|Year : 2020 | Volume
| Issue : 2 | Page : 84-88
Increase in Haemoglobin A2 Window among Healthy Children in Najran Area, Saudi Arabia
Osama Al Shehri1, Jobran M Alqahtani2, Mohamed Samir M. Khalil3, Mohamed Mahmoud El Khawanky4
1 Department of Haematology, College of Applied Medical Sciences, Najran University, Saudi Arabia
2 Department of Pediatrics, College of Medicine, Najran University, Saudi Arabia
3 Department of Clinical Pathology, College of Medicine, Assiut University, Egypt
4 Department of Clinical Haematopathology, College of Medicine, Najran University, Saudi Arabia
|Date of Submission||16-Feb-2020|
|Date of Decision||13-Mar-2020|
|Date of Acceptance||14-Mar-2020|
|Date of Web Publication||28-Jul-2020|
Dr. Mohamed Mahmoud El Khawanky
Department of Clinical Hematopathology, College of Medicine, Najran University, Najran
Source of Support: None, Conflict of Interest: None
INTRODUCTION: A minor increase in hemoglobin (Hb) A2 is common in B thalassemia trait in which Hb A2 usually exceeds 3.5%. Other inherited and acquired disorders result in minor increases that do not often exceed 8%. The remarkable increase (>20%) in Hb A2 may refer to other structural variants that also migrate in the Hb A2 window. The aim of this study was to assess the increased Hb A2 levels and their incidence among schoolchildren in Najran city.
AIMS: The aim of this study was to assess the increased Hb A2 levels and their incidence among schoolchildren in Najran city.
SUBJECTS AND METHODS: This study was conducted on 665 male students of Najran primary schools. All students were subjected to a full history taking and laboratory investigations including a complete blood count, peripheral blood smear examination, measurement of serum iron and ferritin levels, reticulocyte count, and alkaline Hb gel electrophoresis.
RESULTS: Four (0.60%) students had highly significant (P = 0.000) increased Hb A2 levels above 20%. Three of them descend to the same tribe. Two cases had mild anemia and the other two cases had normal Hb levels. All cases showed microcytosis and hypochromia with normal serum iron and ferritin levels.
CONCLUSION: Alkaline Hb electrophoresis showed highly significant increased Hb A2 levels among school children with microcytosis and hypochromia. Further investigations are required to ensure whether this is an increase of Hb A2 or from other Hb variants that take the same window of Hb A2.
Keywords: Anemia, electrophoresis, hemoglobin A2, hypochromia, microcytosis
|How to cite this article:|
Al Shehri O, Alqahtani JM, M. Khalil MS, El Khawanky MM. Increase in Haemoglobin A2 Window among Healthy Children in Najran Area, Saudi Arabia. J Appl Hematol 2020;11:84-8
|How to cite this URL:|
Al Shehri O, Alqahtani JM, M. Khalil MS, El Khawanky MM. Increase in Haemoglobin A2 Window among Healthy Children in Najran Area, Saudi Arabia. J Appl Hematol [serial online] 2020 [cited 2021 Mar 1];11:84-8. Available from: https://www.jahjournal.org/text.asp?2020/11/2/84/290960
| Introduction|| |
Hemoglobin A2 (Hb A2) is a variant of Hb that comprises two alpha and two delta chains. Hb A2 is found in <3.5% of the total Hb in average adults. Hence, it has unknown physiological significance. Various causes explain the reason behind the increased Hb A2 levels. One of them is β-thalassemia trait (BTT) in which Hb A2 usually exceeds 3.5%, and is considered a typical characteristic trait of people belonging to certain communities, for instance, Vietnamese or South East Asian communities. Other inherited factors include sickle cell anemia, some cases of congenital dyserythropoietic anemia, and coexisting sickle cell traits and alpha thalassemia. In addition to this, there are various acquired disorders in which Hb A2 levels increase, for instance, thyrotoxicosis, HIV infection, megaloblastic anemia, lead poisoning, sideroblastic anemia, juvenile myelomonocytic leukemia, and zidovudine therapy administration.
The remarkable increase in Hb A2 may refer to other structural variants that are also migrate in the Hb A2 window. Several Hb disorders, such as Hb C, Hb E, and Hb O-Arab, inundate the Hb A2 position in alkaline Hb electrophoresis and are misunderstood as an increase in Hb A2 levels.,,
Hb A2 can be measured by several laboratorial methods, such as cation exchange high performance liquid chromatography (HPLC), microcolumn chromatography, and cellulose acetate or gel electrophoresis.
It is necessary to have good precision in quantitative Hb A2 methods, so that not to be misdiagnosed as an increased Hb A2 instead of another Hb variant has the same migration place.
| Subjects and Methods|| |
This study was conducted on 665 male students of Najran primary schools as part of a research project aimed at detecting anemia among school children. Twelve primary schools were geographically selected to represent all regions of the Najran area.
Ethical approval from the Ethical Committee of Najran University and from the educational administration in the Najran area was obtained prior to conducting the study, and an informed consent form from students' parents was signed before withdrawing blood samples from the students.
A complete family and personal history for each child was taken in a questionnaire. The children were classified according to age and tribe.
Three milliliter of intravenous blood samples were collected in ethylene diamine tetra acetic acid for complete blood count (CBC) and alkaline Hb electrophoresis. Laboratory tests were carried out following the standard procedures after cross checking for quality control from time to time. CBC was done using an automated blood cell counter (COULTER®LH 500 Hematology Analyzer-Beckman Coulter, USA) and Hb electrophoresis was carried out at alkaline pH 8.6 using SAS-1plus, Helena Biosciences' SAS-2 gel processing system.
The collected data were revised, coded, tabulated, and analyzed statistically using the SPSS software (IBM SPSS Inc., version 20, Chicago, Illinois, USA). Data were presented and suitable analyses were carried out according to the type of data obtained for each parameter. Statistical differences were considered significant if the P < 0.05, and highly significant if the P < 0.001.
| Results|| |
Among the 665 male students of the Najran primary schools, 4 (0.60%) students had highly significant (P = 0.000) increased Hb A2 levels above 20% through alkaline Hb gel electrophoresis [Table 1].
|Table 1: Hemoglobin gel electrophoresis pattern, serum iron profile, and reticulocyte count|
Click here to view
These cases inhabit Najran city but their origins descend to the surrounding areas. Three of them belong to the same tribe and are not siblings. The fourth case descends to Abha. All of these areas lie in the western south of KSA [Table 2].
|Table 2: Complete blood count and the descriptive data of the increased hemoglobin A2 cases|
Click here to view
It is known that Najran city is 1100–1700 m above sea level (National Geodetic Survey, 2019), so we compared the Hb levels of these four students (have increased HbA2 levels) with the mean ± SD Hb levels of 582 healthy students. We have excluded the students with chronic disorders and/or with abnormal iron profile. Two cases of increased Hb A2 had mild anemia (11.1, 11.8 g/dl) and another two cases were of normal Hb (14.2, 12.8 g/dl) according to the age, sex, and altitude [Table 2] and [Figure 1] as the normal ranges were (12.74 ± 0.81, 12.02 ± 0.96, 13.1 ± 0.79, and 13.35 ± 1.0), respectively [Table 3].
|Figure 1: Alkaline hemoglobin electrophoresis of increased hemoglobin A2 case|
Click here to view
|Table 3: Hemoglobin concentration, hemoglobin A2, and hemoglobin F levels of healthy male students in Najran city|
Click here to view
All cases showed microcytosis and hypochromia, however, they had a normal iron profile (serum iron and ferritin levels) and a normal reticulocyte count [Table 1].
| Discussion|| |
Hb disorders, or hemoglobinopathies, present a significant worldwide health problem. The basic defect of hemoglobinopathies is either an altered structure or reduced production of globin chain synthesis. Particular consideration is given to altered levels of Hb A2 as its increase by a minor degree indicates the presence of a Hb disorder. For instance, an elevated Hb A2 with a decreased mean corpuscular volume (MCV) is suggestive of a diagnosis of BTT., Normally, Hb A2 forms about 2%–3% of the total Hb in adults with ambiguous physiological roles.
In our study, we found that 4 of the 655 children had Hb A2 levels ranging from 20% to 35%. Three of them had descended from one tribe, although their blood samples were drawn from three different schools from different residential neighborhoods. Notably, their returning to one origin supported the validity of our results.
BTT is the most common Hb variant with elevated Hb A2 levels (>3.5%). Elevated levels of Hb A2, decreased MCV and relative erythrocytosis are suggestive of BTT. However, BTT may be present with normal levels of Hb A2 if associated with iron deficiency or α- thalassemia. There are also many other inherited and acquired factors that can impede Hb A2 levels.
A remarkable increase (>20%) in Hb A2 may refer to other structural Hb variants that migrate into the Hb A2 window, such as Hb C, Hb E, and Hb O-Arab.,,
In our study, the two cases of increased Hb A2 levels showed mild anemia (11.1 and 11.8 g/dl according to ages 6 and 9 years, respectively) with normal serum iron, ferritin levels and reticulocyte count, but the red blood cell (RBC) indices revealed microcytosis and hypochromia. So far, these characteristics (↑HbA2, ↓ MCV, and ↓ MCH) match the Hb E trait.
Hb E, commonly present in South East Asia, results from the substitution of glutamic acid with lysine at codon 26 of the β globin chain. Hb E disorders may be heterozygous (AE), homozygous (EE) or compound with another Hb variant or thalassemia., Hb E is usually associated with a normal Hb concentration or mild anemia, but it may also be associated with decreased MCV that may be confused with iron deficiency anemia. Although Hb E alone does not cause any significant clinical disorders, its interactions with other hemoglobinopathies produce a wide range of clinical syndromes. Co-inheritance of Hb E and β-thalassemia results in overt disease, ranging from mild anemia to severe β-thalassemia.
It is necessary to diagnostically distinguish Hb E disorders because of their clinical diversity. DNA-based diagnosis of Hb E disorders is important and unlike other laboratory investigations, definite. For instance, when tested with alkaline and acidic Hb electrophoresis, Hb E migrates simultaneously with Hb A2.
In our results, the other two cases of increased Hb A2 levels showed normal Hb levels (14.2 and 12.8 g/dl according to ages 10 and 14 years, respectively) and decreased MCV and MCH with normal serum iron and ferritin levels.
Another Hb variant that migrates simultaneously with Hb A2 during alkaline Hb electrophoresis is Hb C. Hb C is a structural variant of the beta globin chain caused by an amino acid substitution of lysine for glutamic acid at position 6 of the beta chain. Hb C is less soluble than Hb A and crystallizable due to the electrostatic interactions between the positively charged β6-lysyl groups and adjacent negatively charged groups in vivo. Although the crystal formation of Hb C increases blood viscosity and cellular rigidity and decreases RBC lifespan, Hb AC as a trait is clinically silent and Hb CC disease is a mild clinically irrelevant disorder. Moreover, it may protect RBCs against malaria infestation.
Hb C becomes clinically important when co-inherited with either Hb S (Hb SC disease) or β-thalassemia (Hb C–β thalassemia). The clinical manifestations of Hb SC disease are commonly similar to but less severe than those of Hb SS disease. Prominently, two specific complications occur more frequently in Hb SC than Hb SS: Vascular retinopathy and avascular necrosis of the femoral head.,
Heterozygous Hb C patients show 30%–40% Hb C, which has the same mobility as Hb E and Hb O-Arab during alkaline electrophoresis. If co-migration is suspected, a second electrophoretic procedure is usually performed on citrate agar, which can distinguish it from the other variants.
Another Hb variant that migrates with Hb A2 at alkaline pH is Hb O-Arab. It carries an altered amino acid at position 121 of the beta globin chain due to a GAA to AAA point mutation., Hb O-Arab heterozygous has no clinical manifestations, while homozygotes present mild hemolytic anemia with mild splenomegaly, an insignificant clinical disorder, especially where no infection or severe illness is present.
The most characteristic features of Hb O-Arab include mild decreased MCV and elevated mean corpuscular hemoglobin concentration and more severe microcytosis, anisocytosis, poikilocytosis, hypochromia, target cell, and basophilic stippling if combined with β-thalassaemia. In addition, Hb O-Arab enhances the sickling effect of Hb S, leading to sickle crisis and mimicking of SS disease., However, a Hb O-Arab blot coincides with that of Hb A2 in alkaline electrophoresis, but it has unique electrophoretic properties with an acidic pH.
This study was a part of a research project aimed at detecting anemia among schoolchildren. The increased Hb A2 levels were unexpected as such levels had been previously undetected in the Najran area as far as we know. This study will be complemented by a research project that will use more investigatory tools, such as acid Hb electrophoresis, HPLC, and DNA sequencing.
| Conclusion|| |
Alkaline Hb electrophoresis revealed a highly significant increase in Hb A2 levels in cases presenting microcytosis and hypochromia among the schoolchildren. Further DNA laboratory studies are required to reach a definite diagnosis.
Financial support and sponsorship
This work was sponsored and supported by Najran University Program for Health and Medical Research Grants (NU 06/09).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Figueiredo MS. The importance of hemoglobin A2 determination. Rev Bras Hematol Hemoter 2015;37:287-9.
Colah RB, Surve R, Sawant P, D'Souza E, Italia K, Phanasgaonkar S, et al
. HPLC studies in hemoglobinopathies. Indian J Pediatr 2007;74:657-62.
Wong P, Weerakul J, Sritippayawan S. Hemoglobin analysis in the first year of life. Mediterr J Hematol Infect Dis 2016;8:e2016012.
Powars DR, Hiti A, Ramicone E, Johnson C, Chan L. Outcome in hemoglobin SC disease: A four-decade observational study of clinical, hematologic, and genetic factors. Am J Hematol 2002;70:206-15.
Papadopoulos V, Vassiliadou D, Xanthopoulidis G, Petridis D, Agorasti A, Loukopoulos D. The implications of haemoglobin O-Arab mutation. Haematologica 2003;6:479-85.
Dass J, Gupta A, Mittal S, Saraf A, Langer S, Bhargava M. Comparison of the characteristics of two hemoglobin variants, Hb D-Iran and Hb E, eluting in the Hb A2 window. Blood Res 2017;52:130-4.
Higgins TN, Khajuria A, Mack M. Quantification of HbA(2) in patients with and without beta-thalassemia and in the presence of HbS, HbC, HbE, and HbD Punjab hemoglobin variants: Comparison of two systems. Am J Clin Pathol 2009;131:357-62.
Baxi A, Manila K, Kadhi P, Heena B. Carrier screening for β thalassemia in pregnant Indian women: Experience at a single center in Madhya Pradesh. Indian J Hematol Blood Transfus 2013;29:71-4.
Weatherall DJ, Clegg JB. The thalassaemia syndromes. Oxford: Blackwell Science; 2001.
Denic S, Agarwal MM, Al Dabbagh B, El Essa A, Takala M, Showqi S, et al
. Hemoglobin A2 lowered by Iron deficiency and α -thalassemia: Should screening recommendation for β -thalassemia change? ISRN Hematol 2013; Article ID 858294:5. doi:10.1155/2013/858294.
Steinberg MH, Rodgers GP. HbA2: Biology, clinical relevance and a possible target for ameliorating sickle cell disease. Br J Haematol 2015;170:781-7.
Head CE, Conroy M, Jarvis M, Phelan L, Bain BJ. Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of alpha thalassaemia. J Clin Pathol 2004;57:276-80.
Giambona A, Passarello C, Renda D, Maggio A. The significance of the hemoglobin A(2) value in screening for hemoglobinopathies. Clin Biochem 2009;42:1786-96.
Fucharoen S, Winichagoon P. Thalassemia in South East Asia: Problems and strategy for prevention and control. Southeast Asian J Trop Med Public Health 1992;23:647-55.
Kohne E. Hemoglobinopathies: Clinical manifestations, diagnosis, and treatment. Dtsch Arztebl Int 2011;108:532-40.
Park ES, Jung HL, Kim HJ, Park SS, Bae SH, Shin HY, et al
. Hereditary hemolytic anemia in Korea from 2007 to 2011: A study by the Korean hereditary hemolytic anemia working party of the Korean society of hematology. Blood Res 2013;48:211-6.
Bain BJ. Other significant haemoglobinopathies. In: Haemoglobinopathy Diagnosis. 2nd
ed. Malden, MA: Blackwell Publishing; 2006. p. 201-11.
Vichinsky E. Hemoglobin E syndromes. Hematol 2007;1:79-83.
Charache S, Conley CL, Waugh DF, Ugoretz RJ, Spurrell JR. Pathogenesis of hemolytic anemia in homozygous hemoglobin C disease. J Clin Invest 1967;46:1795-811.
Modiano D, Luoni G, Sirima BS, Simporé J, Verra F, Konaté A, et al
. Haemoglobin C protects against clinical Plasmodium falciparum
malaria. Nature 2001;414:305-8.
Tripette J, Alexy T, Hardy-Dessources MD, Mougenel D, Beltan E, Chalabi T, et al
. Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease. Haematologica 2009;94:1060-5.
Wild BJ, Bain BJ. Detection and quantitation of normal and variant haemoglobins: An analytical review. Ann Clin Biochem 2004;41:355-69.
Lacerra G, Fioretti G, De Angioletti M, Pagano L, Guarino E, de Bonis C, et al
. (Alpha) alpha 5.3: A novel alpha(+)-thalassemia deletion with the breakpoints in the alpha 2-globin gene and in close proximity to an Alu family repeat between the psi alpha 2- and psi alpha 1-globin genes. Blood 1991;78:2740-6.
Sabo G, Brodbeck U, Cardile N, Viollier AF, Scheurmann T, Knecht H. Diagnosis of thalassemias and hemoglobinopathies by HPLC (high performance liquid chromatography): Study of 627 patients. Schweiz Med Wochenschr 1999;129:1196-200.
Vassiliadou D, Papadopoulos V, Konstantinidou C, Arnaoutoglou M. HbOThrace trait, HbOThrace hemoglobinopathy and HbOThrace/Hb beta zero hemoglobinopathy: A retrospective study of 118 cases. Folia Med (Plovdiv) 1998;40:46-50.
Rachmilewitz EA, Tamari H, Liff F, Ueda Y, Nagel RL. The interaction of hemoglobin O Arab with Hb S and beta+ thalassemia among Israeli Arabs. Hum Genet 1985;70:119-25.
[Table 1], [Table 2], [Table 3]