What are the key lab tests and diagnostic criteria to differentiate between beta and alpha thalassemia in a patient with a suspected diagnosis and relevant family history and ethnic background?

Laboratory Differentiation of Beta vs Alpha Thalassemia

The key to differentiating beta from alpha thalassemia is hemoglobin electrophoresis or HPLC: elevated HbA2 (>3.5%) confirms beta thalassemia trait, while normal or low HbA2 with microcytosis suggests alpha thalassemia trait, which requires DNA testing for definitive diagnosis. 1, 2

Initial Screening Tests (Both Types)

Complete Blood Count with Red Cell Indices:

• MCV <80 fL is the primary screening indicator for thalassemia carrier status in at-risk populations (Mediterranean, Middle Eastern, South Asian, Southeast Asian, African descent) 1, 2
• RBC count is typically normal or elevated in thalassemia trait, distinguishing it from iron deficiency where RBC count is usually low 1, 3
• RDW ≤14.0% suggests thalassemia trait, while RDW >14.0% suggests iron deficiency anemia 1, 3
• Serum ferritin must be measured to exclude concurrent iron deficiency, as iron deficiency can mask thalassemia trait characteristics and falsely lower HbA2 levels 1, 3

Critical Pitfall to Avoid:

If iron deficiency is present, provide iron replacement therapy before performing hemoglobin analysis, as iron deficiency can falsely lower HbA2 levels and mask beta-thalassemia trait diagnosis 3

Confirmatory Testing to Differentiate Beta from Alpha Thalassemia

Hemoglobin Analysis (HPLC or Capillary Electrophoresis):

Beta Thalassemia Trait:

• HbA2 >3.5% is diagnostic for beta thalassemia trait 1
• HbF may be mildly elevated (1-5%) 1
• This test provides both qualitative and quantitative analysis of hemoglobin components 4

Alpha Thalassemia Trait:

• HbA2 is normal or low (<3.5%) 1, 2
• Hemoglobin H (HbH) detection indicates three-gene deletion alpha thalassemia (Hemoglobin H disease) 1
• In newborns with severe alpha thalassemia, Hemoglobin Bart's may be detected 2, 3
• DNA testing for deletions or point mutations is the definitive diagnostic test to identify the specific genetic defect in alpha thalassemia 2, 4

Ethnic Background Considerations

Alpha thalassemia is most common in Southeast Asian, Mediterranean, Middle Eastern, and African populations, and is the most common cause of non-immune hydrops fetalis in Southeast Asian populations (28-55% of cases) 2, 3

Beta thalassemia predominates in Mediterranean, Middle Eastern, and South Asian populations 3

Severity Assessment Based on Lab Findings

Alpha Thalassemia Severity:

• Silent carrier (one-gene deletion): Normal CBC, no clinical significance 5
• Alpha thalassemia trait (two-gene deletion): Mild microcytic anemia, MCV <80 fL 1, 5
• Hemoglobin H disease (three-gene deletion): Hemolytic anemia with HbH detected on electrophoresis, requires CBC monitoring every 3-6 months 1, 2
• Hemoglobin Bart's hydrops fetalis (four-gene deletion): Typically fatal, detected prenatally 2, 5

Beta Thalassemia Severity:

• Beta thalassemia trait: Mild microcytic anemia, HbA2 >3.5%, asymptomatic 1, 5
• Beta thalassemia intermedia: Variable severity, may require occasional transfusions 1, 5
• Beta thalassemia major: Severe transfusion-dependent anemia, markedly elevated HbF, absent or very low HbA 1, 5

When DNA Testing is Required

DNA analysis is mandatory for:

• Confirming alpha thalassemia when hemoglobin analysis shows normal or low HbA2 with microcytosis 2, 4
• Identifying specific deletions in alpha thalassemia (Southeast Asian deletion is common) 4, 6
• Detecting point mutations in beta thalassemia when diagnosis is unclear 4, 6
• Partner screening when one parent is a carrier to assess risk of severe disease in offspring (25% risk if both carriers) 1, 3
• Prenatal diagnosis via chorionic villus sampling (10-13 weeks) or amniocentesis (15-20 weeks) for at-risk couples 1, 3

Algorithmic Approach

1. Screen with CBC: MCV <80 fL + normal/elevated RBC count + RDW ≤14.0% → suspect thalassemia 1, 3
2. Check ferritin: If low, treat iron deficiency first before proceeding 1, 3
3. Perform hemoglobin electrophoresis/HPLC: 1, 4
   • HbA2 >3.5% → Beta thalassemia trait confirmed
   • HbA2 normal/low + microcytosis → Alpha thalassemia suspected, proceed to DNA testing
   • HbH detected → Hemoglobin H disease (three-gene deletion alpha thalassemia)
4. DNA testing: Required for definitive alpha thalassemia diagnosis and genetic counseling 2, 4
5. Partner screening: Mandatory when carrier identified to assess reproductive risk 1, 3