Centrifugation of blood samples to produce platelet-poor plasma is one of the important steps for coagulation testing. Reduction of the time required for specimen processing without affecting quality of results should be ideal for tests which require immediate results. Centrifugation of platelet-poor plasma (3580 rpm) for 15 minutes performed for routine coagulation tests would prolong the turn-around time for an urgent test (30 minutes). This study was done to determine the effect of reducing centrifugation time for routine coagulation tests in order to meet the turn-around time (TAT) for urgent tests. Seventy-nine blood samples sent for routine coagulation tests, were assayed for prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen level and platelet counts, using two different centrifugation speed for plasma preparation: centrifugation at 3580 rpm for 15 minutes and rapid centrifugation at 4000 rpm for five minutes. Paired sample t-test showed that there was a significant
difference in the platelet count between the two groups (p=0.001). However, there was no significant difference in the normal APTT (p=0.16), abnormal APTT (p=0.80), abnormal PT (p=0.43) and the results of fibrinogen levels (p=0.36). In conclusion, rapid centrifugation at 4000 rpm for five minutes does not modify results of routine coagulation tests (PT, APTT and fibrinogen). It would be beneficial in providing rapid results for urgent coagulation tests.
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.
Leukaemic stem cells have heterogenous differentiation potential. The immunophenotypes of blast cells are usually consistent throughout the disease course even at relapse. Rarely, blast cells may undergo a ‘lineage switch’ during the course of disease especially during relapse. We would like to highlight such a case in a 10- year old boy who presented with a two weeks history of lethargy, poor appetite, low grade fever, respiratory distress, cardiac failure, generalized oedema and hepatosplenomegaly. Full blood count showed a leucocyte count of 41.5x10 9 /L and platelet count of 37x10 9 /L. The peripheral blood film showed presence of numerous blast cells. Bone marrow aspiration revealed a hypercellular marrow, which consisted of mainly blast cells with high nuclear to cytoplasmic ratio and inconspicuous nucleoli. Immunophenotyping and cytochemistry results were consistent with the diagnosis of Tcell acute lymphoblastic leukaemia. The patient achieved remission after treatment with UK ALL 97 protocol, regime B chemotherapy. However, he relapsed seven months after the initial diagnosis with 26% blast cells in the bone marrow aspirate. The majority was L1 blast cells admixed with some L2 blast cells. Immunophenotyping was consistent with common precursor B acute lymphoblastic leukaemia. The treatment was changed to a more lineage specific chemotherapy. Nonetheless, the patient never achieved remission and was planned for palliative management. This case illustrated a unique and rare case of rapid lineage switch from T-cell acute lymphoblastic leukaemia to common precursor B-cell acute lymphoblastic leukaemia.
Chronic myeloid leukemia (CML) patients who have BCR-ABL T315I mutation, usually present in the advance phase of the disease with overall survival (OS) shorter than those without the mutation. This study aimed to determine the prevalence of T315I mutation amongst imatinib mesylate (IM) resistant CML patients and to compare the OS between T315I-mutated and non-T315I-mutated patients. Sixty consecutive CML patients who were treated with IM for at least 18 months and their treatment responses, were recorded. The mutation analysis was done using allele-specific oligonucleotide reverse transcriptase-polymerase chain reaction (RT-PCR) assay followed by direct sequencing technique. Forty-two patients (70%) were found to have IM-resistance. Five out of 42 patients had detectable T315I mutation. Median OS of IM-resistant T315I-mutated patients was 96 months (95% CI:54-138) compared to 84 months (95% CI:48-120) in non T315I-mutated patients, although this was found to be statistically insignificant (p = 0.43). The present study showed a higher prevalence of T315I mutation as compared to a few local studies. Median OS of T315I-mutated patients were observed to be longer than non-T315-mutated patients. Further studies encompassing larger cohort of patients are required to confirm this finding.
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.
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.
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.
Prenatal diagnosis is essential in the new era of diagnosis and management of genetic diseases in obstetrics. Multiple ligation-dependent probe amplification (MLPA) is a recent technique for prenatal diagnosis for the relative quantification of 40 different nucleic acid sequences in one single reaction. We had utilized the MLPA technique in detecting aneuploidies in amniotic fluid samples from 25 pregnant women from the Obstetrics and Gynaecology Department UKMMC, versus the quantitative fluorescent polymerase chain reaction (QF-PCR) method. Conclusive results were obtained in 18 cases and all were concordant with that of the QF-PCR. All four cases of trisomies were correctly identified including one case with maternal cell contamination.
Paraproteinemia is one of the diagnostic features of multiple myeloma. A commonly used method is the detection of paraprotein by agarose gel electrophoresis (AGE) followed by by immunofixation electrophoresis (IFE) to confirm monoclonality. Due to their smaller size, immunoglobulin A (IgA) and light chain only paraproteins may appear at the beta or even alpha 2 protein fractions. Here, we discuss a case report of a 47-year-old man who presented with pathological fracture of third thoracic (T3) vertebra. Serum protein electrophoresis (SPE) was initially reported as no paraprotein detected. However, a bone biopsy was reported to show plasma cell proliferation with light chain restriction. A repeat sample for protein electrophoresis together with IFE revealed lambda light chain paraprotein co-migrating at the beta region. The beta band plus paraprotein was quantitated as 4.3 g/L (7.0%), which was within normal limits of the beta protein fraction. Hence, it has to be remembered that if the SPE is negative, it does not necessarily mean that the paraprotein is absent in cases which are highly suspicious.
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/-Α(3.7) (19.8%), -Α(3.7) /--(SEA) (6.9%), ΑΑ/ΑΑCS (3.0%), --(SEA)/--(SEA) (1.2%), -Α(3.7)/-Α(3.7) (1.1%), ΑΑ/-Α(4.2) (0.7%), -Α(4.2)/--(SEA (0.7%), -Α(3.7)/-Α(4.2) (0.5%), ΑΑ(CS)/-- SEA) (0.4%), ΑΑ(CS)/ΑΑ(Cd59) (0.4%), ΑΑ(CS)/ΑΑ(CS) (0.4%), -Α(3.7)/ΑΑ(Cd59) (0.3%), ΑΑ/ΑΑ(Cd59) (0.1%), ΑΑ(Cd59)/ ΑΑ(IVS I-1) (0.1%), -Α(3.7)/ΑΑ(CS) (0.1%) and --(SEA) /ΑΑ(Cd59) (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.
The capillary electrophoresis (CE) is a new system that utilizes the principle of electrokinetic separation of molecules in eight electrolyte buffer-filled 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 beta thalassaemia and haemoglobin E (HbE) individuals.