What is the role of hemoglobin (Hb) electrophoresis in diagnosing and managing thalassemia?

Role of Hemoglobin Electrophoresis in Thalassemia Diagnosis and Management

Hemoglobin electrophoresis is a critical diagnostic tool for thalassemia that identifies characteristic hemoglobin patterns essential for accurate diagnosis, classification, and management of the disease. 1

Diagnostic Value of Hemoglobin Electrophoresis

Hemoglobin analysis techniques include:

• Hemoglobin electrophoresis: Separates hemoglobin variants based on their electrical charge
• High-performance liquid chromatography (HPLC): Provides quantitative analysis of hemoglobin variants
• Capillary electrophoresis: Offers high resolution separation of hemoglobin variants
• Isoelectric focusing: Separates hemoglobins based on their isoelectric point 1, 2

These techniques reveal characteristic patterns depending on the type of thalassemia:

• Alpha thalassemia: Shows presence of Hemoglobin H (β4) and Hemoglobin Bart's (γ4) in varying percentages
• Beta thalassemia trait: Typically shows elevated HbA2 (>3.5%) and variable HbF
• Beta thalassemia major: Shows predominantly HbF with absent or minimal HbA
• HbE/beta thalassemia: Shows HbE, HbF, and minimal HbA 1, 3

Clinical Applications in Diagnosis

Hemoglobin electrophoresis is indicated when:

1. Microcytic anemia is present with normal or elevated ferritin levels 3
2. Mean cellular hemoglobin (MCH) is <27 pg for beta-thalassemia screening
3. MCH is <25 pg for alpha-thalassemia screening 2

The electrophoresis findings must be interpreted in context with:

• Complete blood count showing microcytic, hypochromic anemia
• Family history of thalassemia
• Ethnic background (Mediterranean, Middle Eastern, African, or Southeast Asian) 1, 3

Diagnostic Limitations and Considerations

Important considerations when interpreting hemoglobin electrophoresis results:

• Co-inheritance of iron deficiency may mask elevated HbA2 in beta thalassemia carriers
• Concurrent alpha and beta thalassemia can normalize MCV and MCH
• HbE can be misidentified as HbA2 on some electrophoresis systems
• Apparent homozygosity for mutations may actually represent compound heterozygosity with a rare variant or deletion 1

In newborns, capillary electrophoresis can detect:

• Hb E (2.6-6.2% in heterozygotes)
• Hb Bart's in alpha thalassemia variants
• Different forms of alpha thalassemia based on the presence and percentage of Hb Bart's 4

Role in Management

Hemoglobin electrophoresis contributes to management by:

1. Classifying disease severity: Determines if the patient has thalassemia trait, intermedia, or major, which guides treatment decisions
2. Monitoring treatment response: Helps assess changes in hemoglobin composition during treatments
3. Genetic counseling: Provides essential information for family planning 1, 2

Integration with Other Diagnostic Methods

For comprehensive diagnosis, hemoglobin electrophoresis should be combined with:

• Molecular genetic testing: PCR-based techniques, gap-PCR, DNA sequencing, and reverse dot blot hybridization to identify specific mutations in the globin genes 1
• Family studies: Testing family members to establish inheritance patterns
• Prenatal diagnosis: Through amniocentesis or chorionic villus sampling with DNA analysis in high-risk pregnancies 1

Practical Recommendations

1. All ethnic groups should be screened for beta-thalassemia trait when MCH is <27 pg
2. All except Northern European Caucasians should be screened for variant hemoglobins
3. Selected ethnic groups should be screened for alpha-thalassemia trait when MCH is <25 pg 2

Pitfalls to Avoid

• Relying solely on electrophoresis: Definitive diagnosis requires genetic testing, especially in complex cases 1, 3
• Misinterpreting results in the presence of iron deficiency: Iron deficiency should be corrected before interpreting hemoglobin electrophoresis for thalassemia 1
• Overlooking the need for family studies: Testing family members provides valuable context for interpretation 1

Hemoglobin electrophoresis remains a cornerstone in thalassemia diagnosis, but its findings must be integrated with clinical data, hematological parameters, and genetic testing for comprehensive patient management.