Evaluation of quantitative point-of-care test for measurement of glucose-6-phosphate dehydrogenase enzyme activity in Malaysia.

INTRODUCTION: The treatment of Plasmodium vivax malaria with 8-aminoquinolines is contraindicated in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals due to the risk of acute haemolytic anaemia. Effective G6PD screening is paramount to avoid adverse drug reactions. This study aimed to evaluate the performance of novel quantitative point-of-care (POC) tests as a new screening method for G6PD deficiency in Malaysia. MATERIALS AND METHODS: A total of 153 neonatal cord blood, 99 peripheral blood of older children aged between 1 month to 12-years old, and 62 peripheral adult blood were screened for G6PD deficiency using two quantitative POC tests, CareStartTM biosensor (Carestart) and CareStartTM Biosensor 1 (S1). The results were compared with OSMMR2000D kit as a reference assay. Two statistical analyses were performed in this study to evaluate the POC test performances, the Spearman's correlation test and the Cohen's kappa method. RESULTS: Both Carestart and S1 tests showed significant positive correlations to OSMMRS000D with r2 = 0.7916 and r2 = 0.7467. Their measurement of agreement showed a kappa (κ) value of 0.805 (p<0.001, 95% CI), and 0.795 (p<0.001, 95% CI), respectively. Analysis of the area under the Receiver Operating Curve (ROC) at 60% cut-off illustrated that the Carestart had 90.2% sensitivity, 98.9% specificity, 98.3% positive predictive value (PPV), and 93.8% negative predictive value (NPV). The corresponding values for the S1 were 95.2%, 100%, 100%, and 96.8%, respectively. CONCLUSION: This study showed that the Carestart and S1 biosensors have high-performance reliability for screening of G6PD deficiency, which can guide safe prescriptions of anti-malaria medications and hence, eradication of Plasmodium vivax malaria.

Evaluation of mean neutrophil volume and immature to total neutrophil ratio as a biomarker for bacterial sepsis in adult patients.

INTRODUCTION: Mean neutrophil volume (MNV) and immature to total neutrophil ratio (IT Ratio) has been found to support the detection of sepsis in elderly and neonates. This study aimed to assess the diagnostic significance of MNV and IT ratio in adult sepsis population. MATERIALS AND METHODS: Sixty-four adult patients presented with suspected bacterial sepsis were included in this study. Relevant cultures and/or pertinent serology tests were performed. Full blood counts were analysed for MNV and IT ratio. RESULTS: Fifty-one patients out of 64 recruited subjects were confirmed sepsis. Twentyfour patients had confirmed bacterial infection by cultivation and two were positive for leptospiral serology. MNV was very good in distinguishing sepsis from non-sepsis group (AUC = 0.80, 95% confidence interval (CI) = 0.69-0.91, Accuracy = 0.72, Kappa = 0.40) with a cut-off value of 153.5 (sensitivity = 67%, specificity = 92%). There was no significant difference in IT ratio between sepsis and non-sepsis group (p-value > 0.05). MNV was superior over IT ratio (AUC = 0.85, 95%CI = 0.76-0.95, and AUC = 0.70, 95% CI = 0.56-0.85, respectively) in diagnosing bacterial infection. The optimum cut-off value for MNV in bacterial infection was 154.5 (sensitivity = 67%, specificity = 89%) and for IT ratio was 0.035 (sensitivity = 45%, specificity = 67%). CONCLUSION: MNV appears to be a very good marker for diagnosing sepsis and bacterial infection. We recommend including MNV into sepsis workup in ED setting, since it can be determined without additional specimen.

H396P mutation in chronic myeloid leukaemia patient on nilotinib - A case report.

INTRODUCTION: The advent of BCR-ABL1-targeted therapy with the tyrosine kinase inhibitor (TKI), for example, imatinib and nilotinib, marked a turning point in the therapy of chronic myeloid leukaemia (CML). However, a substantial proportion of patients experience primary or secondary disease resistance to TKI. There are multifactorial causes contributing to the treatment failure of which BCR-ABL1 kinase domain mutation being the most common. Here, we describe a case of a CML patient with H396P mutation following treatment with nilotinib. CASE: A 60-year-old woman presented with abdominal discomfort and hyperleukocytosis. She was diagnosed as CML in the chronic phase with positive BCR-ABL1 transcripts. Due to the failure to obtain an optimal response with imatinib treatment, it was switched to nilotinib. She responded well to nilotinib initially and achieved complete haematological and cytogenetic responses, with undetectable BCR-ABL1 transcripts. However, in 4 years she developed molecular relapse. Mutation analysis which was done 70 months after commencement of nilotinib showed the presence of BCRABL1 kinase domain mutation with nucleotide substitution at position 1187 from Histidine(H) to Proline(P) (H396P). Currently, she is on nilotinib 400mg twice daily. Her latest molecular analysis showed the presence of residual BCR-ABL1 transcripts at 0.22%. DISCUSSION/CONCLUSION: This case illustrates the importance of BCR-ABL1 mutation analysis in CML patients with persistent BCR-ABL1 positivity in spite of treatment. Early detection and identification of the type of BCRABL1 mutation are important to guide appropriate treatment options as different mutation will have different sensitivity to TKI.

Siriraj I Gγ(Aγδß)0-thalassaemia causing severe thalassaemia intermedia in compound heterozygous state with IVS1-1(G→T) mutation.

The Siriraj I Gγ(Aγδß)0-thalassaemia is a novel mutation involving a 118kb deletion of the ß-globin gene cluster. It was first reported in 2012 in two unrelated families from the southern part of Thailand. The carriers in the heterozygous state are clinically asymptomatic. Nonetheless, its complex interaction with other ß-thalassaemia could give rise to different clinical phenotypes, ranging from mild thalassaemia intermedia to thalassaemia major. We report here a case of a six-year-old Malay boy, presented with pallor, growth failure and hepatosplenomegaly. His haemoglobin at presentation was 9.2g/dL with a mean cell haemoglobin of 22.6pg and a mean cell volume of 69.9fl. His peripheral blood smear showed features of thalassaemia intermedia. Haemoglobin (Hb) analysis revealed markedly raised Hb F (83%), normal HbA2 levels and absent HbA. Deoxyribonucleic acid (DNA) analysis showed compound heterozygous IVS1-1 (G→T) ß-globin gene mutation and Siriraj I Gγ(Aγδß)0-deletion (genotype ßIVS1-1/ ß Siriraj I deletion). Both his father and elder sister are carriers of Siriraj I Gγ(Aγδß)0-thalassaemia while his mother carries IVS1-1 (G→T) gene mutation. Clinically, the patient is transfusion dependent on six weekly regime. To the best of our knowledge, this is the first reported case in Malaysia involving unique Siriraj I Gγ(Aγδß)0-thalassaemia and IVS1-1 (G→T) in a compound heterozygous state. In summary, detection of Siriraj I Gγ(Aγδß)0-thalassaemia is essential as this deletion can lead to severe disease upon interaction with a ß-thalassemia point mutation as demonstrated in our case. The establishment of effective carrier screening and genetic counselling is important to prevent its adverse consequences.

Microcytic to hypochromic ratio as a discriminant index of thalassaemia trait in subjects with hypochromic anaemia.

INTRODUCTION: Differentiating between thalassaemia and iron deficiency anaemia (IDA) in hypochromic anaemia is a challenge to pathologists as it influences the choice of subsequent specialized confirmatory tests. In this study, we aimed to evaluate the performance of microcytic to hypochromic ratio (MicroR/ Hypo-He, M/H ratio) as a discriminant index in hypochromic anaemia. MATERIALS AND METHODS: A retrospective study was carried out on 318 subjects with hypochromic anaemia, which comprised 162 IDA and 156 thalassaemia trait subjects with α-thalassemia, ß-thalassemia and HbE trait. Optimal cut-off value, sensitivity and specificity of M/H ratio for thalassaemia trait discrimination was determined using Receiver Operating Characteristic (ROC) analysis. RESULTS: Subjects with thalassaemia trait showed higher MicroR compared to IDA ( p< 0.001) while subjects with IDA demonstrated higher Hypo-He than thalassaemia trait (p < 0.001). M/H ratio was significantly higher in thalassaemia trait compared to IDA, with medians of 3.77 (interquartile range: 2.57 - 6.52) and 1.73 (interquartile range: 1.27 - 2.38), respectively (p < 0.001). M/H ratio ≥ 2.25 was the optimal cut-off value for discriminating thalassaemia trait from IDA in hypochromic anaemia, with the area under ROC curve (AUC) of 0.83, sensitivity of 80.8% and specificity of 71.6%. CONCLUSIONS: M/H ratio is a useful discriminant index to distinguish thalassaemia trait from IDA in hypochromic anaemia prior to diagnostic analysis for thalassaemia confirmation. High M/H ratio is suggestive of thalassaemia trait than of IDA. However, more studies are required to establish the role of M/H ratio as a screening tool for thalassaemia discrimination in hypochromic anaemia.

Early relapse after complete remission of primary plasma cell leukaemia manifesting clonal evolution: A case report.

INTRODUCTION: Plasma cell leukaemia (PCL) is a rare variant of multiple myeloma. We report a case of PCL to demonstrate the clonal evolution, resulting in disease relapse after achieving complete remission, and its aggressive nature of the disease, leading to poor clinical outcome. CASE REPORT: A 69-year-old man presented with a three-day-history of worsening generalized body weakness, poor oral intake, nausea, significant loss of weight and lower back pain. He was diagnosed as primary PCL, based on hypercalcaemia, renal insufficiency, anaemia, thrombocytopenia, lytic bone lesions, 24% abnormal plasma cells in peripheral blood, immunophenotype of clonal plasma cells which were positive for CD38, CD138 and CD56 markers with kappa light chain restriction, 49% abnormal plasma cells in bone marrow, monoclonal paraprotein (IgG kappa) in serum and urine, and positive IGH rearrangement (Fluorescence in-situ hybridisation, FISH). He achieved complete remission after four cycles of Bortezomib-based therapy. There was a plan for high-dose therapy plus autologous haematopoietic cell transplantation. A month later, the disease relapsed, as evidenced by 94% abnormal plasma cells in his bone marrow aspirate, complex karyotype and abnormal FISH results. He passed away a few days later, from severe septicaemia. Time-to-progression of disease was 1 month and overall survival was 5 months. DISCUSSION: This case report illustrates the clonal evolution and aggressive nature of primary PCL with older age at presentation, leading to a shorter duration of remission and overall survival.

FISH versus real-time quantitative PCR for monitoring of minimal residual disease in chronic myeloid leukaemia patients on tyrosine kinase inhibitor therapy.

INTRODUCTION: BCR-ABL fusion gene, the oncogenic driver of CML, results from a translocation between short arms of chromosome 9 and 22. Monitoring of CML patients during treatment is essential, not only for tailoring the treatment but also to detect early relapse to enable timely intervention. Commonly used methods for detection of residual disease are conventional karyotyping, FISH and molecular methods. In this study, we compared FISH with QRT-PCR for detection of residual disease in CML. MATERIALS AND METHODS: CML patients on tyrosine kinase inhibitor (TKI) therapy and on regular follow up at University Kebangsaan Malaysia Medical Center (UKMMC) were selected. A comparative study was conducted between FISH and QRT-PCR for BCR-ABL transcripts at diagnosis and during follow-up. RESULTS: There was good correlation between FISH and QRT-PCR for BCR-ABL. At 6th month of follow-up post diagnosis, FISH had a sensitivity of 83.3% and specificity of 65.2% (k >0.339, p<0.033). At 12th month, the sensitivity of FISH was 83% and the specificity was 59.1% (k >0.286, p <0.065). Similarly, at the 24th month, FISH had a sensitivity of 100% and specificity of 68.8% (k >0.642, p<0.000). DISCUSSION: Early achievement of major molecular response (MMR) and complete cytogenetic remission (CCyR) were reliable predictors of long-term maintenance of molecular remission.

Genetic Modifiers of Fetal Haemoglobin (HbF) and Phenotypic Severity in ß-Thalassemia Patients.

BACKGROUND: The phenotypic severity of ß-thalassemia is highly modulated by three genetic modifiers: ß-globin (HBB) mutations, co-inheritance of α-thalassemia and polymorphisms in the genes associated with fetal haemoglobin (HbF) production. This study was aimed to evaluate the effect of HbF related polymorphisms mainly in the HBB cluster, BCL11A (B-cell CLL/lymphoma 11A) and HBS1L-MYB (HBS1-like translational GTPase-MYB protooncogene, transcription factor) with regards to clinical severity. METHODS: A total of 149 patients were included in the study. HBA and HBB mutations were characterised using multiplex PCR, Sanger sequencing and multiplex ligationdependent probe amplification. In addition, 35 HbF polymorphisms were genotyped using mass spectrometry and PCR-restriction fragment length polymorphism (PCRRFLP). The genotype-phenotype association was analysed using SPSS version 22. RESULTS: Twenty-one HBB mutations were identified in the study population. Patients with HBB mutations had heterogeneous phenotypic severity due to the presence of other secondary modifiers. Co-inheritance of α-thalassemia (n = 12) alleviated disease severity of ß-thalassemia. In addition, three polymorphisms (HBS1LMYB, rs4895441 [P = 0.008, odds ratio (OR) = 0.38 (0.18, 0.78)], rs9376092 [P = 0.030, OR = 0.36 (0.14, 0.90)]; and olfactory receptor [OR51B2] rs6578605 [P = 0.018, OR = 0.52 (0.31, 0.89)]) were associated with phenotypic severity. Secondary analysis of the association between single-nucleotide polymorphisms with HbF levels revealed three nominally significant SNPs: rs6934903, rs9376095 and rs9494149 in HBS1L-MYB. CONCLUSION: This study revealed 3 types of HbF polymorphisms that play an important role in ameliorating disease severity of ß-thalassemia patients which may be useful as a predictive marker in clinical management.

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

INTRODUCTION: 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 ß-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.

α-Thalassemia with Haemoglobin Adana mutation: prenatal diagnosis.

Thalassaemia carriers are common in the Asian region including Malaysia. Asymptomatic patients can be undiagnosed until they present for their antenatal visits. Devastating obstetric outcome may further complicate the pregnancy if both parents are thalassaemia carriers leading to hydrophic fetus due to haemoglobin Bart's disease. However in certain cases where unexplained hydrops fetalis occur in parents with heterozygous thalassaemia carrier,mutated α genes should be suspected. We report a twenty-nine year old woman in her third pregnancy with two previous pregnancies complicated by early neonatal death at 21 and 28 weeks of gestation due to hydrops fetalis. DNA analysis revealed the patient to have heterozygous (--SEA) α-gene deletion, while her husband has a compound heterozygosity for α(3.7) deletion and codon 59 (GGC → GAC) mutation of the α-gene. This mutation, also known as hemoglobin Adana, can explain hydrops fetalis resulting from two alpha gene deletions from the patient (mother) and a single alpha gene deletion with mutation from the father. The third pregnancy resulted in a grossly normal baby boy with 3 α-gene deletions (HbH disease). We postulate that, in view of heterogenisity of the α-thalassaemia in this patient with severely unstable haemoglobin Adana chains from her husband, there will be a 25% possibility of fetal hydrops in every pregnancy.