Elevated Hemoglobin A₂: A Molecular Revisited, and Implications to β-Thalassemia Screening

In Thailand, the Hb A₂ cut-off value for β-thalassemia carrier has been changed from 4.0% to 3.6% since 2015. We examined the molecular basis of β-thalassemia in a large cohort of Thai subjects with this change.

A total of 36,313 retrospective specimens encountered during January 2016 to June 2025 were reviewed, and 5909 subjects (16.7%) with Hb A₂ ≥ 3.6% were recruited. Diagnosis of thalassemia was based on Hb and DNA analyses.

Among the 5909 subjects, 5395 (91.3%) had Hb A₂A, 115 (1.9%) carried Hb variants, and the remaining 399 (6.8%) resulted from misinterpretations. Of 5395 subjects, 5117 (94.8%) carried β-thalassemia, 189 (3.5%) had normal β-globin genes, and 89 (1.6%) had incomplete molecular analysis. Fifty different β-thalassemia genes were identified, including nine hitherto undescribed in Thailand and two novel mutations, β^{CD81(CTC > CTA)} and β^{IVSII-713(G > A)}. Falsely elevated Hb A₂ was found in 364 of 399 (70.8%) subjects with misinterpretation, 20 (3.9%) represented human errors, and 15 (2.9%) resulted from Hb E blood transfusion. A low proportion of β⁰- and a high proportion of β⁺-thalassemias, a normal β-globin gene, and diagnostic errors were observed at borderline Hb A₂ levels.

A change in the Hb A₂ cut-off can alter the molecular spectrum of β-thalassemia in Thailand. The molecular basis of β-thalassemia was updated to include 50 mutations, both new and known. Misinterpretation at borderline Hb A₂ should be aware and cost-effectiveness should be considered at the screening of β-thalassemia in the region.