Antinuclear Antibody (ANA) Test: Procedure, Patterns, and Clinical Utility

The Antinuclear Antibody (ANA) test detects autoantibodies targeting nuclear components. It is a screening test for systemic autoimmune diseases, especially systemic lupus erythematosus (SLE).

Test Procedure (Indirect Immunofluorescence - IIF)

1. Blood Collection: Venous draw into serum separator tube (SST) or red-top. No fasting required.
2. Serum Separation: Centrifuge → collect serum.
3. Substrate Preparation: HEp-2 cells (human epithelial carcinoma line) fixed on slides—express >100 nuclear/cytoplasmic antigens. (Historically: rodent liver/kidney sections.)
4. Serum Incubation: Patient serum (diluted, typically 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280) applied to slides. Incubate 30 min at RT.
5. Washing: Remove unbound antibodies.
6. Detection: Fluorescein-labeled anti-human IgG (or IgG/IgM/IgA) added. Incubate, wash.
7. Visualization: Fluorescence microscope. Patterns: homogeneous, speckled, nucleolar, centromere, nuclear membrane, cytoplasmic.
8. Interpretation: Titer = highest dilution with visible fluorescence. Pattern suggests specific autoantibodies (e.g., homogeneous → anti-dsDNA, chromatin; speckled → anti-Sm, RNP, SSA/Ro, SSB/La; centromere → anti-centromere; nucleolar → anti-Scl-70, RNA Pol I/III).

Clinical Utility

• SLE: ~95% sensitive; titer ≥1:160 + clinical criteria supports diagnosis.
• Other diseases: Sjögren's, scleroderma, polymyositis/dermatomyositis, MCTD, autoimmune hepatitis, drug-induced lupus.
• False positives: Healthy individuals (up to 15% at 1:40, 5% at 1:160), infections, medications, aging.

Follow-Up

Positive ANA → specific autoantibody panel (EIA, multiplex, line blot) for anti-dsDNA, Sm, RNP, SSA, SSB, Scl-70, centromere, Jo-1, etc.

ANA is a screening tool, not diagnostic alone. Clinical correlation is mandatory.