia. It is maintained by the balance between oxidants, such as reactive oxygen species (ROS), and antioxidants, such as reduced glutathione (GSH). METHODS: We studied peripheral RBC derived from normal and thalassemic donors. Flow cytometric methods were used to measure (1) generation of ROS; (2) the content of reduced GSH; and (3) peroxidation of membrane lipids as an indication of membrane damage. RESULTS: ROS and lipid peroxidation were found to be higher, and GSH lower, in thalassemic RBC compared with normal RBC, both at baseline as well as following oxidative stress, such as exposure to hydrogen peroxide. To simulate a state of iron overload, normal RBC were exposed to extracellular ferric ammonium citrate or hemin, or their Hb was denatured by phenylhydrazine. All these treatments increased ROS and lipid peroxidation and decreased GSH. These effects were reversed by N-acetyl cysteine, a known ROS scavenger. CONCLUSIONS: Flow cytometry can be useful for measuring oxidative stress and its effects on RBC in various diseases and for studying various chemical agents as antioxidants.

Unique features of laparoscopic cholecystectomy in Beta thalassemia patients.

Beta Thalassemia patients suffer from a high incidence of gallstones as well as systemic complications of iron overload. We performed laparoscopic cholecystectomy in 8 beta thalassemia patients and describe their specific features. Diagnosis was based on patients' clinical presentation and sonography. Trocars were readjusted due to the hepatomegaly. No intraoperative cholangiograms were performed. Follow-up included clinical assessment and abdominal sonography. All procedures were completed laparoscopically. Pigment cirrhosis and fibrosis around the cystic duct were noted in all patients. No biliary injury occurred. Post operatively, 4 patients suffered fluid leakage through the trocar site, treated conservatively. No major cardiopulmonary complications occurred. During a mean follow-up time of 65 months, all patients are symptom free and without evidence of biliary lithiasis. Thalassemia patients are difficult and often high-risk patients. Improvement in anesthesia and monitoring enables better management of these patients. Laparoscopic cholecystectomy should be advised in carefully selected thalassemia patients.

Flow cytometric analysis of hydroxyurea effects on fetal hemoglobin production in cultures of beta-thalassemia erythroid precursors.

An increase in fetal hemoglobin (Hb F) ameliorates the clinical symptoms of the underlying disease in the beta hemoglobinopathies-sickle cell anemia and beta-thalassemia (thal). Hydroxyurea (HU) can elevate Hb F production in erythroid cells and is the agent currently in clinical use for patients with sickle cell anemia; it is presently being tested in clinical trials for thalassemia. We have developed a two-phase liquid culture system that mimics the in vivo hematological changes that are observed in patients treated with HU. Adding HU during the second phase of the culture increases the proportion of Hb F, increases the levels of total hemoglobin (Hb) content per cell and increases cell size, but it decreases the numbers of cells and the total amount of Hb produced. In the present study we developed and utilized a double labeling procedure for flow cytometric analysis of the cellular distribution of Hb F. Cells exposed to various concentrations of HU on day 6 of the second phase of the culture were harvested on day 12, and stained simultaneously with fluorochrome-conjugated monoclonal antibodies specific for human glycophorin A, an erythroid specific marker, and human Hb F. Both the percentage of the Hb F-containing cells and their intensity of fluorescence were recorded. The latter value gives a semi-quantitative estimation of the mean cellular Hb F content. The results indicated that cultures derived from different beta-thalassemic patients differ in their response to HU. In most patients, low doses of HU decreased the percentage of Hb F-cells as well as their Hb F content. At high doses, some patients showed an increase in both parameters, while others showed an increase in the percentage of Hb F-cells with minimal increase in their mean Hb F content, while still other patients showed little effect at all. In all patients, high doses of HU caused a decrease in cell numbers. These results suggest that HU has mixed effects on erythroid precursors. Both the two-phase liquid culture and the flow cytometric analysis procedures described herein provide the experimental tools for screening of Hb F-inducing drugs and for evaluating patients' cell response prior to treatment.

Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic red blood cells.

Reactive oxygen species (ROS) contribute to the pathogenesis of several hereditary disorders of red blood cells (RBCs), including thalassaemia. We report here on a modified flow cytometric method for measuring ROS in normal and thalassaemic RBCs. RBCs were incubated with 0.4 mM 2',7'-dichlorofluorescin diacetate (DCFH-DA), then washed and further incubated either with or without 2 mM H2O2. Flow cytometric analysis showed that RBC fluorescence increased with time; it increased faster and reached higher intensity (by 10-30-fold) in H2O2-stimulated RBCs as compared to unstimulated RBCs. In both cases, the antioxidant N-acetyl-l-cysteine reduced fluorescence, confirming previous reports that DCFH fluorescence is mediated by ROS. While the fluorescence of unstimulated RBCs increased with time, probably because of exposure to atmospheric oxygen, in H2O2-stimulated RBCs fluorescence decreased after 30 min. The latter effect is most likely related to H2O2 decomposition by catalase as both sodium azide, an antimetabolite that inhibits catalase and low temperature increased the fluorescence of stimulated RBCs. Washing had a similar effect, suggesting that maintenance of the oxidised DCF requires a constant supply of ROS. We next studied RBCs of beta-thalassaemic patients. The results demonstrated a significantly higher ROS generation by stimulated and unstimulated thalassaemic RBCs compared to their normal counterparts. These results suggest that flow cytometry can be useful for measuring the ROS status of RBCs in various diseases and for studying chemical agents as antioxidants.