Hataichanok Srivorakun

Phenotypic expression of hemoglobins A2, E and F in various hemoglobin E related disorders

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.

Presumptive diagnosis of common haemoglobinopathies in Southeast Asia using a capillary electrophoresis system

Introduction:  This study was conducted to examine ability of the Capillarys 2 haemoglobin (Hb) testing system to assist in presumptive diagnosis of common Hb variants found in Southeast Asia including five α‐chain and nine β‐chain variants.

Thalassemia and hemoglobinopathies in Southeast Asian newborns: diagnostic assessment using capillary electrophoresis system

BACKGROUND We have investigated the Capillarys 2 Hemoglobin testing system to assist in presumptive diagnosis of thalassemia and hemoglobinopathies commonly found in Southeast Asia. METHODS Study was conducted on 226 newborns. Hematological parameters were recorded and Hb profiles were examined on the Capillarys 2 Hemoglobin analyzer (SEBIA). DNA analyses were used to establish the final diagnoses. RESULTS Among 226 newborns examined, 122 had thalassemias with 17 different genotypes. The capillary electrophoresis system could provide useful data for presumptive diagnoses of cases, especially those with Hb E and α-thalassemia. Hb E was found to be 2.6-6.2% in heterozygote whereas Hb Barts were clearly observed in cases with compound heterozygous or homozygous α(+)-thalassemia and heterozygous α(0)-thalassemia. Hb H disease and other forms of α-thalassemia could be differentiated based on the presence of Hb Barts and its percentage. CONCLUSION The capillary electrophoresis system is applicable to newborn screening for common forms of thalassemia in Southeast Asia.

Interactions of hemoglobin Lepore (δβ hybrid hemoglobin) with various hemoglobinopathies: A molecular and hematological characteristics and differential diagnosis

Hemoglobin (Hb) Lepore is a variant consisting of two alpha-globin and two deltabeta-globin chains. In heterozygote, it is associated with clinical findings of thalassemia minor but interactions with other hemoglobinopathies can lead to various clinical phenotypes. Using a combination of Hb-HPLC, Hb-capillary electrophoresis and DNA analyses, we have identified 14 patients with Hb Lepore-Hollandia including eight heterozygotes, two double heterozygotes with alpha(+)-thalassemia, two compound heterozygotes with Hb E (initially diagnosed as Hb E-beta-thalassemia) and two previously undescribed conditions of double heterozygote for Hb Lepore/Hb Constant Spring and Hb Lepore/alpha(0)-thalassemia, both associated with higher levels of Hb F and lower levels of Hb Lepore. Hematological and molecular features of these patients are presented along with those observed in four other Thai individuals encountered with heterozygous Hb Lepore-Washington-Boston. Haplotype analysis of the beta-globin gene cluster showed that all Hb Lepore-Hollandia genes were associated with a single haplotype not described previously in other populations, (- + - + + - +) whereas the four Hb Lepore-Washington-Boston genes were associated with haplotypes (+ - - - - + -/+) (N=1) and (+ - - - - - +) (N=3), data indicating multiple origins of these two variants. Hb Lepore may not be uncommon in the Thai and other Asian populations and both hematological and molecular studies are required for accurate diagnosis. To facilitate rapid epidemiological, diagnostic screening and differentiation of the two Hb Lepore defects, a simple assay based on multiplex PCR has been developed.

A Large Cohort of Hemoglobin Variants in Thailand: Molecular Epidemiological Study and Diagnostic Consideration

Background Hemoglobin (Hb) variants are structurally inherited changes of globin chains. Accurate diagnoses of these variants are important for planning of appropriate management and genetic counseling. Since no epidemiological study has been conducted before, we have investigated frequencies, molecular and hematological features of Hb variants found in a large cohort of Thai subjects. Materials and Methods Study was conducted on 26,013 unrelated subjects, inhabiting in all geographical parts of Thailand over a period of 11 years from January 2002-December 2012. Hb analysis was done on high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Mutations causing Hb variants were identified using PCR and related techniques. Results Among 26,013 subjects investigated, 636 (2.4%) were found to carry Hb variants. Of these 636 subjects, 142 (22.4%) carried α-chain variants with 13 different mutations. The remaining included 451 (70.9%) cases with 16 β-chain variants, 37 (5.8%) cases with Hb Lepore (δβ-hybrid Hb) and 6 (0.9%) cases with a single δ-chain variant. The most common α-globin chain variant was the Hb Q-Thailand (α74GAC-CAC, Asp-His) which was found in 101 cases (15.8%). For β-globin chain variants, Hb Hope (β136GGT-GAT, Gly-Asp) and Hb Tak (β146+AC, Ter-Thr) are the two most common ones, found in 121 (19.0%) and 90 (14.2%) cases, respectively. Seven Hb variants have never been found in Thai population. Hb analysis profiles on HPLC or CE of these variants were illustrated to guide presumptive diagnostics. Conclusions Hb variants are common and heterogeneous in Thai population. With varieties of thalassemias and hemoglobinopathies in the population, interactions between them leading to complex syndromes are common and render their diagnoses difficult in routine practices. Knowledge of the spectrum, molecular basis, genotype-phenotype correlation and diagnostic features should prove useful for prevention and control of the diseases in the region.

Hb Phimai [β72(E16)Ser→Thr]: A Novel β-Globin Structural Variant Found in Association with Hb Constant Spring in Pregnancy

A Thai pregnant woman with α and β hemoglobinopathies is described. Initial hemoglobin (Hb) analysis revealed an unknown variant with a high performance liquid chromatography (HPLC) elution pattern similar to Hb Hope [β136(H14)Gly→Asp]. Subsequent DNA-based diagnostics revealed that she was a carrier of Hb Constant Spring [Hb CS, α142, TAA>CAA (α2)] and a novel β-globin chain variant [β72(E16)Ser→Thr, AGT>ACT] which we named Hb Phimai. Her hematological findings and a simple DNA test for differential diagnosis of Hb Phimai and Hb Hope are presented.

Association of Hb Thailand [α56(E5)Lys→Thr] and Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] with α0-Thalassemia: Molecular and Hematological Features and Differential Diagnosis

We report the molecular and hematological characteristics of two rare hemoglobin (Hb) variants found in associations with a common α0-thalassemia (α0-thal) in Thai patients. The first case (P1) was a generally healthy 27-year-old man discovered during our ongoing thalassemia screening program. Hemoglobin and DNA analyses identified a previously undescribed condition of compound heterozygosity for Hb Thailand [α56(E5)Lys→Thr] and α0-thal (SEA deletion). The second case (P2) was a 4-year-old boy with hypochromic microcytic anemia. Hemoglobin and DNA analyses also identified a compound heterozygosity for Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] in association with α0-thal (SEA deletion). Although Hb H (β4) inclusion bodies were observed in both cases, Hb analysis using both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) did not show Hb H. While the two Hb variants could be recognized on Hb HPLC analysis, their corresponding Hb A2 derivatives: the Hb A2-Thailand and Hb A2-Phnom Penh, were clearly observed on CE. Apparently, combination of these two Hb variants with α0-thal are not expressed as Hb H disease. The two Hb variants could be confirmed by polymerase chain restriction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR assays.

Diagnosis of common hemoglobinopathies among South East Asian population using capillary isoelectric focusing system.

We have evaluated an automated capillary isoelectric focusing (cIEF)‐based Hb analyzer in diagnosis of hemoglobinopathies commonly found among South East Asian population.

ARKRAY ADAMS A1c HA-8180T Analyzer for Diagnosis of Thalassemia and Hemoglobinopathies Common in Southeast Asia

OBJECTIVE To evaluate the ARKRAY ADAMS A1c HA-8180T analyzer for diagnosis of thalassemias and hemoglobinopathies commonly found in the Southeast Asian population. METHODS Our cohort consisted of 557 specimens from adults referred for thalassemia diagnosis. From these, we selected 457 specimens and subjected them to DNA analysis to determine various thalassemia genotypes. Also, to confirm the reference range for HbA2, we obtained an additional 48 specimens from healthy individuals. We estimated the diagnostic range for Hb E from specimens from another 52 subject individuals previously diagnosed with heterozygous HbE. All of these individuals had negative results in DNA testing for all common α-thalassemia alleles found in Thailand. We performed hemoglobin (Hb) analysis and compared the results with those we derived from testing the CAPILLARYS 2 Flex Piercing device. We defined genotypes via by DNA analysis. RESULTS Performance evaluation revealed the within- and between-run precision for analysis of HbA2 and HbE, with coefficients of variation (CVs) ranging from 0.6% to 2.5%. We determined the reference ranges of HbA2 and HbE in the HbE heterozygote to be 2.2% to 3.4 % and 25.7% to 31.0%, respectively. We were able to identify all cases of β-thalassemia and HbE disorders. We coeluted HbH and Hb Bart and interfered with acetylated HbF. CONCLUSION The ARKRAY ADAMS A1c HA-8180T analyzer could accurately identify which individuals had β-thalassemia and HbE disorders. However, compared with other high-performance liquid chromatography instruments in diagnosing α-thalassemia disease with HbH and Hb Bart, this analyzer is relatively difficult to use.

Co‐inheritance of α0‐thalassemia elevates Hb A2 level in homozygous Hb E: Diagnostic implications

Differentiation of homozygous hemoglobin (Hb) E with and without α0‐thalassemia is subtle on routine hematological ground. We examined in a large cohort of homozygous Hb E if the level of Hb A2 is helpful.