Antinuclear Antibody (ANA) With HEp-2 Substrate

Last Literature Review: November 2019 Last Update:
  • Initial screening test for connective tissue diseases (SARDs).
  • One or more reflexive tests may be added, depending on ANA pattern detected (see ANA IFA Reflex Testing Algorithm).
  • Preferred screening test for SARD.
  • Reported patterns may help guide differential diagnosis, but may not be specific for individual antibodies or diseases.
  • Negative results do not necessarily rule out SARD.

Aids in initial diagnosis of connective tissue disease.

Aids in initial diagnosis of connective tissue disease.

Antinuclear antibody (ANA) testing is used in the diagnostic evaluation of various autoimmune diseases, including connective tissue diseases such as systemic lupus erythematosus (SLE), Sjögren syndrome, and systemic sclerosis (SSc).  Initial testing for autoimmune connective tissue diseases (also referred to as systemic autoimmune rheumatic diseases, or SARDs) should include tests for C-reactive protein (CRP), ANAs, rheumatoid factor, and cyclic citrullinated peptide antibodies. If ANA results are positive, follow-up or confirmatory testing may be guided by the pattern(s) observed and/or the patient’s clinical presentation.

Disease Overview

Diagnostic Issues

Autoimmune connective tissue diseases may present with similar features, making diagnosis difficult. Possible diagnoses may include:

ANA with reflex by immunofluorescent assay (IFA) (based on ANA patterns) may help guide differential diagnosis but may not be specific for individual diseases.


Antigen/antibody complexes affect a variety of organs in connective tissue diseases, which frequently leads to a multisystem disease presentation. ANA antibodies are the most common antibodies and may precede the onset of connective tissue disease. Although certain antibodies may show specificity for certain diseases (eg, SSA 52, SSA 60, and SSB antibodies for Sjögren syndrome), ANA antibodies are not specific for connective tissue disease, and may also be associated with infectious diseases, cancers, other autoimmune disorders (eg, autoimmune liver disease), and advanced age, and may even be present in healthy patients.

Test Interpretation


A dual or mixed pattern may indicate disease overlap. Visit the International Consensus on Antinuclear Antibody Patterns website  for additional information about pattern and disease associations.


  • Dual or mixed patterns will not be reflexed; additional testing for dual or mixed patterns should be determined by the ordering physician.
  • A negative ANA by IFA test does not rule out the presence of connective tissue disease.

ANA IFA Reflex Testing Algorithms

ANA IFA Reflex Testing Algorithm

See the following ARUP Consult algorithms for ANA testing and expanded information on cytoplasmic and nuclear patterns:

Antinuclear Antibody Disease Testing Algorithm

Antinuclear Antibody Disease Testing - Nuclear Patterns

Antinuclear Antibody Disease Testing - Cytoplasmic Patterns