Diagnostic Challenges of Borderline A2 Hemoglobin in β-Thalassemia Carriers: Insights from a Bangladeshi Cohort

Background The identification of beta-thalassemia (β-thal) carriers during prenatal screening relies on elevated hemoglobin A2 (HbA2) levels. However, β-thal minor may present as silent carriers, showing no hematologic abnormalities despite carrying a mutation. Borderline HbA2 levels pose a diagnostic challenge. This study aimed to characterize 120 individuals—selected from a pool of 1,650—with borderline HbA₂ levels, to distinguish true carriers from non-carriers. Methods Blood samples were collected in EDTA tubes from Bangladeshi subjects with borderline Hb A2 levels (3.0–3.9%) for this study. The samples were obtained from the Thalassemia Center at Bangladesh Shishu Hospital and Institute. Hematological parameters were measured using an automated blood counter (ADVIA 2120i hematology analyzer; Siemens Healthcare Diagnostics, Deerfield, IL, USA). HBB genotypes were identified using the Sanger sequencing method. Results Among the subjects, pathogenic mutations of the β-globin gene were identified in 3 cases (15%) with HbA₂ levels of 3.3–3.4%, and in 17 cases (85%) with levels between 3.5–3.9%. A total of seven pathogenic mutations were identified, with IVS1-5 (G > C) being the most common (55%, n = 11), followed by FS 41/42 (− CTTT), Codon 30 (G < C), and Codon 30 (G < A), each at 10% (n = 2). FS 8/9 (+ G), Codon 15 (G > A), and FS 16 (− C) were each found in 5% of cases (n = 1). Conclusions This study supports improved identification of β-thal carriers with borderline HbA₂ levels, helping to reduce the risk of misdiagnosis.