What laboratory tests are recommended for an autoimmune workup?

Laboratory Tests for Autoimmune Workup

Begin with antinuclear antibody (ANA) testing by indirect immunofluorescence assay (IIFA) on HEp-2 cells at a screening dilution of 1:160, which provides optimal balance with 95.8% sensitivity and 86.2% specificity for systemic autoimmune rheumatic diseases. 1, 2

Initial Screening Tests

First-Line Testing

• ANA by IIFA on HEp-2 cells is the reference method for screening, with both titer and pattern reported, as these guide subsequent testing 1, 2
• Complete blood count (CBC) with differential to assess for cytopenias (leukopenia, lymphopenia, thrombocytopenia, anemia) seen in autoimmune diseases 1, 3
• Inflammatory markers: Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) to assess systemic inflammation 1, 3
• Comprehensive metabolic panel including liver function tests (AST, ALT, alkaline phosphatase, bilirubin) and kidney function (creatinine, BUN) to evaluate organ involvement 3
• Urinalysis to screen for proteinuria and hematuria suggesting lupus nephritis 3

Additional Initial Screening

• Rheumatoid factor (RF) when rheumatoid arthritis, Sjögren's syndrome, or cryoglobulinemia is suspected 1, 3, 4
• Anti-cyclic citrullinated peptide (anti-CCP) antibodies for suspected rheumatoid arthritis 1, 3
• Creatine kinase (CK) to evaluate for inflammatory myositis 1, 3

Reflex Testing Based on ANA Results

If ANA Positive with Homogeneous Pattern

• Anti-double-stranded DNA (anti-dsDNA) antibodies using a double-screening strategy: last-generation solid phase assay (ELISA, FEIA, or CLIA) first, followed by Crithidia luciliae immunofluorescence test (CLIFT) for confirmation 1, 2
• Anti-histone antibodies particularly if drug-induced lupus is suspected 1, 5
• Anti-nucleosome antibodies for SLE evaluation 2

If ANA Positive with Speckled Pattern

• Extractable nuclear antigen (ENA) panel including 1, 2, 3:
  • Anti-Sm (Smith) antibodies - diagnostic for SLE
  • Anti-U1-RNP antibodies - for mixed connective tissue disease (MCTD) and SLE
  • Anti-SSA/Ro60 and anti-SSB/La antibodies - for Sjögren's syndrome, SLE, subacute cutaneous lupus, neonatal lupus
  • Anti-Scl-70 (topoisomerase I) antibodies - for systemic sclerosis
  • Anti-Jo-1 antibodies - for inflammatory myopathies with pulmonary fibrosis

If ANA Positive with Centromere Pattern

• Anti-centromere antibodies (CENP-B) for limited cutaneous systemic sclerosis (CREST syndrome) 1, 5

If ANA Positive with Nucleolar Pattern

• Anti-RNA polymerase III antibodies for diffuse systemic sclerosis with renal crisis risk 1
• Anti-PM/Scl antibodies for polymyositis/scleroderma overlap syndrome 1

Disease-Specific Additional Testing

For Suspected SLE

• Complement levels (C3, C4) to assess disease activity and lupus nephritis risk 1, 6
• Anti-C1q antibodies found in almost 100% of patients with active lupus nephritis 1
• Anti-ribosomal P antibodies associated with neuropsychiatric lupus 1, 2
• Direct Coombs test if hemolytic anemia is present 6

For Suspected Inflammatory Myopathies

• Myositis-specific antibodies including anti-Jo-1, anti-Mi-2, anti-SRP, anti-MDA5 1
• Troponin to evaluate myocardial involvement 3
• Lactate dehydrogenase (LDH) and aldolase as additional muscle enzyme markers 3

For Suspected Autoimmune Hepatitis

• Anti-smooth muscle antibodies (SMA) at titers ≥1:40 in adults or ≥1:20 in patients under 18 years 3
• Anti-liver/kidney microsomal antibody type 1 (anti-LKM-1) at titers ≥1:40 in adults or ≥1:10 in patients under 18 years 3
• Anti-liver cytosol type 1 (anti-LC1) 3
• Serum immunoglobulins (IgG) typically elevated 3

For Suspected Vasculitis

• Cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA/PR3) highly sensitive and specific for Wegener's granulomatosis 4
• Perinuclear antineutrophil cytoplasmic antibodies (p-ANCA/MPO) for microscopic polyangiitis 4

Critical Interpretation Guidelines

Titer-Specific Management

• ANA 1:40: Seen in 31.7% of healthy individuals; exercise extreme caution and require strong clinical correlation 2, 7
• ANA 1:80: Present in 13.3% of healthy individuals; low positive likelihood ratio, consider anti-SSA/Ro testing specifically 2, 7
• ANA ≥1:160: Found in only 5.0% of healthy individuals; pursue specific antibody testing as positive likelihood ratio is substantially higher 2, 7

Method Reporting Requirements

• The specific method used (IIFA, ELISA, multiplex, chemiluminescence) must be reported, as test characteristics differ significantly between platforms 1
• Results from different methods should be reported separately and cannot be directly compared 1
• For anti-dsDNA monitoring in diagnosed SLE, use the same quantitative method consistently, preferably from the same laboratory 1

High Clinical Suspicion Override

• In cases of high clinical suspicion, specific antibody testing should proceed regardless of ANA result, as anti-Jo-1, anti-ribosomal P, and anti-SSA/Ro antibodies may be present in ANA-negative patients by IIFA [1,2, @23@]
• Request specific ENA testing when clinical features strongly suggest a particular autoimmune disease, even with negative ANA screening 1, 2

Common Pitfalls to Avoid

• Do not repeat ANA testing for disease monitoring once diagnosis is established; ANA is for diagnostic purposes only, not for assessing disease activity 2, 6
• Do not order ANA testing in asymptomatic patients without clinical features suggesting autoimmune disease, as false positives are common 1, 8
• Do not rely on ANA testing alone for diagnosis; compatible clinical symptoms, laboratory abnormalities, and histological findings when appropriate are required 2, 8
• Do not assume all anti-dsDNA assays are equivalent; CLIFT offers highest specificity but lower sensitivity, while ELISA provides higher sensitivity but lower specificity 1, 6
• Do not delay infectious disease screening (HIV, hepatitis B/C, tuberculosis) before initiating immunosuppressive therapy in confirmed autoimmune disease 3
• Do not interpret low-titer ANA (1:40-1:80) as clinically significant without strong supporting clinical and laboratory evidence 2, 7

Referral Indications

• Refer to rheumatology when ANA titer ≥1:160 with compatible clinical symptoms 2
• Early rheumatology referral is essential for suspected systemic autoimmune disease to prevent irreversible organ damage 3
• Refer to appropriate subspecialty (hepatology for autoimmune hepatitis, neurology for inflammatory myopathies with severe weakness) based on predominant organ involvement 1, 3