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Simon Senanu: G6PD Deficiency Testing – What the Lab Must Get Right
Simon Senanu
Mar 20, 2026, 16:19

Simon Senanu: G6PD Deficiency Testing – What the Lab Must Get Right

Simon Senanu, Medical Laboratory Scientist at Perkins Medical Centre, shared a post on LinkedIn:

”G6PD Deficiency Testing – What the Lab Must Get Right

G6PD deficiency is the most common enzymatic disorder of red blood cells worldwide – yet misinterpretation in the lab remains common.

Accurate diagnosis depends on method selection, timing, and clinical context.

Why It Matters
G6PD maintains NADPH production in red cells.
NADPH protects hemoglobin and cell membranes from oxidative damage.

When activity is low:

  • Oxidative stress → Hemoglobin denaturation
  • Heinz body formation
  • Hemolysis

Common triggers:

  • Infections
  • Oxidant drugs (e.g., sulfa drugs, primaquine)
  • Fava beans

Screening Tests (Qualitative)
Fluorescent Spot Test (FST)
G6PD generates NADPH, which fluoresces under UV light.
Reduced fluorescence → deficiency suspected.

  • Rapid
  • Cost-effective
  • Ideal for screening programs

Limitations:

  • May miss intermediate deficiency (heterozygous females)
  • False normal during acute hemolysis
  • Does not quantify activity

Methemoglobin Reduction Test (Sodium Nitrite + Methylene Blue Method)
This is a functional oxidative challenge test performed in a single reaction system.

  • Sodium nitrite oxidizes hemoglobin → methemoglobin (brown color).
  • Methylene blue acts as an artificial electron carrier.
  • G6PD generates NADPH, which reduces methylene blue.
  • Reduced methylene blue facilitates conversion of methemoglobin back to hemoglobin.

Interpretation

  • Restoration of red color indicates Normal
  • Partial restoration(light brown) indicates Partial/intermediate deficiency
  • Persistent brown color indicates Deficiency

A functional redox screening test.

Limitations:

  • Semi-quantitative, subjective, and affected by hemolysis or incubation conditions.
  • Primarily used in low-resource or teaching settings.

Quantitative Enzyme Assay (Diagnostic Standard)
Spectrophotometric Assay
Measures NADPH production rate per gram of hemoglobin.

Provides:

  • Exact enzyme activity
  • Classification (normal, intermediate, deficient)
  • Clinical risk stratification

This is the reference diagnostic method.

Critical Laboratory Pitfalls

  • Acute hemolysis

Older deficient RBCs are destroyed first. Reticulocytes have higher activity → False normal results.
Repeat testing 2-3 months post-episode.

  • Recent transfusion

Donor RBCs can mask deficiency. Always review history.

  • Female heterozygotes

Due to X-linked lyonization, enzyme levels may fall in the intermediate range.
Qualitative tests may miss these cases.

Molecular Testing
Used when:

  • Results are inconclusive
  • Neonatal diagnosis is required
  • Borderline activity with severe hemolysis
  • Genotyping is needed

Laboratory Takeaway

  • Screening ≠ Quantification
  • Timing is critical
  • Always assess hemolysis and transfusion status
  • Quantitative assay remains the diagnostic reference

Accurate G6PD diagnosis prevents drug-induced hemolysis and improves patient safety.”

Simon Senanu: G6PD Deficiency Testing - What the Lab Must Get Right

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G6PD deficiency Health Hematology Hemostasis Hemostasis News Hemostasis Today Medicine NADPH RBCs Simon Senanu
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