Laboratory diagnosis for thalassemia intermedia: Are we there yet?

BACKGROUND: Differentiation between thalassemia major and thalassemia intermedia at presentation is not uniformly characterized, for which an absolute criteria needs to be developed. This study investigated the primary and secondary genetic modifiers to develop a laboratory finding by forming different genetic mutational combinations seen among thalassemia intermedia patients and comparing them with thalassemia major. METHODS: This cross-sectional study analyzed 315 thalassemia intermedia patients. One hundred and five thalassemia major patients were recruited on the basis of documented evidence of diagnosis and were receiving blood transfusion therapy regularly. Various mutational combinations were identified, and comparison was performed between thalassemia intermedia and major using statistical software STATA 11.1. RESULTS: The mean age of the total population was 5.9 ± 5.32 years of which 165 (52%) were males. Of the two groups (thalassemia intermedia and thalassemia major), IVSI-5, IVSI-1, and Fr 8-9 were more prevalent among the thalassemia intermedia cohort. When comparison was performed between the thalassemia intermedia and thalassemia major patients, it showed significant results for the presence of Xmn-1 polymorphism. CONCLUSION: The presence of IVSI-5 homozygous with Xmn-1, IVSI-5 heterozygous with Xmn-1, Cd 30 homozygous with or without Xmn-1 and IVSI-1 homozygous or heterozygous either with or without Xmn-1 prove to be strong indicators towards diagnosis of thalassemia intermedia.

Preparation, characterization and evaluation of HPßCD-PTX/PHB nanoparticles for pH-responsive, cytotoxic and apoptotic properties.

Paclitaxel (PTX) is a potent anticancer drug. However, PTX exhibits extremely poor solubility in aqueous solution along with severe side effects. Therefore, in this study, an inclusion complex was prepared between PTX and hydroxypropyl-ß-cyclodextrin (HPßCD) by solvent evaporation to enhance the drug's solubility. The HPßCD-PTX inclusion complex was then encapsulated in poly-3-hydroxybutyrate (PHB) to fabricate drug-loaded nanoparticles (HPßCD-PTX/PHB NPs) by nanoprecipitation. The HPßCD-PTX/PHB NPs depicted a higher release of PTX at pH 5.5 thus demonstrating a pH-dependent release profile. The cytotoxic properties of HPßCD-PTX/PHB NPs were tested against MCF-7, MDA-MB-231 and SW-620 cell lines. The cytotoxic potential of HPßCD-PTX/PHB NPs was 2.59-fold improved in MCF-7 cells in comparison to free PTX. Additionally, the HPßCD-PTX/PHB NPs improved the antimitotic (1.68-fold) and apoptotic (8.45-fold) effects of PTX in MCF-7 cells in comparison to PTX alone. In summary, these pH-responsive nanoparticles could be prospective carriers for enhancing the cytotoxic properties of PTX for the treatment of breast cancer.

Gγ-Xmn I polymorphism: a significant determinant of ß-thalassemia treatment without blood transfusion.

ß-thalassemia is characterized by impaired ß-chain synthesis leading to ineffective erythropoiesis, severe anemia, and a need for blood transfusion. Presence of Xmn I polymorphism (-158 C-T nucleotide change) in γ-globin gene is associated with a higher fetal hemoglobin and a lesser clinical severity. This prospective study attempted to find out the effect of hydroxyurea (HU) on ß-thalassemia patients in the presence or absence of Xmn I polymorphism. A total of 143 consecutive ß-thalassemia patients received HU (16 mg/kg/d). Sixty-four (44.7%) had Xmn I polymorphism (either homozygous or heterozygous). Patients were evaluated at a median duration of 3 years (range, 6 mo to 9 y). Responders became transfusion independent after 6 months, partial responders had a least 50% reduction in transfusion requirement and nonresponders had no significant reduction. Of the 64 patients with Xmn I polymorphism, 44 (69%) showed response (P<0.01), whereas in those who lacked Xmn I polymorphism (n=79), only 17 (21%) were responders. This study showed that the presence of Xmn I polymorphism in ß-thalassemia is a predictor of response to HU and highlights the possibility of managing this subset of patients without blood transfusion.

Screening immediate family members for carrier identification and counseling: a cost-effective and practical approach.

OBJECTIVE: To screen immediate family members of thalassaemia patients for carrier identification and counselling. METHODS: The cross-sectional study was conducted at an urban thalassaemia treatment and prevention centre in Karachi, Pakistan, from January to December 2008, and involved 188 siblings of 100 thalassaemia patients. Complete blood count, including haemogram, was performed in the siblings. Samples with MCV < 75fl and MCH < 25% were subjected to haemoglobin-electrophoresis. Haemoglobin A2 of 3.5% to 7.0% was labelled as beta-thalassaemia minor. Those with haemoglobin A2 of 3.0-3.4% but red blood cell count of > 4.5 x 1012/L were reported as equivocal and were screened for iron deficiency anaemia and a repeat haemoglobin A2 estimation was done on high performance liquid chromatography. Equivocal results of the chromoatography were screened for thalassaemia mutation. Mean values along with standard deviation were worked out for relevant variables. RESULT: Of the 188 subjects, there were 124 (66%) males and 64 (34%) females. The mean age was 16.5 +/- 6.3 years (range: 3 months to 30 years) and the mean family size was 1.88 +/- 3.8 (range: 1-12) children per family. There were 51 (51%) first-cousin marriages in this group. Of the siblings, 65 (34.5%) were identified as normal, while 117 (62.2%) were reported as beta-thalassaemia carriers. Six asymptomatic siblings were reported as consistent with beta-thalassaemia major. CONCLUSION: There were 62.2% siblings identified as beta thalassaemia carriers in the study as opposed to 5-8% carriers in the general population. We also identified six asymptomatic and unidentified cases of beta-thalassaemia intermedia in these families. Therefore, in our context where both resources and budgets are limited, it is practical to focus on siblings of identified thalasaemia patients.

Molecular epidemiology of ß-thalassemia in Pakistan: Far reaching implications.

BACKGROUND: ß -Thalassaemia, an autosomal recessive hemoglobinopathy, is one of the commonest genetically transmitted disorders throughout the world. Collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing ß-thalassemia. AIM: To achieve this objective, Identification of the spectrum of genetic mutations, especially for various ethnic backgrounds in Pakistan. Therefore, we designed a cross sectional prospective study to identify the frequency of various gene mutations in different ethnic groups of Pakistan. MATERIALS AND METHODS: Over a 5-year period, DNA from 648 blood samples {including specimens of chorionic villus sampling (CVS)} were analyzed for the twelve most common ß-thalassemia mutations found in the Pakistani population by a Multiplex amplification refractory mutation system (ARMS). Each sample was analyzed for the mutation as well as the normal gene, appropriate with negative and positive controls, and reagent blanks. RESULTS: Out of 648 samples mutations were identified in 640 (98.75%) samples by multiplex ARMS. 8 common ß-thalassemia mutations were identified in 8 different ethnic groups accounting for 93.9% of the ß-thalasemia alleles. CONCLUSIONS: Based on the outcome of this study a cost effective proposal is formulated for detection of ß-thalassemia mutations.

Efficacy of hydroxyurea in providing transfusion independence in ß-thalassemia.

BACKGROUND: Packed red blood cell (PRC) transfusion with iron chelation is the mainstay of treatment for ß-thalassemia major. This prospective interventional trial serves as a follow up to our similar earlier study that evaluated the efficacy and safety of hydroxyurea (HU) in minimizing PRC transfusions in patients with ß-thalassemia major. METHODS: One hundred fifty-two patients with ß-thalassemia major received HU at a mean dose of 16 mg/kg/d. The results were analyzed at the end of 24 months. Transfusion requirement during the 6 months preceding the study was considered as the control. RESULTS: One hundred forty-six of 152 patients were evaluated after 24 months of follow up; 6 patients were either lost to follow-up or withdrew consent. Grade 1 myelosuppression was observed in 4 patients and diarrhea in 2 patients. Sixty children (41%) did not require any transfusion after using HU; 57 patients (39%) showed partial response with greater than 50% reduction in PRC transfusion; and 29 patients (20%) were nonresponders with less than 50% reduction in PRC transfusion. The mean volume of PRC transfused was reduced for all patients. CONCLUSIONS: HU was found to be safe in patients with ß-thalassemia major, and resulted in the reduction of transfusion requirement and in an increase in the interval between transfusions.

Preparation, characterization and evaluation of antibacterial properties of epirubicin loaded PHB and PHBV nanoparticles.

Poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) are considered as ideal drug carriers due to their non-toxic, biodegradable and biocompatible nature. In this study, the epirubicin (EPI) was used as a model drug. The blank (PHBo, PHBVo) and drug loaded (EPI-PHB-PEG, EPI-PHBV-PEG) nanoparticles were prepared by nanoprecipitation method. The average particle size, polydispersity index and zeta potential of blank and drug loaded nanoparticles were determined. While, the morphology of blank and drug loaded nanoparticles was evaluated by scanning electron microscopy. The drug loading efficiency of EPI-PHB-PEG nanoparticles was higher in comparison to EPI-PHBV-PEG nanoparticles. A sustained release of EPI was found over a period of 8 days at pH 4 from EPI-PHB-PEG and EPI-PHBV-PEG nanoparticles in comparison to faster drug release at pH 7. The assessment of antibacterial properties of the drug loaded nanoparticles showed significant antibacterial properties against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa bacterial strains as compared to an equivalent amount of free drug.

Seropositivity of hepatitis C, hepatitis B and HIV in chronically transfused ß-thalassaemia major patients.

The purpose of this study was to determine the frequency and trend of transfusion transmitted infections (TTI) in chronically transfused ß-thalassaemia major (TM) patients with reference to the duration of transfusions. A cross-sectional study was done on 160 ß-TM patients and 5517 healthy blood donors to find out the prevalence of HCV, HBV and HIV infections. Out of 160 patients, 21 cases (13.1%) were anti-HCV positive, 2 (1.25%) were HBsAg positive. HIV antibodies were not detected in any sample. However, 109 (1.9%) and 104 (1.8%) of 5517 blood donors were positive for HCV and HBV respectively. No donor showed HIV antibodies. Anti-HCV was positive in 9/111(8.4%) thalassaemics (< 10 years of age) while 11/49 (22%) [> 10 years of age] showing significant difference (p = 0.005) among the two groups. For the past 10 - 12 years the screening of blood has reduced the magnitude of the disease significantly as shown by the trend in two age groups. Further improvements need to be done to implement uniform screening throughout the country.

Molecular epidemiology of ß-thalassemia in Pakistan: far reaching implications.

ß-thalassaemia, an autosomal recessive hemoglobinopathy, is one of the commonest genetically transmitted disorders throughout the world. Collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing ß-thalassemia. To achieve this objective, Identification of the spectrum of genetic mutations, especially for various ethnic backgrounds in Pakistan is necessary. Therefore, we designed a cross sectional prospective study to identify the frequency of various gene mutations in different ethnic groups of Pakistan. Over a 5-year period, DNA from 648 blood samples [including specimens of chorionic villus sampling (CVS)] were analyzed for the twelve most common ß-thalassemia mutations found in the Pakistani population by a Multiplex amplification refractory mutation system (ARMS). The most common mutation identified was Intervening Sequence 1-5 (IVS 1-5 (G-C)); accounting for 40.89% mutated alleles, and was represented in all ethnic groups. 15.7 % of the ß-thalassemia alleles were found to have Frameshift 8-9 (Fr 8-9) as the second most common mutation Other common genetic defects responsible for ß-thalassemia: IVS 1-1 (G-T) was found in 8.17%, Codon-30 (Cd-30 (G-C)) 8.02%, Codon-5(Cd-5 (-CT)) contributed 2.16% and Deletion 619 base pair (Del 619bp) affected 11.11% were found in Pakistan. This large study adds to the pre-existing data in Pakistan. Knowledge of the predominant mutation in a given ethnic group will not only help in developing a short panel of (population-specific) primers of mutations thereby providing a cost-effective method for prenatal diagnosis and also help the clinicians to counsel regarding blood transfusion regimen/ pregnancy termination.