Hematological and Molecular Characterization of Hb J-Buda [α61(E10)Lys → Asn, AAG > AAT].

Hemoglobin (Hb) J-Buda [α61(E10)Lys â†’ Asn, AAG > AAT] is a very rare α-chain variant found in South-East Asia. We analyzed hematological parameters and provided a rapid molecular analysis method for detection of this hemoglobinopathy in two Thai women who had severe microcytic anemia with Hb and MCV <70 g/L and 80 fL, respectively. The HPLC revealed an abnormal Hb peak eluted ahead of HbA at retention time of 1.91-1.98 min. On CE, the abnormal Hb peak was observed at the electrophoretic zone 12 which corresponded to Hb Bart's. The DNA sequencing revealed the AAG â†’ AAT mutation at codon 61 for Hb J-Buda on one allele of the α1-globin gene. The developed Allele-specific PCR (ASPCR) showed the 455 bp amplified fragment from Hb J-Buda allele. Thus, understanding of hematological characterizations and the developed ASPCR for diagnosis of Hb J-Buda are essential for genetic counseling of this hemoglobinopathy.

Development of hemoglobin typing control materials for laboratory investigation of thalassemia and hemoglobinopathies.

BACKGROUND: To date, the hemoglobin (Hb) typing control materials for laboratory investigation of thalassemia with low (1.8%-3.2%) and high (4%-6%) levels of HbA2 are available but there are no Hb typing quality control materials for analysis of thalassemia and hemoglobinopathies which are highly prevalent in South-East Asian countries. The main aim of the present study was to develop the lyophilized Hb typing control materials for laboratory investigation of thalassemia and hemoglobinopathies that are commonly found in South-East Asia. METHODS: Erythrocytes of blood samples containing Hb Bart's, HbH, HbE, HbF, Hb Constant Spring (CS), Hb Hope, and Hb Q-Thailand were washed and dialysed with 0.85% saline solution. The erythrocytes were then lysed in 5% sucrose solution. The lyophilized Hb typing control materials were prepared by using a freeze drying (lyophilization) method. The high performance liquid chromatography (HPLC) analysis of lyophilized Hb was performed after the storage at -20 °C for 1 year and also after reconstitution and storage at 4 or -20 °C for 30 days. In addition, the Hb analysis was compared between the three different methods of HPLC, low pressure liquid chromatography (LPLC) and capillary electrophoresis (CE). RESULTS: Following a year of storage at -20 °C, the HPLC chromatograms of lyophilized Hb typing control materials showed similar patterns to the equivalent fresh whole blood. The stability of reconstituted Hb typing control materials was also observed through 30 days after reconstitution and storage at -20 °C. Moreover, the Hb typing control materials could be analyzed by three methods, HPLC, LPLC and CE. Even a degraded peak of HbCS was found on CE electropherogram. CONCLUSIONS: The lyophilized Hb typing control materials could be developed and used as control materials for investigation of thalassemia and hemoglobinopathies.

Elevated Hb A2 Levels in a Patient with a Compound Heterozygosity for the (ß⁺) -31 (A > G) and (ß°) Codon 17 (A > T) Mutations Together with a Single α-Globin Gene.

We report the molecular and hematological feature of a Thai woman who had clinical diagnosis of ß-thalassemia intermedia (ß-TI). Hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified Hb A (64.4%), Hb F (12.3%) and Hb A2/E (15.9%) with small peaks of Hb Bart's (γ4) and Hb H (ß4). She was initially diagnosed as EA Bart's disease, which occurs from combination of Hb H disease and Hb E (HBB: c.79G > A) trait. However, the Hb analysis using capillary electrophoresis (CE) demonstrated no Hb E, 68.5% Hb A, 15.5% Hb F and 16.0% Hb A2. DNA analysis showed a compound heterozygosity for (ß(+)) -31 (A > G) (HBB: c.-81A > G) and (ß(0)) codon 17 (A > T) (HBB: c.52A > T) mutations and deletional Hb H (- -(SEA)/-α(3.7)). Thus, she was finally diagnosed with a combination of Hb H disease and compound heterozygosity of ß(+)/ß(0)-thalassemia (ß(+)/ß(0)-thal). The ß-globin mutations could affect not only hematological parameters but also elevate the Hb A2 levels. These effects could not be ameliorated by the coinheritance of Hb H disease. Therefore, a better understanding of the effects of this combination on hematological analysis data will be useful for providing accurate diagnosis, genetic counseling, prevention and control programs of ß-thalassemia major (ß-TM).

Red Cell Indices and Formulas Used in Differentiation of ß-Thalassemia Trait from Iron Deficiency in Thai Adults.

ß-Thalassemia (ß-thal) and iron deficiency cause most microcytic anemias. Red cell indices and formulas have been established as simple, fast, and inexpensive in discrimination between these two hematological disorders in school children. However, whether these formulas could be applied to diagnose ß-thal trait and iron deficiency in adult Thai subjects is unclear. The aim of this study was to examine the diagnostic accuracy of five red cell indices [red blood cell (RBC) counts, mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW)] and nine formulas (RDW/RBC, RDW Index, Sirdah, Green and King, Mentzer, England and Fraser, Ehsani, Srivastava and Shine and Lal). Their sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV), efficiency, and Youden's Index were analyzed in 102 ß-thal trait and 64 iron deficiency adult Thai subjects. The RDW/RBC formula proved to be the most reliable index as they had 100.0% specificity and PPV and the highest efficiency (94.58%) and Youden's Index (91.18%), as well as high sensitivity (91.18%) and NPV (87.67%). Therefore, this formula could be used in initial discrimination of ß-thal trait from iron deficiency in adult Thai subjects.

Blood transfusion from a Hb E trait donor can affect ß-thalassemia diagnosis.

A subject with Hb E (HBB: c.79G > A) trait is asymptomatic and can become a blood donor. However, a blood transfusion from a Hb E trait donor can affect ß-thalassemia (ß-thal) diagnosis. Blood samples from three Thai women were sent to the Associated Medical Sciences (AMS) Clinical Service Center, Chiang Mai, Thailand, for thalassemia diagnosis. Their Hb A2 levels, analyzed by high performance liquid chromatography (HPLC), were higher than 4.0%, thus they were diagnosed to have ß-thal. However, elevated Hb A2 levels in these patients were not certain because the Hb A2 levels analyzed at the initial hospitalization and follow-up were controversial. In addition, there were some cases shown to have controversy between the increased Hb A2 level and red cell indices. The blood transfusion history was confirmed and hemoglobin (Hb) analysis was reanalyzed by capillary electrophoresis (CE). On the CE electrophoregram, Hb A2 levels were observed to be normal and Hb E peaks were present. Therefore, to rule out misdiagnosis and unnecessary genetic counseling, Hb analysis should be performed on the recipient prior to blood transfusions. Moreover, CE has a high efficiency to prevent the misinterpretation of Hb analysis in patients who receive blood transfusions from a donor carrying Hb E.

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.

Development of control material for hemoglobin analysis.

Background: There is no certified control material for hemoglobin analysis which has the hemoglobin (Hb)A(2)/E level as high as found in ?-thalassemia trait, HbE trait, ?-thalassemia/HbE disease and homozygote of HbE, the thalassemia types found frequently in the Southeast Asian population. The aim of this study was to prepare the lyophilized hemoglobin control materials for hemoglobin analysis. Methods: Washed and dialysed erythrocytes of normal individuals and patients with ?-thalassemia trait, HbE trait, ?-thalassemia/HbE disease, homozygous HbE were lysed in 5% sucrose solution. The lyophilized hemoglobin control materials were prepared by using a freeze-drying (lyophilization) method. The high performance liquid chromatography (HPLC) analysis of lyophilized hemoglobin was performed after storing at ?20?C for 1, 15 and 30?days and for 3?months. Results: The chromatograms of lyophilized hemoglobin control materials showed similar patterns and similar levels of HbA, HbA(2)/E and HbF when compared with equivalent fresh whole blood. Moreover, the lyophilized hemoglobin presented a good correlation coefficient (r>0.990) of relationships between HPLC, low pressure liquid chromatography (LPLC) and capillary electrophoresis (CE) methods. Conclusions: The lyophilized hemoglobin could be developed and used as control materials for hemoglobin analysis.

Interference of hemoglobin Hope on beta-thalassemia diagnosis by the capillary electrophoresis Method.

The ß-chain hemoglobin (Hb) variants interfere with the diagnosis of ß-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a ß-chain Hb variant frequently found in the Thai population, on ß-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 ß(0)-thalassemia codon 17, 41/42, and 71/72 mutations and ß-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of ß-thalassemia trait analyzed by CE but not by HPLC.