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Hemoglobinopathies are major public health problems worldwide. Accurate laboratory diagnosis of the carrier is essential, which includes initial screening, Hb analysis, and DNA analysis. For the first time, we have developed a single-tube quality control (QC) sample for these laboratory tests.The QC sample was made from a lyophilized mixture of the stabilized hemolysate with carbon monoxide saturation and the white blood cells of known thalassemia mutations. Homogeneity and stability were examined by Hb and DNA analyses on day 0 and every month for 12 months, at room temperature, 4°C, and -20°C. A preliminary proficiency testing (PT) program for hemoglobinopathies using this single QC material was developed.Hemoglobin (Hb) and DNA analyses of a single-tube QC sample demonstrated satisfactory results of Hb analysis for at least five months and DNA analysis for at least one year of storage at -20°C. The results obtained from a preliminary PT program on five expert laboratories confirmed that a single tube QC sample prepared could be used as a PT item with various Hb and DNA analyses methods.A single lyophilized control sample has been generated for use in hemoglobinopathies' internal and external quality control program. Unlike other available control materials, which are used for individual testing, a single-tube QC sample generated can be used to control the pre-analytical and analytical processes of both Hb and DNA analyses and is suitable for use in the PT program of hemoglobinopathies.

The wide variation in hemoglobin (Hb) F levels has been observed in patients with Hb EE disease. This study aimed to describe hematologic features and determine the effect of genetic variants on Hb F expression in young children with Hb EE disease.Hematologic features and Hb profiles of Laotian children aged 6-23 months, who originally enrolled in the Lao-Zinc study, were retrospectively reviewed. Only children with Hb EE disease, as indicated by DNA analysis, were included in this current analysis. Genetic variants, including theIn total, 205 children were diagnosed as having Hb EE disease with Hb F ranged from 1.2 to 43.7%. Most of the children had mild to moderate anemia with a remarkable microcytosis. Analysis of the genetic variants revealed an extremely high frequency of theOur findings provide insight into complex situation of Hb F variability in young children with Hb EE disease; and this can guide to appropriate care and counseling to affected families.

Introduction Determination of hemoglobins (Hbs) F, A2, and E is crucial for diagnosis of thalassemia. This study determined the levels of Hbs F, A2, and E in children aged 6‐23 months and investigated the effect of age, sex, and types of thalassemia on the expression of these Hbs. Methods A total of 698 blood samples of Laotian children including 272 non‐Hb E, 271 Hb E heterozygotes, and 155 Hb E homozygotes were collected. Hb profiles were determined using the capillary zone electrophoresis. Coinheritance of α‐thalassemia and the homozygosity for Hb E mutation were checked by PCR‐based assay. Results Children heterozygous and homozygous for Hb E had significantly higher Hb F and A2 levels than non‐Hb E children (median Hb F = 1.1% for non‐Hb E group, 2.7% for Hb E heterozygotes, and 9.4% for Hb E homozygotes; median Hb A2 = 2.6% for non‐Hb E group, 3.8% for Hb E heterozygotes, and 5.2% for Hb E homozygotes). The median Hb E levels were 21.9% for Hb E heterozygotes and 85.3% for Hb E homozygotes. Comparing within group, there was a statistically significant difference between children with and without an α‐gene defect for Hb A2 and E, but not Hb F. Based on a multiple regression analysis, age and sex were significantly associated with the expression of Hb F and A2 but not Hb E. Conclusions Our findings can guide the development of a diagnostic approach to thalassemia in children aged 6‐23 months.

Objectives: A co-inheritance of α(0)-thalassemia can ameliorate the clinical severity of the hemoglobin (Hb) E-β-thalassemia disease. This information should be provided at prenatal diagnosis. Identification of α(0)-thalassemia in an affected fetus is therefore valuable. We have explored this genetic interaction in a large cohort of affected fetuses with hemoglobin (Hb) E-β-thalassemia in northeast Thailand. Methods: A study was done retrospectively on 1,592 couples at risk of having fetuses with Hb E-β(0)-thalassemia, encountered from January 2011 to December 2019. A total of 415 left-over DNA specimens of the affected fetuses with Hb E-β(0)-thalassemia disease were further investigated. Examination of α(0)-thalassemia was done using gap-PCR or a multiplex PCR assay for simultaneous detection of Hb E and α(0)-thalassemia mutations. Results: Of the 415 affected fetuses, the two most common β(0)-thalassemia genes found were the codons 41/42 (-TTCT) (199/415; 48.0%) and codon 17 (A-T) (115/415; 27.7%). α(0)-thalassemia was found unexpectedly in 21 (5.1%) fetuses. Hematologic phenotypes of the parents indicated that it was impossible to differentiate a pure β(0)-thalassemia carrier from a double β(0)-thalassemia/α(0)-thalassemia heterozygote unless DNA analysis is performed. In contrast, a reduced level of Hb E in the Hb E carrier (

Background To evaluate whether the quantification of fetal hemoglobin (Hb) Bart’s is useful for differentiation of α-thalassemia syndromes in the fetus and to characterize the fetal anemia associated with fetal α-hemoglobinopathy. Methods A total of 332 fetal blood specimens collected by cordocentesis were analyzed using capillary electrophoresis and the amount of Hb Bart’s was recorded. The result was evaluated against thalassemia genotypes determined based on Hb and DNA analyses. Prenatal Hb and DNA characterization of the fetal anemia observed in two families was done. Results Among 332 fetuses investigated, Hb and DNA analyses identified 152 fetuses with normal genotypes. The remaining 180 fetuses carried α-thalassemia with several genotypes. Variable amounts of Hb Bart’s were identified in all fetuses with α-thalassemia, which could be used for simple differentiation of fetal α-thalassemia genotypes. These included α+- and α0-thalassemia traits, homozygous α+-thalassemia and Hb Constant Spring (CS), Hb H disease, Hb H-CS and Hb H-Quong Sze diseases, homozygous α0-thalassemia causing the Hb Bart’s hydrops fetalis and a remain uncharacterized α-thalassemia defect. The previously undescribed interactions of Hb Queens Park and Hb Amsterdam A1 with Hb E were detected in two fetuses with Hb Bart’s of 0.5%. The Hb Queens Park-AEBart’s disease was also noted in one pregnant woman. Prenatal analysis of the fetuses with severe fetal anemia and cardiomegaly with Hb Bart’s of 9.0% and 13.6% revealed unexpectedly the homozygous Hb CS and a compound heterozygosity of Hb CS/Hb Pakse’ with Hb E heterozygote, respectively. Conclusions The usefulness of detecting and differentiation of fetal α-thalassemia syndromes by quantifying of Hb Bart’s was demonstrated. Apart from the fatal condition of Hb Bart’s hydrops fetalis associated with homozygous α0-thalassemia, homozygous Hb CS and a compound Hb CS/Hb Pakse’ could result in severe fetal anemia and fetal complications, prenatal diagnosis is highly recommended. The simple Hb Bart’s quantification of fetal blood should prove helpful in this matter.

Objective The 3.7 kb deletion (-α 3.7 ) is the most common form of α + -thalassemia found in multiple populations which can be classified into three subtypes. In order not to mis-identify it, the molecular information within each population is required. We have addressed this in northeast Thai and Laos populations. Methods Screening for α + -thalassemia was initially done on 1192 adult Thai subjects. In addition, 77 chromosomes of Thai newborns and 26 chromosomes of Laos with -α 3.7 α + -thalassemia were also examined. All subjects were screened for -α 3.7 α + -thalassemia and subtyped by PCR-RFLP assay. Exact deletion breakpoint of each -α 3.7 subtype was determined by DNA sequencing. α-Globin gene haplotypes were determined. Results The proportions of -α 3.7 subtypes found in 216 Thai -α 3.7 chromosomes were 94.9% for -α 3.7I , 4.2% for α 3.7II and 0.9% for -α 3.7III . All 26 Laos -α 3.7 chromosomes were of -α 3.7I variety. At least six α-globin gene haplotypes were associated with the -α 3.7I α + -thalassemia. Conclusion All -α 3.7 subtypes were observed among Southeast Asian population. Haplotype analysis indicated a multiple origin of this common disorder in the region. A multiplex PCR assay has been developed for simultaneous detection of all subtypes of -α 3.7 α + -thalassemia as well as other α + -thalassemia found in the region including -α 4.2 α + -thalassemia, Hb Constant Spring and Hb Pakse.

Objective: Increased hemoglobin (Hb) A(2) level is an important diagnostic marker for β-thalassemia carrier screening. The level of Hb A(2) is also useful for differentiating several thalassemia syndromes. We have examined data bases for reduced Hb A(2) expression in a large cohort of Thai subjects. Methods: A study was done on 1,498 subjects with non-thalassemia and various types of thalassemia and Hb variants to determine the effect of thalassemia genotypes and on 103 women of reproductive age to determine the effect of iron deficiency. Hb analysis was done using capillary electrophoresis, and thalassemia genotypes were defined by DNA analysis. Serum ferritin was measured using chemiluminescent microparticle immunoassay. Results: Subjects were divided into 35 groups based on iron status, Hb, and DNA analysis. Decreased Hb A(2) level was observed in those with Hb Q-Thailand, δ-hemoglobinopathies, δβ(0)-thalassemia, Hb Lepore, iron deficiency, α-thalassemia, and especially Hb Constant Spring (Hb CS). While β-thalassemia carriers with Hb H disease still had elevated Hb A(2) levels, most of the β-thalassemia carriers with Hb H-CS disease had Hb A(2) less than 3.5% as a diagnostic cut-off. The lowest Hb A(2) level was observed in those with Hb H-CS disease. Conclusion: Iron deficiency, Hb CS trait, homozygous Hb CS, and Hb H disease may reduce Hb A(2) level, leading possibly to misdiagnosis of β-thalassemia, especially in carriers with borderline Hb A(2). Hb CS showed the strongest effect on Hb A(2) expression. Understanding the basis for reduced Hb A(2) expression may help reduce the diagnostic pitfalls of β-thalassemia in the region.

Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked enzymopathy, highly prevalent in the areas where malaria is or has been endemic. Prevalence of G6PD deficiency and characterization of G6PD variants in females from previously malaria-endemic areas of northeastern Thailand remain unstudied. Methods Prevalence of G6PD deficiency was determined by a fluorescent spot test (FST), quantitative G6PD activity assay, and multiplex allele-specific (AS)- and restriction fragment length polymorphic (RFLP)-PCR developed for detection of common G6PD variants in the Thai population. Results Prevalence of G6PD deficiency in female samples (n = 355) was 18% by FST, 29.6% by quantitation of G6PD activity, and 28.1% by PCR-based genotyping. The most common variant was G6PD Viangchan (54%), followed by G6PD Canton (11%) and G6PD Union (11%); in addition, a novel heterozygous variant, G6PD Khon Kaen (c.305T>C, p.F102S), was identified. The majority of heterozygotes expressed G6PD activity within the intermediate deficiency range (30-70% median of normal enzyme activity). Conclusion High prevalence of G6PD deficiency was present in females from northeastern Thailand, the majority being due to heterozygosity of G6PD variants. The findings will have a bearing on the inclusion of primaquine in antimalarial-based policies for malaria elimination in populations with a high prevalence of G6PD deficiency.

Objective Prenatal and postnatal diagnosis of hemoglobin E-β 0-thalassemia can be made using polymerase chain reaction (PCR) analysis mostly on purified DNA. We have establihed a direct amplification method without DNA extraction on whole blood (WB) and amniotic fluid (AF) specimens to diagnose the disease. Methods Three reactions of WB PCR assays and 7 reactions of AF PCR tests were developed for postnatal and prenatal diagnosis, respectively. Assays were validated against routine tests in a blinded trial. Results The results showed 100% concordance with routine DNA PCR assays. Among 309 β-thalassemia carriers, 191 patients (61.8%) carried common β-thalassemia mutations. Among 448 AF specimens, 116 (25.9%) fetuses were found to be affected, 247 (55.1%) fetuses were carriers, and 85 (19%) fetuses were unaffected. Conclusion We found that WB and AF PCR assays are simple, rapid, and reliable. The developed techniques could be applicable in routine settings.

Objective To establish a new indicator derived from reticulocyte hemoglobin (Ret-He) content and red blood cell (RBC) indices for screening for iron deficiency anemia (IDA) in an area in whch thalassemia is prevalent. Methods Blood specimens from 304 women aged between 18 and 30 years residing in northeast Thailand were collected and measured for RBC and reticulocyte parameters. Iron deficiency was diagnosed when a participant had a serum ferritin level of less than 15 ng per mL. Thalassemia genotypes were defined by hemoglobin (Hb) and DNA analyses. Results Of the total participants, 25% had iron deficiency (ID) and 50% carried the thalassemia gene. Various mathematical formulas were established and analyzed using the receiver operating characteristic (ROC) curve. The formula derived from Ret-He: (Ret-He/RDW-SD) × 10, was the best predictor for identifying ID among participants (area under the curve [AUC] = 0.812). Further testing of this indicator among individuals with positive thalassemia-screening results revealed stronger performance with an AUC of 0.874. Conclusions The findings indicate that the formula derived from Ret-He might be applicable for screening ID in areas in which thalassemia is prevalent.

Background: The objective of the study was to describe a formula based on hemoglobin (Hb)A2 and HbF levels for differentiation of homozygous HbE and HbE-β-thalassemia. Methods: A total of 1256 subjects suspected for homozygous HbE or HbE-β0-thalassemia were recruited at the ongoing thalassemia screening program at Khon Kaen University, Thailand. Hb analysis was done using capillary electrophoresis. Genotyping was based on DNA analysis. An arbitrary formula based on HbA2 and HbF was developed statistically for differentiation of the two conditions. Validation was carried out prospectively on another 139 subjects encountered at routine laboratory. Results: Among 1256 subjects, Hb and DNA analyses identified cases with homozygous HbE (n=1076, 85.7%), HbE-β0-thalassemia (n=140, 11.1%), HbE-δβ0-thalassemia (n=30, 2.4%) and unknown HbE-related disorder (n=10, 0.8%). An inverse correlation between the amounts of HbA2 and HbF in HbE-β0-thalassemia was observed. With differences in the amounts of HbA2 and HbF between the groups, an arbitrary score (7.3 HbA2+HbF) was developed where score above 60 indicated HbE-β0-thalassemia. Application of this score on another 139 subjects showed accurate prediction of HbE-β0-thalassemia with 100% sensitivity, 96.5% specificity, 85.7% positive predictive value and 100% negative predictive value. Successful application onto couples at risk was demonstrated. Conclusions: An established score should prove useful in the differentiation of homozygous HbE and HbE-β0-thalassemia in routine setting and lead to a significant reduction in number of referring cases for molecular testing.

Haemoglobin (Hb) E is the most common Hb variant in Asia where its gene frequency approaches 0.3 in some areas. We studied genetic background of Hb E genes among Southeast Asian populations. This study examined β-globin gene haplotypes linked to haemoglobin E (Hb E) in diverse groups of Southeast Asian populations. The study was conducted on southern Thai (22 alleles), Cambodian (84 alleles), Laotian (120 alleles), Vietnamese (87 alleles) and Burmese (one allele) subjects. Results were compared with those of previous studies in northeast Thailand, the Yunnan of China, West India and Europe. Ten different haplotypes were observed. The four most common haplotypes were haplotypes 1 (- + - + + + -) and 2 (+ - - - - + -) on chromosomes with framework 2 and haplotypes 6 (- + - + + - +) and 7 (+ - - - - - +) on chromosomes with framework 3 variety. Phylogenetic analysis indicated that haplotype 1 is a relatively recent haplotype found in all populations, whereas haplotype 6 is found predominately in Cambodians. The results indicate that at least two genetic origins of Hb E are responsible for the high prevalence and spread of Hb E among Southeast Asian populations.

INTRODUCTION Thalassemia screening program has been implemented for years in Southeast Asia, but no external quality assessment program has been established. We have developed and initiated the proficiency testing (PT) program for the first time in Thailand with the aim to assess the screening performance of laboratory staff and their competency in interpretation of the screening results. MATERIALS AND METHODS Three PT cycles per year were organized. From the first to the third cycle of the PT scheme, a total number of participant laboratories increased from 59 to 67. In each cycle, 2 PT items (assigned as blood samples of the couple) were provided. Performance evaluation was based on the accuracy of screening results, i.e. mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and the dichlorophenolindophenol (DCIP) test for haemoglobin E, including the competency in interpretation of screening results and assessment of foetal risk. Performance was assessed by comparing the participants' result against the assigned value. RESULTS Of all 3 cycles, most laboratories reported acceptable MCV and MCH values. From the first to the third cycle, incorrect DCIP test and misinterpretation rates were decreased while incorrect risk assessment varied by cycle to cycle. Combining the accuracy of thalassemia screening and the competency in interpretation and risk assessment, approximately half of participants showed excellent performance. CONCLUSION Improved performance observed in many laboratories reflects the achievement and benefit of the PT program which should be regularly provided.

Thalassemia is a genetic condition that can result in long and expensive treatments, and severe thalassemia may lead to death if left untreated. Couples contributing two genes for thalassemia place their children at particular risk for severe thalassemia. Gene frequency of thalassemia varies in Vietnam, but presents remarkably high levels among some ethnic minority groups. Limited information about thalassemia frequency makes prevention and control of thalassemia difficult. This study aimed to determine gene frequency of certain types of thalassemia among 390 women of reproductive age of the Ta-Oi ethnic minority. Hemoglobin and DNA analyses were carried out to diagnose thalassemia and hemoglobinopathies. Of the total participants, 56.1% (95% CI = 51.1–61.1) carried thalassemia genes. A remarkably high frequency of hemoglobin Constant Spring (Hb CS) of 23.8% (95% CI = 19.7–28.4) was noted. The frequency of α+-thalassemia (−3.7 kb deletion) was 26.4% (95% CI = 22.1–31.1), while hemoglobin E (Hb E) and hemoglobin Paksé (Hb Ps) were identified at frequencies of 14.6 (95% CI = 11.2–18.5) and 2.6% (95% CI = 1.4–5.0), respectively. Further analysis of α-globin gene haplotype revealed the same Hb CS haplotype (+ − M + + −) as of the Co-Tu minority, a neighboring minority of the Ta-Oi, indicating that these two minorities may share the same ancestors. This information will be helpful for further studies in population genetics, as well as the development prevention and control program in the region.

α0-thalassemia of SEA deletion (-SEA) is common among Southeast Asian and Chinese. Using haplotype and phylogenetic analyses, we examined the origin of this defect in Southeast Asian populations. Study was done on both normal and α0-thalassemia alleles in 3 ethnic groups including 96 Thai, 52 Laotian and 21 Cambodian. Five SNPs encompassing the (-SEA) including (rs3760053 T>G), (rs1211375 A>C), (rs3918352 A>G), (rs1203974 A>G) and (rs11248914 C>T) were examined using high-resolution melting assays. It was found that 94.0% of Thai, 100% of Laotian and 100% of Cambodian α0-thalassemia alleles were linked to the same haplotype: the haplotype H4 (AAGC), representing an Asian specific origin. An G allele of the (rs3760053) was found to be in strong linkage disequilibrium with the α0-thalassemia allele in these populations. A multiplex PCR assay was developed to detect simultaneously the (-SEA) allele and genotyping of a linked (rs3760053) to improve accuracy of prenatal diagnosis of α0-thalassemia. Application of this multiplex PCR assay for routine prenatal diagnosis of α0-thalassemia in 12 families revealed a 100% concordant result with conventional gap-PCR assay. Therefore, a single genetic origin is responsible for the spread and high prevalence of the (-SEA) in the region. The multiplex PCR assay developed should provide a double-check PCR system for more accurate diagnosis and allow the monitoring of possible maternal contamination at prenatal diagnosis of this important genetic disorder.

AimsThe presence of the ζ-globin chain is a good marker of (--SEA) α0-thalassaemia. We evaluated an immunochromatographic (IC) strip assay for ζ-globin in screening for (--SEA) α0-thalassaemia in a population with a high prevalence and heterogeneity of haemoglobinopathies.MethodsThe study was carried out on 300 screen positive blood samples of Thai individuals. The IC strip assay for the ζ-globin chain was performed on all samples. The results were interpreted with thalassaemia genotyping using standard haemoglobin and DNA analyses.ResultsSeveral thalassaemia genotypes were noted. Among the 300 subjects investigated, 79 had a positive IC strip assay for ζ-globin and (--SEA) α0-thalassaemia was identified in 40 of them. No (--SEA) α0-thalassaemia was detected in the remaining 39 samples with a positive IC strip test result or in the 221 samples with a negative IC strip test result. Further DNA analysis identified α+-thalassaemia in 25 of the 39 (--SEA) α0-thalassaemia negative samples. Using this IC strip assay in combination with a conventional screening protocol for (--SEA) α0-thalassaemia could provide sensitivity and specificity of 100% and 90.4%, respectively.ConclusionsIC strip assay for ζ-globin is simple, rapid and does not require sophisticated equipment. Use of this test in addition to the existing screening protocol could detect potential (--SEA) α0-thalassaemia leading to a significant reduction in the workload of DNA analysis. This could be used in areas where haemoglobinopathies are prevalent and heterogeneous but molecular testing is not available.

Non-transfusion-dependent thalassemia (NTDT) is associated with various forms of thalassemia and genetic modifiers. We report the molecular basis of NTDT in hemoglobin (Hb) E-β-thalassemia disease. This study was done in 73 adult patients encountered at the prenatal diagnosis center of Khon Kaen University, Northeast Thailand. Hematological parameters and Hb patterns were collected, and α- and β-globin gene mutations were determined. Multiple single-nucleotide polymorphisms (SNPs) including the rs7482144/Gγ-XmnI polymorphism, rs2297339, rs2838513, rs4895441, and rs9399137 in the HBS1L-MYB gene, rs4671393 and rs11886868 in the BCL11A gene, and G176AfsX179 in the KLF1 gene were examined. Five β0-thalassemia mutations and a severe β+-thalassemia mutation in trans to the βE gene were identified. No significant difference in hematological parameters was observed among β-thalassemia genotypes. Coinheritance of α-thalassemia was observed in 31 of the 73 subjects (42.5%). Four SNPs including Gγ-XmnI, rs2297339, rs4895441, and rs9399137 of HBS1L-MYB were found to be associated with high Hb F levels in 39 (53.4%) subjects. The molecular basis of NTDT in the remaining 3 (4.1%) cases could not be defined. These results indicate multiple genetic factors in NTDT patients and underline the importance of complete genotyping to provide proper management, make clinical predictions, and improve genetic counseling.

Elevated hemoglobin (Hb) AExamination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb AAmong 202 subjects, DNA analysis identified carriers of αA large proportion of subjects with borderline Hb A

Hemoglobin Bart's hydrops fetalis (homozygous α

Screening for thalassemia carriers should not only be conducted in middle-income countries but also can be possible in low-middle income countries, through cooperation of experienced professionals from middle income countries. We describe a collaborating model between two close neighboring countries in establishing such a screening program for thalassemia. After training and setting up of facilities, a total of 152 out of 187 hospital staff were screened as a pilot activity to encourage community participation. Referring system for sending blood samples to a reference center in Thailand was also established. Among 152 health staff, 12.5% α0-thal, 2% β-thal and 13% Hb E carriers were found. Applying thalassemia screening to 411 pregnant women and 71 spouses, 5 couples at risk of bearing a child of thalassemia disease were identified. The thalassemia screening program has a sensitivity of 99.5%, specificity of 77%, positive predictive value of 73%, and negative predictive value of 99.5%. Thus, it is possible to operate a thalassemia screening program with acceptable performance in a low-middle income country (Lao People’s Democratic Republic) with the cooperation of a referral center located within close proximity in a middle income country (Thailand).

We report the molecular and hematological characteristics associated with a large cohort of β(+)-thalassemia in Thailand. Study was done on 21,068 unrelated subjects referred to our center in northeast Thailand for hemoglobinopathies investigation. Among 21,068 subjects, 2637 (12.5%) were found to carry β-thalassemia. Of these 2637 cases, 705 (26.7%) carried β(+)-thalassemia with eight different mutations including 6 promoter mutations; NT-28 (A-G), NT-31 (A-G), NT-50 (G-A), NT-86 (C-G), NT-87 (C-A) and NT-90 (C-T) and two missense mutations; Hb Malay (codon 19; AAC-AGC) and Hb Dhonburi (codon 126; GTG-GGG). Hematological features of carriers with these β(+)-thalassemia (n=528) were compared with those with β(0)-thalassemia (n=309). Data for Hb E-β(+)-thalassemia (n=177) were also presented along with Hb E-β(0)-thalassemia in our series (n=94). All patients with Hb E-β(+)-thalassemia were associated with mild thalassemia intermedia phenotypes. Most of the β(+)-thalassemia carriers had elevated Hb A2 and mild hypochromic microcytosis, some demonstrated borderline MCV and MCH values which, could compromise carrier screening. Analysis of α/β-globin mRNA ratio in representative cases with normal, Hb E trait, β(+)-thalassemia trait, Hb Dhonburi trait and β(0)-thalassemia trait demonstrated the average values of 1.1, 1.7, 2.1, 1.7 and 3.1, respectively which is helpful in identification and differentiation of the cases.

Background Severe thalassemia diseases are a major health problem in Southeast Asia. In Cambodia, there has never been a significant program for prevention or control of severe thalassemia. We, therefore, studied the effect of a health education program on severe thalassemia prevention and control in Phnom Penh, Cambodia. Methods A quasi-experimental study in several communities around Phnom Penh was done. The respective intervention and control group comprised 124 and 117 people, between 18 and 40 years of age, male and female. Pre- and post-tests using a validated and reliable questionnaire were performed in the intervention group and one test was done in the control group. A health education program was organized to give important information to the intervention group and, at the end of the process, to the control group. The outcomes were evaluations of their knowledge and attitude vis-à-vis severe thalassemia prevention and control, and participating in thalassemia screening. Results Among participants in the intervention group, 105 (84.7%) considered undergoing blood screening vs. 65 (55.6%) in the control group (p-value

This community-based study investigated anemia prevalence and certain hematologic features and their possible relationships to thalassemia and iron deficiency (ID) in a population of older people in Northeastern Thailand. Participants included 319 apparently healthy individuals ranging in age from 60 to 98 years, whose current health status was assessed by means of personal interviews. Blood samples were also collected to determine the following parameters: red blood cell indices, serum ferritin, C-reactive protein, hemoglobin profiles, and the α0-thalassemia gene. Based upon established WHO criteria, the overall prevalence of anemia was found to be 47.7%, increasing from 39% within the age group of 60-70 years to 68% in those >80 years. Factors considered to be significant contributors to anemia were classified as ID (3.6%), thalassemia (56.2%), and “unknown” (40.1%). Overall, only 2.4% of participants exhibited any ID. Hematologic changes appear to correlate with age. Our findings provide not only baseline information, potentially useful for implementing appropriate control measures, but also an enhanced awareness and understanding of the factors contributing to anemia among the elderly in the region.

Background: The finding of many Thai Hb E-β0-thalassemia patients with non-transfusion dependent thalassemia (NTDT) phenotype without co-inheritance of α-thalassemia has prompted us to investigate the existence of other genetic modifying factors. Methods: Study was done on 146 adult Thai patients with NTDT Hb E-β0-thalassemia and a homozygous β-thalassemia patient without co-inheritance of α-thalassemia. Multiple single-nucleotide polymorphisms (SNPs) associated with γ-globin gene expression including the Gγ-XmnI of HBG2 gene, rs2297339, rs4895441, and rs9399137 of the HBS1L-MYB gene, rs4671393 in the BCL11A gene, and G176AfsX179, T334R, R238H and -154 (C-T) in the KLF1 gene were investigated using PCR-and related techniques. Results: Heterozygous and homozygous for Gg-XmnI of HBG2 gene were detected at 68.0% and 6.1%, respectively. Further DNA analysis identified the rs2297339 (C-T), rs4895441 (A-G), and rs9399137 (T-C) of HBS1L-MYB gene in 86.4%, 22.5% and 20.4%, respectively. The rs4671393 (G-A) of the BCL11A gene was found at 31.3%. For the KLF1 gene, the T334R and G176AfsX179 (+/-) were detected at 8.2% and 1.4%, respectively. Conclusion: It was found that these SNPs when analyzed in combination could explain the mild phenotypic expression of all cases. These results underline the importance of these informative SNPs on phenotypic expression of Hb E-β-thalassemia patients.

In a distal renal tubular acidosis, a patient with coexistence of anion exchanger 1 (AE1) mutation and α-thalassemia had more severe clinical phenotype than usually observed. AE1 mutation might therefore modify the hematological and clinical presentations of the hemoglobin (Hb) E-β-thalassemia disease. To examine the role of AE1 mutation on severity difference in Hb E-β-thalassemia disease in northeast Thailand, three common AE1 mutations were investigated in Hb E-β-thalassemia patients with thalassemia major (TM) and thalassemia intermedia (TI) phenotypes. One hundred forty-eight patients including 103 TM and 45 TI were studied. Three common AE1 mutations including Southeast Asian Ovalocytosis (SAO), G701D and A858D were screened for using allele specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays. No any AE1 mutation was detected in all these Thai patients with Hb E-β-thalassemia. This result indicates that AE1 mutation is rare and should not be an important phenotypic modifier in the Hb E-β-thalassemia disease. Therefore, screening of common AE1 mutation in thalassemia patient is not necessary.

α0-thalassemia of SEA deletion (—SEA) is common among Southeast Asian and Chinese. Using haplotype and phylogenetic analyses, we examined the origin of this defect in Southeast Asian populations. Study was done on both normal and α0-thalassemia alleles in 3 ethnic groups including 96 Thai, 52 Laotian and 21 Cambodian. Five SNPs encompassing the (—SEA) including (rs3760053 T>G), (rs1211375 A>C), (rs3918352 A>G), (rs1203974 A>G) and (rs11248914 C>T) were examined using high-resolution melting assays. It was found that 94.0% of Thai, 100% of Laotian and 100% of Cambodian α0-thalassemia alleles were linked to the same haplotype: the haplotype H4 (AAGC), representing an Asian specific origin. An G allele of the (rs3760053) was found to be in strong linkage disequilibrium with the α0-thalassemia allele in these populations. A multiplex PCR assay was developed to detect simultaneously the (—SEA) allele and genotyping of a linked (rs3760053) to improve accuracy of prenatal diagnosis of α0-thalassemia. Application of this multiplex PCR assay for routine prenatal diagnosis of α0-thalassemia in 12 families revealed a 100% concordant result with conventional gap-PCR assay. Therefore, a single genetic origin is responsible for the spread and high prevalence of the (—SEA) in the region. The multiplex PCR assay developed should provide a double-check PCR system for more accurate diagnosis and allow the monitoring of possible maternal contamination at prenatal diagnosis of this important genetic disorder.

Background To evaluate the use of MCV and MCH cutoffs for thalassemia screening in areas with a high frequency of thalassemia and hemoglobinopathies using different hematology analyzers. Methods Blood samples of known α(0)-thalassemia and β-thalassemia carriers were analyzed to establish the appropriate cutoffs for each analyzer. These selected cutoffs were validated prospectively for screening of α-thalassemia, β-thalassemia in combination with the dichlorophenolindophenol test for hemoglobin E on another 288 Thai and 325 Laotian students. Genotypes were defined by standard hemoglobin and DNA analyses. Results The appropriate cutoffs for the Sysmex XS-800i and Sysmex XN-1000, Coulter LH 780, and Pentra ES-60 were found to be 78 fL for MCV and 25 pg for MCH. These were 82 fL and 25 pg for the Cell-Dyn Ruby. Further validation on Thai and Laotian students revealed 100% sensitivity and specificity of higher than 80% for all analyzers. Conclusions While using MCV in screening requires establishment of appropriate cutoffs, MCH appears to be applicable to all analyzers. Each analyzer should be evaluated and appropriate cutoffs should be established before application in the field.

INTRODUCTION We have evaluated an automated capillary isoelectric focusing (cIEF)-based Hb analyzer in diagnosis of hemoglobinopathies commonly found among South East Asian population. METHODS Study was performed on a cohort of 665 adult Thai subjects and 13 fetal blood specimens obtained at routine thalassemia diagnostic laboratory. Hb analysis was performed using the cIEF system. Thalassemia genotypes were defined by DNA analysis. RESULTS The system revealed satisfactorily within-run and between-run precision for quantitation of Hb A2 and Hb E (CV: 0.02-0.09%). The reference ranges of Hb A2 and Hb E were 2.6-4.0% and 25.7-33.1%, respectively. The system identified the cases of β-thalassemia and Hb E disorders correctly. Several thalassemia genotypes and Hb variants were identifiable. However, Hb Constant Spring was separated closely to Hb A2 and Hbs Bart's and H were relatively difficult to be reported due to interfering peaks separating at the same regions. Prenatal diagnosis by fetal blood analysis was found to be accurate for Hb Bart's hydrops fetalis and Hb E-β0 -thalassemia disease. CONCLUSIONS The cIEF system could accurately diagnose β-thalassemia and Hb E carriers and demonstrate many Hb variants found in the region. The system cannot report Hb A2 in the presence of Hb E whereas Hbs Lepore and F are comigrated. Diagnosis of α-thalassemia disease based on Hb H and Hb Bart's might be difficult.

Hemoglobin E is the most common Hb variant found in South East Asia. Variation of Hb F expression in Hb E syndrome is associated with several genetic modifiers. We report several single nucleotide polymorphisms (SNPs), including nine known and five novel mutations of the Kruppel-like factor 1 (KLF1; an erythroid specific transcription factor) gene and determine their associations with phenotypic expression of Hb F in Hb E disorders. KLF1 mutations were examined using high resolution melting (HRM) assay and DNA sequencing in 575 homozygous Hb E, 278 heterozygous Hb E and 100 normal subjects. Fourteen mutations were mostly observed in subjects with elevated Hb F, including nine known mutations (G176AfsX179, T334R, R238H, -154 (C>T), A298P, S270W, R301H, -148 (G>A) and G335R and five novel mutations (Q217X, Q223X, Y290_S293del, K307N, and M358I). None of them, but the -148 (G>A), were observed in normal controls to have Hb F

Up to now, more than 200 different β-thalassemia (β-thal) mutations have been characterized. The majority are point mutations causing expression defects. Only approximately 10.0% of the defects are caused by large deletions involving the β-globin gene cluster causing β(0)-thal, (δβ)(0)-thal, (G)γ((A)γδβ)(0)-thal and other conditions with or without persistence of fetal hemoglobin (Hb). For the prevention of severe forms of β-thal intermedia and β-thal major, it is important to identify carriers of point mutations as well as carriers of deletions. β-Thalassemia and related disorders are most commonly present among populations from all Mediterranean countries as well as Southeast Asia, India, Africa, Central America and the Middle East. Twelve relatively frequently occurring deletion types have been described involving the β-globin gene cluster. These include the 105 bp β(0)-thal deletion, the 619 bp deletion, the 3.5 kb deletion, the Southeast Asian (SEA) deletion, the Filipino deletion, Hb Lepore, the Thai (δβ)(0)-thal, the Siriraj J (G)γ((A)γδβ)(0)-thal, the Chinese (G)γ((A)γδβ)(0)-thal, the Asian Indian deletion-inversion (G)γ((A)γδβ)(0)-thal as well as the (hereditary persistence of fetal hemoglobin) HPFH-6 and HPFH-7 deletions. To improve the rapid detection of the eight common β-globin cluster deletions in Southeast Asian countries, a simple molecular technique based on a single-tube multiplex gap-polymerase chain reaction (PCR) has been developed in this study. This technique provides a fast, simple and cost effective diagnostic test for deletion types of β-thal that can be applied in every molecular diagnostic laboratory having standard PCR equipment.

We describe hematologic and molecular characteristics of a hitherto undescribed interaction between the Filipino deletional β 0 -thalassemia with Hb E and α-thalassemia in a Thai family. This study was conducted during the prenatal screening of a pregnant Thai woman and her family members. A prenatal diagnosis was performed at her second pregnancy by amniocentesis. Laboratory investigations identified that the pregnant woman was Hb E heterozygote with α + -thalassemia, whereas her husband was a double heterozygote for the Filipino deletional β 0 -thalassemia and α + -thalassemia. Their affected son was a patient with a previously undescribed condition of Hb E-β 0 -thalassemia with α + -thalassemia. Both a combined gap-polymerase chain reaction (PCR) and allele-specific PCR were used successfully in the prenatal diagnosis, which identified an affected fetus with Hb E-β 0 -thalassemia without α + -thalassemia. Beta globin gene haplotype analysis indicated the same origin of this Filipino β 0 -thalassemia in Asian populations.

Study on the phenotypic expression of hemoglobin (Hb) A(2) and Hb E in Hb E disorders has been difficult due to the co-separation of Hb A(2) and Hb E in most Hb analysis assays. Because these two Hbs are separated on capillary electrophoresis, we studied phenotypic expression of Hbs A(2), E and F in various Hb E disorders using this system. This was done on 362 subjects with several Hb E disorders including heterozygous Hb E, homozygous Hb E, β-thalassemia/Hb E, δβ-thalassemia/Hb E, and Hb Lepore/Hb E and those of these disorders with several forms of α-thalassemia. Normal controls showed Hb A(2) of 2.7 ± 0.3%. Heterozygous Hb E and homozygous Hb E had elevated Hb A(2) i.e. 3.8 ± 0.3% and 4.8 ± 0.5%, respectively. Further elevations were observed for β(0)-thalassemia/Hb E (6.1 ± 1.9%) and β(+)-thalassemia/Hb E (7.1 ± 1.2%). Interestingly, no elevation of Hb A(2) was found in the δβ-thalassemia/Hb E, and Hb Lepore/Hb E (2.3 ± 0.3%) but higher Hb F levels were noted which could be useful diagnostic markers. The levels of Hb E were variable. Co-inheritance of these Hb E disorders with α-thalassemia were associated with lower outputs of Hb E and Hb F but the levels of Hb A(2) were not altered. Different phenotypic expression of Hb A(2), Hb E and Hb F could help in differential diagnosis of these Hb E related disorders commonly encountered in the regions where access to molecular techniques is limited.

Background and Aims: The study aimed to determine the proportion of iron deficiency (ID) anemia (IDA) among vegans in northeast Thailand and to explore whether mathematical formulas derived from red blood cell (RBC) indices are applicable for IDA screening in the study population. Methods: Blood samples from 234 individuals (age 6–45 years) living in a vegan community were taken. Complete blood cell count, serum ferritin, hemoglobin profiles and DNA analysis for α-thalassemia were determined. Anemia was defined using the WHO criteria adjusted for age and sex. Serum ferritin Results: Anemia was found in 41.5% (95% CI = 35.1–48.1%) of the study participants. The overall proportion of thalassemia and hemoglobinopathies was 56.4% (95% CI = 49.8–62.9%). Of the anemic participants, 45.4% had ID. Based on the receiver-operating characteristic curve analysis, 4 formulas were applicable for predicting ID among anemic individuals (highest sensitivity of 86.4%). Conclusions: The proposed formulas might be used as proxy indicators for the identification of ID among anemic children and adult vegans if more sophisticated laboratory determinations are not available due to limited financial resources.