Sitthichai Panyasai

Comparison Between Capillary Electrophoresis and High Performance Liquid Chromatography for Detection and Quantification of Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA IN α2)]

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is often missed by routine laboratory testing since its mRNA as well as gene product are unstable and presented at a low level in peripheral blood. This study aimed to analyze the efficacy of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) for detecting and quantifying Hb CS in 19 heterozygotes and 14 homozygotes with Hb CS as well as 10 Hb H-CS disease subjects who were detected by molecular analysis. In the CE electrophoregram, Hb CS was seen at zone 2 and was observed in all samples, while the chromatogram of Hb CS peaks was found in 26.32% heterozygotes, 42.86% homozygotes and 90% Hb H-CS disease subjects, respectively. In addition, the Hb CS levels in each group of subjects quantified by CE were significantly higher than those quantified by HPLC. Based on the CE method, the lowest Hb CS level was found in the heterozygotes, whereas the highest level was found in the Hb H-CS disease patients. Therefore, the CE method was superior to the HPLC method for detecting Hb CS. Furthermore, the level of Hb CS quantified by CE proved useful in screening heterozygotes and homozygotes with Hb CS as well as Hb H-CS disease.

Molecular confirmatory testing of hemoglobin Constant Spring by real-time polymerase chain reaction SYBR Green1 with high-resolution melting analysis

To the Editor: Hemoglobin Constant Spring (Hb CS) is the most prevalent non-deletional a-thalassemia in the southeast Asian population. This abnormal hemoglobin results from a point mutation at the stop codon of a2-globin gene (TAA fi CAA), which leads to the addition of 31 amino acids to normal a-globin sequence (1, 2). The Hb CS mRNA is unstable and therefore the rate of a-globin chain synthesis is decreased (3). Although the heterozygote of Hb CS is clinically and hematologically normal, the homozygote shows a clinical picture as thalassemia intermedia with mild anemia, jaundice and hepatosplenomegaly. Moreover, the interaction of Hb CS with a-thalassemia leads to Hb H disease () ⁄ aa), which tends to be more severe than Hb H disease caused by a triple a gene deletion () ⁄ -a) (4). Hb CS diagnosis is often missed by routine laboratory testing, especially in the heterozygote as the Hb CS is unstable and presented at a low level in peripheral blood (5, 6). The present study used the real-time polymerase chain reaction (PCR) with SYBR Green1 and high-resolution melting (HRM) analysis for molecular confirmatory testing of Hb CS. Blood samples were collected from three patients who were diagnosed as heterozygote of Hb CS by using capillary electrophoresis (CAPILLARYS 2; Sebia, Norcross, GA, USA) together with clinical and hematological parameters as shown in Table 1. However, the hemoglobin analysis by high-performance liquid chromatography (HPLC; BIO-RAD, Variant b-thalassemia short program, Hercules, CA, France) of three blood samples showed no peak of Hb CS. The DNA was extracted from 200 lL whole blood sample using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). The amplification was carried out in a reaction volume of 25 lL containing 12.5 lL of 2· SYBR Green1 PCR master mix (QuantiTect SYBR Green PCR Kits; Qiagen), 0.1 lm of internal control forward primer (aG17; 5¢-AGATGGCGCCTTCCTCTCAGG-3¢), 1.2 lm of Hb CS forward primer (aG2; 5¢-GCTGACCTC CAAATACCGTC-3¢) and common reward primer (C3; 5¢-CCATTGTTGGCACATTCCGG-3¢), 0.6 m betaine, 5% DMSO and 5 lL of DNA sample. The multiplex SYBR Green1-PCR was performed on Rotor-Gene 6000 (Corbett Research, Mortlake, NSW, Australia). The mixture was preheated at 95 C for 5 min and then the PCR was cycled 40 times at 94 C for 1 min and 65 C for 1 min 30 s. The amplification cycles were then followed by a HRM cycle from 80 to 95 C at a rate of 0.1 C per 2 s. The DNA amplification fragments of internal control and Hb CS were 391 and 180, respectively, as a previous study reported (7). The amplified internal control fragment had a specific peak height at melting temperature (Tm) of 86 ± 1 C, whereas the amplified fragments of Hb CS had a specific peak height at Tm of 83 ± 1 C (Fig. 1). Both peaks were observed in all three blood samples in which Hb CS could be detected by the capillary electrophoresis method. One DNA sample of three heterozygotes of Hb CS showed a lower peak was because of a lower copy number of the target sequence than the other two. However, when analyzing separately by using the auto analysis program, the two-specific peak heights were clearly observed in this sample (data not shown). Only a specific peak of amplified internal control fragment was found in the blood sample of a normal individual (Fig. 1). This technique was tested further with five DNA samples of normal individuals and five from heterozygotes of Hb CS, and we found that the similar oneand two-specific peak

Unmasking Hb Paksé (codon 142, TAA>TAT, α2) and its Combinations in Patients also Carrying Hb Constant Spring (codon 142, TAA>CAA, α2) in Northern Thailand

The incidence of Hb Paksé (codon 142, TAA>TAT, α2) might have been underestimated due to misidentifying some cases as Hb Constant Spring (Hb CS, codon 142, TAA>CAA, α2) since both abnormal hemoglobins (Hbs) migrate to the same position on Hb electrophoresis or chromatography. Multiplex asymmetric allele-specific polymerase chain reaction (PCR) for identification of Hb CS and Hb Paksé, and a real-time PCR (ReTi-PCR) with SYBR Green1 high resolution melting (HRM) analysis, for detection of the α-thalassemia-1 (α-thal-1) Southeast Asian (– –SEA/) type deletion, were performed on 114 blood samples collected from subjects who lived in northern Thailand. These samples were previously identified as carrying Hb CS by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC). Five out of 114 (4.4%) samples were found to carry Hb Paksé with four different genotypes including Hb Paksé trait, compound Hb CS/Hb Paksé, Hb H-Hb Paksé disease and Hb H-Hb Paksé-Hb E disease. These results suggested that Hb Paksé and its various combinations can be misidentified as Hb CS. Although the clinical symptoms of Hb Paksé and Hb CS are similar, to prevent erroneous epidemiological data on Hb CS as well as underestimating the prevalence of Hb Paksé in northern Thailand, DNA analysis is recommended to be performed in all cases when peaks of Hb CS/Hb Paksé are detected on CE or HPLC.

Interference of Hemoglobin Hope on β-Thalassemia Diagnosis by the Capillary Electrophoresis Method

The β-chain hemoglobin (Hb) variants interfere with the diagnosis of β-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a β-chain Hb variant frequently found in the Thai population, on β-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA(2) in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA(2) level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of β(0)-thalassemia codon 17, 41/42, and 71/72 mutations and β-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of β-thalassemia trait analyzed by CE but not by HPLC.

Quantification of hemoglobin Constant Spring in heterozygote and homozygote by a capillary electrophoresis method

Introduction:  Capillary electrophoresis (CE) is a high‐resolution method for detection of hemoglobin Constant Spring (Hb CS).

Hb Agenogi [β90(F6)Glu→Lys (GAG>AAG) HBB: c.271G>A)] in a Pregnant Thai Woman

Abstract Hb Agenogi [β90(F6)Glu→Lys (GAG>AAG) HBB: c.271G>A)] is a very rare β-globin chain variant. We report for the first time this hemoglobinopathy in a pregnant 20-year-old Thai woman. She was seen by an obstetrician at her 14th week of gestation. She was pale and had an inflammatory lesion of her lower left leg. The hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) and low pressure liquid chromatography (LPLC) showed a peak of abnormal Hb at the C window. On capillary electrophoresis (CE), the abnormal Hb peak was observed at electrophoretic zone 4 that corresponded to the Hb E (HBB: c.79G>A) peak. Direct DNA sequencing revealed a GAG>AAG mutation at codon 90 of the β-globin gene. Thus, even though Hb Agenogi is very rare, it can be found in Thai people. The knowledge and understanding of this hemoglobinopathy will be used to assist in diagnosis, management and counseling for patients.

Comparison of capillary electrophoregram among heterozygous Hb Hope, Hb Hope/α-thalassemia-1 SEA type deletion and Hb Hope/β0-thalassemia

Abstract Background: Hemoglobin (Hb) A2 is artifactually elevated in cases of heterozygous Hb Hope when measured by capillary electrophoresis (CE). However, there is no report of HbA2 levels and capillary electrophoregrams for associations of heterozygote of Hb Hope with α-thalassemia nor β-thalassemia. Methods: Levels of HbA0, HbA2 and Hb Hope in 16 heterozygous Hb Hope, 3 Hb Hope/α-thalassemia-1 SEA type deletion and 2 Hb Hope/β0-thalassemia were measured by CE. Electrophoregram and the levels of those were compared within these three groups. Results: Artifactually elevated HbA2 levels (≥4%) were found in both groups of heterozygous Hb Hope and Hb Hope/α-thalassemia-1 SEA type deletion. Manual corrections were performed by adjusting baselines, and results showed that means of HbA2 in both groups decreased from 4.47% and 4.03% to 1.93% and 1.77%, respectively. The highest levels of HbA2 and Hb Hope were observed in samples with Hb Hope/β0-thalassemia. Moreover, HbA0 was not observed in these cases. Conclusions: The elevation of HbA2 in patients with heterozygous Hb Hope and with Hb Hope/α-thalassemia-1 SEA type deletion measured by CE leads to incorrect β-thalassemia trait diagnosis. However, using CE electrophoregram together with levels of HbA0, HbA2 and Hb Hope would be a more accurate and precise method for diagnosis of Hb Hope/β0-thalassemia.

Complex Interaction of Hb Q-Thailand (HBA1: c.223G>C) with β-Thalassemia/Hb E (HBB: c.79G>A) Disease

Abstract Hb Q-Thailand [α74(EF3)Asp→His (α1), GAC>CAC, HBA1: c.223G>C] is an abnormal hemoglobin (Hb) frequently found in Thailand and Southeast Asian countries. The association of the αQ-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report how to diagnose the coinheritance of Hb Q-Thailand with β-thalassemia (β-thal)/Hb E disease in four Thai samples from high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) testing results. Understanding of the HPLC chromatogram and CE electropherogram patterns of this complex mutation is important for interpretation of testing results and providing genetic counseling.

Co-inheritance of Hb J-Kaohsiung [β59(E3) AAG>ACG, Lys→Thr] and HbE [β26(B8) GAG>AAG, Glu→Lys] in a Thai Woman

Hemoglobin (Hb) J-Kaohsiung [b59(E3) AAG[ACG, Lys?Thr] is the b-globin chain variant that results from a point mutation (AAG[ACG) at codon 59 of b-globin gene where the normally occurring lysine is replaced by threonine. It was found, in 1969, in the Taiwanese male resident of Kaohsiung City, Taiwan, during a population survey for hemoglobin variants [1]. The pure heterozygote of Hb J-Kaohsiung and the combination of Hb J-Kaohsiung heterozygote with a-thalassemia had been also reported in three Thai subjects [2]. We reported here for the first time the combination of Hb J-Kaohsiung heterozygote with HbE [b26 GAG[AAG, Glu?Lys] in a 34-year-old single Thai woman. The subject was seen by a physician at Theppanya Hospital, Chiang Mai, Thailand for her annual health checkup. Upon examination, she was pale and did not have hepatosplenomegaly. She had no history of receiving a blood transfusion. The complete blood count (CBC) was analyzed by using the automated blood counter (Sysmex KX-21, Sysmex Corporation, Kobe, Japan). Laboratory findings are as follows: WBC 9.10 9 10 cells/L, RBC 4.34 9 10 cells/L, Hb 107 g/L, PCV 0.34 L/L, MCV 78 fL, MCH 24.6 pg, MCHC 31.5 g/L, RDW 13.7% and platelet 196 9 10/L. Thus, she was diagnosed as having mild anemia with the red blood cell microcytosis. The blood sample was also sent to the Associated Medical Sciences–Clinical Service Center (AMS–CSC), Chiang Mai University, Chiang Mai, Thailand for the thalassemia diagnosis. The hemoglobin analysis was performed by using the high-performance liquid chromatography (HPLC, VARIANT II, b-thalassemia Short Program, Bio-Rad Laboratories, Hercules, California, USA). There was no peak of HbA on the HPLC chromatogram while her HbF and HbA2/E were 2.5 and 25.9%, respectively. In addition, the abnormal Hb peak with a value of 65.3% of the total Hb was observed at the retention time of 2.06 min (Fig. 1a). The hemoglobin analysis was also performed by using the capillary electrophoresis method (CE, Capillarys 2 Flex Piercing, Sebia, Norcross, Georgia, USA). Her HbE and HbA2 were 22.7 and 3.0 respectively. In addition, the abnormal Hb peak with the value of 74.3% of total Hb was also presented at a migration position of 50–80 s (Zone 13–14) (Fig. 1b). The real-time PCR with SYBR Green1 and high resolution melting (HRM) analysis for detection of the a-thalassemia-1 Southeast Asian (SEA) and Thai type deletions [3] is routinely performed at the same time the hemoglobin analysis is carried out. Furthermore, the multiplex Gap-PCR for detection of a-thalassemia-2 (-a and -a kb deletions) and the multiplex allele specific PCR for diagnosis of Hb Constant Spring (Hb CS) and Hb Pakse’ [4, 5] were also performed in this case. The negative analysis results for a-thalassemia-1 and -2 deletions and Hb CS and Hb Pakse’ mutations were observed. In order to identify the abnormal Hb, the PCR amplification of the bglobin gene was accomplished according to conditions described previously [5] and then the direct DNA sequencing of the amplified product was performed on an ABI PRISM 3130 XL analyzer (Applied Biosystems, Foster City, CA, USA). The results showed the molecular defect causing abnormal hemoglobin resulting in A–C & Sakorn Pornprasert sakornmi001@gmail.com

Hematological Characterizations and Molecular Diagnostic Aspects of Hb Wiangpapao [α44(CE2)Pro→Ser (α1), CCG>TCG; HBA1: c.133C>T], a New α-Globin Variant Found in a Pregnant Thai Woman

Abstract We report the hematological parameters and provide a rapid molecular analysis method for detection of Hb Wiangpapao [α44(CE2)Pro→Ser, CCG>TCG; HBA1: c.133C>T], a new α-globin variant found in a pregnant Thai woman. Her red cell indices were measured by an automated blood counter. The results were: red blood cell (RBC) count 4.03 × 1012/L, Hb 13.1 (g/dL), packed cell volume (PCV) 0.39 L/L, mean corpuscular volume (MCV) 97.0 fL, mean corpuscular hemoglobin (Hb) (MCH) 32.5 pg, mean corpuscular Hb concentration (MCHC) 33.4 g/dL, and RBC distribution width (RDW) 9.4%. The Hb typing by high performance liquid chromatography (HPLC) showed 13.6% abnormal Hb at a retention time of 2.20 min. that was difficult to distinguish from Hb A. On the capillary electrophoresis (CE) electropherogram, this hemoglobinopathy peak did not separate from the Hb A peak. DNA sequencing showed a C>T transition at the first position of codon 44 (CCG>TCG) of the α1-globin gene that led to a substitution of proline for serine. This mutation has not been recorded in the public databases. Therefore, we named it Hb Wiangpapao as it was first discovered in the Wiangpapao District, Chiang Rai, Thailand. The multiplex allele-specific polymerase chain reaction (ASPCR) for detection of Hb Wiangpapao was developed and revealed a 510 bp specifically amplified fragment. The better understanding of hematological characterizations and the newly developed multiplex ASPCR for diagnosis of Hb Wiangpapao are useful for genetic counseling and family education.