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The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations

Teh LK, Lee TY, Tan JA, Lai MI, George E

Int J Lab Hematol, 2015 Feb;37(1):79-89.
PMID: 24725998 DOI: 10.1111/ijlh.12240

In Malaysia, β-thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR-incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β-thalassaemia mutation detection. ARMS allows single-mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β-thalassaemia mutations can be identified in samples with low DNA concentrations.
Molecular study and genotype/phenotype correlation of β Thalassemia in Malaysia

Sivalingam M, Looi ML, Zakaria SZ, Hamidah NH, Alias H, Latiff ZA, et al.

Int J Lab Hematol, 2012 Aug;34(4):377-82.
PMID: 22335963 DOI: 10.1111/j.1751-553X.2012.01405.x

INTRODUCTION: To study the ß-gene mutations spectrum, the genotype/phenotype correlation, the modulatory effect of co-inherited factors such as α-gene mutations and of Xmn1 polymorphism in a large cohort of Malaysian patients.
METHODS: A total of 264 cases clinically diagnosed as Thalassemia major (TM) (111), Thalassemia intermedia (21), HbE-β Thalassemia (131), and 1 HbE homozygous were studied. The detection of α and ß gene mutations and characterization of Xmn1 polymorphism were performed by multiplex PCR, amplification refractory mutation system (ARMS), DNA sequencing, and restriction fragment length polymorphism (RFLP)-PCR.
RESULTS: A total of 19 ß Thalassemia mutations were characterized. CD26 and CD41/42 were the most common found in the Malay and Chinese population, respectively. The sensitivity of the clinical diagnosis for β TM, thalassemia intermedia, and HbE/β thalassemia was 94.0%, 15.2%, and 89.2%, respectively. Patients with Xmn1 heterozygosity [+/-] required less frequent transfusion compared with those without the polymorphism. Co-inheritance of α-thalassemia alleviates the severity of HbE-β thalassemia in our cohort.
CONCLUSION: Molecular analysis should be used for a better diagnosis and management of β thalassemia.
Modification to reporting of qualitative fluorescent spot test results improves detection of glucose-6-phosphate dehydrogenase (G6PD)-deficient heterozygote female newborns

Nadarajan V, Shanmugam H, Sthaneshwar P, Jayaranee S, Sultan KS, Ang C, et al.

Int J Lab Hematol, 2011 Oct;33(5):463-70.
PMID: 21501392 DOI: 10.1111/j.1751-553X.2011.01309.x

INTRODUCTION:
The glucose-6-phosphate dehydrogenase (G6PD) fluorescent spot test (FST) is a useful screening test for G6PD deficiency, but is unable to detect heterozygote G6PD-deficient females. We sought to identify whether reporting intermediate fluorescence in addition to absent and bright fluorescence on FST would improve identification of mildly deficient female heterozygotes.

METHODS:
A total of 1266 cord blood samples (705 male, 561 female) were screened for G6PD deficiency using FST (in-house method) and a quantitative enzyme assay. Fluorescence intensity of the FST was graded as either absent, intermediate or normal. Samples identified as showing absent or intermediate fluorescence on FST were analysed for the presence of G6PD mutations using TaqMan@SNP genotyping assays and direct nucleotide sequencing.

RESULTS:
Of the 1266 samples, 87 samples were found to be intermediate or deficient by FST (49 deficient, 38 intermediate). Of the 49 deficient samples, 48 had G6PD enzyme activity of ≤ 9.5 U/g Hb and one sample had normal enzyme activity. All 38 intermediate samples were from females. Of these, 21 had G6PD activity of between 20% and 60%, and 17 samples showed normal G6PD activity. Twenty-seven of the 38 samples were available for mutation analysis of which 13 had normal G6PD activity. Eleven of the 13 samples with normal G6PD activity had identifiable G6PD mutations.

CONCLUSION:
Glucose-6-phosphate dehydrogenase heterozygote females cannot be identified by FST if fluorescence is reported as absent or present. Distinguishing samples with intermediate fluorescence from absent and bright fluorescence improves detection of heterozygote females with mild G6PD deficiency. Mutational studies confirmed that 85% of intermediate samples with normal enzyme activity had identifiable G6PD mutations.
An assessment of three noncommercial DNA extraction methods from dried blood spots for beta-thalassaemia mutation identification

Karthipan SN, George E, Jameela S, Lim WF, Teh LK, Lee TY, et al.

Int J Lab Hematol, 2011 Oct;33(5):540-4.
PMID: 21884505 DOI: 10.1111/j.1751-553X.2011.01304.x

Dried blood spots (DBS) are currently the recommended sample collection method for newborn screening programmes in America. Early diagnosis of beta-thalassaemia screening is essential as it provides an added advantage especially in sickle cell disease. Beta-thalassaemia frequency is high in many poor countries, and the cost of using commercial DNA extraction kits can be prohibitive. Our study assessed three methods that use minimal reagents and materials to extract DNA from DBS for beta-thalassaemia identification.
Development of an algorithm of satellite markers for monitoring chimerism status in post-allogeneic haematopoietic stem cell transplantation patients

Choong SS, Rosmanizam S, Ibrahim K, Gan GG, Ariffin H

Int J Lab Hematol, 2011 Apr;33(2):182-6.
PMID: 20868447 DOI: 10.1111/j.1751-553X.2010.01264.x

Analysis of variable number tandem repeats (VNTRs) by polymerase chain reaction (PCR) is a common method used to predict engraftment status in post-allogeneic haematopoeitic stem cell transplantation (HSCT) patients. Different populations have different copies of repeated DNA sequence and hence, different percentage of informativeness between patient and donor.
RBC-Y/MCV as a discriminant function for differentiating carriers of thalassaemia and HbE from iron deficiency

Nadarajan VS, Sthaneshwar P, Jayaranee S

Int J Lab Hematol, 2010 Apr;32(2):215-21.
PMID: 19566741 DOI: 10.1111/j.1751-553X.2009.01174.x

Individuals with alpha-thalassaemia (ATT), beta-thalassaemia (BTT) and HbE trait (HET) are often initially identified based on haematological parameters. However, the values of these parameters usually overlap with iron deficiency anaemia (IDA) and anaemia of chronic disease (ACD). We evaluated the use of RBC-Y in 156 normal individuals and 332 patients; ATT (n = 37), BTT (n = 61), HET (n = 25), HbH disease (n = 5), ACD (n = 67), IDA (n = 83) and ACD with IDA (n = 54). Diagnostic efficiency was analysed by receiver operating characteristics (ROC). MCH was better compared with RBC-Y in discriminating normal from abnormal with sensitivity and specificity of 94% at a cut-off of 26 pg. The Green and King (G&K) index performed the best in discriminating carriers from IDA and ACD with area under the ROC curve (AUC(ROC)) of 0.81. However, if ACD was excluded, RBC-Y/MCV was a good discriminator for carriers from IDA with AUC(ROC) = 0.845. In general screening of populations with ATT, BTT and HET, we propose that hypochromic individuals be first identified by MCH <26 pg and carriers distinguished within these hypochromic individuals from IDA by using RBC-Y/MCV. However, if the prevalence of ACD were high within the screening population, G&K index would be a more suitable discriminator.
RET-Y and RBC-Y in the diagnosis of iron deficiency associated with anaemia of inflammation

Jayaranee S, Sthaneshwar P

Int J Lab Hematol, 2010 Oct;32(5):512-8.
PMID: 20109166 DOI: 10.1111/j.1751-553X.2009.01215.x

We evaluated the usefulness of RET-Y and RBC-Y in distinguishing functional iron deficiency from iron-deficiency anaemia (IDA) in patients with anaemia of inflammation (AI). Sixty healthy blood donors constituted the control group. We studied RET-Y and RBC-Y in 115 patients with hypochromic/microcytic anaemia. Of these 42 patients had uncomplicated IDA and 73 had AI. The AI patients were further subdivided into AI with IDA and AI with functional IDA based on soluble transferrin receptor (sTfR) levels. The mean RBC-Y and RET-Y values in iron-deficient patients were 122.4 and 119.8, respectively, which were significantly lower than the control (P < 0.001). The mean level of RET-Y in patients with AI associated with IDA was 149.3 and this level in AI patients with functional iron deficiency was 147.4. RET-Y levels in both subgroups of AI patients were significantly lower than control but no significant difference was observed between the two subgroups. Similar findings were observed for RBC-Y. Receiver operating characteristic analysis also showed lower specificity for RBC-Y and RET-Y compared with that of sTfR and its log ferritin ratio (F-index). RET-Y and RBC-Y are useful in the diagnosis of simple IDA but have limited utility in the diagnosis of IDA with AI.
The utility of immature reticulocyte fraction as an indicator of erythropoietic response to altitude training in elite cyclists

Nadarajan VS, Ooi CH, Sthaneshwar P, Thompson MW

Int J Lab Hematol, 2010 Feb;32(1 Pt 2):82-7.
PMID: 19170774 DOI: 10.1111/j.1751-553X.2008.01132.x

Altitude training is sometimes employed by elite endurance athletes to improve their sea level performance. This improvement results from the increased red cell mass consequent upon the boost in erythropoietin (EPO) level that occurs as a response to the relatively hypoxic environment at high altitudes. We measured serum EPO levels together with various red cell and reticulocyte parameters including immature reticulocyte fraction (IRF) in eight national track-endurance cyclists, resident at sea-level, prior to and upon return from an altitude of approximately 1905 m. Reticulocytes and soluble transferrin receptor (sTfR) were significantly increased with reduction in ferritin levels immediately on return from high altitude indicating increased erythropoietic activity. IRF in particular showed a significant peak immediately on return but decline to sub-baseline levels by day 9, and recovery to baseline by day 16. Our results indicate that IRF is a sensitive marker of erythropoietic status in athletes undergoing altitude training and subsequent loss of EPO stimuli on return to sea level.
Hematological reference values of healthy Malaysian population

Roshan TM, Rosline H, Ahmed SA, Rapiaah M, Wan Zaidah A, Khattak MN

Int J Lab Hematol, 2009 Oct;31(5):505-12.
PMID: 18498389 DOI: 10.1111/j.1751-553X.2008.01068.x

Health and disease can only be distinguished by accurate and reliable reference values of a particular laboratory test. It is now a proven fact that there is considerable variation in hematology reference intervals depending on the demographic and preanalytical variables. There are evidences that values provided by manufacturers do not have appropriate application for all populations. Moreover, reference ranges provided by different laboratory manuals and books also do not solve this problem. We are presenting here normal reference ranges of Malaysian population. These values were determined by using Sysmex XE-2100 and ACL 9000 hematology and coagulation analyzers. Results from this study showed that there were considerable differences in the reference values from manufacturers, western population or laboratory manuals compared with those from the local population.
Age- and gender-related reference ranges for thromboelastography from a healthy Indian population

Ahammad J, Kurien A, Shastry S, Shah HH, Nayak D, Kamath A, et al.

Int J Lab Hematol, 2020 Apr;42(2):180-189.
PMID: 31889401 DOI: 10.1111/ijlh.13148

INTRODUCTION: Thromboelastography (TEG) is a whole blood clotting assay largely used in major surgeries and trauma to monitor patients' in vivo hemostatic status. Standardization of kaolin-activated citrated whole blood thromboelastography is not done in the Indian population. This study primarily aims to derive reference ranges of kaolin-activated TEG for healthy volunteers in the Indian population. Secondarily, it aims to study the age- and gender-related hemostatic changes in the study population.
METHODS: A total of 120 healthy volunteers were enrolled (55 adult males, 32 adult females, and 33 children). The volunteers were interviewed for any bleeding history or drug intake which affects coagulation. Kaolin-activated TEG was performed on citrated whole blood, and parameters including R-time, K-time, angle, MA, LY30, and CI were analyzed.
RESULTS: Derived reference range for total volunteers irrespective of age and sex were as follows: R-time: 3.8-10.6, K-time: 1.2-3.1, angle: 44.9-72.0, MA: 41.2-64.5, LY30: 0-9.9, and CI: -3.7 to 3.4. Statistically significant difference was observed in different age and sex groups for R-time, K-time, and angle. About 40% of the volunteers had at least one abnormal parameter according to the manufacturer's reference range which decreased to 12.5% when the derived reference ranges were considered.
CONCLUSION: Gender- and age-related variances were observed in reference ranges of our population and which was also differed from the other ethnic population. Many of our healthy volunteers were categorized as coagulopathic when manufacturer's reference range was considered. So, it is important to derive the reference range of the target population before using the TEG into clinical practice.
Rapid detection of α-thalassaemia variants using droplet digital PCR

Lee TY, Lai MI, Ramachandran V, Tan JA, Teh LK, Othman R, et al.

Int J Lab Hematol, 2016 Aug;38(4):435-43.
PMID: 27349818 DOI: 10.1111/ijlh.12520

INTRODUCTION: Alpha thalassaemia is a highly prevalent disease globally and is a well-known public health problem in Malaysia. The deletional forms of the mutation are the most common forms found in alpha thalassaemia. The three most common deletional alpha thalassaemia found in this region include --(SEA) deletion, -α(3.7) rightward and -α(4.2) leftward deletions. The prevalence rate of triplication alpha cases such as ααα(anti3.7) and ααα(anti4.2) is not known in Malaysia although it plays a role in exacerbating the clinical phenotypes in beta thalassaemia carriers. Recently, there have been more reported cases of rare alpha thalassaemia mutations due to the advancement of molecular techniques involved in thalassaemia detections. Therefore, it is essential to develop a new method which allows the detection of different alpha thalassaemia mutations including the rare ones simultaneously and accurately.
METHODS: The purpose of this study was to design an assay for the detection of triplications, common and rare deletional alpha thalassaemia using droplet digital PCR (ddPCR).
RESULTS: This is a quantitative detection method to measure the changes of copy number which can detect deletions, duplications and triplications of the alpha globin gene simultaneously.
CONCLUSION: In conclusion, ddPCR is an alternative method for rapid detection of alpha thalassaemia variants in Malaysia.
Percentage of hypochromic red cells as a potential screening test to evaluate iron status in blood donors

Amir N, Md Noor S, Subbiah I, Osman M, Seman Z

Int J Lab Hematol, 2019 Jun;41(3):418-423.
PMID: 30938931 DOI: 10.1111/ijlh.13009

INTRODUCTION: Haemoglobin (Hb) levels are used to assess eligibility for blood donation but are not correlated with iron status. The percentage of hypochromic red cells (%Hypo-He) has been suggested as a useful screening parameter for iron deficiency. The aim of this study was to determine the cut-off level and accuracy of %Hypo-He screening among blood donors.
MATERIALS AND METHODS: A total of 170 blood donors were recruited into the study. Blood donors were classified into three groups: normal, latent iron deficiency and iron deficiency anaemia based on their Hb, serum ferritin and transferrin saturation (TSAT) levels. The diagnostic performance of %Hypo-He was evaluated with a validation group comprising 160 blood donors.
RESULTS: Receiver operating characteristic (ROC) curve analysis showed that %Hypo-He is an excellent parameter for detecting iron deficiency, with an area under the curve (AUC) of 0.906, a confidence interval (CI) of 0.854-0.957 at a cut-off of 0.6%, and 74.51% sensitivity and 88.24% specificity. A moderate negative correlation between %Hypo-He and TSAT (ρ = -0.576 [P
Conformation sensitive gel electrophoresis for the detection of calreticulin mutations in BCR-ABL1-negative myeloproliferative neoplasms

Zakaria NA, Rosle NA, Siti Asmaa MJ, Aziee S, Haiyuni MY, Samat NA, et al.

Int J Lab Hematol, 2021 Dec;43(6):1451-1457.
PMID: 34125992 DOI: 10.1111/ijlh.13628

INTRODUCTION: Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients.
METHODS: Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing.
RESULTS: CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2.
CONCLUSION: CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.
The magnitude of tyrosine kinase inhibitor induced cytopaenia on normal haematopoietic tissues in chronic myeloid leukaemia patients

Kuan JW, Ng SY, Hon SL, Lim SM, Chai AJM, Teo HG, et al.

Int J Lab Hematol, 2023 Feb;45(1):e1-e5.
PMID: 35896146 DOI: 10.1111/ijlh.13940

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