What is the purpose of the nitroblue tetrazolium (NBT) test in diagnosing chronic granulomatous disease (CGD)?

Nitroblue Tetrazolium (NBT) Test

Primary Purpose and Clinical Application

The nitroblue tetrazolium (NBT) test serves as the primary screening test for chronic granulomatous disease (CGD) by detecting defective oxidative burst in phagocytic cells. 1, 2

The test works by measuring the ability of neutrophils and other phagocytic cells to reduce NBT dye to blue formazan after oxidative stimulation—normal cells turn blue, while CGD cells remain colorless due to their inability to produce superoxide. 2

Diagnostic Mechanism

Test Principle

• Normal phagocytes: When stimulated (typically with phorbol myristate acetate), neutrophils readily reduce NBT to blue formazan through superoxide production, resulting in intense blue staining throughout the cell cytoplasm. 2, 3
• CGD phagocytes: Cells remain colorless or show minimal staining due to deficiencies in the NADPH oxidase complex, which prevents the oxidative burst necessary for microbial killing. 2, 4

Technical Considerations

• The improved microscopic NBT slide test uses purified granulocyte suspensions incubated with NBT, fixed in suspension, centrifuged onto microscope slides, and stained with nuclear fast red. 3
• This method prevents cell selection bias from adherence and washing, allowing semiquantitative assessment of formazan grains per cell. 3
• Parallel staining with May-Grünwald/Giemsa allows identification of both formazan-positive and formazan-negative cells. 3

Clinical Context for CGD Diagnosis

When to Order NBT Testing

Order NBT testing in patients presenting with: 1

• Deep-seated infections with abscess formation and granuloma development
• Recurrent serious bacterial and fungal infections affecting respiratory tract, skin, and viscera
• Gingivostomatitis with severe pyogenic infections
• Mild eosinophilia (often 4-5% in CGD patients)

Diagnostic Algorithm Position

The NBT test functions as a screening test that should be followed by more definitive testing when positive or equivocal. 1, 4, 5

Confirmatory testing includes: 1, 4, 5

• Dihydrorhodamine (DHR) flow cytometry with PMA stimulation: More sensitive and specific than NBT, now considered the gold standard functional assay
• Flow cytometry for NADPH oxidase components: Helps identify the specific affected gene (CYBB, NCF1, NCF2, CYBA)
• Molecular genetic testing: Identifies specific mutations in the five genes encoding NADPH oxidase components

Important Diagnostic Pitfalls

False Positive Results

Eosinophil staining can cause false-positive NBT results in CGD patients. 2

• In approximately 50% of CGD patients tested, phorbol myristate acetate-stimulated eosinophils show blue staining (75.5 ± 5.3% of purified eosinophils), despite these cells being unable to produce superoxide. 2
• The staining pattern in CGD eosinophils appears most intense between nuclear lobes with minimal peripheral staining, unlike the diffuse cytoplasmic staining in normal cells. 2
• This phenomenon may account for the small number of positive NBT cells reported in some CGD patients and can lead to diagnostic confusion. 2

Heterozygote Detection

The NBT test can discriminate between normal individuals, CGD patients, and heterozygotes for X-linked CGD, though heterozygotes show intermediate NBT-reducing activity. 3

Relationship to Other Phagocytic Defect Testing

The NBT test is specifically listed as a screening test for phagocytic cell defects in the diagnostic workup of primary immunodeficiencies. 1

Other phagocytic function tests include: 1

• DHR reduction (preferred over NBT)
• Flow cytometry for adhesion molecules (for leukocyte adhesion defects)
• Chemotaxis and phagocytosis assays
• Enzyme assays (myeloperoxidase, G6PDH)
• Bacterial or fungal killing assays

Clinical Significance

Early diagnosis of CGD through NBT testing (followed by confirmatory studies) leads to improved survival and disease prognosis by enabling: 4

• Prompt initiation of antibacterial and antifungal prophylaxis
• IFN-γ therapy for chronic granulomatous disease 1
• Consideration for hematopoietic stem cell transplantation in severe cases 1

The test remains valuable as an initial screening tool despite being largely superseded by DHR flow cytometry in specialized centers, particularly in resource-limited settings where flow cytometry may not be readily available. 4, 5