Is there a role for Antinuclear Antibody (ANA), perinuclear Anti-Neutrophil Cytoplasmic Antibody (pANCA), and cytoplasmic Anti-Neutrophil Cytoplasmic Antibody (cANCA) testing in diagnosing autoimmune conditions?

Role of ANA, pANCA, and cANCA Testing in Autoimmune Conditions

ANA, pANCA, and cANCA testing have distinct diagnostic roles: ANA is essential for screening systemic autoimmune rheumatic diseases, cANCA (PR3) is highly specific for granulomatosis with polyangiitis, and pANCA has limited specificity but can support diagnosis in specific clinical contexts including autoimmune hepatitis, vasculitis, and inflammatory bowel disease. 1, 2

ANA (Antinuclear Antibody) Testing

Primary screening tool for systemic autoimmune diseases:

• ANA testing by indirect immunofluorescence (IIFA) on HEp-2 cells is the recommended first-line screening test for systemic autoimmune rheumatic diseases 1
• Positive ANA requires follow-up with specific extractable nuclear antigen (ENA) testing depending on pattern, titer, and clinical context 1
• The recommended cut-off for positivity is 1:40 in adults, though any level of positivity from 1:10 may be clinically relevant in children and adolescents up to age 18 1
• In autoimmune hepatitis (AIH-1), ANA is detected in 8-77% of patients, though it lacks specificity 1

Critical caveat: High clinical suspicion warrants specific antibody testing even with negative ANA results, as anti-Jo-1, anti-ribosomal P, and anti-SS-A/Ro antibodies can be present in ANA-negative patients 1

cANCA (Cytoplasmic ANCA) Testing

Highly specific for ANCA-associated vasculitis:

• cANCA with PR3 specificity has 99% specificity and 73% sensitivity for granulomatosis with polyangiitis (GPA, formerly Wegener's granulomatosis) when using combined IIFA and ELISA 2
• Present in 80-90% of GPA patients with active systemic disease, confirming diagnosis in up to 95% of cases 2
• The American College of Rheumatology and European Renal Association recommend antigen-specific ELISA for PR3-ANCA as the primary screening method, with confirmatory IIFA if positive 2

Monitoring utility: Sequential cANCA monitoring can predict relapse in GPA, though not all patients show classical fluctuation with disease activity 2

pANCA (Perinuclear ANCA) Testing

Limited specificity but useful in specific contexts:

In ANCA-Associated Vasculitis:

• pANCA with MPO specificity is associated with microscopic polyangiitis (MPA), pauci-immune crescentic glomerulonephritis, and eosinophilic granulomatosis with polyangiitis (30-40% of cases) 3, 2
• Screening should use antigen-specific ELISA for MPO-ANCA 2

In Autoimmune Liver Disease:

• "Atypical" pANCA is found in 26-94% of primary sclerosing cholangitis (PSC) patients and 50-96% of AIH-1 patients 1
• The pANCA pattern in PSC and AIH is atypical because the antigen is nuclear rather than cytoplasmic, and the perinuclear pattern persists on formaldehyde-fixed cells (unlike classical pANCA) 1
• Routine autoantibody screening including pANCA is NOT required to establish PSC diagnosis 1
• pANCA testing may provide additional diagnostic support in AIH when conventional autoantibodies (ANA, SMA, anti-LKM1) are negative 1

In Inflammatory Bowel Disease:

• pANCA is present in 60-87% of ulcerative colitis and 5-25% of Crohn's disease cases 1, 3

Critical Diagnostic Pitfalls

ANA interference with pANCA interpretation:

• ANA-positive sera can produce false-positive pANCA patterns on ethanol-fixed granulocyte substrates 4
• Nuclear homogenous ANA pattern shows the strongest association with false pANCA interpretation (31.5% of cases) 4
• About 19.2% of ANA-positive samples may display pANCA patterns without true MPO reactivity 4

False positives leading to misdiagnosis:

• Positive ANCA results in patients without vasculitis have led to inappropriate immunosuppressive treatment with significant morbidity and mortality 5
• pANCA occurs in high titers (>1:160) in 62.3% of various systemic autoimmune diseases, but MPO specificity is infrequent outside vasculitides 6
• In most non-vasculitis autoimmune diseases with positive pANCA, the target antigens remain unknown 6

Recommended Testing Algorithm

For suspected systemic autoimmune disease:

1. Start with ANA by IIFA on HEp-2 cells at 1:40 dilution (1:10 in children) 1
2. If positive, perform specific ENA testing based on pattern and clinical suspicion 1
3. Proceed with disease-specific antibody testing regardless of ANA result if clinical suspicion is high 1

For suspected ANCA-associated vasculitis:

1. Order antigen-specific ELISA for both PR3-ANCA and MPO-ANCA simultaneously 2
2. If ELISA is positive, confirmatory IIFA can assess pattern 2
3. Interpret pANCA results cautiously in ANA-positive patients to avoid false positives 4

For suspected autoimmune hepatitis or PSC:

1. ANA and SMA testing by IIFA on rodent tissue (liver, kidney, stomach) at 1:40 dilution 1
2. pANCA testing may provide additional support but is not required for diagnosis 1
3. Consider anti-LKM1, anti-LC1, and anti-SLA/LP testing in appropriate clinical contexts 1

Never rely on a single positive ANCA or ANA result alone—always interpret within the full clinical context including symptoms, physical findings, and other laboratory abnormalities. 1, 5