HbA2 levels in β-thalassaemia carriers with the Filipino β0-deletion: are the levels higher than what is found with non-deletional forms of β0-thalassaemia?

AIMS: Classical carriers of β-thalassaemia are identified by a raised HbA2 level. Earlier studies indicated that the Filipino β-deletion has high raised HbA2 levels. The introduction of automated high performance liquid chromatography (HPLC) for thalassaemia screening is an important advance in technology for haematology laboratories. The BioRad Variant II Hb analyser is a common instrument used to quantify HbA2 levels in thalassaemia screening. This study aimed to determine HbA2 levels in carriers of Filipino β-mutation using the BioRad Variant II Hb analyser. METHODS: The Filipino β-deletion was identified using gap-polymerase chain reaction (PCR) in the parents of transfusion dependent β-thalassaemia patients who were homozygous for the Filipino β-deletion in the indigenous population of Sabah, Malaysia. Hb subtypes were quantified on the BioRad Variant II Hb analyser. Concurrent α-thalassaemia was identified by multiplex gap-PCR for deletions and amplification refractory mutation system (ARMS)-PCR for non-deletional mutations. RESULTS: The mean HbA2 level for Filipino β-thalassaemia trait was 5.9 ± 0.47 and with coinheritance of α-thalassaemia was 6.3 ± 0.44 (-α heterozygous) and 6.7 ± 0.36 (-α homozygous). The HbA2 levels were all >4% in keeping with the findings of classical β-thalassaemia trait and significantly higher than levels seen in non-deletional forms of β-thalassaemia. CONCLUSION: The HbA2 level measured on the BioRad Variant II Hb analyser was lower than the level in the first description of the Filipino β-thalassaemia. β-thalassaemia trait with coinheritance of α-thalassaemia (-α) is associated with significantly higher HbA2 level

Correlation of BACH1 and hemoglobin E/Beta-thalassemia globin expression

Objective: The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia

Concurrent inheritance of deletional a-thalassaemia in Malays with HbE trait

Introduction: HbE is the commonest beta haemoglobin (Hb) variant in Southeast Asia. It causes a reduction in synthesis of the beta-globin E chain. Studies indicate HbE coinherited with a-thalassaemia leads to milder clinical phenotype. This study investigates the commitant inheritance of a-thalassaemia in Malays with HbE. Methods: Four hundred and fourteen (414) blood samples were screened for haemoglobinopathy using primarily the first three steps of the BHES [ (B) blood counts, blood film; (H), HPLC; (E) electrophoresis; (S), stability ] protocol. Complete blood cell analyser, Hb typing with cation exchange high-performance liquid chromatography (HPLC) and Hb electrophoresis at an alkaline pH (pH 8.5) Forty-five (10.9%) were identified as HbE trait and DNA analysis was done for deletional a-thalassaemia using a single-tube multiplex-PCR assay. Results: Among the 45 subjects with HbE trait, 4 (8.9%) were found to have alpha-thalassaemia -2 (a) (a-37 kb deletion) and 1 (2.2%) the alpha-thalassaemia-1 (a0) (---SEA 20.5 kb deletion) defects respectively. Discussion: These findings show that 11.1% of Malays with HbE inherit alpha-thalassaemia concurrently. The most prevalent interaction found was a double heterozygote for HbE /a-thalassaemia 2, followed by HbE/a-thalassaemia 1. Conclusion: Molecular screening of deletional a-thalassaemia identified its concurrent inheritance in 11.1%o of Malays who were HbE carriers. This information will guide genetic counseling and the planning of treatment modalities in patients with HbE alpha-thalassaemia

Non-deletional alpha thalassaemia: a review of emerging therapy

More than seventy non-deletional α-thalassaemia mutations have been reported and they usually exhibit more severe clinical presentations compared to their deletional counterparts. Conventional treatment aims to manage the symptoms of the disease through red blood cell transfusion, but this has its own set of complications. For the time being, the only cure for thalassaemia is bone marrow transplantation, hence, it is a priority to explore other potential treatment methods. Novel gene editing methods could potentially be a long-term treatment option for this single gene disorder. This manuscript provides an overview of recent breakthroughs in non-deletional α-thalassaemia treatment, including intrauterine transfusion, cord blood transplantation, gene therapy, and several genome modification techniques, to contribute to the overall knowledge in not only ameliorating the condition of α-thalassaemia, but also to find a cure

Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells

Haplotype analysis of β-Thalassaemia major and carriers with Filipino β°-deletion in Sabah, Malaysia

Objective: The Filipino β°-deletion has been reported as a unique mutation in East Malaysia with a severe phenotype due to the complete absence of β-globin chain synthesis. In this study, the haplotype patterns of the β-globin gene cluster were used to relate the human genetic variation to this specific β-thalassaemia mutation. Methods: The 376 study subjects included 219 β-thalassaemia major (β-TM) patients with homozygous Filipino β°-deletion and 157 carriers with heterozygous Filipino β°-deletion from 10 government hospitals in different regions of Sabah. Genomic DNA was isolated from whole blood using silica membrane based DNA purification protocol. Polymerase chain reaction restriction fragment length polymorphism analysis (PCR-RFLP) was conducted on five markers within the β-globin gene cluster to construct the haplotype patterns.Results: Four haplotypes (Haplotype I–IV) were identified with Haplotype I as the predominant haplotype with the highest frequency of 0.98, followed by Haplotype II, III and Haplotype IV with 0.02. Haplotype I was strongly linked with the Filipino β°-deletion among the indigenous population. Conclusion: Haplotype I as the predominant haplotype suggests the patients with the Filipino β°-deletion in Sabah have a similar origin

Screening for intermediate and severe forms of thalassaemia in discarded red blood cells: optimization and feasibility

Detection and quantification of Hb subtypes of human blood is integral to presumptive identification of thalassaemias. It has been used in neonatal screening of thalassaemia and Hb variants. The use of discarded red blood cells following processing of the cord blood for stem cells provides readily available diagnostic material for thalassaemia screening. In this study, we determined the range of Hb subtypes in 195 consecutive cord blood samples collected for cord blood banking. The 'cord blood samples' analysed were those of the remaining red blood cells after the cord blood was processed for stem cell storage. Quantification of Hb subtypes by high performance liquid chromatography (HPLC) was done on BioRad Variant II Hb testing system. Only 73 (36.5%) of the samples could be analyzed neat without dilution. With a 1:300 dilution with wash solution the acceptable area as recommended by the manufacturer for reading of a C-gram within the 1 to 3 million ranges were achieved in all. Eighteen (9%) 12 showed classical Hb Barts (γ4) prerun peaks were confirmed by Sebia Hydrasys automated Hb gel electrophoresis and quantified by Sebia Capillarys 2 capillary electrophoresis. Only 1 (0.5%) was presumptively identified with HbH disease. Due to the limited number of samples no beta-thalassaemia major, Hb E beta-thalassaemia and Hb Barts hydrops fetalis were found. The HPLC assay was possible at a cost US$ 5 per sample and a turnover time of 10 samples per hour without technical difficulties. This study reports an effective and valuable protocol for thalassaemia screening in red blood cells which would otherwise be discarded during cord blood processing. Cord blood with severe and intermediate forms of thalassaemia can be preselected and not stored

Effects of transfusion and splenectomy on globin chain expression in NTDT HbE/β-thalassaemia

Introduction: Majority of HbE/β-thalassaemia patients resembles the phenotype of non-transfusion dependent thalassaemia (NTDT). Current management strategies are highly diverse, and the objective of this study is to examine the effects of different treatments on multiple parameters in NTDT HbE/β-thalassaemia to further streamline the management of this disorder. Methods: In this cross-sectional study, we analysed the correlation between different treatment strategies with variable parameters including haematological parameter and globin gene expression. Statistical analyses were carried out using non-parametric tests such as Kruskal-Wallis and Mann-Whitney tests. Results: A total of 29 HbE/β-thalassaemia patients were included in the study. Data showed statistically significant differences were observed in the MCV, MCHC levels, reticulocyte count and log α/β fold change between the groups. Further analysis showed higher log α/β fold change in the transfusion only group compared to the non-treated group. Red blood cell count was found to be lower in transfused and splenectomised group compared to transfusion only. Significantly higher MCV level and reticulocyte count was seen in transfusion and splenectomised group compared to both non-treated and transfusion only groups and higher MCH level in the transfusion and splenectomised group compared to transfusion only group. Conclusion: In general, regardless of single or double combined therapies, HbE/β-thalassaemia showed variable changes in laboratory parameters to the therapies received particularly splenectomy

Primary plasma cell leukaemia

Plasma cell leukemia (PCL) is a rare form of malignant plasma cell dyscrasia. It can occur as a primary form without prior evidence of multiple myeloma or as a secondary form which is a terminal event in multiple myeloma. It is characterised by a proliferation of plasma cells in blood and the bone marrow. The outcome of plasma cell leukemia is poor with conventional therapy. Here we illustrate a case of primary plasma cell leukemia complicated by paraplegia. The patient initially responded to combination chemotherapy but succumbed to the disease two months after presentation