|Year : 2012 | Volume
| Issue : 2 | Page : 92-98
Frequency of platelet aggregation defects in children suffering fromg β-thalassemia
Hammad Tufail Chaudhary, Nisar Ahmad
Department of Haematology and Transfusion Medicine, Children's Hospital and The Institute of Child Health, Lahore, Pakistan
|Date of Web Publication||13-Sep-2012|
Hammad Tufail Chaudhary
Assistant Professor of Pathology, Taif University, Taif, Saudi Arabia, 267 Gulshan Block, Allama Iqbal Town, Lahore
Source of Support: None, Conflict of Interest: None
Background: β-thalassemia is a genetic disorder of hemoglobin synthesis, which is not uncommon in world's population. According to international studies, platelet aggregation is increased in splenectomized patients while it is decreased or normal in non-splenectomized adult patients and children. These findings are one of the indications of hypercoagulable state in β-thalassemia patients, which leads to thromboembolic disease. Aims: To determine the frequency of platelet aggregation defects in children (aged 6 months to 5 years) suffering from β-thalassemia. Settings and Design: This study was conducted in Department of Hematology and Transfusion Medicine of the Children's Hospital and Institute of Child Health, Lahore. It was a cross-sectional study. Subjects and Methods: We selected 100 β-thalassemia patients of age between 0 and 5 years. History taking, examination, and investigations like complete blood count (CBC), Hb-electrophoresis on High Performance Liquid Chromatography (HPLC), serum iron, and platelet aggregation were performed on these patients. Results: During the study period, 100 children fulfilling the inclusion criteria were selected, and platelet aggregation with ADP, collagen, and ristocetin, was performed. It was found out that 66, 62, and 69% of the children showed decreased aggregation with ADP, collagen, and ristocetin, respectively, while 34, 38, and 31% children were found to have normal aggregation with ADP, collagen, and ristocetin, respectively. Conclusions: It was found that majority of the children showed decreased aggregation with ADP, collagen and ristocetin.
Keywords: Aggregation, platelet, thalassemia
|How to cite this article:|
Chaudhary HT, Ahmad N. Frequency of platelet aggregation defects in children suffering fromg β-thalassemia. Saudi J Health Sci 2012;1:92-8
| Introduction|| |
Thalassemia refers to a heterogeneous group of genetic disorders of hemoglobin synthesis, which results from lack or reduced rate of production of one or more of globin chains of hemoglobin.  Thalassemia is mainly divided into alpha (α)-thalassemia and beta (β)-thalassemia, depending upon reduced or absent synthesis of α-globin chain or β-globin chain of hemoglobin, respectively.  Clinically, β-thalassemia is divided into major, intermediate, and minor forms.  β-thalassemia is associated with many complications.  Defect in platelet aggregation, i.e. joining together of platelets to form a plug for stopping a bleed, is one of the complications of β-thalassemia. ,
A vast majority of population of the world, mainly in broad belt, ranging from the Mediterranean and parts of North and West Africa through the Middle East and Indian subcontinent to South-East Asia, is suffering from β-thalassemia. 
Pakistan is also affected by β-thalassemia. In a study, it was found that about 66% of hemolytic anemia was constituted by β-thalassemia in some areas of Pakistan. Majority of them were β-thalassemia major, i.e. about 41%.  In another study in Punjab, prevalence of β-thalassemia trait was reported to be 8% in the general population and 58% in siblings of children with β-thalassemia major. 
There are many complications related to β-thalassemia and hypercoagulable state is one of them.  In β-thalassemia, platelets are activated by free radical from hemoglobin, , RBC phospholipids,  thrombin,  and microparticles.  Then, after their activation, platelets bind to proteins S and C,  release thromboxane A2 (TXA2),  and form microparticles of platelets.  Simultaneously, iron overload is also present in β-thalassemia.  Vitamin C is decreased due to this iron overload, which may result in platelet defect.  On the other hand, this iron overload decreases lipid peroxide, which leads to release of TXA2 from platelets.  Iron overload worsens the situation by creating oxidative stress which causes vascular injury,  which is itself increased by hemolysis due to hypoxia.  As a result of vascular injury, prostacyclin is released. Prostacyclin and TXA2 result in clumping of platelets with vessel wall,  and hence hypercoagulable state on one hand and hemorrhagic tendency due to overusage of platelets on the other hand. , The hemorrhagic tendency is enhanced by decreased platelet life.  Splenectomy enhances hypercoagulable state in thalassemia by increasing the damaged RBCs. ,, Damaged RBCs have more phospholipids on their membrane, they act themselves as activated platelets, and they also cause platelet activation. ,,,,, Children are no exception to this hypercoagulable state  and no thromboembolic events have been documented in children. 
Hussain et al.,  Eldor et al., , and Domopoulas  found decreased platelet aggregation in β-thalassemia patients in their studies. Isarangkura and other researchers also came across decreased platelet aggregation in vitro in non-splenectomized β-thalassemia patients. ,,,, On the other hand, increased platelet aggregation in splenectomized patients was observed in some studies. ,,,,,
The importance of this study lies in the fact that cure of β-thalassemia is quite expensive and available to minority of patients. Researchers are striving hard to know maximum about the complications of β-thalassemia so that they can be prevented.  In this regard, knowledge about the frequency of platelet aggregation defects, a complication of β-thalassemia, among β-thalassemia patients, is significant, especially when β-thalassemia is not uncommon in Pakistan. ,
| Subjects and Methods|| |
This study was carried out in Department of Hematology and Transfusion Medicine of the Children's Hospital and the Institute of Child Health, Lahore. It is a tertiary care hospital providing state-of-the-art facilities in child health.
Duration of study
This study was completed in 6 months after the approval of synopsis.
A total of 100 diagnosed patients were studied (as the average number of β-thalassemia patients presenting to the hospital in 6 months was 100).
Non-probability purposeful sampling.
Children with hemoglobin <10 g/dL, mean corpuscular volume (MCV) <76 fL, mean corpuscular hemoglobin (MCH) <26 pg, microcytic hypochromic anemia with anisopoikilocytosis on peripheral smear, HbF >10%, reticulocyte count >2%, serum iron >11 μmol/L, and platelet count > 150 × 10 9 /L.
All patients who:
- had hemoglobinopathies except β-thalassemia major or intermedia;
- had taken blood transfusion within a month prior to platelet aggregation studies;
- were diagnosed cases of acquired or hereditary platelet aggregation defect;
- had taken any medication within 15 days before platelet aggregation studies;
- had taken diet affecting platelet aggregation, e.g. garlic, within a week prior to platelet aggregation; and
- were symptomless carriers of β-thalassemia, i.e. β-thalassemia minor.
It was a cross-sectional study.
Data collection procedure
Informed consent was taken from guardians of the patients (as the patients were children) included in this study. Patients who fulfilled the inclusion criteria were asked to provide information on history and examination of β-thalassemia related to the study, according to the variables mentioned in proforma.
Specimens were drawn with a minimum of trauma or stasis at the venipuncture site and anticoagulated with 3.8% sodium citrate, in the ratio of 1 part anticoagulant to 9 parts of blood. Specimens were kept at room temperature. Testing was started 30 min after venipuncture and completed within 2½ h after venipuncture. Sample was mixed by gentle inversion. Platelet-rich plasma (PRP) was prepared by centrifuging plasma at approximately 100 g for 15 min. Platelet-rich plasma was taken with propylene transfer pipette and transferred into polypropylene plastic tube. Tubes were properly labeled. Then, platelet-poor plasma (PPP) was prepared by centrifuging one sample tube at approximately 2400 g for 20 min. 250 μL platelet-rich plasma was added to P/N 312 cuvettes, with P/N 365 spacer attached below the cuvette. Sample was pre-warmed in incubation well of the aggregometer. P/N stir bar was added to cuvettes. 500 μL platelet-poor plasma was added to P/N 312 cuvette. Platelet-poor plasma cuvette and platelet-rich plasma cuvettes were placed in their respective positions in aggregometer. Then, 2.5 μL ADP, 1 μL collagen, and 2.0 μL ristocetin were added to 250 μL of PRP to achieve concentrations of 10 μM, 4 mg/μl, and 10 μM, respectively.
Aggregation was observed for 3 min at least against ADP, collagen, and ristocetin in percentages and compared with controls. Results of aggregation were mentioned as abnormal (increased or decreased) or normal after comparison with controls.
The main confounding factors which could interfere with platelet aggregation results were platelet count, recent blood transfusion, and drugs. These factors were taken care of as follows:
- Platelet count of PRP was kept between 150 and 200 × 10 9 /L by dilution with PPP of the same patient, if the platelet count of PRP was found to be greater than 200 ×10 9 /L.
- Patients who had received blood transfusions within a month prior to platelet aggregation studies were excluded from the study.
- Patients who had received any drug within 15 days prior to platelet aggregation studies were also excluded.
Blood was also taken and anticoagulated with ethylenediamine tetraacetic acid (EDTA). Complete blood count was done by using Sysmex SF3000. Sysmex SF 3000 counts the blood cells by aperture impedance as well as light scattering technology.
Serum iron was measured by Hitachi 902 Automatic Analyzer using commercial kit based on photometric technique supplied by Roche Diagnostics GmbH, Mannheim, Germany.
Hemoglobin electrophoresis was carried out by High Performance Liquid Chromatography (HPLC) on a fully automated HPLC system. The variant analyzer by Bio-Rad Laboratories Inc, Hercules, California, USA, using the β-thalassemia short program, was used for the estimation of HbF. This program uses the principle of cation-exchange HPLC. Whole blood samples were collected into a vial containing EDTA.
5 μL whole blood from each patient was pipetted into separate 1.5-ml sample vials. 1.0-ml of hemolysis reagent was added to each sample. Sample vials were then placed into variant. Patient hemolysates remained stable for 24 h when stored at 2-8°C. Area percentages for hemoglobin adult (A), fetal (F), and A2 were separated and determined by HPLC. At the end of each sample analysis, a copy of chromatogram and report data was automatically printed. The report table included the corrected area for hemoglobins A2 and F for all subsequent samples in the run.
Data analysis procedure
The data were entered in computer and statistically analyzed by using SPSS-10. Results were calculated and reported as mean, frequency, percentages, and standard deviation. Variables from demographic data, e.g. age, sex, and all the variables of history, examination, and investigation, as mentioned in the proforma, were correlated with platelet aggregation results. Ranges, means, and standard deviations of numerical data, e.g. age, hemoglobin etc., were found out. The results were compared with those of national and international studies on the same topic.
| Results|| |
During the study period, 100 children fulfilling the inclusion criteria were selected and platelet aggregation with ADP, collagen, and ristocetin was performed. It was found out that percentage of children showing decreased aggregation were 66% with ADP, 62% with collagen, and 69% with ristocetin, while percentage of children showing normal aggregation was 34% with ADP, 38% with collagen, and 31% with ristocetin [Table 1].
In our analysis, patients were divided into two groups, i.e. patients with reduced aggregation and those with normal aggregation.
Majority of the patients in our study were males, i.e. 80% in comparison to 20% of females [Figure 1]. Male to female ratio was 4:1.
Mean age at presentation, age at onset, and age at diagnosis were 41.48, 9.18, and 6.07 months, respectively. Mean age at presentation, age at onset, and age at diagnosis are given in [Table 2], with their standard deviations and ranges.
|Table 2: Distribution of cases by age at presentation, age at diagnosis, and age at onset of pallor (N = 100)|
Click here to view
Pallor was present in all patients of the study. No patient was found with leg ulcers. Four percent of the patients were found with nose bleed and bruises. All of them belonged to the group of patients with decreased aggregation.
Mean spleen and liver size, below costal margin, was 4.22 and 2.12 cm, respectively [Table 3].
|Table 3: Distribution of cases according to liver and spleen size (N = 100)|
Click here to view
Mean hemoglobin, MCH, and MCV are given in [Table 4], with their standard deviations and ranges. Mean hemoglobin, MCV, and MCH were 5.8 g/dL, 72.4 fL, and 24.21 pg, respectively [Table 4].
[Table 5] shows the mean HbF, HbA, and HbA2, with their standard deviations and ranges. Mean HbF, HbA, and HbA2 were 53.87%, 43.13%, and 2.99%, respectively (% is the unit of measurement of HbF, HbA, and HbA2) [Table 5].
Mean serum iron was 175.4 mg/dL. Mean serum iron with standard deviation and ranges are given in [Table 6].
| Discussion|| |
The present study was carried out in Department of Hematology and Transfusion Medicine Division of Children's Hospital and Institute of Child Health on a sample of 100 patients who presented with history of β-thalassemia.
Platelet aggregation was done on β-thalassemia patients. Decreased aggregation was found in majority of patients, i.e. 66% with ADP, 62% with collagen, and 69% with ristocetin.
These results were comparable to the other studies in which it was mentioned that platelet aggregation was decreased or normal in children with β-thalassemia.  A study by Hussain et al. reported that 44% of β-thalassemia patients showed decreased platelet aggregation with ADP, collagen, and ristocetin.  Another study by Eldor et al. reported 93% of β-thalassemics showed reduced platelet aggregation with ADP, collagen, and ristocetin. , Also, the study by Domopoulas showed decreased or normal platelet aggregation in homozygous β-thalassemics with ADP.  Isarangkura also concluded that 28% of his study patients had decreased platelet aggregation and majority of them were non-splenectomized.  This fact of decreased platelet aggregation in non-splenectomized patients was also supported by other studies. ,,,
Whereas in splenectomized β-thalassemics, increased platelet aggregation was observed in many international studies. ,,,,,
So, all of the above studies reported the same conclusion that platelet aggregation is reduced or normal in children or non-splenectomized β-thalassemic patients, which is also found in our study.
Continuous research has been going on to correlate this data and its relationship with thalassemia and its complications. It has been observed that platelets are activated in β-thalassemics by free radicals from Hb as a result of hemolysis, , by RBC phospholipids ,  thrombin,  and microparticles of platelets, which are circulating in blood and formed themselves by platelet activation.  Then, these activated platelets bind to proteins S and C,  release TXA2,  and form microparticles of platelets. 
Iron overload is present in β-thalassemia.  This iron overload results in decrease in vitamin C which may result in platelet defect.  This iron overload also causes decreased lipid peroxide, which ultimately results in release of TXA2 from platelets.  Iron overload exuberates the situation by creating oxidative stress which causes vascular injury.  This oxidative stress is handled by vitamin E through its antioxidant activity.  Vascular injury is enhanced by hemolysis due to hypoxia.  As a result of vascular injury, prostacyclin is released. Prostacyclin and TXA2, which are released by platelet activity, act on platelets, which results in its clumping with vessel wall.  This clumping of platelets with vessel wall causes hypercoagulable state in thalassemia on one side  and hemorrhagic tendency due to overusage of platelets on the other side,  as observed in four patients of our study and in other international studies.  The hemorrhagic tendency is enhanced by decreased platelet life. 
The role of splenectomy in creating hypercoagulable state in thalassemia is mainly through increase in damaged RBCs and thrombocytosis. ,, Damaged RBCs have more phospholipids on their membrane , they act themselves as activated platelets, and they also cause platelet activation. ,,,,
This hypercoagulable state also exists in children,  but there are no thromboembolic events documented in children.  There were no ulcers reported in the study by Eldor et al.,  which is comparable to our study, in which no ulcers were present in the children included.
Several answers have been suggested about decreased aggregation in non-splenectomized β-thalassemics. Hussain  had the opinion that because of reduced size and volume of red cells in β-thalassemia major, it was difficult to prepare PRP free of red cell. If they are free of red cells, then aggregability can be improved, particularly to ADP and collagen. Secondly, he thought that decrease in platelet aggregability might be due to platelet refractoriness caused by in vitro manipulations of platelets. 
Eldor et al. had the view that chronic platelet activation results in decreased platelet aggregation in vitro because activated platelets become refractory to additional stimulation. 
But why is platelet aggregation in vitro increased in splenectomized β-thalassemia patients? Because platelet refractoriness by in vitro manipulations or chronic activation of platelets is also present in splenectomized patients. If these would have been the cause of decreased platelet aggregation, they should have caused decreased or normal platelet aggregation in splenectomized patients.
In our view, after considering the above literature, increased platelet clumping as a result of platelet activation resulted in bruising as it was suggested by Eldor et al. Mild bleeding might be due to vitamin C deficiency which was present in β-thalassemics. 
Increased platelet aggregates present in splenectomized thalassemics  result in the formation of relatively big aggregates, which shows increased aggregation in optical aggregometer. Increased platelet number and increased oxidative stress due to splenectomy also causes increased aggregation.  Increased, normal, or decreased aggregation may also be affected by homozygous or heterozygous state of thalassemia patients. 
Further studies should be carried out to prove the above hypothesis.
As population of the study consisted of children (6 months to 5 years) and children have less chances of being splenectomized,  lack of splenectomized patients was one of the limitations in our study. As a result, we could not correlate this parameter with platelet aggregation results.
Age of onset of pallor was 14 months in the study by Özkan et al.  while it was 6 months in our study. This difference might be due to difference of genetic mutations in our population in children presenting with β-thalassemia and lack of adequate primary and secondary health facilities in our country.
Age at diagnosis was 29 months in the study by El-Harth et al.,  while it was 9.2 months in our study. This difference may be the result of early age of onset of pallor in our country as mentioned above.
Male to female ratio in our study was 4:1 which was comparable to the study by Hashmi et al. in which it was 3:1. 
Splenomegaly, measured as spleen enlargement below costal margin, was present in 96% of patients, while the study by Shah et al.  showed that spleen was enlarged in β-thalassemia patients in 63.2% of cases. Hepatomegaly, measured as liver enlargement below costal margin, was present in 79% of patients in our study, which was comparable to the study by Shah et al.  in which it was found that hepatomegaly was a common finding in β-thalassemia patients, as it was present in 36.8% of patients. Splenomegaly and hepatomegaly were found more in patients having decreased platelet aggregation than those having normal aggregation. This might give the reason of decreased aggregation in patients with increased spleen and liver size, as more platelets are pooled in bigger spleen. Secondly, platelet aggregates, which had been proven in thalassemic patients,  were captured more in larger spleen, which resulted in decreased platelet aggregation in vitro.
None of the patients underwent splenectomy in our study as it was mentioned in the study by Shah et al.  and the study by Pattanapanyasat et al.  that splenectomy is rare before 5 years of age.
4% of patients got bruises and nosebleeds in our study. Increased platelet clumping as a result of platelet activation resulted in bruising as it was suggested by Eldor et al.  Mild bleeding might also be due to vitamin C deficiency which was present in β-thalassemics. Bleeding tendency was also observed in β-thalassemia patients in the study by Eldor et al.,
Bleeding time should be included in future studies to correlate bruises and bleeding in β-thalassemics with bleeding time.
Leg ulcers, sign of oxidative stress and hypercoagulability in β-thalassemia,  were absent in our study as it was observed by Shah et al.  and Eldor et al.  in their studies. The reason was that signs of thromboembolism had not been recorded in children, even though hypercoagulability was present. 
In an Indian study by Özkan et al., the mean Hb was 9 g/dL in β-thalassemia patients of the age group 0-5 years,  in comparison to our study in which the mean Hb was 5.83 g/ dL. This simply highlights the lack of care and severity of disease of β-thalassemics in Pakistan. 
Mean HbA2 was 2.99% in this study, while it was 2.9% in the study by El-Harth et al.  Mean HbF in our study was 53.84%, while it was 33% in the study by Ahmad et al.  No relationship was found between mean Hb, HbA2, and HbF, and platelet aggregation defects in our study as well as in other studies. ,,,,,,,,
Mean MCV and MCH values in our study were 72.4 fL and 24.21 pg, respectively. These findings were comparable to that reported by Ahmad et al. who proved that MCV and MCH did not decrease too much in transfused β-thalassemia patients.  No significant correlation between MCV and MCH and platelet aggregation defects in β-thalassemia patients was established in our study and other international studies. ,,,,,,,,
Mean serum iron was 175.4 mg/dL, which is comparable to the study by Pearson, which also showed increased serum iron of β-thalassemia patients.  There was no significant difference in the levels of serum iron among decreased and normal aggregation groups of β-thalassemia patients. This was probably due to the fact that iron causes oxidative stress to the platelets, and as a result, platelet activation.  So, platelets of both the groups were activated, but some patients showed decreased platelet aggregation due to the larger spleen than in those patients who showed normal platelet aggregation. In other words, serum iron is reduced in all patients of β-thalassemia regardless of whether they have normal or decreased platelet aggregation. It is spleen and liver size which may be the contributing factor in decreased or normal aggregation, as discussed before. Serum ferritin is a better indicator of body iron stores , and should be correlated with platelet aggregation in future studies.
Although basic health facilities are still an issue in Pakistan,  drugs like anti-platelet drugs and hydroxyurea should be kept under consideration while planning for care of thalassemic children in the future. 
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]