What is ANCA (Antineutrophil Cytoplasmic Antibodies)?

What is ANCA (Antineutrophil Cytoplasmic Antibodies)?

ANCA are autoantibodies directed against proteins in neutrophil cytoplasm that serve as highly specific diagnostic biomarkers for a group of life-threatening systemic small-vessel vasculitides, including granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). 1

Classification and Target Antigens

ANCA are classified based on two main staining patterns detected by indirect immunofluorescence (IIF) and their specific target antigens:

Cytoplasmic ANCA (c-ANCA)

• Produces diffuse cytoplasmic staining pattern on ethanol-fixed neutrophils 2, 3
• Primarily targets proteinase 3 (PR3), a 29 kDa serine protease located in azurophilic granules 4, 5
• Found in 77-90% of patients with active GPA 4
• PR3-ANCA is the hallmark serologic marker for GPA 4

Perinuclear ANCA (p-ANCA)

• Produces perinuclear/nuclear staining pattern (an artifact of ethanol fixation) 2, 6
• Primarily targets myeloperoxidase (MPO), a myeloid lysosomal enzyme 7, 5
• More commonly associated with MPA and EGPA 1, 7
• Detected in 30-40% of EGPA patients 7

Atypical ANCA (a-ANCA)

• Rarely seen in systemic small-vessel vasculitis but can occur in other inflammatory conditions 2

Clinical Associations

Granulomatosis with Polyangiitis (GPA)

• PR3-ANCA (c-ANCA) positive in 80-90% of cases with generalized disease 1, 4
• Clinical features include destructive sinonasal lesions, pulmonary nodules, and pauci-immune glomerulonephritis 1
• 10-20% of GPA cases are ANCA-negative, particularly in limited disease 4, 6

Microscopic Polyangiitis (MPA)

• MPO-ANCA (p-ANCA) positive in 75-97% of cases 1
• Characterized by rapidly progressive pauci-immune glomerulonephritis and alveolar hemorrhage 1
• PR3-ANCA can be found in 2-27% of MPA patients 1

Eosinophilic Granulomatosis with Polyangiitis (EGPA)

• Only 40% of patients produce detectable ANCA 1, 4
• MPO-ANCA is the predominant serotype when present 1
• ANCA-positive patients more frequently have vasculitic features (glomerulonephritis, peripheral neuropathy, purpura) 1, 7
• ANCA-negative patients more commonly have cardiac involvement and gastroenteritis 1

Diagnostic Testing Methodology

Recommended Testing Approach

The 2024 EULAR guidelines recommend testing for both PR3-ANCA and MPO-ANCA using high-quality antigen-specific immunoassays as the primary method of testing in patients with suspected AAV. 1

Testing Algorithm

• Perform both IIF for pattern determination and ELISA for specific antigens (MPO and PR3) simultaneously 1, 7
• If immunoassay is negative but clinical suspicion remains high, perform a second test (another immunoassay and/or IIF) 1
• Best diagnostic performance is achieved when IIF is combined with PR3 and MPO-specific ELISAs 3, 6

Important Testing Caveats

• A negative ANCA does not exclude AAV diagnosis, as 10-20% of GPA/MPA and 60% of EGPA patients are ANCA-negative 1, 4
• ANCA can be found in other inflammatory diseases, infections, and drug-induced conditions 1
• The diagnosis should never be made on ANCA serology alone 1

Pathophysiologic Significance

ANCA induce systemic vasculitis by binding to and activating neutrophils, causing release of oxygen radicals, lytic enzymes, and inflammatory cytokines. 4

• Both MPO and PR3 are sequestered in primary granules and translocated to the cell surface during neutrophil activation 1, 5
• Defective neutrophil apoptosis or impaired clearance of apoptotic cell fragments may lead to prolonged immune system exposure to these antigens 1
• Infection may play a role through molecular mimicry 1

Clinical Utility Beyond Diagnosis

Disease Monitoring

• ANCA levels can be useful to monitor disease activity but should not be used alone to guide treatment 6
• Sequential monitoring of ANCA titers can predict relapse in some patients, though not all show this pattern 7
• A significant increase in ANCA titers or reappearance of ANCA should prompt stricter patient monitoring 6

Prognostic Implications

• ANCA-positive patients tend to have more frequent relapses than ANCA-negative patients 7
• In EGPA, overall survival appears worse in ANCA-negative patients, while relapses are more frequent in ANCA-positive patients 7
• The more widespread and severe the clinical presentation, the more likely ANCA is to be positive 4

Genetic Associations

There is a strong relationship between PI*Z alpha-1 antitrypsin (AAT) deficiency alleles and PR3-ANCA-positive vasculitis. 4

• Approximately 2% of all patients with anti-PR3-positive multisystemic vasculitis are PI*Z homozygotes 4
• The Z allele frequency in PR3-ANCA-positive patients ranges from 5.6-17.6% compared to 0.9-2.4% in controls 4
• Diagnostic testing for AAT deficiency is indicated for patients with anti-PR3-positive vasculitic syndromes 4

Non-Vasculitic Conditions with ANCA Positivity

P-ANCA can be found in various conditions beyond vasculitis, including autoimmune hepatitis, primary sclerosing cholangitis, and inflammatory bowel disease. 7

• These atypical ANCA often target antigens other than MPO or PR3 2
• Clinical context is essential for interpretation 1