A 2-month-old infant born to parents of West African descent is screened as part of a newborn screening program. The initial test shows an abnormal hemoglobin pattern. Which investigation is most appropriate for confirming the diagnosis and determining the genotype (HbSS, HbAS, or HbSC)?

Question

Accepted Answer

D. High-performance liquid chromatography (HPLC). ## Newborn Screening and Genotyping in Sickle Cell Disease

### Newborn Screening Context

**Key Point:** Newborn screening programs detect abnormal hemoglobin patterns early, but confirmation and genotyping require a definitive test. HPLC is the gold standard for both diagnosis and accurate phenotyping in the neonatal period.

### Why HPLC is Optimal for Neonatal Diagnosis

**High-Yield:** HPLC offers:
1. **Quantification of all hemoglobin variants:** Accurately measures HbF (fetal hemoglobin, still elevated in infants), HbA, HbS, HbC, and others.
2. **Definitive genotyping:** Can distinguish:
   - HbSS (sickle cell disease): ~85% HbS, ~15% HbF
   - HbAS (sickle cell trait): ~40% HbS, ~60% HbA
   - HbSC (sickle-hemoglobin C disease): ~50% HbS, ~50% HbC
3. **Neonatal-specific advantage:** Accurately identifies patterns even when HbF is elevated (normal in infants <6 months).
4. **Single test for diagnosis and counseling:** Provides information for both medical management and genetic counseling.

### Comparison of Confirmatory Tests

| Test | Principle | Accuracy in Neonates | Genotyping Capability | Time | Cost |
| --- | --- | --- | --- | --- | --- |
| **HPLC** | Separation by charge & hydrophobicity | >99% | Excellent (HbSS, HbAS, HbSC) | 30–45 min | Moderate |
| **Isoelectric focusing** | Separation by isoelectric point | >95% | Good (but less precise than HPLC) | 45–60 min | Moderate |
| **Sickle solubility test** | Polymerization in reducing conditions | ~90% | Poor (only detects HbS, not trait vs. disease) | 10 min | Low |
| **Peripheral smear** | Morphology of RBCs | ~70% | None (visual identification only) | 5 min | Very low |

**Clinical Pearl:** In India, the Indian Academy of Pediatrics recommends HPLC or cellulose acetate electrophoresis for all newborns with abnormal screening results. HPLC is preferred because it provides quantitative data and is less affected by HbF levels.

### Diagnostic Pathway in Newborn Screening

```mermaid
flowchart TD
  A[Newborn screening: Abnormal Hb pattern]:::outcome --> B[Repeat test at 2-3 months]:::action
  B --> C[HPLC for confirmation & genotyping]:::action
  C --> D{HPLC Result}:::decision
  D -->|HbSS| E[Sickle Cell Disease - Start prophylaxis]:::urgent
  D -->|HbAS| F[Sickle Cell Trait - Counseling only]:::outcome
  D -->|HbSC| G[Sickle-Hb C Disease - Monitor closely]:::outcome
  D -->|Other pattern| H[Refer for further evaluation]:::action
```

**Mnemonic: HPLC = **H**igh **P**recision **L**iquid **C**hromatography** — the most precise method for hemoglobin variant identification and quantification.

Answer

A. Peripheral blood smear examination

Answer

B. Repeat hemoglobin electrophoresis with isoelectric focusing

Answer

C. Sickle solubility test (dithionite test)

A 2-month-old infant born to parents of West African descent is screened as part of a newborn screening program. The initial test shows an abnormal hemoglobin pattern. Which investigation is most appropriate for confirming the diagnosis and determining the genotype (HbSS, HbAS, or HbSC)?

Explanation

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Newborn Screening and Genotyping in Sickle Cell Disease

Newborn Screening Context

Why HPLC is Optimal for Neonatal Diagnosis

Comparison of Confirmatory Tests

Diagnostic Pathway in Newborn Screening

Test	Principle	Accuracy in Neonates	Genotyping Capability	Time	Cost
HPLC	Separation by charge & hydrophobicity	>99%	Excellent (HbSS, HbAS, HbSC)	30–45 min	Moderate
Isoelectric focusing	Separation by isoelectric point	>95%	Good (but less precise than HPLC)	45–60 min	Moderate
Sickle solubility test	Polymerization in reducing conditions	~90%	Poor (only detects HbS, not trait vs. disease)	10 min	Low
Peripheral smear	Morphology of RBCs	~70%	None (visual identification only)	5 min	Very low