How to diagnose Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency?

Diagnostic Testing for G6PD Deficiency

G6PD deficiency testing should be performed using quantitative enzyme activity assay as the gold standard, with fluorescent spot test (FST) serving as an acceptable rapid screening method, particularly when reporting includes intermediate fluorescence to detect female heterozygotes. 1, 2, 3

Diagnostic Methods for G6PD Deficiency

Primary Testing Options

1. Quantitative Enzyme Activity Assay

   • Gold standard for diagnosis
   • Measures G6PD activity in U/g Hemoglobin
   • Can detect heterozygous females with intermediate deficiency
   • Normal range: approximately 4.6-13.5 U/g Hb (laboratory reference ranges may vary)
   • Deficiency typically defined as <30% of normal activity 2, 4

2. Fluorescent Spot Test (FST)

   • Widely used screening method
   • Based on the generation of NADPH which fluoresces under UV light
   • Results interpretation:
     • Absent fluorescence: severe deficiency
     • Intermediate fluorescence: partial deficiency (important for detecting heterozygous females)
     • Bright fluorescence: normal activity 3
   • High sensitivity (95-100%) for severe deficiency 4

3. Point-of-Care Rapid Diagnostic Tests

   • CareStart G6PD RDT has similar performance to FST
   • 100% sensitivity for detecting severe and moderate deficiency (<30% activity)
   • Specificity >97% 4
   • Particularly useful in resource-limited settings

Special Considerations for Testing

Timing of Testing

• Avoid testing during acute hemolytic episodes as results may be falsely normal
• If G6PD deficiency is strongly suspected but initial test is normal during hemolysis, repeat testing should be performed 3 months after the hemolytic episode resolves 1

Female Heterozygotes

• Due to X-linked inheritance and random X-chromosome inactivation, heterozygous females may have normal, intermediate, or deficient enzyme activity
• Standard FST may miss heterozygous females if only reporting "deficient" or "normal" results
• Modified FST reporting with "intermediate" category improves detection of heterozygotes 3

Newborn Screening

• Automated methods like the SPOTCHECK Microflow Analyzer can be used for newborn screening 5
• Fluorometric assays on dried blood spots are effective for mass screening 6

Testing Algorithm

1. Initial Screening:

   • FST with three-category reporting (absent/intermediate/normal fluorescence)
   • Point-of-care rapid tests in field settings

2. Confirmation:

   • Quantitative enzyme activity assay for:
     • All patients with absent or intermediate fluorescence
     • Patients with high clinical suspicion despite normal screening
     • Female relatives of affected males

3. Special Situations:

   • For patients requiring methylene blue treatment (e.g., methemoglobinemia):
     • G6PD testing is essential as methylene blue is contraindicated in G6PD deficiency 1
   • For patients with significant hyperbilirubinemia:
     • Test for G6PD deficiency as these patients may experience sudden increases in bilirubin levels 1

Common Pitfalls and Caveats

• False normal results can occur during acute hemolysis due to selective destruction of G6PD-deficient cells and reticulocytosis (young RBCs have higher G6PD activity)
• Sample storage conditions affect enzyme activity - samples should be processed promptly or stored at -20°C or 6°C to maintain stability 6
• Hemoglobin levels can affect fluorescence readings but should not cause false negatives 6
• Cytofluorometric methods may provide more accurate assessment of heterozygous females by measuring G6PD at the individual cell level 7

By following this diagnostic approach, clinicians can accurately identify G6PD deficiency and prevent potentially life-threatening hemolytic episodes by avoiding oxidative stressors in affected individuals.