The importance of hemoglobin A₂ determination^☆

Hemoglobin (Hb) A₂ (a₂d₂) constitutes less than 3% of the total hemoglobin (Hb) in adults and has almost no physiological importance.¹ On the other hand, the determination of Hb A₂ is an important tool to diagnose the beta-thalassemia trait (BTT).^{1 and 2} Although individuals with BTT do not need treatment, the accurate detection of the carrier state is important in genetic counseling to determine risk of having a child with a major disease.³

Elevated levels of Hb A₂ and microcytosis are suggestive of the diagnosis of BTT. However, BTT may be present with normal levels of Hb A₂ as a few cases of ß-thalassemia are not associated with elevated Hb A₂, and because of the association of BTT with iron deficiency or with a-thalassemia (a-Thal).^{1 , 2 , 4 , 5 and 6} There are many other factors, inherited or acquired, that can interfere in Hb A₂ levels (Table 1).^{3 and 4}

Table 1
- Causes of variation in the percentage of hemoglobin A₂.

• Hb A₂ - hemoglobin A₂; HPFH: hereditary persistence of fetal hemoglobin; HIV: human immunodeficiency virus; HbH: hemoglobin H; Hb F: hemoglobin F or fetal hemoglobin. Source: Modified from Bain et al. 4

Hb A₂ can be measured by several laboratorial methods, but these methods have differences in accuracy.⁷ Cation exchange high performance liquid chromatography (HPLC), microcolumn chromatography, and cellulose acetate electrophoresis with elution are considered acceptable methods to diagnose BTT, whereas cellulose acetate electrophoresis followed by scanning densitometry is not.² The accuracy of cellulose acetate electrophoresis with elution depends on the training and experience of the laboratory technician who performs the test, and microcolumn chromatography can give problems with co-elution of some Hb variants.⁷

Recent studies have confirmed the higher quality of automated HPLC in the measurement of Hb A₂ compared to the other methods,^{8 and 9} which is why this has become the method of choice. On the other hand, in automated HPLC, the measurement of Hb A₂ is inaccurate when Hb S is present.^{2 , 3 and 10} As the amount of Hb S is related to the degree of inaccuracy, levels are higher in patients with sickle cell anemia (SCA) or Hb S/ß-thalassemia (S-ßThal) than in sickle cell trait.² Thus, the amount of Hb A₂ does not indicate BTT when Hb A and beta gene variants are found together.¹¹ Furthermore, when beta gene variants are present without Hb A, the diagnosis of concomitant BTT is not necessarily associated to the elevation of Hb A₂ and so further investigations using family studies or DNA analysis are necessary.¹⁰

As mentioned above, a-Thal is capable of interfering in the determination of Hb A₂.⁴ Individuals with the a⁰-thalassemia trait or homozygous for a⁺-thalassemia have lower levels of Hb A₂, but the influence of the coinheritance of a-Thal and BTT on Hb A₂ levels is uncertain.⁶

In Brazil, the incidence of a-Thal varies from 0.11 to 0.22% depending on the geographical region studied.^{12 , 13 , 14 and 15} It is well known that the association of a-Thal and SCA is common in Brazil.^{16 , 17 and 18} Since SCA is considered a public health problem in Brazil and due to the clinical significance of a-Thal in respect to this anemia, diagnosis is important.^{19 , 20 and 21} However, diagnosis is mainly achieved by molecular techniques that are expensive and not easily accessible. It is also important to remember that the co-inheritance of a-Thal and SCA results in increased levels of Hb A₂ as measured by automated HPLC, and could result in a misdiagnosis of S-ßThal.⁴

There lies the importance of the paper entitled "Hemoglobin A₂ values quantified by high performance liquid chromatography in patients with sickle cell disease, and the influence of the presence of alpha-thalassemia" written by Fonseca et al. and published in this edition of the Revista Brasileira de Hematologia e Hemoterapia.²² The authors demonstrate that Hb A₂ was overestimated not only in individuals with Hb S but also in patients with Hb C, and that the Hb A₂level was influenced by the genotype of a-Thal.

In conclusion, in a country with a high degree of miscegenation such as Brazil, not only the diagnosis of double heterozygous states, such as S-ßThal, but also the diagnosis of co-inheritance of SCA with a-Thal should be carried out carefully, taking into consideration the limitations of the available laboratory techniques. Family studies or DNA analysis, when possible, are desirable to confirm the correct diagnosis.

References

• ¹
  Steinberg MH, Adams JG. Hemoglobin A2 : origin, evolution, and aftermath. Blood. 1991;78(9):2165-77.
• ²
  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(3):276-80.
• ³
  Giambona A, Passarello C, Renda D, Maggio A. The significance of the hemoglobin A(2) value in screening for hemoglobinopathies. Clin Biochem. 2009;42(18):1786-96.
• ⁴
  Bain BJ, Wild BJ, Stephens AD, Phelan LA. Variant hemoglobins: a guide to identification. 1st ed. West Sussex, UK: Wiley-Blackwell; 2010.
• ⁵
  Saleh-Gohari N, Khademi Bami M, Nikbakht R, Karimi-Maleh H. Effects of alpha-thalassaemia mutations on the haematological parameters of beta-thalassaemia carriers. J Clin Pathol. 2015;68(7):562-6.
• ⁶
  Denic S, Agarwal MM, Al Dabbagh B, El Essa A, Takala M, Showqi S, et al. Hemoglobin A2 lowered by iron deficiency and alpha-thalassemia: should screening recommendation for beta-thalassemia change? ISRN Hematol. 2013;2013:858294.
• ⁷
  Colah RB, Surve R, Sawant P, D'Souza E, Italia K, Phanasgaonkar S, et al. HPLC studies in hemoglobinopathies. Indian J Pediatr. 2007;74(7):657-62.
• ⁸
  Anagnostopoulos K, Tentes I, Kalleas C, Margaritis D, Toli A, Pendilas D, et al. Effect of HbS in the determination of HbA2 with the Menarini HA-8160 analyzer and comparison with other instruments. Int J Lab Hematol. 2009;31(6):665-72.
• ⁹
  Paleari R, Gulbis B, Cotton F, Mosca A. Interlaboratory comparison of current high-performance methods for HbA2 . Int J Lab Hematol. 2012;34(4):362-8.
• ¹⁰
  Shokrani M, Terrell F, Turner EA, Aguinaga MD. Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin. Ann Clin Lab Sci. 2000;30(2):191-4.
• ¹¹
  Giordano PC. Editorial: measurement of HbA2 . Int J Lab Hematol. 2012;34(4):335.
• ¹²
  Silva Cde A, Baldim LB, Nhoncanse GC, Estevao Ida F, Melo DG. Neonatal screening for hemoglobinopathies in Sao Carlos, Sao Paulo, Brazil: analysis of a series of cases. Rev Paul Pediatr. 2015;33(1):19-27.
• ¹³
  de Medeiros Alcoforado GH, Bezerra CM, Araújo Moura Lemos TM, de Oliveira DM, Kimura EM, Ferreira Costa F, et al. Prevalence of alpha-thalassemia 3.7 kb deletion in the adult population of Rio Grande do Norte, Brazil. Genet Mol Biol. 2012;35(3):594-8.
• ¹⁴
  Wagner SC, de Castro SM, Gonzalez TP, Santin AP, Filippon L, Zaleski CF, et al. Prevalence of common alpha-thalassemia determinants in south Brazil: importance for the diagnosis of microcytic anemia. Genet Mol Biol. 2010;33(4):641-5.
• ¹⁵
  Adorno EV, Couto FD, Moura Neto JP, Menezes JF, Rêgo M, Reis MG, et al. Hemoglobinopathies in newborns from Salvador, Bahia, Northeast Brazil. Cad Saude Publica. 2005;21(1):292-8.
• ¹⁶
  Lyra IM, Gonçalves MS, Braga JA, Gesteira Mde F, Carvalho MH, Saad ST, et al. Clinical, hematological, and molecular characterization of sickle cell anemia pediatric patients from two different cities in Brazil. Cad Saude Publica. 2005;21(4):1287-90.
• ¹⁷
  Figueiredo MS, Kerbauy J, Gonçalves MS, Arruda VR, Saad ST, Sonati MF, et al. Effect of alpha-thalassemia and beta-globin gene cluster haplotypes on the hematological and clinical features of sickle-cell anemia in Brazil. Am J Hematol. 1996;53(2):72-6.
• ¹⁸
  De Lemos Cardoso G, Guerreiro JF. Molecular characterization of sickle cell anemia in the Northern Brazilian state of Para. Am J Hum Biol. 2010;22(5):573-7.
• ¹⁹
  de Paiva e Silva RB, Ramalho AS, Cassorla RM. Sickle cell disease as a public health problem in Brazil. Rev Saude Publica. 1993;27(1):54-8.
• ²⁰
  Domingos IF, Falcão DA, Hatzlhofer BL, Cunha AF, Santos MN, Albuquerque DM, et al. Influence of the betas haplotype and alpha-thalassemia on stroke development in a Brazilian population with sickle cell anaemia. Ann Hematol. 2014;93(7):1123-9.
• ²¹
  Camilo- Araujo RF, Amancio OM, Figueiredo MS, Cabanas- Pedro AC, Braga JA. Molecular analysis and association with clinical and laboratory manifestations in children with sickle cell anemia. Rev Bras Hematol Hemoter. 2014;36(5):334-9.
• ²²
  Fonseca SF, Amorim T, Purificação A, Goncalves MS, Boa-Sorte N. Hemoglobin A2 values quantified by high performance liquid chromatography in patients with sickle cell disease, and the influence of the presence of alpha-thalassemia. Rev Bras Hematol Hemoter. 2015;37(5):296-301.

• ☆See paper by Fonseca et al. on pages 296-301.

Publication Dates

• Publication in this collection
  Sep-Oct 2015

History

• Received
  24 June 2015
• Accepted
  07 July 2015