## Investigation Strategy for Thalassemia Diagnosis ### Diagnostic Hierarchy **Key Point:** While HPLC and hemoglobin electrophoresis confirm the hemoglobin phenotype (elevated HbF and HbA2 in β-thalassemia major), DNA sequencing of the β-globin gene is the gold standard for identifying the specific molecular defect and enabling genetic counseling and prenatal diagnosis. ### Role of Each Investigation | Investigation | Purpose | Limitation | |---|---|---| | **HPLC** | Quantifies HbA, HbF, HbA2 levels; confirms thalassemia phenotype | Does not identify the underlying mutation | | **Hemoglobin electrophoresis** | Separates hemoglobins by charge; detects abnormal variants | Less sensitive than HPLC; does not identify molecular defect | | **DNA sequencing (β-globin gene)** | Identifies point mutations, deletions, insertions; enables genetic counseling | Gold standard for molecular diagnosis and family planning | | **Osmotic fragility test** | Assesses RBC membrane integrity | Not specific for thalassemia; used for hereditary spherocytosis | ### Clinical Context **High-Yield:** In β-thalassemia major, HPLC will show: - Elevated HbF (fetal hemoglobin, 50–90%) - Elevated HbA2 (α2δ2, >3.5%) - Absent or severely reduced HbA (normal adult hemoglobin) However, the question asks for confirmation AND determination of the **molecular defect** — only DNA sequencing provides this. **Clinical Pearl:** Knowing the specific β-globin mutation is essential for: - Genetic counseling of parents - Prenatal diagnosis in future pregnancies - Identifying patients eligible for gene therapy trials - Predicting severity (some mutations cause milder disease) **Warning:** Do not confuse HPLC (phenotypic diagnosis) with DNA sequencing (genotypic diagnosis). NEET PG frequently tests this distinction.
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