What is the most appropriate diagnostic test for a patient with suspected hemoglobinopathy, presenting with exertional dyspnea, easy fatiguability, pallor, jaundice, hepatosplenomegaly, and a history of frequent blood transfusions?

Hemoglobin Electrophoresis is the Highest Diagnostic Value Test

For this 16-year-old with chronic hemolytic anemia, history of frequent transfusions, consanguineous parents, pallor, jaundice, hepatosplenomegaly, and prior cholecystectomy, hemoglobin electrophoresis (Option D) has the highest diagnostic value because it definitively identifies and quantifies abnormal hemoglobin variants and confirms the specific hemoglobinopathy diagnosis.

Clinical Reasoning

This patient's presentation strongly suggests a hereditary hemoglobinopathy, most likely β-thalassemia major or β-thalassemia/Hb E disease:

• Chronic transfusion history from childhood indicates severe hemolytic anemia requiring regular blood product support 1
• Consanguineous parents dramatically increases the likelihood of autosomal recessive hemoglobinopathies 1
• Cholecystectomy scar suggests chronic hemolysis with pigmented gallstone formation, a classic complication of thalassemia 1
• Hepatosplenomegaly results from extramedullary hematopoiesis and iron overload from chronic transfusions 1, 2
• Pallor and jaundice reflect ongoing hemolysis with elevated unconjugated bilirubin 1

Why Hemoglobin Electrophoresis is Superior

Hemoglobin electrophoresis (or its modern equivalent, high-performance liquid chromatography) provides both qualitative identification and quantitative measurement of hemoglobin variants, which is essential for definitive diagnosis 3, 4:

• It directly identifies abnormal hemoglobin species (Hb E, Hb S, Hb C, Hb D-Punjab, Hb Lepore) and quantifies Hb A2 and Hb F levels 4, 5
• Elevated Hb A2 (>3.5%) confirms β-thalassemia trait in carriers, while absent or markedly reduced Hb A with elevated Hb F characterizes β-thalassemia major 3, 4
• The test distinguishes between different thalassemia syndromes and compound heterozygous states like β-thalassemia/Hb E 3, 5

Why Other Options Are Less Valuable

Peripheral Blood Film (Option A)

While useful as an initial screening tool, the peripheral smear in hemoglobinopathies typically shows non-specific findings 1:

• Red cell morphology in pyruvate kinase deficiency and many hemoglobinopathies is "usually unremarkable, generally displaying some degree of anisocytosis and poikilocytosis" 1
• Microcytosis and hypochromia are common to both thalassemia and iron deficiency, lacking diagnostic specificity 3
• The blood film cannot definitively distinguish between different hemoglobinopathies or confirm the specific diagnosis 1

Sickle Cell Saturation/Solubility Test (Option B)

This test is too narrow in scope 4:

• It only detects Hb S (sickle hemoglobin) and provides no information about thalassemias or other hemoglobin variants 4
• Given this patient's clinical picture suggesting thalassemia rather than sickle cell disease, this test would likely be negative and non-contributory 4, 5

Bone Marrow Biopsy (Option C)

This invasive procedure is unnecessary for hemoglobinopathy diagnosis 1:

• Hemoglobinopathies are diagnosed through peripheral blood testing, not bone marrow examination 1, 3
• Bone marrow may show dyserythropoietic features that could lead to misdiagnosis as congenital dyserythropoietic anemia 1
• The risks of an invasive procedure are not justified when non-invasive testing provides the diagnosis 1

Diagnostic Algorithm

Step 1: Obtain complete blood count showing microcytic hypochromic anemia with elevated reticulocyte count 1

Step 2: Perform hemoglobin electrophoresis or HPLC as the definitive diagnostic test 3, 4:

• Quantifies Hb A, Hb A2, Hb F, and identifies variant hemoglobins
• Results available within 2-10 working days 1
• Modern HPLC systems provide both qualitative and quantitative analysis simultaneously 3

Step 3: Confirm with molecular genetic testing (DNA analysis of globin genes) if hemoglobin electrophoresis results are equivocal or for genetic counseling 1, 3

Step 4: Assess markers of hemolysis (LDH, unconjugated bilirubin, haptoglobin, reticulocyte count) to confirm chronic hemolytic process 1

Critical Pitfalls to Avoid

• Recent transfusion interference: Hemoglobin electrophoresis should ideally be performed at least 50 days after the last transfusion to minimize donor red blood cell contamination, though results can still be interpreted with caution in recently transfused patients 1
• Misinterpreting normal Hb A2 in α-thalassemia: Alpha-thalassemia trait typically shows normal Hb A2 levels, unlike β-thalassemia trait where Hb A2 is elevated 3, 4
• Overlooking compound heterozygous states: Patients may have combinations like β-thalassemia/Hb E, which requires careful interpretation of electrophoresis patterns 3, 5