How do you diagnose Glucose-6-phosphate dehydrogenase (G6PD) deficiency in individuals, particularly those of African, Asian, or Mediterranean descent, with a history of jaundice, anemia, or symptoms suggestive of hemolysis?

How to Diagnose G6PD Deficiency

Quantitative G6PD enzyme activity assay is the gold standard for diagnosing G6PD deficiency, measuring enzyme activity in units per gram of hemoglobin (IU/gHb), but the type of assay and timing of testing are critical—qualitative tests work only for males and homozygous females with severe deficiency, while quantitative assays or molecular testing are necessary for heterozygous females and borderline cases. 1

Initial Screening Approach

Who Should Be Tested

• Screen all patients of Mediterranean, African, Indian, or Southeast Asian descent before starting oxidant drugs (dapsone, primaquine, sulfonamides, rasburicase, methylene blue) 1, 2, 3
• Test infants with jaundice appearing in the first 24 hours of life 2
• Test infants with rapidly rising total serum bilirubin approaching exchange transfusion levels or not responding to phototherapy 2
• Evaluate patients presenting with acute hemolytic anemia, particularly after exposure to fava beans, infections, or certain medications 4, 5
• Screen first-degree relatives of known G6PD-deficient patients 1

Clinical Presentation Clues

• Classic triad: pallor (100%), dark red urine (89.6%), and jaundice (87.8%) occurring 24-72 hours after exposure to precipitating factors 5
• Neonatal jaundice, often severe enough to risk kernicterus (G6PD deficiency accounts for 31.5% of kernicterus cases in one series) 2
• Acute hemolytic episodes triggered by fava beans (most common dietary trigger at 32.6%), infections (12.4%), or drugs (4.2%) 5

Laboratory Testing Strategy

Timing Considerations (Critical for Accuracy)

• Avoid testing during or immediately after acute hemolytic episodes—reticulocytes and young RBCs contain near-normal enzyme levels, potentially masking deficiency 1
• 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 acute hemolysis, repeat testing after 3 months for accurate diagnosis 2

Sample Handling Requirements

• Buffy coat removal or filtration is essential—testing whole blood without removing white cells and platelets 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

Choosing the Right Assay

For Males and Homozygous Females:

• Qualitative fluorescent spot tests are appropriate for detecting severe G6PD deficiency (enzyme activity <10% of normal) 1
• These rapid screening tests are sufficient for initial assessment in this population 2

For Heterozygous Females:

• Quantitative assays or flow cytometry are necessary—qualitative tests are inadequate due to X-chromosome inactivation creating a mosaic of normal and deficient cells 1
• Molecular analysis may be required when enzymatic testing is inconclusive 2

For Borderline Cases (30-70% activity):

• Quantitative testing is mandatory to determine the precise degree of deficiency 1
• Calculate the PK/G6PD ratio or compare to controls with similar reticulocyte counts when interpreting borderline results 1
• Consider genetic testing to identify specific variants (Mediterranean vs. African), which determines hemolytic risk severity 1

Interpreting Results

Normal G6PD enzyme activity: 3.50 ± 0.8 μmol/min/g tissue in liver; specific reference ranges vary by laboratory for blood samples 6

Severity Classification:

• Severe deficiency: <10% of normal activity—qualitative tests detect this level 1
• Moderate deficiency: 10-60% of normal activity—requires quantitative testing 1
• Mild deficiency: 60-80% of normal activity—may be missed by qualitative screening 1

For tafenoquine use: Quantitative G6PD testing is required—do not use if activity is <70% 1

Calculate percentage of normal activity: Divide patient's G6PD level by laboratory upper limit of normal and multiply by 100 1

Molecular Genetic Testing

When to Use Genetic Testing

• Heterozygous females with inconclusive enzymatic testing 2
• Borderline enzymatic activity (30-70% of normal) to determine specific variant and predict severity 2
• Prenatal diagnosis and carrier testing for patients with known family mutations 6
• When enzymatic testing is unavailable or results are equivocal 7

Variant-Specific Information

Mediterranean variant (G6PD Mediterranean 563T):

• Causes life-threatening hemolysis requiring strict oxidant drug avoidance 1
• Found in Southern Europe, Middle East, and Indian subcontinent 8

African variant (G6PD A- 202A376G):

• Produces milder, self-limited hemolysis 1
• Most common African mutation 8

Asian variants:

• Multiple mutations common in Asia, including G6PD Canton and others 8, 9
• Require molecular characterization for precise identification 9

Supporting Laboratory Findings

Complete Blood Count During Hemolysis

• Normocytic normochromic anemia with reticulocytosis in all acute cases 5
• Heinz bodies visible on peripheral smear 5
• Most common G6PD enzyme level at presentation: 3-4 U/gm Hb (59.2% of cases) 5

Additional Laboratory Markers

• Elevated indirect bilirubin (>1.0 mg/dL when total bilirubin is ≤5 mg/dL) suggests hemolysis 2
• Baseline hematocrit and hemoglobin must be checked before treatment if G6PD deficiency is suspected 3
• Close hematological monitoring (at day 3 and 8) is required if oxidant drugs are used in deficient patients 3

Common Pitfalls to Avoid

• Never test immediately after transfusion—wait minimum 50 days to avoid false-negative results from donor RBCs 1
• Never rely on qualitative tests alone for females—they will miss heterozygotes who are still at risk 1
• Never test during acute hemolysis without planning repeat testing—young RBCs have higher enzyme activity and mask deficiency 1, 2
• Never assume neutropenia indicates G6PD deficiency—it is not a reliable screening marker 1
• Do not use whole blood without buffy coat removal—this prevents accurate detection 1

When G6PD Status is Unknown and Testing Unavailable

If primaquine or other oxidant drugs must be considered when G6PD testing is unavailable:

• Assess risk factors for G6PD deficiency or favism based on ethnicity and family history 3
• Check baseline hematocrit and hemoglobin before treatment 3
• Implement close hematological monitoring (at day 3 and 8) 3
• Ensure adequate medical support to manage hemolytic risk is available 3
• Discontinue drug promptly if signs of hemolytic anemia occur (darkening of urine, marked fall of hemoglobin or erythrocyte count) 3