What is the recommended test for diagnosing alpha thalassemia?

Diagnostic Testing for Alpha Thalassemia

DNA testing for deletions or point mutations is the definitive diagnostic test to identify the specific genetic defect in alpha thalassemia. 1

Initial Screening Approach

Begin with complete blood count (CBC) focusing on mean corpuscular volume (MCV):

• MCV <80 fL suggests possible alpha thalassemia carrier status 1, 2
• Red blood cell count is typically normal or elevated, which helps distinguish from iron deficiency anemia where RBC count is usually low 2
• Red blood cell distribution width (RDW) ≤14.0% suggests thalassemia trait, while RDW >14.0% suggests iron deficiency anemia 2

Critical pitfall to avoid: Always measure serum ferritin to exclude concurrent iron deficiency, as iron deficiency can mask thalassemia trait characteristics 2. If iron deficiency is present, provide iron replacement therapy before proceeding with further testing 2.

Hemoglobin Analysis

After initial screening suggests thalassemia, hemoglobin analysis should be performed using either:

• High-performance liquid chromatography (HPLC) 3
• Capillary zone electrophoresis (CE) 3

Both systems provide qualitative and quantitative analysis of hemoglobin components 3. However, hemoglobin analysis alone may not detect all alpha thalassemia cases, particularly silent carriers with single gene deletions 4.

Definitive Molecular Testing

DNA analysis is essential for confirming alpha thalassemia carrier status and determining the specific mutation. 1, 5

Available Molecular Techniques:

For common deletions:

• Multiplex gap-polymerase chain reaction (PCR) 4
• Single-tube multiplex PCR 4
• Multiplex ligation-dependent probe amplification 4
• Loop-mediated isothermal amplification 4

For non-deletion mutations (point mutations):

• Reverse dot blot analysis 4
• Single tube multiplex PCR 4
• Sanger sequencing (widely used standard) 4

For comprehensive analysis:

• Next-generation sequencing (NGS) can detect both deletions and point mutations with high accuracy 4, 3. This technique may identify mutations missed by conventional methods, though cost remains a limitation 3.

Validated PCR Techniques Include:

• Restriction fragment length polymorphism (RFLP) 6
• Sequence-specific primer (SSP) 6
• Real-time (RT) PCR 6
• Bead-chip techniques 6

Ethnicity Considerations

Failure to consider ethnicity can lead to missed diagnoses. 1 Alpha thalassemia is most common in:

• Southeast Asian populations 1, 2
• Mediterranean populations 1
• Middle Eastern populations 1
• African populations 2

Prenatal Diagnosis

When both parents are carriers, prenatal diagnosis is recommended to detect severe forms like Hemoglobin Bart's hydrops fetalis 1:

Invasive testing options:

• Chorionic villus sampling (CVS) at 10-13 weeks gestation for DNA-based testing 2
• Amniocentesis at 15-20 weeks gestation for DNA analysis 2
• Fetal blood sampling (cordocentesis) to evaluate for abnormal Bart's hemoglobin, though less commonly used than DNA-based methods 2

Non-invasive options:

• Non-invasive prenatal diagnosis using NGS, RT-PCR, or droplet digital PCR from maternal circulation 6

Ultrasound surveillance:

• Middle cerebral artery Doppler to assess for fetal anemia 1
• Ultrasound in late second and early third trimester can detect hydrops fetalis in severe alpha thalassemia 2

Laboratory Selection Considerations

The routine method employed should be based on facility resources, expertise, available equipment, and economic conditions 4. Most service laboratories should select the technique(s) they are most familiar with and most economical for routine use 3.