Connective Tissue Diseases - Systemic Autoimmune Rheumatic Diseases

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 syndromemixed 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

Who should be tested for connective tissue or systemic autoimmune rheumatic diseases?

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.

Which lab tests should be used if systemic autoimmune rheumatic disease is suspected?

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. 

How are antinuclear antibody tests interpreted?

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.

Which testing algorithms are related to this topic?

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 

Laboratory Testing

Diagnosis

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.

In cases of high clinical suspicion for SARDs, specific autoantibody testing should be performed, even if ANA testing is negative. 

ANA Patterns, Autoantibody Targets, and Clinical Associations
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

Sources: Tebo, 2017 ; Agmon-Levin, 2014 

In addition to IFA ANA testing, other testing options are available. See table below for comparison of ANA testing methods.

Comparison of ANA Testing Methods
Method Advantages Disadvantages
IFA Preferred method

Strong screening tool; resulting patterns can guide confirmatory testing for specific diagnosis or prognosis

Labor intensive

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

ELISA Commonly available

Specificities for CTDs may be affected by limited antigenic targets

Offers faster TAT

More objective than IFA

High throughput

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

High throughput

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

Faster TAT

More objective than IFA

High throughput

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

Source: Tebo, 2017 

Monitoring

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 Laboratory Tests

Preferred Initial Screening Tests for SARDs
Confirmatory Tests for Specific and More Common SARDs

Confirmatory for specific SARD

First-line testing for connective tissue disease

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

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

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

Disease-Specific Panels
Method

Quantitative Immunoturbidimetry/Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody/Quantitative Chemiluminescent Immunoassay/Semi-Quantitative Multiplex Bead Assay

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References

  1. Nuclear Patterns ICAP

    International Consensus on Antinuclear Antibody Patterns. Nuclear patterns. International Consensus on ANA Patterns. Gainesville, FL. [Updated: 2019; Accessed: Jun 2020]

    Online
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