Recommended Test for Beta Thalassemia
The recommended initial test for beta thalassemia is a complete blood count (CBC) with red blood cell indices, followed by hemoglobin electrophoresis or high-performance liquid chromatography (HPLC) with quantitation of HbA2 and HbF—but only after iron deficiency has been excluded or corrected. 1, 2
Initial Screening: CBC with Red Blood Cell Indices
The first step involves analyzing specific red blood cell parameters that distinguish beta thalassemia trait from other causes of microcytic anemia:
- Mean Corpuscular Volume (MCV) is characteristically low in beta thalassemia trait, typically <80 fL and often <76 fL in all cases 1, 3
- Mean Corpuscular Hemoglobin (MCH) is reduced, with mean values around 19.3 g/dL; MCH <26 provides 99% sensitivity for detecting beta thalassemia trait 3
- Red Blood Cell (RBC) count is normal or elevated in thalassemia trait, which contrasts with iron deficiency where it is typically low 1, 4
- Red Blood Cell Distribution Width (RDW) is a critical differentiating factor: RDW ≤14.0% suggests thalassemia trait, while RDW >14.0% suggests iron deficiency anemia 1
Essential Pre-Testing Requirement: Exclude Iron Deficiency
A critical pitfall is performing hemoglobin analysis before correcting iron deficiency, as concurrent iron deficiency can falsely lower HbA2 levels and mask the diagnosis of beta thalassemia trait. 1
- Serum ferritin must be measured simultaneously with the CBC to rule out concurrent iron deficiency 1, 2
- If iron deficiency is present (ferritin below reference range), iron replacement therapy should be provided before proceeding with definitive hemoglobin analysis 1
- Iron deficiency can mask typical laboratory findings of thalassemia trait, particularly HbA2 elevation 1
Definitive Diagnostic Test: Hemoglobin Analysis
Once iron deficiency has been excluded or corrected, proceed with hemoglobin analysis:
- Hemoglobin electrophoresis or HPLC with quantitation of HbA2 and HbF is the definitive test 2, 5
- Both HPLC and capillary zone electrophoresis (CE) provide qualitative and quantitative analysis of hemoglobin components 5
- Elevated HbA2 (typically >3.5%) is the hallmark finding in beta thalassemia trait 2
- These systems enable both prenatal and postnatal thalassemia diagnosis within a short period 5
Additional Diagnostic Considerations
- Peripheral blood smear examination should be performed to confirm the size, shape, and color of red blood cells 1
- Reticulocyte count helps distinguish between decreased production and increased destruction or loss of red blood cells 1
- If microcytosis/hypochromia is present with normal hemoglobin electrophoresis, a brilliant cresyl blue stained blood smear should be performed to identify H bodies (to exclude alpha thalassemia) 2
When to Proceed to Molecular Testing
- DNA analysis for specific beta-globin gene mutations is indicated when both partners are carriers or when hemoglobin analysis results are inconclusive 2, 6
- Molecular techniques are necessary for definitive mutation identification and prenatal diagnosis 5, 6
- Next-generation sequencing (NGS) provides the most accurate diagnosis and can detect mutations that may be missed by conventional techniques, though cost remains a limitation 5
Common Pitfalls to Avoid
- Never interpret HbA2 levels without first confirming normal iron stores, as this is the most common cause of false-negative results 1
- Lead poisoning, anemia of chronic inflammation, and sickle cell trait can present with similar red cell indices and must be excluded 1
- In the presence of chronic disease, vitamin B12/folate deficiency, or other hemoglobinopathies, MCV and hypochromia lose sensitivity for distinguishing between conditions 7
- When using capillary electrophoresis, interpretation requires caution because HbA2 is clearly separated from HbE, and alpha thalassemia gene interactions can affect HbA2/E quantitation 5