Antinuclear Antibodies

Antinuclear antibodies (ANA) are autoantibodies directed against components of the cell nucleus (DNA, histones, ribonucleoproteins, centromere, nucleolar proteins). The reference method is indirect immunofluorescence on HEp-2 cells, which reports a titre (the highest serum dilution giving a positive signal) and a pattern (homogeneous, speckled, centromere, nucleolar, nuclear dots, cytoplasmic).

ANA is the screening test for systemic autoimmune rheumatic disease: systemic lupus erythematosus (SLE), systemic sclerosis, Sjögren syndrome, mixed connective tissue disease, inflammatory myopathies and autoimmune hepatitis. Positive results are followed by specific antibody tests (anti-dsDNA, extractable nuclear antigens).

ANA is very sensitive for SLE (over 95% at a titre of 1 in 80 or more) but poorly specific. About 20 to 30% of healthy people have a titre of 1 in 40, 10 to 15% at 1 in 80, 5% at 1 in 160, and 3% at 1 in 320, with higher rates in older adults and women. The test is used to exclude rather than diagnose: a negative ANA by immunofluorescence makes SLE very unlikely. Only 1 in 10 to 1 in 20 people with a positive ANA has a rheumatic disease, so it is requested only when there is clinical suspicion.

Indications. Suspected SLE (malar rash, photosensitivity, arthritis, serositis, cytopenias, nephritis, neuropsychiatric symptoms), systemic sclerosis (Raynaud phenomenon, sclerodactyly, digital ulcers), Sjögren syndrome (dry eyes and mouth), inflammatory myopathy, mixed connective tissue disease, unexplained arthritis with systemic features, autoimmune hepatitis, drug induced lupus, unexplained pericarditis or pleurisy, and juvenile idiopathic arthritis with uveitis risk. Not for screening of asymptomatic people or for fatigue and non-specific pain.

Specimen. Serum 1 mL, no fasting. Stable for several days at 2 to 8 °C. Immunofluorescence is the reference method. Solid phase assays (ELISA, multiplex bead, chemiluminescence) are used as screening in many laboratories but have different sensitivity and specificity.

Titre.

• Negative: under 1 in 40 (some laboratories use 1 in 80 as the positive cut off to improve specificity).
• Low titre: 1 in 40 to 1 in 80, common in healthy people, with low clinical significance.
• Moderate: 1 in 160 (positive predictive value rises).
• High: 1 in 320 or more, 1 in 640 or more, more likely linked to autoimmune disease.
• A titre of 1 in 160 or more on HEp-2 cells is the entry criterion for the SLE classification criteria.

Patterns and associations.

Clinical interpretation.

• SLE: sensitivity over 95%, specificity about 65 to 80% (lower at low titre). A negative ANA makes SLE very unlikely, but ANA-negative lupus with anti-Ro antibodies is described.
• Systemic sclerosis: ANA positive in 90 to 95%.
• Sjögren syndrome: ANA positive in 50 to 80%, with anti-Ro and anti-La.
• Inflammatory myopathy: ANA positive in 30 to 80% depending on type.
• Drug induced lupus (hydralazine, procainamide, isoniazid, minocycline, anti-TNF drugs): ANA positive with anti-histone antibodies, anti-dsDNA usually negative.
• Rheumatoid arthritis: ANA positive in 30 to 40%, juvenile idiopathic arthritis in 40 to 60% (oligoarticular, uveitis risk).
• Infection (hepatitis C, Epstein-Barr virus, HIV, endocarditis, tuberculosis), malignancy (lymphoma, solid tumours), autoimmune thyroid disease and chronic liver disease also yield positive results.

Critical values and findings needing immediate action.

• Positive ANA with new renal impairment, proteinuria or haematuria: suspected lupus nephritis, urgent urinalysis, protein to creatinine ratio, complement, anti-dsDNA and nephrology referral.
• Positive ANA with cytopenias, serositis, seizures or psychosis: suspected severe SLE.
• High titre ANA with Raynaud phenomenon and digital ischaemia or ulcers: systemic sclerosis with vascular crisis.
• Positive ANA with dyspnoea and cough: interstitial lung disease or pulmonary hypertension, high resolution CT and echocardiography.
• Pregnant woman with positive anti-Ro: foetal heart block surveillance.

Interferences and pitfalls.

• A positive ANA alone does not diagnose an autoimmune disease, and the clinical picture determines further testing.
• Low titre positives (1 in 40 to 1 in 80) are frequent in healthy people, relatives of patients with autoimmune disease, and older adults.
• Different methods are not interchangeable: immunofluorescence is more sensitive than ELISA, and ELISA may miss Ro, centromere and Jo-1 antibodies and nucleolar patterns.
• Specific antibodies (anti-Ro, Jo-1) can be present with negative ANA, so extractable nuclear antigen testing is considered in suspected disease despite negative ANA.
• Results depend on the reader and the substrate, and the pattern should be recorded.
• The titre does not correlate with disease activity, and ANA should not be repeated to monitor SLE. Anti-dsDNA and complement are used.
• A positive result in a patient with fatigue and arthralgia but no objective features often reflects fibromyalgia or non-specific findings.
• Drugs (anti-TNF, hydralazine, procainamide, isoniazid, minocycline, interferon) and infections induce positive ANA.

Special groups.

• Children: ANA is positive in 20 to 30% of healthy children at low titre, and is used in juvenile idiopathic arthritis for uveitis risk stratification.
• Pregnancy: anti-Ro and anti-La antibodies are linked to neonatal lupus and congenital heart block.
• Older adults: low titre positives are frequent (up to 30%), and the significance is lower.
• Women: ANA is more frequent, reflecting the higher prevalence of autoimmune disease.
• Regions: infections such as leprosy, tuberculosis and hepatitis can produce positive ANA.

Follow up tests. Extractable nuclear antigen panel (Sm, RNP, Ro, La, Scl-70, Jo-1), anti-dsDNA, complement (C3, C4), full blood count, creatinine, urinalysis and protein to creatinine ratio, antiphospholipid antibodies, rheumatoid factor and anti-CCP, anti-centromere and anti-Scl-70, myositis panel, creatine kinase, liver autoantibodies, inflammatory markers, and organ specific imaging.