What lab tests are used to assess for autoimmune disorders?

Laboratory Testing for Autoimmune Disease Assessment

First-Line Screening Test

ANA testing by indirect immunofluorescence assay (IIFA) on HEp-2 cells at a screening dilution of 1:160 is the reference standard for initial evaluation of suspected systemic autoimmune rheumatic diseases. 1, 2

Why IIFA is the Gold Standard

• IIFA offers superior sensitivity (>95% for SLE) compared to automated methods like ELISA or multiplex assays 3
• Both the titer AND pattern (nuclear, cytoplasmic, or mitotic) must be reported, as patterns provide critical diagnostic clues 1, 2
• The 1:160 dilution represents the 95th percentile of healthy controls in adult populations 1
• If alternative automated methods are used and negative, but clinical suspicion remains high, IIFA must still be performed due to its superior sensitivity for SLE and systemic sclerosis 1

Critical Pitfall to Avoid

• Do not use limited antigen panels (ELISA with restricted antigens) as initial ANA screening—they miss important patterns and have lower sensitivity 1
• A negative ANA does not exclude autoimmune disease, as sensitivity is not 100% even at 1:160 dilution 1, 3
• ANA positivity occurs in up to 31.7% of healthy individuals at 1:40 dilution and 5% at 1:160, so clinical context is essential 1

Core Laboratory Panel (Baseline Assessment)

Complete Blood Count and Metabolic Assessment

• Complete blood count with differential to detect cytopenias, anemia, or abnormal cells indicating disease activity 1
• Comprehensive metabolic panel including serum creatinine (or eGFR) and serum albumin to assess renal and hepatic function 1, 2
• Inflammatory markers (ESR and CRP) to evaluate acute phase response and disease activity 1, 2

Immunologic Baseline

• Quantitative immunoglobulin levels (IgG, IgA, IgM) to identify immunodeficiency states 1
• Urinalysis with urine protein/creatinine ratio to detect renal involvement 1

Pre-Treatment Screening

• Infectious disease screening (HIV, hepatitis B and C) based on risk factors, especially before immunosuppressive therapy 1
• Tuberculosis screening according to local guidelines before immunosuppression 1

Reflex Testing Based on ANA Pattern

The specific pattern observed on IIFA determines which disease-specific antibodies should be ordered next. 3

Pattern-Directed Reflex Algorithm

• Homogeneous pattern → anti-dsDNA and anti-histone antibodies 3
• Speckled pattern → anti-Sm, anti-RNP, anti-SSA/Ro, anti-SSB/La antibodies 3
• Nucleolar pattern → anti-Scl-70/topoisomerase-1 antibodies (associated with systemic sclerosis) 2, 3
• Centromere pattern → anti-centromere antibodies (associated with limited systemic sclerosis/CREST) 2, 3

Disease-Specific Antibody Testing When ANA Positive

For Suspected SLE

• Anti-dsDNA antibodies (high specificity for SLE) 1, 2
• Complement levels (C3, C4) essential for SLE evaluation and monitoring 1
• Farr assay or Crithidia luciliae immunofluorescence test (CLIFT) offer high clinical specificity for anti-dsDNA 2

For Suspected Systemic Sclerosis

• Anti-Scl-70/topoisomerase-1 (associated with diffuse cutaneous systemic sclerosis) 2
• Anti-centromere antibodies (associated with limited cutaneous systemic sclerosis/CREST) 2

For Suspected Sjögren's Syndrome

• Anti-SSA/Ro and anti-SSB/La antibodies 2
• These should be checked before pregnancy due to risk of congenital heart block 1

For Suspected Inflammatory Myopathies

• Myositis-specific antibodies including anti-Jo-1 and other antisynthetase antibodies 2

For Suspected Antiphospholipid Syndrome

• Anti-phospholipid antibodies (lupus anticoagulant, anticardiolipin, anti-β2-glycoprotein I) if thrombosis, recurrent pregnancy loss, or thrombocytopenia is present 1
• Should be measured before pregnancy, surgery, transplant, or estrogen-containing treatments 1

Autoimmune Hepatitis-Specific Testing

For suspected autoimmune hepatitis, a different antibody panel is required beyond standard ANA testing. 4

AIH-Specific Autoantibodies

• ANA and SMA (smooth muscle antibodies) detected by IFL—ELISA alone is inappropriate as primary screening 4
• Anti-LKM1 and anti-LC1 antibodies (ELISA results are interchangeable with IFL for these specific antibodies) 4
• Anti-SLA/LP antibodies using recombinant/purified target antigens 4

Important AIH Testing Considerations

• Autoantibody titers may vary during disease course, and seronegative individuals at diagnosis may express antibodies later 4
• Repeated testing may allow autoantibody detection and correct disease classification 4
• In pediatric patients specifically, autoantibody titers correlate with disease activity and can monitor treatment response 4
• Anti-LC1 antibodies correlate well with disease activity, showing >50% decrease or disappearance during remission 4

Special Population Considerations

Pediatric Patients

• No consensus exists for screening dilution in children under 16 years—some laboratories use 1:40 1, 3

Pre-Pregnancy Evaluation

• Anti-Ro and anti-La antibodies should be checked before pregnancy due to risk of congenital heart block 1
• Anti-phospholipid antibodies should be measured before pregnancy 1

Before Immunosuppression

• Complete infectious disease screening (HIV, hepatitis B/C, tuberculosis) 1
• Autoantibody testing should be performed before initiating immunosuppressive therapy when possible, as treatment may affect results 2

Critical Testing Pitfalls

Common Errors to Avoid

• Low-titer ANA can be clinically significant—titers above the screening threshold do not correlate with disease activity 1
• Only 10% of individuals have matched serum and CSF evaluation when appropriate, leading to missed diagnoses 5
• Overlapping panel evaluations (ordering multiple panels within 14 days) occur frequently but add limited value 5
• Repeat autoantibody testing yields novel information in only a minority of cases and changes clinical decision making in <1% of cases 5
• Bone marrow biopsy is only indicated for unexplained cytopenias or abnormal peripheral blood cells, not routine screening 1

Interpretation Caveats

• ANA testing is primarily for diagnostic purposes, not for monitoring disease progression 2
• Tests must be ordered specifically based on reliable clinical data and results must not be interpreted outside the specific clinical context 4
• Complete autoimmune serology work-up is not available in all laboratories—patient sera should be sent to reference laboratories for full evaluation in cases of diagnostic uncertainty 4