What labs should be drawn for a patient with a high titer for Antinuclear Antibody (ANA)?

Laboratory Testing for High-Titer ANA

For a patient with a high-titer ANA, order specific extractable nuclear antigen (ENA) antibodies and anti-dsDNA antibodies as the essential next step, with the specific panel guided by the ANA pattern observed on immunofluorescence. 1, 2

Pattern-Directed Testing Approach

The ANA pattern determines which specific antibodies to pursue:

Homogeneous Pattern

• Anti-dsDNA antibodies are the priority test, using both Crithidia luciliae immunofluorescence test (CLIFT) for high specificity and solid-phase assays (ELISA/FEIA) for sensitivity 3
• Anti-histone antibodies to evaluate for drug-induced lupus 3
• Anti-nucleosome antibodies for additional SLE evaluation 3
• Anti-Smith (Sm) antibodies which are highly specific for SLE 3, 4

Speckled Pattern

• Anti-SSA/Ro and anti-SSB/La for Sjögren's syndrome and SLE 3, 5
• Anti-RNP antibodies for mixed connective tissue disease and SLE 3
• Anti-Sm antibodies for SLE 3
• Anti-Scl-70 (topoisomerase-1) for systemic sclerosis 3
• Anti-Jo-1 if inflammatory myopathy is suspected 3

Nucleolar Pattern

• Anti-centromere antibodies (CENP-A, B, C, F) for limited cutaneous systemic sclerosis 5
• Anti-fibrillarin (U3-RNP) for systemic sclerosis 5
• Anti-PM/Scl, anti-To/Th for overlap syndromes 5

Dense Fine Speckled Pattern

• Anti-DFS70/LEDGF antibodies to confirm this pattern, which is more common in healthy individuals than autoimmune disease 2, 3
• Still pursue anti-RNP, anti-SSA/Ro, anti-SSB/La, and anti-dsDNA to rule out true autoimmune conditions 2

Additional Essential Testing

Beyond pattern-specific antibodies, obtain:

• Complete blood count to assess for cytopenias seen in autoimmune disease 1
• Comprehensive metabolic panel including liver and kidney function 1
• Complement levels (C3, C4) which are often low in active SLE 3
• Urinalysis to screen for proteinuria and hematuria suggesting lupus nephritis 1
• Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) for inflammation assessment 6

Specialized Testing Based on Clinical Context

If Systemic Sclerosis Suspected

• Anti-centromere, anti-Scl-70, anti-RNA polymerase III 5
• Pulmonary function tests and DLCO to assess for interstitial lung disease 1

If Autoimmune Hepatitis Suspected

• Anti-smooth muscle antibodies (SMA) 1, 5
• Anti-liver/kidney microsomal antibody type 1 (anti-LKM-1) 1, 5
• Anti-mitochondrial antibodies (AMA) to exclude primary biliary cirrhosis 5
• Anti-liver cytosol type 1 (anti-LC1) 1

If Vasculitis Suspected

• Anti-neutrophil cytoplasmic antibodies (ANCA) 6

If Antiphospholipid Syndrome Suspected

• Lupus anticoagulant, anticardiolipin antibodies, anti-β2-glycoprotein I 1

If Pulmonary Arterial Hypertension Context

• HIV serology (all patients should be tested) 1
• Thrombophilia screen including antiphospholipid antibodies 1

Critical Interpretation Points

• Titer significance: A screening dilution of 1:160 provides optimal balance with 95.8% sensitivity and 86.2% specificity for systemic autoimmune rheumatic diseases 1, 3
• Age considerations: In patients under 18 years, any positivity at 1:20 for ANA/SMA or 1:10 for anti-LKM-1 is clinically relevant 1
• Method matters: Always specify whether results are from indirect immunofluorescence (IIFA, the reference standard) versus automated methods, as they may yield different results 1, 2

Common Pitfalls to Avoid

• Do not repeat ANA testing for monitoring - ANA is for diagnosis, not disease activity monitoring 2, 3
• Do not skip specific antibody testing in high clinical suspicion - Some patients with autoimmune disease may have negative ANA by certain methods but positive specific antibodies 3
• Do not assume low titers are insignificant in children - Pediatric patients have lower background ANA positivity than adults 1
• Do not use ANA alone for diagnosis - A positive ANA requires clinical correlation and specific antibody confirmation 2, 3
• Beware of false negatives with automated methods - IIFA remains the gold standard 5

Reporting Requirements

• Report both titer and pattern together, as both influence interpretation 1, 2
• Report specific ENA results separately, including negative results 2
• Specify the detection method used (IIFA vs. solid-phase assays) 1, 2
• For anti-dsDNA, report results quantitatively and use the same method for serial monitoring 3