What are the initial lab tests for diagnosing Glucose-6-phosphate dehydrogenase (G6PD) deficiency?

Initial Laboratory Testing for G6PD Deficiency Diagnosis

The initial diagnostic test for G6PD deficiency should be a quantitative spectrophotometric assay measuring G6PD enzyme activity in whole blood, which can be performed directly from standard collection tubes and provides definitive diagnosis in most cases. 1, 2

Primary Diagnostic Approach

First-Line Testing: Quantitative Enzyme Activity Measurement

• Spectrophotometric testing is the gold standard for detecting G6PD enzymatic activity and should be the initial test of choice 1
• Quantitative assays measure G6PD activity in units per gram of hemoglobin (U/gHb), with normal values typically around 6.6 μmol/min/gHb and deficient samples showing values around 2.0 U/gHb or lower 3
• Modern automated platforms (such as Hitachi P modular systems) can analyze whole blood directly from original collection tubes, dramatically reducing analysis time and cost 2

Alternative Screening Methods

• Fluorescence-based testing offers a rapid, economical alternative that can detect G6PD deficiency from dried blood spot specimens within 30 minutes 3
• Formazan-based spot testing provides qualitative screening but lacks the precision needed for heterozygous females 1
• Cytofluorometric assays using flow cytometry can assess G6PD deficiency at the individual erythrocyte level, making them particularly useful for detecting female heterozygotes 4

Critical Timing Considerations

When NOT to Test

• Avoid testing during acute hemolytic episodes, as G6PD levels can be falsely elevated during active hemolysis because young reticulocytes have higher enzyme activity 5, 6
• If testing must occur during hemolysis and results are normal but clinical suspicion remains high, repeat testing after 3 months when the red blood cell population has normalized 5, 6

When to Test

• Screen before initiating oxidant drugs in patients of Mediterranean, African, Indian, or Southeast Asian descent 6, 7
• Test any patient presenting with unexplained hemolytic anemia, neonatal jaundice, or hemolysis following fava bean ingestion or medication exposure 1

Supporting Laboratory Findings

Indirect Evidence of G6PD Deficiency

• Complete blood count may show anemia with hemoglobin drop during hemolytic episodes 8
• Peripheral blood smear can reveal bite cells, blister cells, and Heinz bodies during oxidative stress 1
• Elevated indirect bilirubin (>1.0 mg/dL when total bilirubin is ≤5 mg/dL) suggests hemolysis 5
• Hemoglobinuria and elevated lactate dehydrogenase indicate active red blood cell destruction 1

Advanced Testing for Specific Scenarios

Molecular Genetic Analysis

• Gene sequencing of G6PD can detect up to 100% of mutations in homogeneous populations but may be required when enzymatic testing is inconclusive, particularly in heterozygous females 1
• Molecular analysis is most useful for prenatal diagnosis and carrier testing when family mutations are known 5
• Consider genetic testing when enzymatic activity is borderline (30-70% of normal) to determine specific variant and predict severity 5

Functional Neutrophil Testing

• In patients with severe G6PD deficiency and recurrent infections, measure reactive oxygen species production and neutrophil extracellular trap (NET) formation to assess for chronic granulomatous disease-like phenotype 8
• This additional testing determines whether antibiotic and antimycotic prophylaxis is warranted 8

Common Pitfalls to Avoid

• Do not rely solely on qualitative screening in females, as heterozygotes may have mixed populations of normal and deficient red blood cells requiring quantitative or cytofluorometric methods 1, 4
• Do not assume normal G6PD levels rule out deficiency during acute hemolysis—always plan for repeat testing 5, 6
• Do not delay treatment while awaiting G6PD results if hemolysis is suspected; remove the offending agent immediately and manage supportively 1
• Remember that hemoglobin concentration can affect fluorometric assays, causing approximately 1.1 fluorescence unit decrease per g/dL of hemoglobin, though this does not cause false negatives 3

Sample Handling Requirements

• 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 3
• Samples stored at 37°C lose approximately 31% activity by 14 days, so refrigeration is preferred if testing is delayed 3
• For enzymatic activity testing in liver tissue (relevant for glycogen storage disease differential diagnosis), snap-frozen samples in liquid nitrogen are required 5