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Tests for Autoimmune Diseases

Test code(s): 249, 16814, 19946, 94954, 36378

Panel components may be ordered separately. Please see the Quest Diagnostics Test Center for ordering information.

  • What’s new in this FAQ (version 3):
    • Question 8: 14.3.3eta is replaced with a new marker, Mutated Citrullinated Vimentin (MCV).

“Autoimmune disease” refers to a diverse group of disorders that can involve almost every one of the body’s organs and systems. It encompasses diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood, and blood vessels. In all of these autoimmune diseases, the underlying problem is “autoimmunity”—the body’s immune system becomes misdirected and attacks the very organs it was designed to protect.

Diagnosis is challenging for several reasons:

  1. Patients initially present with nonspecific symptoms such as fatigue, joint and muscle pain, fever, and/or weight change.
  2. Symptoms often flare and remit.
  3. Patients frequently have more than 1 autoimmune disease.

According to a survey by the Autoimmune Association, it takes up to 4 years and 4 doctors, on average, for a patient to receive a proper autoimmune disease diagnosis.1

An estimated 23.5 million Americans2 suffer from 1 or more of over 801,2 autoimmune diseases.

On average, autoimmune diseases affect 2 times more women than men.2,3 Autoimmune diseases are one of the top 10 leading causes of death among women aged 65 and under2 and represent the fourth-largest cause of disability among women in the United States.3

Autoimmune diseases commonly occur in multiple members of a family, indicating a genetic predisposition. Family members are often affected by various autoimmune disorders rather than a single specific disorder.

When evaluating a patient for autoimmune diseases, an antinuclear antibody (ANA) test is typically performed first. The ANA by immunofluorescence assay (IFA) screens for approximately 150 autoantibodies that can occur in various autoimmune diseases. The American College of Rheumatology (ACR) recommends IFA as the gold standard method for ANA testing.4

A negative ANA IFA result suggests that ANA-associated autoimmune diseases are not present but does not rule out the possibility. Patients with negative results on the ANA IFA usually also have negative results for specific ANA antibodies. However, Jo-1 antibody may be detected in ANA IFA-negative patients with some types of myositis, and SSA antibody may be present in some ANA IFA-negative patients with SLE or Sjögren syndrome.5

A positive result on the ANA IFA screen suggests the presence of autoimmune disease and will reflex to titer and pattern. A low ANA titer (1:40 to 1:80) may be associated with preclinical disease or lack of disease. Titers >1:80 are consistent with autoimmune disease. When a result is positive, the staining pattern helps predict the disease type.

Specific antibody testing, if clinically indicated, is often useful for diagnosis of specific autoimmune disorders. Quest Diagnostics offers testing options for early diagnosis and prognosis of autoimmune diseases. Please read questions 7, 8, and 9 for more information. 

As noted above, the ANA staining pattern can be helpful in suggesting a diagnosis but does not provide definitive evidence. The significance of various patterns is summarized below (click on the table to enlarge).

 Table showing the significance of various ANA staining patterns

Note: At Quest Diagnostics, all patterns and corresponding titers are reported when more than one pattern is observed.

In recent years, Quest Diagnostics has been following the recommendations of the International Consensus of ANA Patterns (ICAP)6 and reporting nuclear, cytoplasmic, and mitotic patterns when observed in a patient sample. There are times when more than one pattern is observed in a sample, and each will be reported with a corresponding titer.  The interpretation of these patterns (and the associated titers) requires clinical context as they may represent the same disease, represent 2 different diseases, or have limited clinical significance, particularly if a titer is low.  

Yes. Quest Diagnostics offers ANA Screen, IFA, Reflex Titer/Pattern, Reflex Multiplex 11-Ab Cascade (test code 16814). This test begins with an ANA screen using IFA technology as well as the IdentRA panel. A positive ANA result reflexes to an ANA titer and pattern and to a 3-tiered, 11-antibody cascade.  These 3 tiers include groups of autoantibodies associated with rheumatic diseases; the test progresses through the 3 tiers in order of highest to lowest relative prevalence for associated rheumatic diseases. The first tier includes chromatin, dsDNA, RNP, Sm, and Sm/RNP antibodies. If all 5 antibodies are negative, testing proceeds to the second tier, which includes Jo-1, Scl-70, SS-A, and SS-B antibodies. If all 4 of these antibodies are negative, testing proceeds to the final tier, which includes centromere B and ribosomal P antibodies.

Note that if the ANA IFA result is positive, but all 11 specific antibody results are negative, an autoimmune disease may still be present. The disease may be associated with an antibody not tested in the cascade. Diseases to be considered include rheumatoid arthritis, autoimmune hepatitis, primary biliary cholangitis, autoimmune thyroiditis, Addison disease, pernicious anemia, autoimmune neuropathies, vasculitis, celiac disease, and bullous disease, among others.

Yes. Quest Diagnostics offers an integrated panel to facilitate timelier diagnosis of rheumatoid arthritis in patients who present with undifferentiated arthritis: the ANA, IFA Cascade and Rheumatoid Arthritis Panel 2, with reflexes (test code 94954).

For patients with positive ANA IFA results, the reflex pattern and testing for specific autoantibodies is identical to that of the ANA Screen, IFA, Reflex Titer/Pattern and Reflex to Multiplex 11 Ab Cascade (test code 16814). The Rheumatoid Arthritis Panel 2 component of this assay includes tests for rheumatoid factor (RF), cyclic citrullinated peptide (CCP) antibody, and mutated citrullinated vimentin (MCV) antibody.

Adding MCV antibody improves diagnostic sensitivity for early rheumatoid arthritis relative to testing for RF and CCP antibodies alone. American College of Rheumatology guidelines indicate that disease-modifying anti-rheumatic drug therapy should be prescribed for patients who receive a diagnosis of early RA and are experiencing active disease at the time of testing.7

Yes. Testing for each of the autoantibodies included in the 11-antibody cascade may be ordered separately or by using the ANA Multiplex with Reflex to 11 Antibody Cascade (test code 19946). The 11-antibody reflex cascade is triggered if one of the 11 antibodies included in the multiplex bead immunoassay screen is positive. This ANA screen is less sensitive, as it tests for only 11 autoantibodies in comparison to ANA IFA screen, which tests for approximately 150 autoantibodies. The 3-tier, 11-antibody reflex cascade is identical to that of ANA Screen, IFA, Reflex Titer/Pattern and Reflex to Multiplex 11 Ab Cascade (test code 16814; see Question 7). This test may be used when looking for a tiered approach to diagnosis of autoimmune conditions on the patient who has had a previous positive ANA by IFA screening test result.

A negative result on the multiplex screen does not rule out autoimmune disease. In addition, a disease associated with an antibody not tested in the cascade may be present, especially if the patient has previously tested positive on an ANA IFA screen. Diseases to be considered include rheumatoid arthritis, autoimmune hepatitis, primary biliary cholangitis, autoimmune thyroiditis, Addison disease, pernicious anemia, autoimmune neuropathies, vasculitis, celiac disease, bullous disease, and others.

For a more comprehensive (non-tiered) approach to testing for one or more rheumatic diseases, Quest offers ANAlyzeR™ ANA, IFA with Reflex Titer/Pattern, Systemic Autoimmune Panel 1 (test code 36378).  

The presence of a specific antibody is highly suggestive of the associated autoimmune disease. However, these antibodies are not completely specific for a particular disease; thus, results need to be interpreted in context of the clinical information and considering the following antibody prevalence.

Prevalence of Tier 1 Antibodies8-11

  • Double-stranded DNA (dsDNA) antibodies are present in 57% to 62% of systemic lupus erythematosus (SLE) cases, 10% to 43% of polymyositis, 11% to 20% of Sjögren syndrome, 8% of systemic sclerosis (scleroderma), and 0% to 8% of mixed connective tissue disease (MCTD).
  • Chromatin antibody is present in >80% of MCTD cases, 37% to 73% of SLE, 14% of systemic sclerosis, 12% of Sjögren syndrome, and 8% of polymyositis.
  • Ribonucleoprotein (RNP) antibodies are present in >80% of MCTD cases, 22% to 48% of SLE, 14% of systemic sclerosis, 12% of Sjögren syndrome, and 8% of polymyositis.
  • Sm/RNP antibodies are present in 54% to 94% of MCTD cases, 30% of SLE, 4% of systemic sclerosis, and 9% of Sjögren syndrome and polymyositis.
  • Sm antibody is present in 20% to 30% of SLE cases, 8% of MCTD, 10% of polymyositis, 0% of systemic sclerosis, and 4% of Sjögren syndrome.
  • Double-stranded DNA, chromatin, ribonucleoprotein, Sm/RNP complex, and Sm antibodies are also present in <2% of normal blood donors.

Prevalence of Tier 2 Antibodies10-11

  • SS-A and SS-B antibodies are present in >80% of Sjögren syndrome cases and are considered a diagnostic indicator for this autoimmune disease. However, these antibodies are also present in other autoimmune disorders. SS-A antibodies are seen in 33% to 52% of SLE cases, 42% of polymyositis, 23% of systemic sclerosis (scleroderma), and 13% of MCTD. SS-B antibody is present in 13% to 27% of SLE cases, 5% of systemic sclerosis, <2% of polymyositis, and <2% of MCTD.
  • Scl-70 antibody is present in 16% of systemic sclerosis cases, 7% of MCTD (especially those with features of systemic sclerosis), 2% to 3% of SLE, <2% of Sjögren syndrome, and <2% of polymyositis.
  • Jo-1 antibody is present in 17% of polymyositis cases, 7% of MCTD (especially in those with features of muscle inflammation), <2% of SLE, Sjögren syndrome, and systemic sclerosis.

Tier 2 antibodies are also present in <2% of normal blood donors.

Prevalence of Tier 3 Antibodies10-13

  • Centromere B antibody is present in 27% of systemic sclerosis (scleroderma) cases, 66% of CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), 3% to 12% of SLE, 7% of MCTD (typically with features of polymyositis), and <2% of Sjögren syndrome, polymyositis, and normal blood donors.
  • Ribosomal P antibody is present in 9% to 30% of SLE cases (often with neurological manifestations), 7% of MCTD, and <2% of Sjögren syndrome, systemic sclerosis, polymyositis, and normal blood donors.

Note: The prevalence levels listed above may vary with the population studied and methods used.7 Values given are for general guidance.


  1. Autoimmune diagnosis tips. Autoimmune Association Web site. Accessed April 6, 2023.
  2.  U.S. Department of Health and Human Services Office on Women’s Health Autoimmune Diseases web site. Updated February 22, 2021. Accessed April 6, 2023.
  3. Angum F, Khan T, Kaler J, et al. The prevalence of autoimmune disorders in women: a narrative review. Cureus. 2020;12(5):e8094. doi:10.7759/cureus.8094
  4. American College of Rheumatology Position Statement: Methodology of testing for antinuclear antibodies. Published January 2009. Updated August 2015. Accessed February 3, 2016.
  5. Yazdany J, Schmajuk G, Robbins M, et al. Choosing wisely: the American College of Rheumatology's Top 5 list of things physicians and patients should question. Arthritis Care Res (Hoboken). 2013;65(3):329-339. doi:10.1002/acr.21930
  6.  International Consensus on ANA Patterns (ICAP). Published May 19, 2015. Accessed March 14, 2023.
  7. Singh JA, Saag KG, Bridges SL, Jr, et al. 2015 American College of Rheumatology guideline for the treatment of rheumatoid arthritis. Arthritis Rheumatol. 2016;68(1):1-26. doi: 10.1002/art.39480
  8. Scholz J, Grossmann K, Knütter I, et al. Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing. Clin Chem Lab Med. 2015;53:1991-2002. doi: 10.1515/cclm-2015-0083
  9. Colglazier CL, Sutej PG. Laboratory testing in the rheumatic diseases: a practical review. South Med J. 2005;98(2):185-191.
  10. Moder KG, Wener MH, Weisman MH, et al. Measurement of antinuclear antibodies by multiplex immunoassay: a prospective, multicenter clinical evaluation. J Rheumatol. 2007;34(5):978-986.
  11. Binder SR, Genovese MC, Merrill JT, et al. Computer-assisted pattern recognition of autoantibody results. Clin Diagn Lab Immunol. 2005;12(12):1353-1357. doi:10.1128/CDLI.12.12.1353–1357.2005
  12. Mouthon L, Dunogue B, Guillevin L. Diagnosis and classification of eosinophilic granulomatosis with polyangiitis (formerly named Churg-Strauss syndrome). J Autoimmun. 2014;48-49:99-103. doi: 10.1016/j.jaut.2014.01.018
  13. Tur BS, Süldür N, Ataman S, et al. Anti-neutrophil cytoplasmic antibodies in patients with rheumatoid arthritis: clinical, biological, and radiological correlations. Joint Bone Spine. 2004;71(3):198-202. doi: 10.1016/j.jbspin.2003.04.002

This FAQ is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on the physician’s education, clinical expertise, and assessment of the patient.

Document FAQS.177 Version 3

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Version 2: Effective 06/28/2023 to 10/30/2023

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