What tests are used to diagnose autoimmune diseases?

Diagnostic Testing for Autoimmune Diseases

Begin with antinuclear antibody (ANA) testing by indirect immunofluorescence assay (IIFA) on HEp-2 cells at a screening dilution of 1:160, as this is the mandatory reference standard for initial screening of systemic autoimmune rheumatic diseases. 1, 2

Initial Screening Approach

Primary Screening Test

• ANA by IIFA on HEp-2 cells is the mandatory first test, with both titer and pattern (nuclear, cytoplasmic, or mitotic) reported, as patterns provide critical diagnostic information for guiding subsequent testing. 1, 2
• If automated methods (ELISA, multiplex) are used and negative but clinical suspicion remains high, IIFA must be performed due to superior sensitivity for SLE and systemic sclerosis. 2
• Autoantibody positivity alone does not make a diagnosis, and absence of autoantibodies does not exclude diagnosis—clinical context is paramount. 3

Essential Baseline Laboratory Panel

• Complete blood count with differential to detect cytopenias, anemia, or abnormal cells indicating disease activity. 2, 4
• Comprehensive metabolic panel including serum creatinine (or eGFR), serum albumin, and liver function tests to assess organ involvement. 2, 4
• Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) to assess acute phase response and disease activity. 2, 5
• Quantitative immunoglobulin levels (IgG, IgA, IgM) to identify immunodeficiency states. 2
• Urinalysis with urine protein/creatinine ratio to detect renal involvement. 2

Disease-Specific Reflex Testing

For Suspected Systemic Lupus Erythematosus (SLE)

• Anti-dsDNA antibodies should be tested using Crithidia luciliae immunofluorescence test (CLIFT) or Farr assay for high specificity when SLE is clinically suspected. 1, 2
• Complement levels (C3, C4) are essential for SLE evaluation and monitoring. 2
• Anti-Sm and anti-RNP antibodies provide additional diagnostic specificity. 1, 2
• Anti-phospholipid antibodies (lupus anticoagulant, anticardiolipin, anti-β2-glycoprotein I) if thrombosis, recurrent pregnancy loss, or thrombocytopenia are present. 2

For Suspected Sjögren's Syndrome

• Anti-SSA/Ro and anti-SSB/La antibodies should be tested, even if ANA is negative, as these can be present in ANA-negative patients. 1, 2

For Suspected Systemic Sclerosis

• Anti-Scl-70/topoisomerase-1 (indicates diffuse disease) and anti-centromere (indicates limited disease/CREST) should be tested. 2

For Suspected Inflammatory Myopathies

• Myositis-specific antibodies including anti-Jo-1 and other antisynthetase antibodies should be tested, even if ANA is negative. 1, 2

For Suspected Autoimmune Hepatitis

• Immunofluorescence on rodent tissue is the best technique for detecting autoantibodies (ANA, SMA, anti-LKM1, anti-LC1), though ELISA using recombinant antigens (CYP2D6, FTCD, SLA/LP, F-actin) can supplement but not replace IFL. 1
• ELISA should not be used as the sole primary screening test because there is no useful combination of molecular specificities for dependable detection of ANA and SMA. 1
• Atypical pANCA (perinuclear anti-neutrophil nuclear antibodies) can be an additional diagnostic element, particularly if other autoantibodies are negative. 1
• Liver biopsy is considered a prerequisite for diagnosis and should be performed before starting treatment to guide treatment decisions. 1

For Suspected Type 1 Diabetes

• Multiple islet autoantibodies (GAD65, insulin, IA-2, IA-2b, ZnT8) define stage 1 type 1 diabetes when combined with normoglycemia. 1
• Standardized islet autoantibody tests are recommended for classification in adults with phenotypic overlap (younger age, unintentional weight loss, ketoacidosis, short time to insulin treatment). 1
• Thyroid autoantibodies (antithyroid peroxidase and antithyroglobulin) and celiac disease screening (IgA tissue transglutaminase with total IgA levels) should be performed soon after diagnosis. 2

For Suspected Autoimmune Encephalitis

• Brain MRI with contrast to evaluate hippocampus, striatum, or other brain areas is recommended. 6
• Lumbar puncture for CSF analysis including cell count, protein, glucose, and IgG index is essential. 6
• Panel of neuronal antibodies in both CSF and serum (anti-NMDAR, anti-VGKC, anti-LGI1, anti-CASPR2) should be tested, as sensitivity varies by antibody type and specimen. 6
• PCR for viral infections (HSV1/2, VZV) to exclude infectious causes. 6
• Collect blood samples before administering immunotherapy to avoid false results. 6

Critical Testing Principles

Timing and Interpretation

• Autoantibody titers and specificity may vary during disease course, and seronegative individuals at diagnosis may express autoantibodies later—repeated testing may allow correct diagnosis. 1
• In adults with autoimmune hepatitis, autoantibody titers correlate only roughly with disease activity and do not need regular monitoring unless clinical phenotype changes. 1
• In pediatric patients, autoantibody titers (particularly anti-LC1) correlate well with disease activity and can monitor treatment response. 1

When Clinical Suspicion Remains High

• In cases of high clinical suspicion, specific antibody testing should be performed regardless of negative ANA results, as anti-Jo-1, ribosomal P, or SS-A/Ro may be detected in ANA-negative patients. 1, 2
• Complete autoimmune serology workup is not available in all laboratories—patient sera should be sent to reference laboratories for full evaluation in cases of diagnostic uncertainty. 1

Pre-Treatment Considerations

• Before initiating immunosuppressive therapy, infectious disease screening for HIV, hepatitis B and C should be performed based on risk factors. 2
• Tuberculosis screening should be performed according to local guidelines. 2

Special Population Considerations

• In young females, anti-Ro and anti-La antibodies should be checked before pregnancy due to risk of congenital heart block. 2

Common Pitfalls to Avoid

• Do not use ELISA as sole screening for autoimmune hepatitis—IFL remains superior for ANA and SMA detection. 1
• Do not skip IIFA if automated methods are negative when clinical suspicion for SLE or systemic sclerosis remains high. 2
• Do not delay antibody testing in autoimmune encephalitis if CSF shows normal cell counts—autoimmune encephalitis can present with normal routine CSF studies. 6
• Do not rely solely on commercial assays for neuronal antibodies—false negatives occur frequently, especially in CSF samples. 6
• Do not interpret test results outside specific clinical context—tests must be ordered based on reliable clinical data. 1