How is Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency diagnosed?

Diagnosis of G6PD Deficiency

For most patients, a quantitative G6PD enzyme activity assay is the gold standard diagnostic test, measuring enzyme activity in units per gram of hemoglobin (IU/gHb), though qualitative fluorescent spot tests are acceptable for screening males and homozygous females with severe deficiency. 1, 2

Primary Diagnostic Approach

Quantitative Testing (Preferred Method)

• Quantitative G6PD enzyme activity assay is the WHO-recommended gold standard for confirming G6PD deficiency, measuring activity in IU/gHb 2
• Normal G6PD activity ranges from 11 ± 2.5 IU/gHb in females and 10.9 ± 0.6 IU/gHb in males 3
• Severe deficiency is defined as <10% of normal mean (approximately <0.95 IU/gHb), while partial deficiency is <60% of normal mean (approximately <5.7 IU/gHb) 3
• Calculate the percentage of normal activity by dividing the patient's G6PD level by the laboratory upper limit of normal and multiplying by 100 1

Qualitative Screening Tests

• Qualitative fluorescent spot tests effectively detect severe G6PD deficiency (<10% of normal activity) in males and homozygous females 1
• These screening tests are appropriate for initial assessment in resource-limited settings but miss many heterozygous females 1, 3
• The fluorescent spot test detected only 6.1% prevalence compared to 14.2% by molecular analysis in one study 3

Patient Selection for Testing

High-Risk Populations Requiring Screening

• Screen all patients of Mediterranean, African, Indian, or Southeast Asian descent before initiating oxidant drugs (dapsone, primaquine, sulfonamides) 1, 2
• Screen first-degree relatives of G6PD-deficient patients 1

Clinical Scenarios Requiring Testing

• Infants with rapidly rising total serum bilirubin approaching exchange transfusion levels or not responding to phototherapy 1
• Infants with jaundice appearing in the first 24 hours of life 1
• Patients being considered for tafenoquine therapy for P. vivax (requires activity >70%) 1
• Any patient with unexplained hemolytic anemia 1, 2

Critical Timing Considerations

When NOT to Test

• Avoid testing during or immediately after acute hemolytic episodes, as reticulocytes and young RBCs contain near-normal enzyme levels, potentially causing false-negative results 1, 2
• Wait at least 50 days after RBC transfusion before performing enzyme assays, as donor RBC contamination of 6-12% causes false-negative results 1
• Testing 120 days post-transfusion is ideal but often impractical in frequently transfused patients 1
• If testing must occur during these periods, repeat testing after 3 months for accurate diagnosis 2

Sample Handling Requirements

• Remove buffy coat (white cells and platelets) before testing, as whole blood without this step prevents detection of deficiency in all cases 1
• 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 2, 4

Special Populations and Advanced Testing

Heterozygous Females

• Quantitative assays or flow cytometry are necessary for heterozygous females, as qualitative tests frequently miss partial deficiency 1
• Cytochemical methods using a cut-off of >20% negative cells detected 12.9% prevalence, closest to molecular analysis results 3
• Only 14.3% of heterozygous females show severe enzyme deficiency (<10% activity), while most have intermediate levels 3

Molecular/Genetic Testing Indications

• Required when enzymatic testing is inconclusive, particularly in heterozygous females 1, 2
• Helpful when enzymatic activity is borderline (30-70% of normal) to determine specific variant and predict severity 2
• Identifies Mediterranean variant (Gdmed) versus African variant (GdA-), which determines hemolytic risk severity—Mediterranean causes life-threatening hemolysis requiring strict oxidant drug avoidance, while African produces milder, self-limited hemolysis 1
• Molecular analysis can detect up to 100% of mutations in homogeneous populations 2

Borderline Results Management

• Consider calculating the PK/G6PD ratio or comparing to controls with similar reticulocyte counts when interpreting borderline results 1
• Patients with 30-70% activity and non-Mediterranean variants can receive modified primaquine regimens with close monitoring 1

Common Pitfalls to Avoid

• Never rely on neutropenia as a screening marker—it may be present but is not reliable for G6PD deficiency detection 1
• Elevated indirect bilirubin (>1.0 mg/dL when total bilirubin is ≤5 mg/dL) suggests hemolysis, which should prompt consideration of G6PD testing 2
• Hemoglobin quenches fluorescence by approximately 1.1 fluorescence units/g/dHb but does not cause false negatives 4
• G6PD deficiency was identified as the cause in 31.5% of infants who developed kernicterus, highlighting the critical importance of testing in neonatal hyperbilirubinemia 1