What is the complete panel of Antinuclear Antibody (ANA) profile for diagnosing autoimmune diseases, specifically for conditions like Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Sjögren's syndrome, and scleroderma in adult patients, particularly women?

Complete ANA Profile Panel for Diagnosing Autoimmune Diseases

Overview of the ANA Testing Algorithm

The diagnosis of systemic autoimmune rheumatic diseases requires a structured panel approach starting with ANA screening by indirect immunofluorescence assay (IIFA), followed by specific antibody testing based on the pattern and titer, with anti-dsDNA and extractable nuclear antigen (ENA) antibodies forming the core diagnostic panel. 1

First-Level Screening: ANA by IIFA

• IIFA on HEp-2 cells is the reference standard method for ANA screening, with sensitivity of 90-100% for systemic autoimmune rheumatic diseases 1, 2
• The screening dilution should be 1:160 on conventional HEp-2 substrates for adult populations, representing the 95th percentile cutoff with 95.8% sensitivity and 86.2% specificity 1, 2
• Both the titer and immunofluorescence pattern must be reported, as different patterns indicate different autoantibodies and associated diseases 1, 2
• Alternative automated methods (ELISA, multiplex assays) may have different false negative and false positive ratios; if clinical suspicion is strong and the alternative method is negative, IIFA must be performed 1

Critical Interpretation Points for ANA Titers

• At 1:40 dilution, 31.7% of healthy individuals test positive 2
• At 1:80 dilution, 13.3% of healthy individuals test positive with specificity of only 74.7% for SLE 2
• At 1:160 dilution, only 5.0% of healthy individuals test positive, with specificity improving to 86.2% while maintaining 95.8% sensitivity 2
• Titers ≥1:160 warrant specific antibody testing regardless of symptoms due to substantially higher positive likelihood ratio 2

Second-Level Testing: Pattern-Directed Specific Antibodies

Homogeneous Pattern

Order anti-dsDNA antibodies first when homogeneous pattern is present, especially if SLE is clinically suspected 1, 2

• Anti-dsDNA antibodies (diagnostic for SLE):

  • Use double-screening strategy: solid phase assay (ELISA/FEIA) first for sensitivity, followed by Crithidia luciliae immunofluorescence test (CLIFT) for confirmation and high specificity 1, 2
  • Report results quantitatively for monitoring purposes 1
  • Found in 3.2% of patients with suspected autoimmune disease who have SLE 3

• Anti-histone antibodies: specific for drug-induced lupus 4

• Anti-nucleosome antibodies: associated with SLE 3

• Anti-Sm antibodies: highly specific for SLE, found in 22% of SLE patients 3, 4

Speckled Pattern (Fine or Coarse)

Order complete ENA panel for speckled patterns, as this pattern is associated with multiple autoimmune diseases 1, 2

Fine Speckled Pattern Antibodies:

• Anti-SSA/Ro antibodies:

  • Most common ANA-specific antibody (50% of positive cases) 5
  • Associated with SLE (48% of cases), Sjögren's syndrome (86% of cases), neonatal lupus, and congenital heart block 2, 5
  • Can be present in ANA-negative patients (5 out of 11 ANA-negative cases) 5
  • Associated with vasculitis and nephritis 4

• Anti-SSB/La antibodies:

  • Associated with Sjögren's syndrome and SLE 2, 3
  • Associated with milder clinical disease 4

• Anti-Scl-70 (topoisomerase-1) antibodies:

  • Specific for diffuse cutaneous systemic sclerosis 2, 3
  • Found in 19% of scleroderma patients 3

Coarse Speckled Pattern Antibodies:

• Anti-U1-RNP antibodies:

  • Diagnostic for mixed connective tissue disease (MCTD) when present in isolation 2, 4
  • Found in 40% of MCTD patients 3
  • Associated with milder clinical disease 4

• Anti-Sm antibodies:

  • Highly specific for SLE (diagnostic marker) 1, 4
  • More common in younger SLE patients 3

Centromere Pattern

• Anti-centromere antibodies (CENP-A, B, C, F):
  • Highly specific for limited cutaneous systemic sclerosis (CREST syndrome) 2, 4
  • Associated with Raynaud's phenomenon 2

Nucleolar Pattern

Order nucleolar-specific antibodies when this pattern is present, as it strongly suggests systemic sclerosis or overlap syndromes 2, 6

• Anti-PM/Scl antibodies: associated with polymyositis-scleroderma overlap syndrome 2
• Anti-RNA polymerase III antibodies: associated with diffuse systemic sclerosis with renal crisis 2
• Anti-fibrillarin (U3-RNP) antibodies: associated with systemic sclerosis 2
• Anti-Th/To antibodies: associated with limited systemic sclerosis 6

Cytoplasmic Patterns

Cytoplasmic patterns must be reported and specified, as they indicate specific autoimmune conditions 1, 6

• Anti-Jo-1 antibodies:

  • Most common myositis-specific antibody 2
  • Associated with polymyositis/dermatomyositis (PM/DM) and antisynthetase syndrome 2, 3
  • Associated with pulmonary fibrosis and poor prognosis 4

• Anti-ribosomal P antibodies:

  • Specific for SLE, particularly with neuropsychiatric manifestations 2, 4
  • May be present in ANA-negative patients by standard IIFA 2
  • More common in younger SLE patients 3

Disease-Specific Antibody Profiles

Systemic Lupus Erythematosus (SLE)

SLE patients frequently have multiple types of ANA, but anti-dsDNA and anti-Sm are diagnostic 4

• Diagnostic antibodies: anti-dsDNA, anti-Sm 1, 4
• Common antibodies: anti-SSA/Ro (found in 48% of SLE patients), anti-SSB/La, anti-RNP, anti-histone, anti-nucleosome, anti-ribosomal P 3, 5
• Monitoring: Use quantitative anti-dsDNA with the same method consistently; do not repeat ANA testing 1, 2
• Additional testing: Anti-C1q antibodies (present in almost 100% of active lupus nephritis), complement levels (C3, C4) 2
• Prevalence: 3.2% of patients with suspected autoimmune disease; 6% in females 3

Sjögren's Syndrome

• Primary antibodies: anti-SSA/Ro (40-60% of primary Sjögren's), anti-SSB/La 2, 5
• Anti-SSA found in 86% of Sjögren's syndrome patients in one study 5
• Prevalence: 1.7% of patients with suspected autoimmune disease 3

Systemic Sclerosis (Scleroderma)

• Diffuse cutaneous: anti-Scl-70 (topoisomerase-1), anti-RNA polymerase III 2, 3
• Limited cutaneous (CREST): anti-centromere antibodies 2, 4
• Overlap syndromes: anti-PM/Scl, anti-U3-RNP (fibrillarin), anti-Th/To 2, 6
• Prevalence: 19% of scleroderma patients have anti-Scl-70 3

Mixed Connective Tissue Disease (MCTD)

• Diagnostic antibody: anti-U1-RNP in isolation (restricted to this antibody) 4
• Anti-SSA may also be present, more common in younger patients 3
• Prevalence: 40% of MCTD patients have anti-RNP 3

Polymyositis/Dermatomyositis

• Myositis-specific antibodies: anti-Jo-1 (most common), anti-Mi-2, anti-SRP, anti-MDA5 2, 3
• Overlap antibodies: anti-PM/Scl 2
• Anti-Jo-1 associated with pulmonary fibrosis and poor prognosis 4

Rheumatoid Arthritis (RA)

• ANA profile has limited significance for RA diagnosis 3
• When present: anti-SSA, anti-hnRNP A1 (50% of RA patients in one study) 3, 7
• Prevalence: 2.5% of patients with suspected autoimmune disease 3

Special Considerations for Autoimmune Hepatitis

For patients under 18 years, any positivity at 1:20 for ANA/anti-smooth muscle antibodies or 1:10 for anti-LKM-1 is clinically relevant 2

• Type 1 autoimmune hepatitis: anti-smooth muscle antibodies (SMA), atypical p-ANCA (50-96% of cases) 2
• Type 2 autoimmune hepatitis: anti-liver/kidney microsomal type 1 (anti-LKM-1), anti-liver cytosol type 1 (anti-LC1) 2

Diseases Where ANA Profile Has Limited Utility

ANA profile testing is of little significance for the following conditions 3:

• Ankylosing spondylitis (AS) - prevalence 1.5%, highest in males at 1.9% 3
• ANCA-associated vasculitis (AAV) 3
• Polymyalgia rheumatica (PMR) 3
• Adult-onset Still's disease (ASD) 3
• Behçet's disease (BD) 3

Critical Pitfalls and Caveats

Testing Methodology Issues

• Always specify the testing method in reports, as IIFA and automated methods have fundamentally different test characteristics 1, 6
• Use the same laboratory and method for serial monitoring to avoid discrepant results 2
• In-house assays must be standardized according to international standards (WHO, CDC/IUIS) 1

Interpretation Errors to Avoid

• Never diagnose based on ANA alone; diagnosis requires compatible clinical symptoms, laboratory abnormalities, and when appropriate, histological findings 2
• Do not repeat ANA testing for monitoring disease activity once diagnosis is established; ANA is for diagnostic purposes only 1, 2
• In high clinical suspicion cases, order specific antibody testing regardless of ANA result, as some autoantibodies (anti-Jo-1, anti-ribosomal P, anti-SSA/Ro) may be present in ANA-negative patients 1, 2
• Testing for ANA-specific antibodies cannot be gated on ANA patterns alone, as there is redundancy with various clinical diagnoses 5

Age and Sex Considerations

• Younger patients with SLE are more likely to have anti-dsDNA, anti-Sm, anti-histone, or anti-ribosomal P antibodies 3
• Younger patients with RA or MCTD are more likely to have anti-SSA antibodies 3
• No significant sex differences exist for most autoantibodies, except in RA and AS 3
• Prevalence of most autoimmune diseases is closely associated with age, with different variation characteristics among age groups for different diseases 3

Clinical Context Requirements

• Provide detailed clinical information on laboratory requisitions so the laboratory can assess results and decide on subsequent reflex testing 2
• Only 11.8% of patients suspected of having systemic autoimmune rheumatic diseases actually have them, emphasizing the importance of clinical correlation 3
• Up to 25% of apparently healthy individuals can be ANA-positive by IIFA, and many will never develop autoimmune disease 2, 6

Reflex Testing Strategy

Recognition of both nuclear and cytoplasmic patterns enables automated reflex testing and improves diagnostic utility 6

• Homogeneous pattern: automatically reflex to anti-dsDNA and anti-histone 6
• Speckled pattern: automatically reflex to anti-Sm, anti-RNP, anti-SSA/Ro, anti-SSB/La 6
• Nucleolar pattern: automatically reflex to anti-Scl-70 6
• Centromere pattern: automatically reflex to anti-centromere antibody quantification 6

Monitoring Established Disease

• For SLE monitoring: Use quantitative anti-dsDNA assays with the same method consistently, plus complement levels (C3, C4) 1, 2
• Never repeat ANA testing once diagnosis is established 1, 2
• Anti-C1q antibodies are useful for monitoring lupus nephritis activity (present in almost 100% of active cases) 2