In Malaysia, alpha-thalassaemia, beta-thalassaemia, haemoglobin (Hb) E, deltabeta-thalassaemia and Hb Constant Spring are prevalent. It has been estimated that 1 in 4 persons carries one of the above genetic abnormalities. In clinical practice, the major problems are: Hb Bart's hydrops fetalis (homozygous alpha(o)thalassaemia), homozygous 3(o)-thalassaemia, E-alpha thalassaemia and HbH disease. The laboratory procedures for diagnosis are standardised and the molecular basis of most of these genetic abnormalities are characterised. Thus it is possible to formulate a strategy for the detection and prevention of these disorders. The steps include the setting-up of population screening and genetic counselling service for the affected individuals, Society of Thalassaemias for public education and group support, and prenatal diagnosis with selective abortion of affected pregnancies. We embarked on such a programme between 1988 and 1992 in Kuala Lumpur General Hospital and hope to kindle similar effort in other state hospitals.
Myelodysplastic syndromes appear to be a rare group of blood disorders in Malaysia. It usually affects the elderly and some of the cases were reported to evolve into acute leukemia. We describe our experience with five cases, seen and managed by us over a 5-year period. All patients receiving supportive therapy died with one of them definitely known to transform to acute leukemia. The patient who survives till the time of writing received low dose continuous cytosine arabinoside infusion.
Hemoglobin S (HbS, α2β26GluVal) merupakan variasi hemoglobin yang terbentuk hasil daripada mutasi GAG GTG pada kodon 6 gen β-globin. Hemoglobinopati haemoglobin S (HbS) jarang ditemui di kalangan penduduk Malaysia tetapi selalunya dijumpai di kalangan pendatang asing dari Afrika. Walau bagaimanapun beberapa kes didapati dalam kaum India dan Melayu. Kajian ini meninjau keputusan makmal pesakit HbS dan penggunaan “multiplex ligation-dependent probe amplification” (MLPA) dan “flow-through hybridization” (FTH) dalam mengesan mutasi HbS. HbS dikenalpasti melalui kromatografi cecair prestasi tinggi (HPLC) dan/atau elektroforesis kapilari serta elektroforesis hemoglobin. Analisis molekul dijalankan menggunakan kaedah MLPA, FTH dan penjujukan Sanger. Dua warga Afrika, tiga Melayu dan dua India berusia antara 2-31 tahun telah dikenalpasti. Lima pesakit adalah HbS homozigot, seorang kompaun heterozigot HbS/β-talasemia dan seorang lagi pembawa HbS. Tahap hemoglobin (Hb) kes HbS homozigot adalah antara 7.4-10.2 g/dL dengan aras HbS dan HbF diantara 58.3-94.7% dan 1.5-35.5%. Hb untuk kes kompaun heterozigot HbS/β-talasemia adalah 5.8 g/dL dan normal pada pembawa HbS. Aras HbS, HbF dan HbA2 untuk HbS/β-talasemia dan pembawa HbS adalah 67%, 27.2% dan 4.2%, dan 38.6%, 0.1% and 2.8% setiap satu. Kedua-dua kaedah MLPA dan FTH berjaya mengesan mutasi HbS dalam semua kes, manakala cuma FTH dapat menentukan zygositi mutasi HbS dan β-talasemia dalam satu ujian yang sama.
We report two cases of biphenotypic acute leukaemia diagnosed in Hospital Universiti Kebangsaan Malaysia (HUKM), their clinical, haematological characteristics and response to chemotherapy. Both patients are middle-aged ladies who presented with hepatosplenomegaly and high white cell count, mainly composed of blast cells. Their bone marrow aspirations were hypercellular comprising of more than 90% heterogenous blast cells. Cytochemical analyses show more than 3% positivity towards peroxidase, with smaller blasts showing block positivity towards PAS. Immunophenotypically, the blasts showed dual expression of CD 33 and CD 19, CD 19 and CD34, intra CD22, intra TdT and intraMPO. One of the patients showed presence of the Philadelphia chromosome on cytogenetic analysis which was confirmed by Fluorecsence In Situ Hybridisation (FISH). Molecular analysis also showed presence of the BCR-ABL fusion protein. Both patients were given combination chemotherapy consisting of daunorubicin and cytosine arabinoside.However, the patient with positive Philadelphia chromosome BCR-ABL did not achieve morphological remission after induction chemotherapy. In view of the poor prognosis of this disease, both the patients were planned for upfront peripheral blood stem cell transplantation.
A number of genetic risk factors have been implicated in the development of neonatal severe hyperbilirubinaemia. This includes mutations in the uridine glucoronosyl transferase 1A1 (UGT1A1) gene which is responsible for unconjugated hyperbilirubinemia in Gilbert's Syndrome. We studied the prevalence of UGT1A1 gene mutations in a group of Malay neonates to determine whether they are risk factors to severe neonatal jaundice. One hundred and twenty-five Malay neonates with severe hyperbilirubinemia were studied. Ninety-eight infants without severe hyperbilirubinaemia were randomly selected from healthy Malay term infants (controls). DNA from EDTA cord blood samples were examined for UGT1A1 mutations nt211G > A and nt247T > C using established Taqman SNP genotyping assays and the UGT1A1*28 variant was detected by the Agilent 2100 bioanalyzer. All samples were also screened for common Malay G6PD variants using established techniques. The frequency of UGT1A1 211G > A mutation is significantly higher in the severely hyperbilirubinemic group (13%) than the control group (4%; p = 0.015) and all the positive cases were heterozygous for the mutation. There was no significant difference in the frequency of UGT1A1*28 mutation between the severely hyperbilirubinemic (3.5%) and the control group (0.01%; p = 0.09). None of the neonates in both groups carried the nt247 T > C mutation. The prevalence of G6PD mutation was significantly higher in the severely jaundiced group than control (9% vs 4%; p = 0.04). In conclusion, nt 211 G > A alleles constitute at least 12% of UGT1A1 mutations underlying unconjugated hyperbilirubinemia and appears to be a significant independent risk factor associated with severe neonatal hyperbilirubinemia in the Malay newborns.
All-trans retinoic acid therapy induces maturation in acute promyelocytic leukaemia. We document in vivo evidence of differentiation by all-trans retinoic acid in a case of acute promyelocytic leukaemia which was characterized by cytoplasmic vacuolations.
DNA analysis for the diagnosis of beta-thalassaemia is a relatively new technique in Malaysia. This, combined with chorionic villus sampling, has enabled us to offer prenatal diagnosis in the first trimester for this common condition. To the best of our knowledge, this has not hitherto been reported in Malaysia.
Most methods used in double esterase cytochemistry for the diagnosis and classification of acute myeloid leukaemias require double incubation and staining, using separate coupling reagents. We evaluated a method by Swirsky on our normal and abnormal blood and bone marrow smears where only a single incubation and the use of a single coupling reagent is required. Its short incubation period and its strong positive reaction for butyrate esterase in demonstrating cells of monocytic lineage gives it an advantage over the conventional double incubation technique.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide including Malaysia. Screening of cord blood for partial G6PD deficiency is important as they are also prone to develop acute haemolysis. In this study, we determined the prevalence of partial G6PD deficient in paediatric population aged 1 month-12 years and normal term female neonates using OSMMR-D kit with haemoglobin (Hb) normalization and compare it with florescence spot test (FST). A total of 236 children, aged between between 1 month-12 years and 614 normal term female neonates were recruited for this study. Determination of normal means for G6PD activity and; cut-off points for partial and severe deficiency were determined according to WHO Working Group (1989). Determination of prevalence for partial deficiency for both groups (female patient) was done using this enzyme assay kit and findings were compared with FST. In this study, 15.7% (18/115) female children were classified as partial G6PD deficient by quantitative enzyme method (G6PD activity: 4.23-5.26U/gHb). However, FST only detected 0.9% (1/115) with minimal G6PD activity. The prevalence of partial G6PD deficiency in female neonate group was 3.42% (21/614) by enzyme assay versus 0.49% (3/614) by FST. This study concluded that our routine screening method using FST was unable to diagnose female heterozygotes. We recommend using this quantitative enzyme assay method by OSMMR-D kit since it was more sensitive in detecting G6PD deficiency in female neonates compared to FST.
Mesenchymal stromal cells (MSC) are an attractive cell-targeting vehicle for gene delivery. MIDGE (an acronym for Minimalistic, Immunologically Defined Gene Expression) construct is relatively safer than the viral or plasmid expression system as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. The objective of this study was to test the ability of the human MSC (hMSC) to deliver the erythropoietin (EPO) gene in a nude mice model following nucleofection using a MIDGE construct. hMSC nucleofected with MIDGE encoding the EPO gene was injected subcutaneously in Matrigel at the dorsal flank of nude mice. Subcutaneous implantation of nucleofected hMSC resulted in increased hemoglobin level with presence of human EPO in the peripheral blood of the injected nude mice in the first two weeks post-implantation compared with the control groups. The basal layer of the hair shaft in the dermal layer was found to be significantly positive for immunohistochemical staining of a human EPO antibody. However, only a few basal layers of the hair shaft were found to be positively stained for CD105. In conclusion, hMSC harboring MIDGE-EPO could deliver and transiently express the EPO gene in the nude mice model. These cells could be localized to the hair follicle and secreted EPO protein might have possible role in hair regeneration.
Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.
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.
This study aimed to determine the prevalence of four variants of organic anion transporter polypeptide 2 (OATP2) gene, and their association with severe hyperbilirubinemia.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the commonest causes of neonatal jaundice in Malaysia. Screening of cord blood for G6PD deficiency by the semiquantitative fluorescent spot test (FST) is performed in Malaysia but this test can miss cases of partial G6PD deficiency. The OSMMR-D kit assay measures G6PD activity and hemoglobin (Hb) concentration, allowing direct expression of results in U/gHb. We evaluated this method and established the normal range for G6PD activity in normal term neonates and adults. EDTA blood from 94 neonates and 295 adults (age 15-59 years old) with normal Hb and FST were selected. The normal means for G6PD activity for neonates and adults were 12.43 +/- 2.28 U/gHb and 9.21 +/- 2.6 U/gHb, respectively; the reference ranges for normal G6PD activity in neonates and adults were 10.15-14.71 U/gHb and 6.61-11.81 U/gHb respectively. There were no significant differences in mean normal G6PD activity between the Malays and Chinese racial groups or between genders. The upper and lower limit cut-off points for partial deficiency in neonates were 7.4 U/gHb (60% of the normal mean) and 2.5 U/gHb (20% of the normal mean), respectively. For adults, the upper and lower limit cut-off points for partial deficiency in adults were 5.52 U/gHb (60% of the normal mean) and 1.84 U/gHb (20% of the normal mean), respectively. The quantitation of G6PD enzymes using this OSMMR-D kit with Hb normalization was simple since the Hb was analyzed simultaneously and the results were reproducible with a CV of less than 5%.
Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder arising from somatic mutation of the X-linked PIG-A gene which leads to deficiency of the glycosylphosphatidylinositol (GP1) membrane anchor proteins such as CD 59 (MIRL: membrane inhibitor of reactive lysis) and CD 55 (DAF: decay accelerating factor). Allogeneic peripheral blood stem cell transplant (PBSCT) is a curative mode of treatment in symptomatic PNH patients. Assessment of donor chimerism for PBSCT can be performed by various methods including short tandem repeat loci (STR) and variable number of tandem repeats (VNTR). Flow cytometry, which is much cheaper and faster, also can be used to assess engraftment in patients with PNH. Engrafted patients will show the presence of CD 55 and CD 59 on their red cells and white cells. We describe here the usefulness of flow cytometry in the assessment of donor chimerism following allogeneic PBSCT, in a case of PNH.
A 2-year-old Chinese boy was referred to Hospital UKM for investigation of recurrent episodes of dark-coloured urine and pallor since birth. He was born prematurely at 34 weeks gestation and developed severe early-onset neonatal jaundice requiring exchange blood transfusion. Screening at birth showed Glucose-6-phosphate dehydrogenase (G6PD) deficiency. On admission, physical examination revealed pallor, jaundice and mild hepatomegaly. Results of laboratory investigations showed a hemoglobin level of 11.0 g/dl with a hemolytic blood picture, reticulocytosis of 20% and red cell G6PD activity reported as undetectable. The patient's DNA was analysed for G6PD mutations by PCR-based techniques and DNA sequencing and results showed a 24 bp deletion of nucleotide 953-976 in the exon 9 of the G6PD gene. DNA analysis was also performed on blood samples of the patient's mother and female sibling confirming their heterozygous status, although both showed normal red cell G6PD activity levels. The patient was discharged well and his parents were appropriately advised on the condition and the importance of taking folic acid regularly. This is a first case report in Malaysia of G6PD deficiency causing chronic-hemolytic anemia. The rare 24 bp deletion causes the G6PD Nara variant, previously reported only in two other unrelated males, a Japanese and a Portuguese both with chronic hemolytic anemia.
Rearrangement of the immunoglobulin heavy chain (IgH) gene has been used as a marker of lineage and clonality in the diagnosis of B lymphoproliferative disorders. A number of PCR-based techniques have been developed to overcome the disadvantages of Southern blotting, the standard technique in detecting IgH gene rearrangement. Using an established seminested PCR technique with consensus primers to the V and J regions of the IgH gene, we analysed DNA prepared from peripheral blood and/or bone marrow specimens from 30 cases of known B cell malignancies (16 chronic lymphocytic leukemia, 11 acute lymphoblastic leukemia and 3 Non-Hodgkin Lymphoma), 3 cases of T lymphoproliferative disease and 3 cases of reactive lymphocytosis diagnosed in Hospital UKM to detect rearranged IgH gene. We found that monoclonality as represented by the presence of rearranged IgH gene were demonstrated in all the 30 cases. The PCR findings showed 100% concordance with the Southern blot analysis results which also showed rearranged IgH bands in all the 30 cases. We also found that none of the cases of T lymphoproliferative diseases and reactive lymphocytosis showed presence of rearranged IgH band, suggesting that the amplification using the IgH primers is lineage-specific. In conclusion, we find the PCR a useful method to detect IgH gene rearrangement in peripheral blood and bone marrow specimen. Since the PCR results are comparable to that of the Southern blotting in demonstrating B cell monoclonality and owing to its many advantages we feel that it can replace the Southern blot technique for the diagnosis of B cell malignancies.
A comparative study was conducted to evaluate three different permeabilization methods: FACS Permeabilizing Solution (FPerm), CytoFix/CytoPerm Kit (CFP) and Paraformaldehyde-Tween 20 (PFT) reagents, in cytoplasmic labeling of myeloperoxidase (MPO). Peripheral blood cells from 23 healthy subjects were fixed and permeabilized according to the proposed procedures, prior to direct immunofluorescence staining with CD14, CD45, IgG1, IgG2 and MPO monoclonal antibodies (McAb). Subsequent flow cytometric analysis was performed on FACSCalibur flow cytometer (Becton Dickinson, BD). As far as the antigenic expression of MPO in normal samples is concerned, FPerm and CFP demonstrated better cytoplasmic staining by inducing minor effects on light-scattering properties of the cell populations, whereas PFT-treated samples showed a diminished ability to distinguish the cell types. However, the simple and rapid FPerm method required an earlier processing of samples since the stored whole blood samples (for more than 8 hours) tended to show a significant decrease of fluorescence intensity. We also have demonstrated that P/N ratio possesses added value in evaluation of cell reactivity in immunophenotyping, based upon the apparent nonspecific cytoplasmic staining of MPO in the lymphocyte population.
A study was undertaken to evaluate the ability of flow cytometric analysis of intracellular myeloperoxidase (MPO) in differentiating populations of lymphocytes (L), monocytes (M) and granulocytes (G), by means of lysed whole blood method. Anticoagulated blood from 23 normal individuals was lysed with FACS lysing solution and permeabilized with FACS permeabilizing solution before subjected to direct immunofluorescence staining. The geometric means of the fluorescence intensity were measured using FACSCalibur flow cytometer (Becton Dickinson). Populations of L, M and G were gated based on their light scatter characteristics and expression of CD14 and CD45. Then, the fluorescence intensity of MPO expression was studied in these individual cell populations. The results showed that fluorescence intensity of MPO was the strongest in G and weakest in L, whereas M showed intermediate fluorescence intensity. Our findings reveal that discrimination of these three cell types is achievable based upon the sole expression of intracellular MPO.