What is the difference between Hemoglobin (Hb) analysis and Deoxyribonucleic Acid (DNA) analysis for ruling out thalassemia, and which test comes first?

Diagnostic Approach: Hemoglobin Analysis vs DNA Analysis for Thalassemia

Which Test Comes First

Hemoglobin (Hb) analysis should always come first as the initial diagnostic test for thalassemia, followed by DNA analysis only when needed for confirmation, carrier identification, or prenatal diagnosis. 1, 2, 3

The Sequential Testing Algorithm

Step 1: Initial Screening with CBC and Red Cell Indices

• Complete blood count (CBC) with red blood cell indices is the mandatory first test, looking specifically for microcytosis (MCV <80 fL) and hypochromia 1, 3
• Critical pitfall: You must exclude or correct iron deficiency before proceeding to hemoglobin analysis, as iron deficiency can mask or confuse thalassemia diagnosis 1
• Measure serum ferritin simultaneously with CBC to rule out concurrent iron deficiency 1
• If iron deficiency is present, provide iron replacement therapy before proceeding with definitive hemoglobin analysis 1

Step 2: Hemoglobin Analysis (HPLC or Capillary Electrophoresis)

• After excluding iron deficiency, perform hemoglobin electrophoresis or high-performance liquid chromatography (HPLC) with quantitation of HbA2 and HbF 1, 2
• This provides both qualitative and quantitative analysis of hemoglobin components and enables both prenatal and postnatal thalassemia diagnosis 2
• HbA2 determination is the most decisive test for β-thalassemia carrier detection 3
• Both HPLC and capillary zone electrophoresis (CE) systems have good correlation, though CE interpretation requires caution as HbA2 is clearly separated from HbE 2

Step 3: DNA Analysis (When Indicated)

DNA analysis is not required for routine β-thalassemia carrier confirmation but is performed in specific situations 3:

Mandatory Indications for DNA Testing:

• Confirming α-thalassemia carrier status (molecular analysis is necessary because hemoglobin analysis alone cannot definitively diagnose α-thalassemia carriers) 3
• Prenatal diagnosis when both parents are carriers to detect severe forms like Hb Bart's hydrops fetalis 4
• Predicting disease severity to distinguish between severe transfusion-dependent and intermediate-to-mild non-transfusion-dependent cases 3
• Cascade family testing after identifying the specific mutation in an affected family member 4

Key Differences Between the Two Tests

Hemoglobin Analysis:

• Purpose: Identifies abnormal hemoglobin patterns and quantifies HbA2/HbF levels 2, 3
• Detects: β-thalassemia carriers definitively through elevated HbA2 (most decisive test) 3
• Limitations: Cannot definitively diagnose α-thalassemia carriers; HbA2 can be lower than normal in α-thalassemia but this is not diagnostic 3
• Cost: Less expensive and faster than DNA analysis 2
• Availability: Widely available in most laboratories 2

DNA Analysis:

• Purpose: Identifies specific genetic mutations causing thalassemia 3
• Detects: Both α- and β-thalassemia mutations with precision; can identify large deletions in α-thalassemia and point mutations in β-thalassemia 2, 3
• Essential for: α-thalassemia carrier confirmation, prenatal diagnosis, and predicting disease severity 3
• Limitations: More expensive; requires specialized laboratory facilities and expertise 2, 3
• Advantage: Can detect mutations that may be missed by conventional hemoglobin analysis techniques 2

Common Diagnostic Pitfalls to Avoid

• Performing hemoglobin analysis before excluding iron deficiency will lead to misdiagnosis, as iron deficiency can lower HbA2 levels and mask β-thalassemia trait 1
• Failing to consider ethnicity in the diagnostic approach (thalassemia is more common in Southeast Asian, Mediterranean, Middle Eastern, and African populations) can lead to missed diagnoses 4, 1
• Assuming normal HbA2 rules out all thalassemia: α-thalassemia carriers may have normal or low HbA2, requiring DNA confirmation 3
• Ordering DNA analysis as a first-line test is unnecessary for β-thalassemia carrier detection and wastes resources 3

When Both Tests Are Needed

In clinical practice, you will need both tests sequentially when 3:

• Hemoglobin analysis suggests α-thalassemia (requires DNA confirmation)
• Both parents are carriers and prenatal diagnosis is needed
• The couple is at risk of having severe thalassemia disease in offspring
• Family cascade screening is being performed after identifying an affected individual 4