Association of Hb Shenyang [α26(B7)Ala→Glu, GCG>GAG, HBA2: c.80C>A (or HBA1)] with Several Types of α-Thalassemia in Thailand.

Hb Shenyang [α26(B7)Ala→Glu, HBA2: c.80C>A (or HBA1)] is a rare α chain variant. Its genotype-phenotype relationship and origin have not been described in Thailand before. Three Thai subjects (P1-P3) carrying this variant were studied. Hemoglobin (Hb) analysis was performed by capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) as well as molecular characterization using appropriate polymerase chain reaction (PCR) techniques and DNA sequencing. Hemoglobin analysis by HPLC revealed fast-moving abnormal peaks at a retention time (RT) of 1.59-1.62 min., while CE revealed a fast-moving abnormal Hb at zone 12 and ahead of Hb A2 in three subjects. DNA analysis revealed a C>A transition at codon 26 of the α2-globin gene glutamic acid to replace alanine, corresponding to Hb Shenyang. The Southeast Asian [- -SEA α-thalassemia-1 (α-thal-1)] deletion was also identified in P1 and his mother, while Hb Constant Spring (Hb CS, HBA2: c.427T > C) was identified in P2. The Hb Shenyang concentration measured by CE revealed 5.1-17.2% heterozygosity with normal red blood cell (RBC) parameters. The α haplotype [+ - S + - + -] [S signifies the inter ζ hypervariable region (HVR)] was associated with the Thai Hb Shenyang. The genotype-phenotype relationship indicates Hb Shenyang is likely a non pathological Hb variant that has neither dramatic clinical symptoms nor hematological anomalies. A simple multiplex allele-specific PCR for rapid diagnosis of Hb Shenyang has been developed.

A Formula to Identify Potential Cases of ß-Thalassemia/HbE Disease Among Patients With Absent HbA, HbE >75% and HbF Between 5 and 15.

OBJECTIVE: To establish a simple formula to be used for discrimination between ß-thalassemia/hemoglobin E (ß-thal/HbE) and homozygous hemoglobin (Hb)E in specimens with absent hemoglobin (Hb)A, HbE of greater than 75%, and HbF between 5% and 15%. METHODS: We analyzed laboratory results from February 2015 through February 2018. Molecular analysis for diagnosis of ß-thal mutation and HbE was performed in specimens that contained HbE of greater than 75% and HbF from 5% to 15%, as measured by high-performance liquid chromatography (HPLC). HbA2 and HbF levels were also measured by capillary electrophoresis. Then, the formula (6 × HbA2 + HbF)/MCV was developed. RESULTS: The score of 0.9 or higher was found in all 19 ß-thal/HbE specimens (100%) and only 8 of 65 homozygous HbE specimens (12.3%). Also, the formula yielded 90.5% efficiency in identifying ß-thal/HbE disease, and the efficiency was found to be higher compared with when the HbA2 value of greater than 6% was used by itself (85.4%). CONCLUSION: The formula (6 × HbA2 + HbF)/MCV, with a cutoff point at 0.9, could identify the potential cases of ß-thal/HbE disease among patients with absent HbA, HbE of greater than 75%, and HbF between 5% and 15%.

Comparison of HbA2, E, F and Red Cell Parameters in Homozygous HbE With and Without α0-Thalassemia Trait.

OBJECTIVES: To compare levels of HbA2, HbE, HbF, and red cell parameters (total Hb, PCV, MCV, and MCH) and also determine their appropriated cut-off points for initial discrimination between homozygous HbE with and without α0-thalassemia trait. METHODS: Hb analysis results from capillary electrophoresis (CE) and red cell parameters of homozygous HbE without α0-thalassemia trait (n = 41) and with α0-thalassemia trait (n = 17) were reviewed. RESULTS: The MCV, MCH, and HbE of homozygous HbE with α0-thalassemia trait were significantly lower than those of homozygous HbE without α0-thalassemia, while HbA2 levels of the former were significantly higher than those of the latter. HbA2 at a cut-off point of 5.3% had 69.0% efficiency in discrimination between the 2 groups. It could also reduce 56.1% of homozygous HbE samples for α0-thalassemia testing. CONCLUSIONS: The elevated HbA2 ≥5.3% is a useful marker for initial discrimination between homozygous HbE with and without α0-thalassemia trait.

Detection of beta-thalassemia/hemoglobin E disease in samples which initially were diagnosed as homozygous hemoglobin E.

BACKGROUND: Differentiation of beta-thalassemia/HbE disease from homozygous HbE in samples containing HbA2/E > 75% and HbF < 15% is difficult. The aim of this study is to observe the possibility of using Hb typing and hematological parameters to identify both disorders. METHODS: Multiplex amplification refractory mutation system (MARMS)-PCR for beta-thalassemia codons 17 (A > T), 41/42 (-TCTT), 71/72 (+A), and IVSI-ntl (G > T) mutations and ARMS-PCR for HbE were performed in 67 samples that contained HbA2/E > 75% and HbF < 15%. RESULTS: Beta-thalassemia/HbE disease was identified in 10 of 67 (14.93%) samples. Levels of hemoglobin, hematocrit, and mean corpuscular volume (MCV) of beta-thalassemia/HbE disease were significantly lower than those of homozygous HbE whereas, levels of HbF were significantly higher. CONCLUSIONS: In places where the molecular analysis is not available, HbF > 5% in combination with MCV < 55 fL, hemoglobin < 100 g/L, and hematocrit < 0.30 L/L could be used for screening of beta-thalassemia/HbE disease.

Diagnosis of alpha-thalassemia-1 Southeast Asian type deletion and fetal gender by single-tube multiplex real-time PCR.

BACKGROUND: The combination of an x-linked hematologic disorder with a heterozygous alpha-thalassemia-1 Southeast Asian (SEA) type deletion might lead to severe anemia in male infants. This study is to develop a simple and cost-effective single tube multiplex real-time PCR for the diagnosis of alpha-thalassemia-1 SEA type deletion and detect fetal gender. METHODS: Multiplex real-time polymerase chain reaction (PCR) for the detection of alpha-thalassemia-1 SEA type deletion gene, wild type alpha-globin gene, and sex-determining region Y (SRY) gene was validated by analysis of 95 cord blood samples (60 normal individuals, 28 heterozygous of alpha-thalassemia-1 SEA type deletion and 7 Bart's hydrops fetalis). The change in threshold cycle (deltaC(T)) was analyzed by subtracting the C(T-mutant) from C(T-wild) type. RESULTS: Mean deltaC(T) values were significantly different among these three groups, and a SRY gene determination was 100% in concordance with fetal genders. Furthermore, analysis of fetal gender did not affect the deltaC(T) of alpha-thalassemia-1 SEA detection. CONCLUSIONS: Combined alpha-thalassemia-1 SEA type detection and fetal gender determination in a single-tube multiplex real-time PCR is an alternative assay for a conventional method for the diagnosis of alpha-thalassemia-1 SEA deletion and fetal gender.

Comparison of capillary electrophoregram among heterozygous Hb Hope, Hb Hope/α-thalassemia-1 SEA type deletion and Hb Hope/ß(0)-thalassemia.

BACKGROUND: Hemoglobin (Hb) A(2) is artifactually elevated in cases of heterozygous Hb Hope when measured by capillary electrophoresis (CE). However, there is no report of HbA(2) levels and capillary electrophoregrams for associations of heterozygote of Hb Hope with α-thalassemia nor ß-thalassemia. METHODS: Levels of HbA(0), HbA(2) and Hb Hope in 16 heterozygous Hb Hope, 3 Hb Hope/α-thalassemia-1 SEA type deletion and 2 Hb Hope/ß(0)-thalassemia were measured by CE. Electrophoregram and the levels of those were compared within these three groups. RESULTS: Artifactually elevated HbA(2) 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 HbA(2) in both groups decreased from 4.47% and 4.03% to 1.93% and 1.77%, respectively. The highest levels of HbA(2) and Hb Hope were observed in samples with Hb Hope/ß(0)-thalassemia. Moreover, HbA(0) was not observed in these cases. CONCLUSIONS: The elevation of HbA(2) in patients with heterozygous Hb Hope and with Hb Hope/α-thalassemia-1 SEA type deletion measured by CE leads to incorrect ß-thalassemia trait diagnosis. However, using CE electrophoregram together with levels of HbA(0), HbA(2) and Hb Hope would be a more accurate and precise method for diagnosis of Hb Hope/ß(0)-thalassemia.

Rapid identification of heterozygous and homozygous hemoglobin constant spring by SYTO9 with a high resolution melting analysis.

BACKGROUND: Gel-electrophoresis and ethidium bromide are not ideally suited to large scale analysis in clinical laboratories. METHODS: Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) specific for Hb CS was performed in 10 blood samples from normal individuals and 61 samples containing a peak of Hb CS when analyzed by capillary electrophoresis. Heterozygosity of Hb CS was identified using SYTO9 and high resolution melting (HRM) analysis method. RESULTS: Specific peak heights of amplified fragments of wild type and Hb CS alleles were observed in the heterozygote. Only one peak height of amplified fragments of the wild type allele was observed in the normal individual while only one peak height of amplified fragments of Hb CS allele was observed in the homozygote. HRM analysis interpretation results were completely consistent with the interpretation results from gel-electrophoresis. CONCLUSIONS: SYTO9 HRM analysis may be used as an alternative for rapid diagnosis of heterozygosity of Hb CS.

Coinheritance of Hb S [ß6(A3)Glu→Val, GAG>GTG] with ß0-thalassemia codon 17 (A>T) in a Thai patient.

Hb S [ß6(A3)Glu→Val, GAG>GTG] is a ß-globin gene variant that has a very low incidence in the Thai population. Coinheritance of Hb S and ß(0)-thalassemia (ß-thal) can result in severe clinical conditions. This study reports the case of a Thai patient with a compound heterozygosity for Hb S and ß(0)-thal codon 17 (A>T). His hemoglobin (Hb), hematocrit (packed cell volume, PCV), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) levels were all less than the lower limits, while red cell distribution width (RDW) was higher than the upper limit. Levels of Hbs S, F and A(2) detected by high performance liquid chromatography (HPLC) were comparable to those from capillary electrophoresis (CE). As Hb S has a similar electrophoretic mobility and the HPLC profile is also similar to those of Hb Tak [ß147, Term→Thr (+AC)] and Hb D-Punjab [ß121(GH4)Glu→Gln, GAA>CAA], DNA sequencing was then performed. This was to detect ß(0)-thal, and to differentiate Hb S from the Hb Tak and Hb D-Punjab mutations. The ß(0)-thal codon 17 and Hb S mutations were detected indicating that coinheritance of these two mutations can be found in the Thai population. Therefore, to provide proper clinical management and genetic counseling of this rare case, DNA analysis should be performed in all cases when a peak at the S-window is detected by HPLC or CE.

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.