How is thalassemia diagnosed?

Diagnosis of Thalassemia

Thalassemia diagnosis begins with complete blood count showing microcytic hypochromic anemia with low MCV and MCH, followed by hemoglobin analysis using HPLC or capillary electrophoresis to quantify hemoglobin fractions, and confirmed by molecular DNA testing to identify specific gene mutations or deletions. 1, 2

Initial Screening Tests

Complete Blood Count (CBC) Analysis

• MCV is typically below 80 fL (often 60-70 fL in β-thalassemia trait), which serves as the primary screening parameter 3, 2
• MCH is markedly reduced (19-23 pg in β-thalassemia carriers) 2
• RBC count is normal or elevated, distinguishing thalassemia from iron deficiency anemia where RBC count is typically low 4
• RDW ≤14.0% suggests thalassemia trait, while RDW >14.0% suggests iron deficiency anemia 4

Peripheral Blood Smear

• Peripheral blood smear examination confirms microcytosis, hypochromia, and characteristic red blood cell morphology 4
• Reticulocyte count helps distinguish between decreased production versus increased destruction 4

Iron Status Assessment

• Serum ferritin must be measured to exclude iron deficiency, as concurrent iron deficiency can mask thalassemia findings 4, 2
• Iron deficiency should be corrected before proceeding with definitive hemoglobin analysis, as it can falsely lower HbA2 levels 4

Hemoglobin Analysis

High-Performance Liquid Chromatography (HPLC) or Capillary Electrophoresis (CE)

• HPLC or CE provides both qualitative and quantitative analysis of hemoglobin fractions and is the standard method for hemoglobin characterization 1, 5
• HbA2 measurement is the most decisive test for β-thalassemia carrier detection, with elevated HbA2 (>3.5%) being diagnostic 2
• Important caveat: HbA2 measurement should only be performed after iron deficiency has been corrected, as iron deficiency falsely lowers HbA2 levels 4
• In α-thalassemia, HbA2 can be lower than normal, which assumes diagnostic significance when iron deficiency is excluded 2
• CE interpretation requires caution because HbA2 is clearly separated from HbE, and α-thalassemia gene interactions can affect HbA2/E quantities 1

Molecular DNA Testing

Confirmatory Diagnosis

• DNA analysis is essential for confirming α-thalassemia carrier status, as hemoglobin analysis alone cannot reliably detect α-thalassemia 2, 6
• Molecular testing is not required to confirm β-thalassemia carrier status but is necessary to predict disease severity and for prenatal diagnosis 2

Detection Methods for α-Thalassemia Deletions

• Multiplex gap-PCR, single-tube multiplex PCR, multiplex ligation-dependent probe amplification (MLPA), and loop-mediated isothermal amplification detect common α-globin gene deletions 6
• DNA testing for deletions or point mutations is the definitive diagnostic test to identify specific genetic defects 3

Detection Methods for β-Thalassemia Point Mutations

• Reverse dot blot analysis and single-tube multiplex PCR detect non-deletion mutations 6
• Sanger sequencing is widely used for detecting point mutations in β-thalassemia 6

Next-Generation Sequencing (NGS)

• NGS provides accurate diagnosis of both α- and β-thalassemia that may be misdiagnosed by conventional techniques, detecting both deletions and point mutations 1, 6
• The major limitation is cost, which remains expensive for routine screening 1

Prenatal Diagnosis

When Both Parents Are Carriers

• Prenatal diagnosis using amniocentesis or chorionic villus sampling with DNA analysis is recommended when both parents are carriers to detect severe forms 3, 7
• DNA analysis on chorionic villi uses the same methods as postnatal mutation detection 2
• Middle cerebral artery Doppler assesses for fetal anemia in suspected hydrops fetalis cases 3

Cascade Testing Strategy

• Test the affected family member first to identify the specific genetic mutation, followed by targeted testing of at-risk family members 3

Critical Diagnostic Pitfalls

• Concurrent iron deficiency masks thalassemia trait characteristics, particularly HbA2 elevation in β-thalassemia trait; always correct iron deficiency before final assessment 4, 2
• Lead poisoning, anemia of chronic inflammation, and sickle cell trait can present with similar red cell indices and must be excluded 4
• Failure to consider ethnicity leads to missed diagnoses; α-thalassemia is most common in Southeast Asian, Mediterranean, Middle Eastern, and African populations, while β-thalassemia is prevalent in Mediterranean, Middle Eastern, and Asian populations 3, 5
• Mathematical formulas (discriminant indices) to distinguish thalassemia from iron deficiency should be used cautiously, as RBC count is the only truly discriminating parameter 2