Laboratory investigations - antinuclear antibodies (ANA)
What it is
- Autoantibodies to nuclear (and some cytoplasmic) antigens, detected by indirect immunofluorescence on HEp-2 cells - the reference method
- Reported as titre + pattern
- Titre 1:40 or 1:80 is often a normal finding
- Clinically significant from 1:160, and rises in significance with titre
- A screening test with high sensitivity and poor specificity - it excludes, it does not diagnose
The problem with ordering it
- Positive in 20-30% of healthy people at 1:40, 5% at 1:160, 3% at 1:320
- Higher in women, the elderly, and relatives of patients with autoimmune disease
- -> Only order when there is a clinical syndrome to explain. In an unselected patient the post-test probability of lupus remains negligible
Epidemiology
- ANA positivity in the general population rises with age; F>M
- SLE prevalence ~50-100/100,000, so most positive ANAs are false positives
- Population ANA positivity appears to be increasing over recent decades
Mechanism
- Loss of tolerance to nucleic acid-protein complexes
- Impaired clearance of apoptotic debris -> nuclear antigen exposure -> B-cell activation, often via TLR7/9 and type I interferon
- Early complement deficiency (C1q, C4, C2) impairs clearance -> lupus
- Drug-induced: hydralazine, procainamide, isoniazid, minocycline, anti-TNF agents, methyldopa -> typically anti-histone
Pattern -> antigen -> disease
| Pattern | Antigen | Association |
|---|---|---|
| Homogeneous (diffuse) | dsDNA, histone, nucleosome | SLE; drug-induced lupus (anti-histone) |
| Speckled | ENA - Ro, La, Sm, RNP | SLE, Sjogren, MCTD, myositis |
| Nucleolar | Scl-70 (topoisomerase I), RNA pol III, fibrillarin, PM-Scl | Diffuse systemic sclerosis, scleromyositis |
| Centromere | CENP-B | Limited cutaneous systemic sclerosis (CREST), pulmonary arterial hypertension |
| Cytoplasmic | Jo-1 and other tRNA synthetases, ribosomal P, AMA-M2 | Antisynthetase syndrome, NPSLE, PBC |
| Nuclear dots / rim | Sp100, gp210 | Primary biliary cholangitis |
| Dense fine speckled (DFS70) | LEDGF/p75 | **Argues against connective tissue disease - the useful negative** |
- A strongly positive anti-dsDNA with a negative ENA screen corresponds to a homogeneous (diffuse) IIF pattern - the classic SLE picture
The ENA panel
| Antibody | Association | Note |
|---|---|---|
| Anti-dsDNA | SLE - highly specific (~95%) | Titre tracks disease activity, especially lupus nephritis |
| Anti-Sm | SLE - the most specific antibody (~99%), but sensitivity only 20-30% | Does not track activity |
| Anti-Ro (SSA) | Sjogren, SLE, subacute cutaneous lupus | Congenital heart block and neonatal lupus |
| Anti-La (SSB) | Sjogren | Usually with Ro |
| Anti-U1RNP | MCTD - high titre, defining | Raynaud, puffy hands, myositis |
| Anti-Scl-70 | Diffuse SSc | ILD risk |
| Anti-RNA pol III | Diffuse SSc | Scleroderma renal crisis; malignancy association |
| Anti-centromere | Limited SSc | PAH risk |
| Anti-Jo-1 | Antisynthetase | ILD, mechanic's hands, arthritis |
| Anti-histone | Drug-induced lupus (>95%) | Also in idiopathic SLE |
Testing strategy
- ANA first. If negative and the pretest probability of SLE is low, stop - ANA-negative SLE is <5%
- Anti-Ro-only disease can be ANA-negative on some solid-phase assays - request specific Ro if subacute cutaneous lupus or Sjogren is suspected
- Proceed to ENA and anti-dsDNA only when ANA is positive at a meaningful titre with a compatible syndrome
- Do not repeat ANA to monitor disease - titre does not track activity. Monitor with anti-dsDNA, C3/C4, urinalysis
Acting on a positive result
- A positive ANA is not a diagnosis and not a treatment indication
- Isolated positive ANA with no clinical features
- Explain; do not repeat reflexively
- Baseline: FBE, UEC, urinalysis with sediment, CRP/ESR
- Ask specifically about Raynaud phenomenon - with an abnormal nailfold capillaroscopy and a specific antibody it predicts evolution to a defined CTD
- Otherwise review only if symptoms develop
- Drug-induced lupus: cease the drug; resolves over weeks to months. Renal and CNS involvement is rare
Associations
- SLE, Sjogren, systemic sclerosis, MCTD, inflammatory myositis, autoimmune hepatitis, PBC
- Rheumatoid arthritis (30-50% low titre), juvenile idiopathic arthritis (uveitis risk)
- Autoimmune thyroid disease
- Chronic infection - hepatitis C, EBV, TB, endocarditis
- Malignancy, especially with nucleolar or RNA pol III patterns
- Drugs - hydralazine, procainamide, minocycline, anti-TNF
- Healthy elderly women
Natural history
- Most isolated positive ANAs never evolve
- Predictors of progression to a defined CTD: high titre, a specific antibody (dsDNA, Sm, Scl-70, centromere), Raynaud with abnormal capillaroscopy, multiple positives
- Antibodies may precede clinical SLE by several years (military serum cohorts)
- DFS70 alone, with no other antibody, argues strongly against evolution
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