Anti-Neutrophil Cytoplasmic Antibodies (ANCA: PR3, MPO)

ANCA are autoantibodies against neutrophil granule and lysosomal proteins, found in small vessel vasculitis. The two relevant targets are proteinase 3 (PR3), giving a cytoplasmic (c-ANCA) immunofluorescence pattern, and myeloperoxidase (MPO), giving a perinuclear (p-ANCA) pattern.

Testing combines indirect immunofluorescence on ethanol fixed neutrophils with antigen specific immunoassay (ELISA, chemiluminescence or multiplex), and both are requested because immunofluorescence alone has false positives.

ANCA-associated vasculitis includes granulomatosis with polyangiitis (PR3-ANCA in 70 to 80%), microscopic polyangiitis (MPO-ANCA in 50 to 60%) and eosinophilic granulomatosis with polyangiitis (MPO-ANCA in 30 to 40%). Annual incidence is 15 to 25 in every million people, with a peak at 55 to 75 years.

Untreated, rapidly progressive glomerulonephritis and pulmonary haemorrhage carry a mortality over 80% within a year, and early recognition changes outcome. PR3-ANCA carries a higher relapse risk than MPO-ANCA.

Indications. Rapidly progressive glomerulonephritis. Pulmonary renal syndrome (haemoptysis with renal failure). Chronic sinusitis, nasal crusting, saddle nose, subglottic stenosis, pulmonary nodules or cavities, scleritis. Mononeuritis multiplex, palpable purpura, unexplained fever with raised inflammatory markers. Asthma with eosinophilia and vasculitic features. Haematuria with proteinuria of unknown cause. Monitoring in confirmed vasculitis (limited value).

Specimen. Serum 1 to 2 mL, no fasting. Stable for days at 2 to 8 °C and frozen for long storage.

Reference values (assay dependent).

• Immunofluorescence: negative at screening dilution 1 in 20.
• Anti-PR3: under 2 to 3 U/mL (some assays under 5 U/mL) negative.
• Anti-MPO: under 3.5 to 5 U/mL negative.
• Moderate and high titres (over 3 times the cut off) are more specific.

Interpretation.

• PR3-ANCA with c-ANCA: granulomatosis with polyangiitis in the right clinical setting (positive predictive value over 90% with pulmonary renal or upper airway disease). Sensitivity for active generalised disease is 90%, and for limited upper airway disease 60 to 70%.
• MPO-ANCA with p-ANCA: microscopic polyangiitis or eosinophilic granulomatosis with polyangiitis, and renal limited vasculitis.
• Double positive (PR3 and MPO) or atypical ANCA: drug induced (hydralazine, propylthiouracil, minocycline, levamisole adulterated cocaine), infection (endocarditis, tuberculosis), inflammatory bowel disease, primary sclerosing cholangitis, autoimmune hepatitis, SLE, rheumatoid arthritis.
• Immunofluorescence positive with negative PR3 and MPO: other targets (elastase, lactoferrin, cathepsin G), usually non-vasculitic.
• Negative ANCA does not exclude vasculitis (10% of generalised, 30% of limited or localised disease), and tissue biopsy decides.
• Rising titres after remission may predict relapse but should not drive treatment on their own. Persistent positivity after remission is common.

Biopsy and associated tests. Renal biopsy shows pauci-immune necrotising crescentic glomerulonephritis. Complement is usually normal, and a low C3 or C4 points to another diagnosis.

Critical values and findings needing immediate action.

• Positive ANCA with rapidly rising creatinine, haematuria and red cell casts: rapidly progressive glomerulonephritis, urgent nephrology review, biopsy, and consideration of high dose methylprednisolone, cyclophosphamide or rituximab and plasma exchange for severe disease. Treatment is started on strong clinical suspicion without waiting for biopsy.
• Positive ANCA with haemoptysis and falling haemoglobin: diffuse alveolar haemorrhage, intensive care availability.
• Positive ANCA with mononeuritis multiplex, foot drop or ischaemic digits: urgent immunosuppression.
• Positive ANCA with sudden visual loss or scleritis: urgent ophthalmology and rheumatology.
• Positive ANCA with fever, cough and new murmur: exclude infective endocarditis before immunosuppression.

Interferences and pitfalls.

• ANCA testing in patients with low pre-test probability gives mostly false positives (low positive predictive value), so testing is for compatible syndromes.
• Immunofluorescence alone cannot distinguish clinically relevant patterns, and antigen specific assays are required for diagnosis.
• Cocaine levamisole produces high titre PR3 and MPO ANCA with skin necrosis, neutropenia and a positive lupus anticoagulant.
• ANCA does not reliably reflect disease activity, and treatment decisions rely on clinical assessment, creatinine, urinalysis, C-reactive protein and imaging.
• Drug induced ANCA vasculitis improves after withdrawal of the drug.
• Antinuclear antibodies can mimic p-ANCA pattern on immunofluorescence, and a negative MPO result in a p-ANCA pattern with positive ANA is common.
• Assay differences between manufacturers affect numerical comparison.
• Cyclophosphamide, rituximab and steroids lower titres, and false negatives may occur once treatment has started.

Special groups.

• Children: ANCA vasculitis is rare, with renal disease prominent, and MPO-ANCA more frequent in adolescent girls.
• Pregnancy: active disease carries risk of foetal loss and maternal morbidity, and cyclophosphamide is avoided. Transplacental IgG ANCA can occur.
• Older adults: MPO-ANCA vasculitis with renal failure is the typical presentation, and infection risk from immunosuppression is high.
• Regions: PR3-ANCA vasculitis is more frequent in Europe, MPO-ANCA in East Asia. Tuberculosis, leprosy and infections cause false positives and are screened for before immunosuppression.

Follow up tests. Creatinine and eGFR, urinalysis with microscopy for red cell casts, urine protein to creatinine ratio, full blood count with eosinophils, C-reactive protein, ESR, complement (C3, C4), anti-GBM antibody, ANA and anti-dsDNA, cryoglobulins, hepatitis B and C, HIV, blood cultures, chest radiograph and CT, bronchoalveolar lavage, renal biopsy, nasal or tissue biopsy, nerve conduction studies, and tuberculosis screening before immunosuppression.