Connective tissue or systemic autoimmune rheumatic diseases (SARDs) are characterized by autoantibodies that can affect tissues and organs throughout the body. Laboratory testing for antinuclear antibodies (ANAs) and specific autoantibodies associated with the presence of SARDs may be useful in the evaluation of these diseases. The SARDS include systemic lupus erythematosus (SLE), Sjögren syndrome, mixed connective tissue disease/undifferentiated connective tissue disease, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (including polymyositis, dermatomyositis, and necrotizing autoimmune), and overlap syndromes.
Quick Answers for Clinicians
Patients with connective tissue or systemic autoimmune rheumatic diseases (SARDs) may present with very different signs and symptoms, depending on disease type and which tissues and organ systems are affected. For example, patients with systemic lupus erythematosus (SLE) may present with rash or photosensitivity, whereas those with Sjögren syndrome may report dry mouth and dry eyes. See Indications for Testing below for common indications of various SARDs.
Testing is particularly important in children with suspected SARDs because of the growth and development issues associated with these diseases.
Tests for antinuclear antibodies (ANAs) and autoantibodies associated with ANA presence are appropriate and are best used in individuals with a high pretest probability of having a systemic autoimmune rheumatic disease (SARD). The gold standard ANA test is the indirect fluorescent antibody (IFA) assay. Although labor intensive and subjective, it offers high sensitivity and is recommended by the American College of Rheumatology (ACR) as important for the evaluation of a number of SARDs. Alternative methods (immunoassays) to IFA for the detection of ANAs are generally less sensitive for SARDs but offer greater specificities, faster turnaround times, and are more objective. (See table below comparing ANA testing methods.) If ANA testing using an immunoassay is negative and IFA is positive, the reported ANA pattern and titer, along with the patient’s clinical history and presentation, may be useful in determining which autoantibody test(s) to perform for disease confirmation. If ANA testing using an immunoassay is positive and the IFA is negative, the positive predictive value for SARDs is generally low.
Test interpretation of antinuclear antibody (ANA) tests is based on common ANA patterns that have shown consistent correlation with systemic autoimmune rheumatic diseases (SARDs). The International Consensus on ANA Patterns has comprehensive information on patterns.
Indications for Testing
The following SARDs may manifest with these indications:
- SLE: rash, photosensitivity, oral ulcers, polyarticular arthritis, proteinurea or cellular casts, pleuritis, pericarditis, interstitial nephritis, fatigue, cytopenia, seizures or psychosis
- Sjögren syndrome: dry mouth, dry eyes
- Mixed or undifferentiated connective tissue disease: Raynaud syndrome, joint aches, puffy fingers, low-grade myositis
- Systemic sclerosis: Raynaud syndrome, sclerodactyly, interstitial lung disease/pulmonary hypertension, gastroesophageal reflux
- Inflammatory myopathies (including polymyositis, dermatomyositis, and necrotizing autoimmune myopathies): progressive muscle weakness (usually proximal), with or without rashes
Antinuclear Antibody Testing
A starting point for testing for SARDs is ANA testing. Antibodies in SARDs can target the nucleus as well as any intracellular components of the cell, most notably the cytoplasm. ANA tests are best used for individuals with a high pretest probability of having a SARD because ANA reactivity may be present in healthy patients as well as those with certain infections, cancer, and autoimmune diseases.
The gold standard for ANA testing is the indirect fluorescent antibody (IFA) assay, performed in a lab experienced with ANA testing. ANA test interpretation is based on common ANA patterns that have shown consistent correlation with SARDs. Patterns reported by ARUP Laboratories include centromere, homogeneous, nuclear dots, nucleolar, speckled, and cytoplasmic (see table below). The International Consensus on ANA Patterns has comprehensive information on patterns, targets, and clinical associations. ANA IFA reports should include the method, type of substrate, results obtained (including qualitative response [positive or negative]), pattern(s) observed, and their titers.
|Patterns||Primary Autoantibody Targets||Clinical Associations|
|Centromere||Centromere A/B(C)||SSc, PBC|
|Homogeneous||dsDNA, histones, chromatin (nucleosomes)||SLE, drug-induced SLE, JIA|
|Nuclear dots||NXP-2, Sp100||PBC, DM, SjS, SLE, SSc, PM|
|Nucleolar||PM/Scl, RNA polymerase, URNP, U3-RNP (fibrillarin), Th/To, NOR-90||SSc, SSc/PM overlap, SjS|
|Speckled||SSA-52 (Ro52), SSA-60 (Ro60), SSB/LA, Topo-1 (Scl-70), Smith, Sm/RNP, U2-RNP, Mi-2, TIF1g, Ku, RNA polymerase, DFS70/LEDGF-P75||SLE, SSc, SjS, DM, PM, MCTD, UCTD (may also be found in healthy individuals)|
|Cytoplasmic||Ribosomal P, tRNA synthetase (Jo-1, PL-7, PL-12, EJ, OJ), SRP, mitochondria (AMA)||ARS, ILD, IM,a SLE, SSc, SjS, RA, MCTD, PBC, AIH, infectious, neurologic, other inflammatory conditions|
|aIM includes DM, PM, and NAM
AIH, autoimmune hepatitis; ARS, antisynthetase syndrome; DM, dermatomyositis; ILD, interstitial lung disease; IM, inflammatory myopathies; JIA, juvenile idiopathic arthritis; MCTD, mixed connective tissue disease; NAM, necrotizing autoimmune myopathy; PBC, primary biliary cholangitis; PM, polymyositis; RA, rheumatoid arthritis; SjS, Sjögren syndrome; SRP, signal recognition particle; SSc, systemic sclerosis; UCTD, undifferentiated connective tissue disease
In addition to IFA ANA testing, other testing options are available. See table below for comparison of ANA testing methods.
Strong screening tool; resulting patterns can guide confirmatory testing for specific diagnosis or prognosis
Subjective (in titer and pattern recognition)
Reagents not well standardized
Has limited diagnostic specificity
Requires significant expertise to interpret
Appropriate cutoff value for positivity not established
Specificities for CTDs may be affected by limited antigenic targets
Offers faster TAT
More objective than IFA
|Has lower sensitivity than IFA
Clinical performances of commercial ELISAs vary
May not be useful in assessing ALDs or SARDs that target complex autoantigens
|CLIA||More sensitive than ELISA
Easier to automate than ELISA
Wide analytical measurement range (vs ELISA)
Offers faster TAT
More objective than IFA
|May not be useful in assessing ALDs or SARDs that target complex autoantigens
Has lower sensitivity than IFA
|Multiplex assay (LIA, MBA)||Can detect multiple independent antibodies associated with ANAs
Can be automated
Can provide quantitative or qualitative results
More objective than IFA
Useful in analyzing autoantibody clusters and evaluating diseases with multiple autoantibody specificities (vs. ELISA)
|Has lower sensitivity than IFA
Diagnostic accuracies differ among immunoassays
LIA is not as technologically sophisticated as MBA
|ALDs, autoimmune liver diseases; CLIA, chemiluminescence immunoassay; ELISA, enzyme-linked immunosorbent assay; LIA, line immunoassay; MBA, multiplexed bead assay; TAT, turnaround time|
Once a SARD has been diagnosed, monitoring tests should be based on organ involvement. If cytopenias are present, the patient should be monitored with sequential CBCs. Certain drugs used for treatment require testing for liver function. In addition, patients should be monitored for malignancy. Dermatomyositis and mixed connective tissue disease have shown increased risk for malignancy. Sjögren syndrome is specifically associated with an increased risk for hematologic malignancy.
ARUP Lab Tests
Semi-Quantitative Indirect Fluorescent Antibody
Semi-Quantitative Indirect Fluorescent Antibody/Qualitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Multiplex Bead Assay/Qualitative Immunoblot
Confirmatory for specific SARD
First-line testing for connective tissue disease
Qualitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody (IFA)/Semi-Quantitative Multiplex Bead Assay
Panel includes dsDNA, IgG; Smith/RNP, IgG; Smith (ENA), IgG; SSA 52 and 60, IgG; SSB, IgG; Jo-1, IgG; Scl-70, IgG
Useful in the differential diagnosis of SARDs with or without myopathy
Recommended to differentiate SLE and Sjögren syndrome
Semi-Quantitative Multiplex Bead Assay
Recommended first-line test to evaluate polymyositis or inflammatory myopathies
Secondary screen based on ANA test results or if ANA IFA is negative but Sjögren syndrome, SLE, systemic sclerosis, or myositis is strongly suspected
May be useful when evaluating for systemic sclerosis or SARD associated with overlapping features of systemic sclerosis and/or myositis
May be useful in differential diagnosis of SLE and Sjögren syndrome
Secondary screen for SLE based on ANA results
Qualitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody
Confirmatory test for SLE based on ANA results
May be useful in detecting central nervous system SLE (somewhat rare) or renal involvement in SLE
Aid in diagnosis of systemic sclerosis; preferred test is Comprehensive Systemic Sclerosis Panel
Recommended for diagnosis of systemic sclerosis in patients negative for centromere, SCl-70, or RNA polymerase antibodies
Qualitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody/Semi-Quantitative Multiplex Bead Assay
Qualitative Immunoprecipitation/Semi-Quantitative Multiplex Bead Assay/Qualitative Immunoblot/Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Quantitative Immunoturbidimetry
Gunnarsson R, Hetlevik SO, Lilleby V, et al. Mixed connective tissue disease. Best Pract Res Clin Rheumatol. 2016;30(1):95-111.
International Consensus on Antinuclear Antibody Patterns. Nuclear patterns. International Consensus on ANA Patterns. Gainesville, FL. [Updated: 2019; Accessed: Jun 2020]
Pisetsky DS. Antinuclear antibody testing - misunderstood or misbegotten? Nat Rev Rheumatol. 2017; 13 (8): 495-502.
Pope JE, Johnson SR. New Classification Criteria for Systemic Sclerosis (Scleroderma). Rheum Dis Clin North Am. 2015;41(3):383-398.
Tebo AE. Recent approaches to optimize laboratory assessment of antinuclear antibodies. Clin Vaccine Immunol. 22017;24(12):e00270-17.