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Quest Diagnostics

Tests for Autoimmune Diseases (ANA)

Test Codes: 249, 16814, 19946, 36378, 94954

Panel components are listed in Q13. Each component may be ordered separately. Please see the Quest Diagnostics Test Directory for ordering information.

  • What’s new in this FAQ (version 4):
    • Question 11: What are the differences between the major approaches to ANA testing at Quest?
    • Question 12: What is the screening titer used for ANA testing?

“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 80 autoimmune diseases.1,2 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.

ANAs are antibodies directed against a variety of nuclear antigens, and ANA testing may also detect other autoantibodies directed at cytoplasmic antigens.

ANA screening is often the first test for suspected systemic autoimmune rheumatic diseases (SARDs), with 2 approaches commonly employed.4 The traditional approach uses fixed cells (typically human epithelial cells called HEp-2) and indirect immunofluorescence assay (IFA). Another approach is screening with a combination of individual antigen tests using immunoassay. International guidelines have stated that combining the IFA with individual antigen screening adds value to the interpretation of both assays.5 Many studies support this statement6; combined, the 2 approaches can significantly improve sensitivity and specificity over each alone for detecting SARDs.7

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.8

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 (<1:80) 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.

Table showing the significance of various ANA staining patterns.

Click the table to open up in new window (enlarged)

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)9 and reporting nuclear, cytoplasmic, and mitotic patterns when observed in a patient sample. There are times when more than 1 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 immunofluorescence assay (IFA) technology. 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 antirheumatic drug therapy should be prescribed for patients who receive a diagnosis of early RA and are experiencing active disease at the time of testing.10

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 for 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 Antibodies11-14

  • Double-stranded DNA (dsDNA) antibodies are present in 57% to 62% of systemic lupus erythematosus (SLE) patients, 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 patients, 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 patients, 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 patients, 30% of SLE, 9% of Sjögren syndrome, 5% of polymyositis, and 4% of systemic sclerosis,
  • Sm antibody is present in 20% to 30% of SLE patients, 10% of polymyositis, 8% of MCTD, 4% of Sjögren syndrome, and 0% of systemic sclerosis.
  • Double-stranded DNA, chromatin, ribonucleoprotein, Sm/RNP complex, and Sm antibodies are also present in <2% of normal blood donors.

Prevalence of Tier 2 Antibodies13-14

  • SS-A and SS-B antibodies are present in >80% of Sjögren syndrome patients 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 patients, 42% of polymyositis, 23% of systemic sclerosis (scleroderma), and 13% of MCTD. SS-B antibody is present in 13% to 27% of SLE patients, 5% of systemic sclerosis, <2% of polymyositis, and <2% of MCTD.
  • Scl-70 antibody is present in 16% of systemic sclerosis patients, 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 patients, 7% of MCTD (especially in those with features of muscle inflammation), and <2% of SLE, Sjögren syndrome, and systemic sclerosis.

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

Prevalence of Tier 3 Antibodies13-16

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

249 - ANA Screen,IFA, with Reflex to Titer and Pattern

This test code includes a screen for the presence of antinuclear antibodies using indirect immunofluorescence, with reflex to titer and pattern if the result is positive.

16814 - ANA Cascade (ANA, IFA with Reflex to Titer and Pattern, and Reflex to 11 Ab Cascade)

This test code includes a screen for the presence of antinuclear antibodies using indirect immunofluorescence, with reflex to titer and pattern if the result is positive. In addition, immunoassays for specific nuclear antibodies will be performed using a three tier approach. If one or more antibodies associated with systemic lupus erythematosus or MCTD are positive, testing will STOP. If these are negative, testing for antibodies associated with Sjogren syndrome, progressive systemic sclerosis or inflammatory myositis will proceed. If one or more of these are positive, testing will STOP. If these are negative, testing for CREST syndrome and neuropsychiatric lupus will proceed. Note that some of the important specific antinuclear antibodies associated with a positive ANA and SARDs may not be tested or reported with this test code.

19946 - ANA Multiplex with Reflex to 11 Antibody Cascade

This test code includes a screen for the presence of antinuclear antibodies using an immunoassay approach in which all of the antibodies are tested simultaneously. If one or more are positive, the screening result is reported as “positive” and the individual test results are reported in a fashion identical to that of 16814 - ANA Cascade (ANA, IFA with Reflex to Titer and Pattern, and Reflex to 11 Ab Cascade). Note that some of the important specific antinuclear antibodies associated with a positive ANA and SARDs may not be tested or reported with this test code.

37521 - ANA Multiplex, Panel 1 with Reflexes

This test code includes a screen for the presence of antinuclear antibodies using an immunoassay approach in which all of the antibodies are tested simultaneously. If one or more are positive, all 11 antibody results are reported. In addition, if the immunoassay screen is positive, ANA by indirect immunofluorescence (with reflex to titer and pattern) will also be performed.

94954 - ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, with Reflexes

This test code is similar to 16814 - ANA Cascade (ANA, IFA with Reflex to Titer and Pattern, and Reflex to 11 Ab Cascade) - except that, in addition to testing and reporting some or all of 11 specific antinuclear antibodies if the ANA screen is positive, three tests for rheumatoid arthritis will also be performed if the ANA screen is positive.

36378 - ANAlyzeR™ ANA, IFA with Reflex Titer/Pattern, Systemic Autoimmune Panel 1

This test code includes a screen for the presence of antinuclear antibodies using indirect immunofluorescence, with reflex to titer and pattern if the result is positive. It also includes testing and reporting of 11 specific antinuclear antibodies, whether the ANA screen by indirect immunofluorescence is positive or not. This strategy (combining both approaches to ANA screening) has been shown to enhance both sensitivity and specificity over either approach alone.17 This test code also includes additional testing for antibodies associated with rheumatoid arthritis, anti-phospholipid antibody syndrome, and autoimmune thyroid disease. 

Worldwide, the majority of clinical laboratories (60%) use a screening dilution of 1:8018 and this is also the recommendation of the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) 2019 classification criteria for systemic lupus erythematosus (SLE)19. In 2026, Quest moved from screening samples for ANA at a titer of 1:40 to 1:80. This change was made to increase the specificity of the test and reduce the number of false positive results.

 A significant percentage of patients with a variety of non-autoimmune disorders (and even healthy people) may have a positive ANA when screened at a low titer. Studies have shown that, at a screening dilution of 1:40, approximately 20% of a healthy population will be positive. This percentage falls to 10% when a 1:80 dilution is used.3    Of course, with increased specificity (fewer false positives), some sensitivity is lost.

The balance between sensitivity and specificity has been best studied for SLE. In a general population, a screening titer of 1:40 may have a sensitivity of 98% but a specificity of 67%. Increasing the screening titer to 1:80 increases the specificity to 75% but results in a decrease in sensitivity of less than 1%.5 Nevertheless, a negative ANA test in a patient with high clinical suspicion of a systemic rheumatic disorder is a concern and such cases should be carefully investigated.

Quest test panels that include ANA (not all-inclusive; see Quest Diagnostics Test Directory for more options).

  • ANA Screen, IFA, with Reflex to Titer and Pattern and Reflex to Multiplex 11 Antibody Cascade (TC 16814) includes ANA Screen, IFA, with Reflex to Titer and Pattern (249) DNA (ds) Antibody (255), Sm/RNP, Antibody (38567) RNP Antibody (19887), Sm Antibody (37923), Chromatin (Nucleosomal) Antibody (34088), Sjogren's Antibody (SS-A) (38568), Sjogren's Antibody (SS-B) (38569), Scleroderma Antibody (Scl-70) (4942), Jo-1 Antibody (5810), Ribosomal P Antibody (34283), Centromere B Antibody (16088)
  • ANA Multiplex with Reflex to 11 Antibody Cascade (test code 19946) includes ANA (bead immunoassay) (not available singly) DNA (ds) Antibody (255), Sm/RNP, Antibody (38567) RNP Antibody (19887), Sm Antibody (37923), Chromatin (Nucleosomal) Antibody (34088), Sjogren's Antibody (SS-A) (38568), Sjogren's Antibody (SS-B) (38569), Scleroderma Antibody (Scl-70) (4942), Jo-1 Antibody (5810), Ribosomal P Antibody (34283), Centromere B Antibody (16088)
  • ANAlyzeR™ ANA, IFA with Reflex Titer/Pattern, Systemic Autoimmune Panel 1 (test code 36378) includes ANA Screen, IFA, with Reflex to Titer and Pattern (249), DNA (ds) Antibody, Crithidia IFA with Reflex to Titer (37092) Sm/RNP, Antibody (38567) RNP Antibody (19887), Sm Antibody (37923), Chromatin (Nucleosomal) Antibody (34088), Sjogren's Antibody (SS-A) (38568), Sjogren's Antibody (SS-B) (38569), Scleroderma Antibody (Scl-70) (4942), Jo-1 Antibody (5810), Centromere B Antibody (16088), Cyclic Citrullinated Peptide (CCP) Antibody (IgG) (11173), Mutated Citrullinated Vimentin (MCV) Antibody (13238), Complement C3c (351), Complement C4c (353), Thyroid Peroxidase Antibody (TPO) (5081), Cardiolipin Antibodies (IgA, IgG, IgM) (7352), Beta-2-Glycoprotein I Antibodies (IgG, IgA, IgM) (30340), Rheumatoid Factor (IgA, IgG, IgM) (19705)
  • ANA, IFA, Cascade and Rheumatoid Arthritis Panel 2, with Reflexes (test code 94954) includes ANA Screen, IFA, with Reflex to Titer and Pattern (249), DNA (ds) Antibody, Crithidia IFA with Reflex to Titer (37092), DNA (ds) Antibody (255), Sm/RNP, Antibody (38567) RNP Antibody (19887), Sm Antibody (37923), Chromatin (Nucleosomal) Antibody (34088), Sjogren's Antibody (SS-A) (38568), Sjogren's Antibody (SS-B) (38569), Scleroderma Antibody (Scl-70) (4942), Jo-1 Antibody (5810), Ribosomal P Antibody (34283), Centromere B Antibody (16088), Cyclic Citrullinated Peptide (CCP) Antibody (IgG) (11173), Rheumatoid Factor (RF) (4418), Mutated Citrullinated Vimentin (MCV) Antibody (13238)

References

  1. Autoimmune Association. Autoimmune diagnosis tips. Accessed June 2, 2026. https://autoimmune.org/resource-center/diagnosis-tips/
  2. U.S. Department of Health and Human Services Office on Women’s Health Autoimmune Diseases web site. Updated February 22, 2021. Accessed June 2, 2026. https://www.womenshealth.gov/a-z-topics/autoimmune-diseases
  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. Agmon-Levin N, Damoiseaux J, Kallenberg C, et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis. 2014;73(1):17-23. doi:10.1136/annrheumdis-2013-203863
  5. Bonroy C, Vercammen M, Fierz W, et al. Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP. Clin Chem Lab Med. 2023;61(7):1167-1198. doi:10.1515/cclm-2023-0209
  6. Fritzler MJ, Choi MY. Antinuclear antibody testing: gold standard revisited. J Appl Lab Med. 2022;7(1):357-361. doi:10.1093/jalm/jfab129
  7. Bizzaro N, Brusca I, Previtali G, et al. The association of solid-phase assays to immunofluorescence increases the diagnostic accuracy for ANA screening in patients with autoimmune rheumatic diseases. Autoimmun Rev. 2018;17(6):541-547. doi:10.1016/j.autrev.2017.12.007
  8. 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
  9. International Consensus on ANA Patterns (ICAP). Published May 19, 2015. Accessed June 2, 2026. https://anapatterns.org/
  10. 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
  11. 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
  12. Colglazier CL, Sutej PG. Laboratory testing in the rheumatic diseases: a practical review. South Med J. 2005;98(2):185-191.
  13. 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.
  14. 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
  15. 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
  16. 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.2003.04.000
  17. Bizzaro N, Brusca I, Previtali G, et al. The association of solid-phase assays to immunofluorescence increases the diagnostic accuracy for ANA screening in patients with autoimmune rheumatic diseases. Autoimmunity Rev 2018;17:541-547
  18. Vercammen M, Bonroy C, Broeders S et al. Analytical aspects of the antinuclear antibody test by HEp-2 indirect immunofluorescence: EFLM report on an international survey. Clin Chem Lab Med 2023; 61:1199-1208
  19. Aringer M, Costenbader KH, Daikh DI et al. 2019 EULAR/ACR classification criteria for systemic lupus erythematosus. Arthritis Rheumatol 2019; 71:1400-1412
  20. Wener MH, Fink SL, Morishima C, et al. Anti-nuclear antibody quantitation: Calibration and harmonization adjustment via population interrogation. J Appl Lab Med 2022; 7:46-56
  21. Leuchten N, Hoyer A, Brinks R et al. Performance of antinuclear antibodies for classifying systemic lupus reythematosus: A systemic literature review and meta-regression of diagnostic data. Arthritis Care Res (Hoboken) 2018; 70:428-438

 

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 4

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Version 3: Effective 10/30/2023 to 05/08/2026 

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