A 3-year-old boy from rural Maharashtra presents with recurrent infections, jaundice, and dark urine after consuming fava beans. On examination, he has pallor and splenomegaly. Hemoglobin is 7.2 g/dL with elevated indirect bilirubin and reticulocytosis. Which investigation is most appropriate to confirm the underlying enzyme deficiency?

Explanation

## Clinical Context This is a classic presentation of **G6PD deficiency**, an X-linked disorder affecting the first enzyme of the pentose phosphate pathway. The patient exhibits acute hemolytic anemia triggered by oxidative stress (fava bean exposure), with hemoglobinuria (dark urine), jaundice, and reticulocytosis. ## Diagnostic Approach **Key Point:** G6PD deficiency is diagnosed by direct measurement of G6PD enzyme activity in red blood cells, NOT by indirect markers. ### Why G6PD Activity Assay? 1. **Gold standard confirmatory test** — directly measures the deficient enzyme 2. **Timing consideration** — must be done after hemolytic crisis resolves (during crisis, young RBCs with higher enzyme activity mask deficiency) 3. **Specificity** — differentiates G6PD deficiency from other causes of hemolysis 4. **Quantification** — can classify severity (Class I–V) ## Pathophysiology Reminder ```mermaid flowchart TD A[G6PD Deficiency]:::outcome --> B[Reduced NADPH production]:::outcome B --> C[Decreased glutathione reduction]:::outcome C --> D[Oxidative stress in RBCs]:::urgent D --> E[Hemoglobin denaturation<br/>Heinz body formation]:::outcome E --> F[Hemolysis triggered by<br/>oxidative agents]:::urgent F --> G[Anemia + Jaundice + Dark urine]:::outcome ``` **High-Yield:** The pentose phosphate pathway's primary function is **NADPH generation** for antioxidant defense (glutathione reduction cycle), not carbohydrate synthesis in RBCs. ## Investigation Comparison | Investigation | Purpose | Diagnostic Value in G6PD | |---|---|---| | G6PD activity assay | Measure enzyme directly | **Gold standard** | | Heinz body stain | Visualize denatured Hb | Supportive, not specific | | Osmotic fragility | RBC membrane integrity | Non-specific | | Reticulocyte count | Bone marrow response | Supportive only | **Clinical Pearl:** Diagnosis made during hemolytic crisis may be falsely reassuring because young RBCs (reticulocytes) have higher G6PD activity; repeat testing 3–4 weeks post-crisis is recommended.