Detection of α-thalassaemia in neonates on cord blood and dried blood spot samples by capillary electrophoresis

Haemoglobin Bart’s (Hb Bart’s) level is associated with α-thalassaemia traits in neonates, enabling early diagnosis of α-thalassaemia. The study aimed to detect and quantify the Hb Bart’s using Cord Blood (CB) and CE Neonat Fast Hb (NF) progammes on fresh and dried blood spot (DBS) specimen respectively by capillary electrophoresis (CE). Methods: Capillarys Hemoglobin (E) Kit (for CB) and Capillarys Neonat Hb Kit (for NF) were used to detect and quantify Hb Bart’s by CE in fresh cord blood and dried blood spot (DBS) specimens respectively. High performance liquid chromatography (HPLC) using the β-Thal Short Programme was also performed concurrently with CE analysis. Confirmation was obtained by multiplex ARMS Gap PCR. Results: This study was performed on 600 neonates. 32/600 (5.3%) samples showed presence of Hb Bart’s peak using the NF programme while 33/600 (5.5%) were positive with CB programme and HPLC methods. The range of Hb Bart’s using NF programme and CB programme were (0.5–4.1%) and (0.5-7.1%), respectively. Molecular analysis confirmed all positive samples possessed α-thalassaemia genetic mutations, with 23/33 cases being αα/--SEA, four -α3.7/-α3.7, two αα/-α3.7 and three αα/ααCS. Fifty Hb Bart’s negative samples were randomly tested for α-genotypes, three were also found to be positive for α-globin gene mutations. Thus, resulting in sensitivity of 91.7% and 88.9% and specificity of 100% for the Capillarys Cord Blood programme and Capillarys Neonat Fast programme respectively. Conclusion: Both CE programmes using fresh or dried cord blood were useful as a screening tool for α-thalassaemia in newborns. All methods show the same specificity (100%) with variable, but acceptable sensitivities in the detection of Hb Bart.

Differential Expression Patterns of Leukaemia Associated Genes in Leukaemia Cell Lines Compared to Healthy Controls

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/ hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages.

Hb Lepore/β0-Thalassaemia With α+-Thalassaemia Interactions, A Potential Diagnostic Pitfall

Haemoglobin (Hb) Lepore is a variant Hb consisting of two α-globin and two δβ-globin chains. In a heterozygote, it is associated with clinical findings of thalassaemia minor, but interactions with other haemoglobinopathies can lead to various clinical phenotypes and pose diagnostic challenges. We reported a pair of siblings from a Malay family, who presented with pallor and hepatosplenomegaly at the ages of 21 months and 14 months old. The red cell indices and peripheral blood smears of both patients showed features of thalassaemia intermedia. Other laboratory investigations of the patients showed conflicting results. However, laboratory investigation results of the parents had led to a presumptive diagnosis of compound heterozygote Hb Lepore/β-thalassaemia and co-inheritance α+-thalassaemia (-α3.7). Hb Lepore has rarely been detected in Southeast Asian countries, particularly in Malaysia. These two cases highlight the importance of family studies for accurate diagnosis, hence appropriate clinical management and genetic counseling.

Comparison of Abtectcell III and Diamed red cell antibody screening kit for detection of clinically significant red cells alloantibody

Antibody screening is important for the antenatal screening and pre-transfusion tests. This study aimed to compare the MUT/Mur kodecytesAbtectcell III (CSL Abtectcell III) red cell antibody screening kit with DiaMed ID-Dia Cell I-II-III Asia that was then used in our laboratory. In this study, 125 samples were randomly chosen, with 67 samples of known antibody specificities and 58 samples identified as negative for antibody, as the negative control. Concordant negative results were obtained in 57 out of 58 antibody negative samples. Concordant antibody positive results with both reagents were seen in 49 out of 67 samples. There were 18 discrepant results of antibody screening with CSL Abtetcell III (16/18 for vMNS antibodies). The sensitivity and specificity for CSL Abtectcell III were 73.0% and 98.3% respectively. In conclusion, the CSL Abtectcell III reagent would be an acceptable alternative for screening of red cell alloantibodies. It was able to detect all the clinically significant alloantibodies.

Molecular characteristic of alpha thalassaemia among patients diagnosed in UKM Medical Centre

Alpha (α) thalassaemia is the most common inherited disorder in Malaysia. The clinical severity is dependant on the number of α genes involved. Full blood count (FBC) and haemoglobin (Hb) analysis using either gel electrophoresis, high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) are unable to detect definitively alpha thalassaemia carriers. Definitive diagnosis of α-thalassaemias requires molecular analysis and methods of detecting both common deletional and non-deletional molecular abnormailities are easily performed in any laboratory involved in molecular diagnostics. We carried out a retrospective analysis of 1623 cases referred to our laboratory in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for the diagnosis of α-thalassaemia during the period October 2001 to December 2012. We examined the frequency of different types of alpha gene abnormalities and their haematologic features. Molecular diagnosis was made using a combination of multiplex polymerase reaction (PCR) and real time PCR to detect deletional and non-deletional alpha genes relevant to southeast Asian population. Genetic analysis confirmed the diagnosis of α-thalassaemias in 736 cases. Majority of the cases were Chinese (53.1%) followed by Malays (44.2%), and Indians (2.7%). The most common gene abnormality was αα/--SEA (64.0%) followed by αα/-α3.7 (19.8%), -α3.7 /--SEA (6.9%), αα/ααCS (3.0%), --SEA/--SEA (1.2%), -α3.7/-α3.7 (1.1%), αα/-α4.2 (0.7%), -α4.2/--SEA (0.7%), -α3.7/-α4.2 (0.5%), ααCS/-- SEA (0.4%), ααCS/ααCd59 (0.4%), ααCS/ααCS (0.4%), -α3.7/ααCd59 (0.3%), αα/ααCd59 (0.1%), αα Cd59/ ααIVS I-1 (0.1%), -α3.7/ααCS (0.1%) and --SEA /ααCd59 (0.1%). This data indicates that the molecular abnormalities of α-thalassaemia in the Malaysian population is heterogenous. Although α-gene deletion is the most common cause, non-deletional α-gene abnormalities are not uncommon and at least 3 different mutations exist. Establishment of rapid and easy molecular techniques is important for definitive diagnosis of alpha thalassaemia, an important prerequisite for genetic counselling to prevent its deleterious complications.

HbA2 levels in normal, B-thalassaemia and haemoglobin E carriers by capillary electrophoresis

Objective: The capillary electrophoresis (CE) is a new system that utilizes the principle of electrokinetic separation of molecules in eight electrolyte buffer-fi lled silica capillaries. In this study, we established the normal ranges of haemoglobin A2 (HbA2) and haemoglobin F (HbF) levels for normal individuals using this system and also the HbA2 level in β thalassaemia and haemoglobin E (HbE) individuals. Materials and Methods: 154 samples from normal individuals, 218 samples from β thalassaemia heterozygotes and 91 samples from HbE heterozygotes were subjected to high performance liquid chromatography (HPLC) and CE analysis. Results: The normal ranges for HbA2 and HbF by CE were 2.75% (SD 0.26%) and 0.03% (SD 0.24%) respectively, which were signifi cantly lower than that of HPLC 2.88% (SD 0.25%) and 0.58% (SD 0.61%) (p <0.001). The HbA2 level for HbE heterozygotes was 3.58% (SD 0.44%), which was signifi cantly higher than normal (p <0.001) but lower than that of β-thalassaemia heterozygotes (p<0.001) and the true HbE level was 24.28% (SD 3.38%). Conclusion: The CE system provided a fully automated and high throughput system for haemoglobin analysis. We established the normal ranges for HbA2 and HbF levels by CE. We also determined the ranges for HbA2 in beta thalassaemia and HbE heterozygotes using this system.

Co-inheritance of compound heterozygous Hb Constant Spring and a single –α3.7 gene deletion with heterozygous δβ thalassaemia: A diagnostic challenge

Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (α) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (β) and delta (δ) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave’s disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for α globin gene mutations showed a single -α-3.7 deletion and Hb CS mutation. These fi ndings were suggestive of compound heterozygosity of Hb CS and a single -α-3.7 deletion with a concomitant heterozygous δβ thalassaemia. Co-inheritance of Hb CS and a single -α-3.7 deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modifi cation of α thalassemia presumptively as a result of co-inheritance with δβ chain abnormality as suggested by the high Hb F level.

Myeloid sarcoma of the urinary bladder with cutaneous tumour seeding after percutaneous suprapubic catheterization

Myeloid sarcoma (MS) is a rare extramedullary myeloid tumour. It has been reported in various sites, including lymph node, bone, skin, soft tissue, various organs and the CNS. It may precede or occur concurrently with acute myeloid leukemia. Urinary bladder involvement is extremely uncommon. We report a 70-year-old female who had MS of the urinary bladder, presented with frank and persistent hematuria associated with lower abdominal pain. She subsequently had tumour seeding in the abdominal skin via percutaneous suprapubic catheter. Tumours from both the urinary bladder and skin showed immature cells that were immunoreactive toward LCA (focal), MPO (strong), CD99 (weak) and CD117 (weak). Summary of cases in the literature is presented. The potential of its misdiagnosis and the useful markers for the diagnosis of MS are discussed

A family study of HbS in a Malay family by molecular analysis

Sickle cell disease (SCD) is an inherited red cell disorder, characterized by the tendency of haemoglobin S or sickle haemoglobin to polymerize and assume a characteristic sickle shape. Molecular analysis has been the mainstay of detection method when confirmation is required. Previously a polymerase chain reaction (PCR)-based restriction enzyme analysis was used for this purpose. A simple bidirectional allele-specific amplification, recently described by Waterfall in 2001 was used to detect the GAG --> GTG mutation on codon 6 of the beta globin gene. Two sets of primers for the mutant and the wild type alleles were used in a single PCR reaction to amplify the regions of interest. The resultant PCR products will produce two fragments at 517 and 267 base pair (bp) respectively. This report highlights the investigations for SCD in the family of a 16-year old girl with recurrent painful crisis affecting the lower limbs whereby the family members are asymptomatic for the disease. Her haemoglobin electrophoresis at an alkaline pH showed dense bands at the HbS and HbF regions, while her father and two sisters had bands at HbS, HbF and HbA. The PCR analysis showed that she was homozygous for the mutation by the presence of only one band at 267 bp fragment, while the father and her sisters were heterozygotes, with the presence of two bands at 267 as well as 517 bp fragments. DNA sequencing of the sample confirmed the mutation. In conclusion, this case report highlighted the simple and cheap yet practical method for molecular confirmation of the presence of HbS gene in subjects with homozygous or heterozygous state of the condition.

Bone marrow necrosis preceding infantile acute lymphoblastic leukaemia

We report a case of bone marrow necrosis preceding infantile acute lymphoblastic leukaemia (ALL). Bone marrow necrosis is a rare antemortem event and has been known to be present in many conditions, notably in haematological malignancies like acute lymphoblastic leukaemia. This case was a 6-month-old Chinese boy who was referred to Hospital Universiti Kebangsaan Malaysia for further investigation of pancytopaenia, high-grade fever, bloody diarrhoea and petechial rashes for one week. His first bone marrow aspirate revealed bone marrow necrosis. His clinical condition improved after ten days. However, his full blood picture then revealed the presence of 5% blast cells. His subsequent marrow 2 weeks later revealed acute lymphoblastic leukaemia (FAB-L1) and immunophenotyping showed precursor B acute lymphoblastic leukaemia-null type. He was started on United Kingdom Acute Lymphoblastic leukaemia (UK ALL) Infantile Leukaemia protocol, however, he defaulted treatment after 3 days. Mode of presentation, mechanism of disease and laboratory investigations and outline of treatment will be discussed.