Manoo Punyamung

Red Cell Indices and Formulas Used in Differentiation of β-Thalassemia Trait from Iron Deficiency in Thai School Children

Abstract Red cell indices and formulas have been established as simple, fast, and inexpensive means for discrimination between the β-thalassemia (β-thal) trait and iron deficiency. However, there were no reports of the diagnostic reliability of different red cell indices and formulas in discrimination of β-thal trait from iron deficiency in the Thai population. The aim of this study was to examine the diagnostic accuracy of five red cell indices [red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW)] and eight formulas (Sirdah, Green & King, RDW Index, Menzler, England & Fraser, Ehsani, Srivastava, and Shine & Lal). Their sensitivity, specificity, positive and negative prognostic value and efficiency, were analyzed in 77 Thai school children, 21 with the β-thal trait and 56 with iron deficiency. The Sirdah and Srivastava formulas proved to be the most reliable indexes as they had 100.0% sensitivity and negative predictive value, the highest efficiency (97.4%), and the highest Youden’s Index value (96.4%). Therefore, these formulas could be used in initial discrimination of the β-thal trait from iron deficiency in Thai school children.

Hb A2/E Levels Found in Co-Inheritance with the α-Thalassemia-1 – –SEA/ Type Deletion and Either Hb E or β-Thalassemia

The α-thalassemia-1 (α-thal-1) Southeast Asian (– –SEA) type deletion, β-thalassemia (β-thal) and Hb E [β26(B8)Glu→Lys, GAG>AAG] are the most common genetic disorders in Southeast Asian populations. Mean corpuscular volume (MCV) <80.0 fL with normal hemoglobin (Hb) is used for screening α- and β-thal, and a Hb E level of less than 25.0% is used for predicting α-thal-1 in Hb E trait. Thus, levels of Hb, MCV and Hb A2/E were reviewed and compared between the SEA type deletion co-inherited with β-thal trait (n = 61), with Hb E trait (n = 102) or homozygous Hb E (n = 13) and β-thal trait (n = 636), Hb E trait (n = 544) or homozygous Hb E (n = 83), respectively. When comparing the values of all three analyzed hematological parameters, only the – –SEA/βE values were shown to be significantly lower than those of Hb E trait. Furthermore, at a cut-off value of Hb A2/E of 21.54%, 95.0% of the – –SEA/βE had Hb A2/E levels lower than this cut-off value, while 94.0% of Hb E trait had Hb A2/E at higher levels. Accordingly, the Hb A2/E level at 21.54% is the best indicator for predicting co-inheritance of the α-thal-1 – –SEA/ deletion and Hb E trait.

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

Abstract β-Thalassemia (β-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 β-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 β-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 β-thal trait from iron deficiency in adult Thai subjects.

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

Abstract 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.

Proficiency testing program for hemoglobin E, A2 and F analysis in Thailand using lyophilized hemoglobin control materials

Abstract Background: There is no external quality assessment (EQA) program for hemoglobin analysis that uses lyophilized hemoglobin control materials with HbA2/E in levels as high as those found in people with the β-thalassemia trait, HbE trait, β-thalassemia/HbE disease or homozygous HbE; these are all found frequently in the southeast Asian population. The aim of this study was to evaluate the efficiency of the control materials used in the established proficiency testing (PT) program at the Associated Medical Sciences-Clinical Service Center (AMC-CSC), Chiang Mai University, Chiang Mai, Thailand. Methods: The PT program for Hb analysis and the thalassemia interpretation was established in compliance with ISO/IEC17043:2010. Three cycles per year were performed in 2015 and 2016. In each cycle, three different types of control material were provided to the participants. Each participant analyzed the control materials in the same manner as in their routine practices. Hb analysis results and their thalassemia interpretation codes were entered into the report form and sent back to AMC-CSC. Results: The number of participants increased from 63 in 2015 to 76 in 2016. In addition, the number of participants who took part in all three cycles increased from 95.2% (60/63) in 2015 to 100% (76/76) in 2016. All participants reported the correct Hb measurement and type; however, misinterpretations in thalassemia diagnosis were noted. Conclusions: The lyophilized hemoglobin control materials prepared at AMC-CSC were used successfully in our PT program. However, the study results indicate the need for further improvement in thalassemia interpretation skills for laboratory staff.

Detection of Hb Constant Spring (HBA2: c.427T>C) Heterozygotes in Combination with β-Thalassemia or Hb E Trait by Capillary Electrophoresis.

Abstract Hb Constant Spring (Hb CS; HBA2: c.427T>C) is often missed by routine laboratory testing as 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) for detecting and quantifying of Hb CS in β-thalassemia (β-thal) trait or Hb E (HBB: c.79G>A) trait samples with reduced β-globin chain expression. Thalassemia diagnostic data were reviewed in 2524 blood samples that were submitted to the laboratory of the Associated Medical Sciences Clinical Service Center, Chiang Mai, Thailand for hemoglobinopathy and thalassemia diagnosis. DNA analysis for Hb CS was performed in 322 β-thal trait and 397 Hb E trait samples using the amplification refractory mutation system (ARMS). The CE electropherogram of Hb CS at zone 2 was observed in all five samples with β-thal trait and nine samples with Hb E trait with levels varying from 0.1–2.8 and 0.1–2.3%, respectively. Thus, the CE method proved useful for screening of Hb CS in samples with β-thal trait or Hb E trait, which is essential for providing accurate diagnosis, genetic counseling, prevention and control programs of Hb H-CS disease.

Hematological Analysis in Thai Samples With Deletional and Nondeletional HbH Diseases

Objectives To compare hematological parameters between deletional and nondeletional HbH diseases, and to investigate the correlation between HbH levels and hematological parameters within these 2 groups. Methods Samples of 43 deletional HbH diseases, which included 39 --SEA/-α3.7, 4 - -SEA/-α4.2, and 22 nondeletional HbH diseases (- -SEA/αcsα), were used in this study. Correlations between HbH levels and hematological parameters within these 2 groups were analyzed. Results The deletional HbH disease had higher levels of RBC counts, total Hb, pack cell volume (PCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), HbA, and HbA2 than did the nondeletional HbH disease. A negative correlation between HbH and RBC counts was detected in the group of deletional HbH disease, while a positive correlation between HbH and RBC counts, total Hb, and PCV was found in the group of nondeletional HbH disease. Conclusions These results reflected that samples with nondeletional HbH showed more anemic features than those with the deletional HbH.

A Formula to Identify Potential Cases of β-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 β-thalassemia/hemoglobin E (β-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 β-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 β-thal/HbE specimens (100%) and only 8 of 65 homozygous HbE specimens (12.3%). Also, the formula yielded 90.5% efficiency in identifying β-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 β-thal/HbE disease among patients with absent HbA, HbE of greater than 75%, and HbF between 5% and 15%.