What lab test confirms Glucose-6-phosphate dehydrogenase (G6PD) deficiency?

Confirming G6PD Deficiency: Diagnostic Testing

Primary Diagnostic Test

Quantitative G6PD enzyme activity assay is the gold standard confirmatory test for G6PD deficiency, measuring enzyme activity in units per gram of hemoglobin (IU/gHb). 1, 2, 3

Diagnostic Thresholds

• Normal G6PD activity: 11 ± 2.5 IU/gHb in females and 10.9 ± 0.6 IU/gHb in males 3
• Severe deficiency: <10% of normal mean (approximately <1.0 IU/gHb) 3
• Partial deficiency: <60% of normal mean (approximately <5.7 IU/gHb) 3
• Intermediate deficiency: 30-70% of normal activity 2, 4

Alternative Screening Methods

Qualitative Tests (For Initial Screening)

• Fluorescent spot test: Appropriate for detecting severe G6PD deficiency (enzyme activity <10% of normal) in males and homozygous females 2, 5
• Methaemoglobin reduction test: Recommended for detection of severe G6PD deficiency in resource-limited settings 3
• Cytochemical assay: Using a cut-off of >20% negative cells provides prevalence closest to molecular analysis (12.9% detection rate) 3

Advanced Diagnostic Methods

• Flow cytometry (fluorocytometric assay): Allows quantification of the percentage of G6PD-deficient red blood cells, particularly useful in heterozygous females due to X-chromosome inactivation 6
• Molecular genetic testing: Can detect up to 100% of mutations in homogeneous populations and is required when enzymatic testing is inconclusive, particularly in heterozygous females 1

Critical Testing Considerations

Timing of Testing

Avoid testing during acute hemolytic episodes, as G6PD levels can be falsely elevated during active hemolysis because young reticulocytes have higher enzyme activity. 1

• If testing occurs during hemolysis, repeat testing after 3 months for accurate diagnosis 1
• G6PD levels may be falsely normal in heterozygous females with recent hemolysis 6

Sample Handling

• Whole blood samples remain relatively stable at -20°C and 6°C for up to 14 days, losing only about 20% activity over 48 hours 5
• Samples should be processed promptly to avoid preanalytic variability 5

Testing Algorithm

1. Initial screening: Qualitative fluorescent spot test or quantitative G6PD enzyme activity assay 1, 2
2. If borderline results (30-70% activity): Consider genetic testing to determine specific variant and predict severity 1
3. If testing during acute illness: Repeat quantitative testing after 3 months to confirm diagnosis 1
4. For heterozygous females with normal enzyme activity but clinical suspicion: Flow cytometry or molecular genetic analysis 6, 1

Common Pitfalls to Avoid

• Do not rely on neutropenia as a screening marker - while it may be present in G6PD deficiency, it is not reliable for diagnosis 2
• Do not use qualitative tests alone in females - heterozygous females may have normal or borderline enzyme activity despite carrying mutations 6
• Do not test during transfusion or immediately post-transfusion - this will give falsely elevated results 7
• Flow cytometry may show false negatives in patients with sickle cell disease - use caution when interpreting results in patients with other hematological conditions 6