What are the recommended investigations for thalassemia?

Recommended Investigations for Thalassemia

Begin with complete blood count (CBC) showing microcytosis (MCV <80 fL), followed by hemoglobin analysis using high-performance liquid chromatography (HPLC) or electrophoresis, and confirm with DNA-based molecular genetic testing for definitive diagnosis.

Initial Screening Tests

Complete Blood Count (CBC)

• MCV <80 fL is the key screening parameter that suggests possible thalassemia carrier status and warrants further investigation 1, 2
• Look for microcytic hypochromic anemia with disproportionately low MCV relative to the degree of anemia 3
• Red cell indices show characteristic patterns: reduced MCV, reduced MCH, and elevated red blood cell count 4

Hemoglobin Analysis

• HPLC (high-performance liquid chromatography) is the first-line method for detecting abnormal hemoglobin patterns and quantifying hemoglobin fractions 4, 5
• Hemoglobin electrophoresis can be used as an alternative method for identifying structural variants 5
• HbA2 levels are critical: elevated HbA2 (>3.5%) indicates beta thalassemia trait, while normal or low HbA2 suggests alpha thalassemia 6, 7
• HbF (fetal hemoglobin) quantification helps classify severity, particularly in beta thalassemia major 6

Common pitfall: Borderline HbA2 values (3.0-3.5%) can occur with concurrent iron deficiency or alpha thalassemia gene coinheritance, requiring iron repletion before repeat testing 7

Definitive Diagnostic Testing

Molecular Genetic Testing

• DNA testing for deletions or point mutations is the definitive diagnostic test to identify the specific genetic defect 1
• Alpha thalassemia: primarily caused by gene deletions in the alpha-globin gene cluster, requiring deletion analysis 3
• Beta thalassemia: caused by point mutations (>200 identified), requiring sequencing of the HBB gene 6
• Massively parallel sequencing (next-generation sequencing) facilitates more precise molecular diagnosis and can detect rare variants 5

Cascade Family Testing

• Test the affected family member first to identify the specific mutation, then perform targeted testing of at-risk family members 1
• Screen parents with MCV measurement; values <80 fL warrant further investigation 1

Assessment of Disease Severity and Complications

For Established Thalassemia Major/Intermedia

Iron Overload Monitoring:

• Cardiac MRI with T2 measurement* is essential for assessing cardiac iron overload; T2* <6 ms confers 47% risk of heart failure within one year 2
• Liver MRI for liver iron concentration measurement (non-invasive alternative to liver biopsy) 8
• Serum ferritin every 3 months (though less accurate than MRI for organ-specific iron) 2

Viral Hepatitis Screening:

• HBsAg (hepatitis B surface antigen) testing using enzyme immunoassay to detect HBV infection 8
• Anti-HCV antibodies to screen for hepatitis C virus exposure 8
• HCV RNA qualitative/quantitative testing using PCR or transcription-mediated amplification to confirm active viremia (critical because only ~50% of anti-HCV-positive thalassemia patients have active infection) 8
• HCV genotyping to predict treatment response 8

Endocrine Complications:

• Annual screening for diabetes mellitus, thyroid dysfunction (TSH, free T4), and hypogonadotropic hypogonadism (LH, FSH, testosterone/estradiol) 2
• Growth hormone assessment in children with growth failure 2

Hepatic Monitoring:

• Liver function tests (ALT, AST) every 3 months 2
• Liver ultrasound every 6 months for hepatocellular carcinoma surveillance in patients with cirrhosis or chronic HBV 2

Prenatal Diagnosis (When Both Parents Are Carriers)

• Amniocentesis or chorionic villus sampling with DNA analysis to detect severe forms like Hb Bart's hydrops fetalis (four-gene deletion alpha thalassemia) 1
• Middle cerebral artery Doppler ultrasound to assess for fetal anemia in suspected hydrops fetalis 1
• This is critical as Hb Bart's hydrops fetalis typically results in fetal demise and accounts for 28-55% of non-immune hydrops fetalis in Southeast Asian populations 1

Ethnicity-Based Considerations

Failure to consider ethnicity leads to missed diagnoses 1:

• Alpha thalassemia: highest prevalence in Southeast Asian, Mediterranean, Middle Eastern, and African populations 1
• Beta thalassemia: most common in Mediterranean, Middle Eastern, and South Asian populations 6
• Adjust screening threshold and testing strategy based on ethnic background 1

Monitoring Schedule for Hemoglobin H Disease (3-Gene Deletion Alpha Thalassemia)

• CBC every 3-6 months to assess for worsening hemolytic anemia and determine transfusion needs 1