What causes false negative Glucose-6-phosphate dehydrogenase (G6PD) deficiency testing?

Causes of False Negative G6PD Deficiency Testing

The most critical cause of false negative G6PD testing is performing the test during or shortly after a hemolytic episode, when reticulocytosis and young red blood cells with higher enzyme activity mask the deficiency.

Primary Causes of False Negative Results

Recent Hemolytic Episode and Reticulocytosis

• Testing during acute hemolysis produces false negatives because young red blood cells and reticulocytes contain significantly higher G6PD enzyme activity than older cells, masking the underlying deficiency 1
• The test should be delayed until the reticulocyte count normalizes, typically 2-3 months after a hemolytic episode, to avoid missing the diagnosis 1
• This is the single most important timing consideration—testing too early after hemolysis is the most common preventable cause of false negatives 2

Recent Blood Transfusion

• Transfused normal donor red blood cells contain normal G6PD enzyme activity, which dilutes and masks the patient's deficient cells 3
• Wait at least 120 days (the full red blood cell lifespan) after transfusion before performing enzyme testing 3
• If testing cannot be delayed, proceed directly to molecular genetic testing rather than enzyme assay 3
• Even 50 days post-transfusion, donor cell contamination of 6-12% can elevate enzyme activity enough to produce borderline or false negative results 3

Heterozygous Females (Mosaic Pattern)

• Female carriers with X-chromosome inactivation may have normal or borderline enzyme activity on standard spectrophotometric testing, despite having a significant population of G6PD-deficient cells 2
• Flow cytometry can detect the percentage of deficient red blood cells in heterozygous females, identifying cases missed by standard enzyme assays 2
• Standard enzyme testing measures average activity across all cells and cannot distinguish the mosaic pattern in female carriers 2

Testing Method Limitations

Spectrophotometric Assay Issues

• Standard spectrophotometric methods measure total enzyme activity and may miss deficiency when compensatory mechanisms are present 2
• Flow cytometry detected several cases of G6PD deficiency that were completely missed by spectrophotometry, particularly in heterozygous females with normal or subnormal enzyme activity 2

Interference from Other Hematological Conditions

• Patients with sickle cell disease showed false negative results on flow cytometry testing, likely due to altered red blood cell characteristics 2
• The presence of other hemolytic anemias with reticulocytosis can confound interpretation of enzyme activity levels 3

Clinical Algorithm for Avoiding False Negatives

Timing Considerations

• Never test during acute hemolysis or within 2-3 months of a hemolytic episode 1
• Wait minimum 120 days after any blood transfusion before enzyme testing 3
• Document recent transfusions, infections, or drug exposures that may have triggered hemolysis 1, 4

Patient-Specific Approach

• For males with suspected deficiency but normal enzyme activity: repeat testing after reticulocyte count normalizes 1
• For females with clinical suspicion but normal enzyme activity: proceed to flow cytometry or molecular genetic testing to detect heterozygous state 2
• In patients with sickle cell disease or other hemoglobinopathies: use molecular genetic testing rather than enzyme assays 2

When Clinical Suspicion Remains High

• If enzyme testing is normal but clinical history strongly suggests G6PD deficiency (hemolysis after oxidant drug exposure, neonatal jaundice, family history): proceed directly to molecular genetic testing 3
• Consider testing family members, particularly the mother for male patients, to establish genetic diagnosis 3, 5
• Qualitative screening is sufficient for initial assessment, but quantitative testing may be needed in borderline cases 3, 5

Key Pitfalls to Avoid

• Do not rely on a single normal enzyme test to exclude G6PD deficiency in patients with recent hemolysis, recent transfusion, or females with suggestive clinical history 1, 2
• Do not test immediately after oxidant drug exposure or infection-triggered hemolysis—wait for hematologic recovery 1, 4
• In urgent clinical situations where testing cannot be delayed (e.g., before starting primaquine for malaria), assume deficiency is present if the patient has high-risk ethnic background and avoid oxidant drugs 3, 5