A 6-year-old boy of African descent presents with acute chest pain, fever, and shortness of breath. Clinical examination reveals hepatosplenomegaly and bone tenderness. A peripheral blood smear shows sickle-shaped RBCs. Which investigation is most appropriate to confirm the diagnosis of sickle cell disease?

Question

Accepted Answer

B. High-performance liquid chromatography (HPLC). ## Diagnostic Confirmation in Sickle Cell Disease

### Why HPLC is the Gold Standard

**High-Yield:** HPLC (High-Performance Liquid Chromatography) is the gold standard and most specific test for confirming sickle cell disease. It definitively identifies and quantifies hemoglobin variants (HbS, HbA, HbF, HbA2) and can distinguish between sickle cell disease (HbSS), sickle cell trait (HbAS), and other hemoglobinopathies.

### Diagnostic Algorithm for SCD

```mermaid
flowchart TD
  A[Clinical suspicion of SCD]:::outcome --> B[Peripheral smear + CBC]:::action
  B --> C{Sickle cells seen?}:::decision
  C -->|Yes| D[Hemoglobin electrophoresis/HPLC]:::action
  D --> E[Confirms HbS pattern]:::outcome
  C -->|No| F[Rule out other hemolysis]:::action
  E --> G{HbSS vs HbAS vs HbC-S?}:::decision
  G -->|HbSS| H[Sickle Cell Disease]:::outcome
  G -->|HbAS| I[Sickle Cell Trait]:::outcome
```

### Comparison of Investigations

| Investigation | Principle | Specificity | Use in SCD |
| --- | --- | --- | --- |
| **HPLC** | Separates Hb variants by charge & hydrophobicity | Very high (>99%) | Gold standard for diagnosis & typing |
| **Hemoglobin electrophoresis** | Separates Hb by electrical charge | High | Confirmatory; less precise than HPLC |
| **Sickle solubility test** | Hb polymerization in reducing conditions | Moderate | Screening test; positive in HbS trait & disease |
| **Osmotic fragility** | RBC membrane integrity | Low | Not specific for SCD; used in hereditary spherocytosis |
| **Reticulocyte count** | Immature RBC percentage | Low | Indicates hemolysis severity, not diagnostic |

**Key Point:** While a sickle solubility test (dithionite) is a rapid screening tool, it cannot distinguish between sickle cell disease and sickle cell trait. HPLC provides both diagnosis AND phenotyping in a single test.

**Clinical Pearl:** In India, HPLC or cellulose acetate electrophoresis at pH 8.6 is recommended by the Indian Academy of Pediatrics for all suspected cases of hemoglobinopathy.

Answer

A. Osmotic fragility test

Answer

C. Reticulocyte count

Answer

D. Bone marrow aspiration

Explanation

Diagnostic Confirmation in Sickle Cell Disease

Why HPLC is the Gold Standard

Diagnostic Algorithm for SCD

Comparison of Investigations

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Investigation	Principle	Specificity	Use in SCD
HPLC	Separates Hb variants by charge & hydrophobicity	Very high (>99%)	Gold standard for diagnosis & typing
Hemoglobin electrophoresis	Separates Hb by electrical charge	High	Confirmatory; less precise than HPLC
Sickle solubility test	Hb polymerization in reducing conditions	Moderate	Screening test; positive in HbS trait & disease
Osmotic fragility	RBC membrane integrity	Low	Not specific for SCD; used in hereditary spherocytosis
Reticulocyte count	Immature RBC percentage	Low	Indicates hemolysis severity, not diagnostic