Testing for Thalassemia
Thalassemia testing begins with a Complete Blood Count (CBC) showing microcytosis (low MCV) and normal or elevated red blood cell count, followed by hemoglobin analysis (HPLC or capillary electrophoresis) to measure HbA2 and HbF levels, and confirmed with DNA analysis for specific mutations when needed.
Initial Screening: Red Blood Cell Indices
The first-line approach uses automated hematology analyzers to identify characteristic red cell abnormalities 1, 2:
- Mean Corpuscular Volume (MCV): Typically low, often below the 5th percentile for age in thalassemia trait 1
- Red Blood Cell (RBC) count: Normal or elevated in thalassemia trait, distinguishing it from iron deficiency where RBC count is typically low 1
- Red Blood Cell Distribution Width (RDW): Critical differentiating factor where RDW ≤14.0% suggests thalassemia trait, while RDW >14.0% suggests iron deficiency anemia 1
- Mean Reticulocyte Volume (MRV): Values <100 fL support thalassemia diagnosis 3
The combination of MCV, MRV, and reticulocyte parameters achieves 99.5% sensitivity for thalassemia screening 3.
Hemoglobin Analysis
After initial screening suggests thalassemia, hemoglobin analysis is performed using either 2, 4:
- High-Performance Liquid Chromatography (HPLC): Provides quantitative measurement of HbA2, HbF, and abnormal hemoglobins
- Capillary Zone Electrophoresis (CE): Offers similar quantitative analysis but requires cautious interpretation as HbA2 is clearly separated from HbE 2
Both systems provide qualitative and quantitative hemoglobin component analysis within a short timeframe 2.
Critical Pre-Testing Requirement
Iron deficiency must be corrected before measuring HbA2 levels 1:
- Serum ferritin should be measured to rule out concurrent iron deficiency 1
- Iron deficiency falsely lowers HbA2 levels, masking beta-thalassemia trait 1
- If ferritin is below reference range, provide iron replacement therapy before final thalassemia assessment 1
This is a common pitfall that leads to missed diagnoses, as concurrent iron deficiency can mask typical laboratory findings of thalassemia trait 1, 5.
Confirmatory DNA Testing
Molecular analysis is required for 2, 6:
- Beta-thalassemia: PCR-based techniques identify specific point mutations
- Alpha-thalassemia: Gap-PCR detects large deletions and discriminates between heterozygous and homozygous states 6
- Prenatal diagnosis: Essential for couples at risk 4, 6
- Prediction of clinical severity: Identifies mutation type (β⁰ vs β⁺) 6
Next-generation sequencing (NGS) provides the most accurate diagnosis and can detect mutations missed by conventional techniques, though cost remains a limitation 2.
Additional Diagnostic Components
- Peripheral blood smear: Essential to confirm red blood cell size, shape, and color characteristics 1
- Reticulocyte count: Distinguishes between decreased production versus increased destruction/loss of red blood cells 1
Common Diagnostic Pitfalls
Several conditions can mimic or mask thalassemia 1, 5:
- Concurrent iron deficiency: Most common confounding factor, present in 22.3% of beta-thalassemia carriers with co-inherited alpha-thalassemia 5
- Lead poisoning: Can present with similar red cell indices 1
- Anemia of chronic inflammation: May show overlapping hematological features 1
- Sickle cell trait: Must be excluded through hemoglobin analysis 1
In regions with high prevalence of both alpha- and beta-thalassemia (such as Southeast Asia), the combination of hemoglobin analysis and DNA testing is necessary for reliable carrier determination, as hematological phenotypes alone cannot reliably distinguish between different thalassemia types 5.
Algorithmic Approach
- Order CBC with red cell indices (MCV, RBC count, RDW, MRV) 1, 3
- Check serum ferritin; if low, treat iron deficiency first 1
- Perform hemoglobin analysis (HPLC or CE) after iron repletion 1, 2
- If thalassemia trait suspected, proceed to DNA analysis for specific mutation identification 2, 6
- For prenatal diagnosis or severity prediction, molecular testing is mandatory 4, 6