A 38-year-old woman presents with progressive fatigue and dyspnea. Peripheral blood smear shows microcytic hypochromic RBCs with target cells and occasional nucleated RBCs. Hemoglobin is 7.2 g/dL with MCV 62 fL. Which is the most appropriate next investigation to confirm the diagnosis?

Explanation

## Clinical Context The peripheral blood smear findings of microcytic hypochromic RBCs with target cells and nucleated RBCs, combined with severe anemia (Hb 7.2 g/dL) and low MCV (62 fL), point toward a hemoglobinopathy rather than simple iron deficiency. ## Why Hemoglobin Electrophoresis is Correct **Key Point:** Target cells on smear + nucleated RBCs + microcytic anemia = thalassemia until proven otherwise. Hemoglobin electrophoresis is the gold standard confirmatory test for hemoglobinopathies. The presence of nucleated RBCs (immature RBCs released prematurely) is a hallmark of thalassemia major, reflecting severe ineffective erythropoiesis. Target cells result from the abnormal hemoglobin composition and reduced hemoglobin content relative to cell volume. **High-Yield:** Hemoglobin electrophoresis separates hemoglobin variants (HbA, HbF, HbS, HbC, HbE) and quantifies their proportions. In β-thalassemia major, you will see: - Elevated HbF (fetal hemoglobin) - Elevated HbA2 (α2δ2) - Absent or severely reduced HbA (α2β2) ## Differential Approach | Investigation | When Used | Limitation | |---|---|---| | Hemoglobin electrophoresis | Confirm hemoglobinopathy | Gold standard; definitive | | Iron studies | Assess iron stores | Won't differentiate thalassemia from iron deficiency | | Bone marrow biopsy | Assess cellularity/dysplasia | Invasive; not first-line for diagnosis | | Reticulocyte count | Assess RBC production | Elevated in both hemolysis and thalassemia; non-specific | **Clinical Pearl:** The combination of target cells + nucleated RBCs on smear is virtually pathognomonic for thalassemia. Iron studies would be normal or elevated (due to transfusions and hemolysis), not low as in iron deficiency anemia. ## Mnemonic: THALASSEMIA RED FLAGS **T**arget cells **H**emolysis (elevated LDH, bilirubin) **A**bnormal Hb on electrophoresis **L**ow MCV (microcytic) **A**nemia (severe) **S**plenomegaly (extramedullary hematopoiesis) **S**evere ineffective erythropoiesis (nucleated RBCs) **E**levated HbF and HbA2 **M**icrocytic hypochromic RBCs **I**ron overload (secondary) **A**bnormal RBC morphology