What are the diagnostic tests for alpha thalassemia trait in adults?

Diagnostic Tests for Alpha Thalassemia Trait in Adults

DNA testing for deletions or point mutations is the definitive diagnostic test for alpha thalassemia trait in adults, as recommended by the American College of Medical Genetics. 1

Initial Screening Tests

Complete Blood Count (CBC) with Red Cell Indices

• Obtain CBC focusing on mean corpuscular volume (MCV), red blood cell count, and red blood cell distribution width (RDW) as the first-line screening test 1
• MCV <80 fL suggests possible alpha thalassemia carrier status 1
• RDW ≤14.0% suggests thalassemia trait, while RDW >14.0% suggests iron deficiency anemia 1
• Alpha thalassemia trait typically shows microcytosis (low MCV) with normal or elevated red blood cell count 2

Serum Ferritin

• Measure serum ferritin to exclude concurrent iron deficiency, which can mask thalassemia trait characteristics 1
• Provide iron replacement therapy before proceeding with further testing if iron deficiency is present, as iron deficiency can obscure the typical findings of thalassemia 1

Hemoglobin Analysis

• Perform hemoglobin quantification using high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) 2, 3
• These systems provide both qualitative and quantitative analysis of hemoglobin components 2
• Critical caveat: Hemoglobin analysis alone may not detect all alpha thalassemia cases, particularly silent carriers with single gene deletions 2, 3
• In adults with alpha thalassemia trait, hemoglobin electrophoresis is typically normal or shows only subtle changes 4

Definitive Molecular Testing

DNA Analysis for Deletions

• Multiplex gap-polymerase chain reaction (Gap-PCR) is the primary method to detect common alpha-globin gene deletions 5, 3
• Multiplex ligation-dependent probe amplification (MLPA) should be used for diagnosis of rare or novel deletions not detected by Gap-PCR 5, 3
• Loop-mediated isothermal amplification can also detect common deletions 3
• Most alpha thalassemia cases (75.3%) are caused by deletions 5

DNA Analysis for Point Mutations

• Sequencing of the alpha-globin gene is necessary to identify known and novel point mutations 5
• Reverse dot blot analysis and single-tube multiplex PCR can detect non-deletion mutations 3
• Sanger sequencing is widely used to detect non-deletion mutations 3
• Point mutations comprise approximately 25% of alpha thalassemia cases 5

Next-Generation Sequencing (NGS)

• NGS can detect both deletion and point mutations of alpha thalassemia in a single test 2, 3
• NGS provides accurate diagnosis that may be missed by conventional techniques 2
• Major limitation: Cost remains prohibitively expensive for routine screening 2

Ethnicity-Based Considerations

• Failure to consider ethnicity can lead to missed diagnoses 1
• Alpha thalassemia is most common in Southeast Asian, Mediterranean, Middle Eastern, and African populations 1
• Muslim Arabs and certain Jewish populations (Sephardic origin) present with higher rates of point mutations and hemoglobin H disease 5

Diagnostic Algorithm

1. First: Obtain CBC with indices (MCV, RBC count, RDW) and serum ferritin 1
2. Second: If MCV <80 fL and ferritin is normal, proceed with hemoglobin analysis by HPLC or CE 2, 3
3. Third: Perform Gap-PCR to detect common deletions 5, 3
4. Fourth: If Gap-PCR is negative but clinical suspicion remains high, perform alpha-globin gene sequencing for point mutations 5, 3
5. Fifth: If sequencing is negative, use MLPA to detect rare or novel deletions 5, 3

Common Pitfalls to Avoid

• Do not rely solely on hemoglobin electrophoresis: Silent carriers (single alpha gene deletion) and alpha thalassemia trait (two gene deletions) typically show normal or near-normal hemoglobin patterns in adults 2, 3, 4
• Do not proceed with molecular testing if iron deficiency is present: Iron deficiency must be corrected first, as it can mask thalassemia findings 1
• Do not assume negative Gap-PCR excludes alpha thalassemia: Point mutations and rare deletions require additional testing 5, 3
• Do not overlook ethnicity: Testing strategy should be guided by the patient's ethnic background, as mutation prevalence varies significantly 1, 5