A Practical Guide to Autoantibody Testing in Rheumatic Diseases

Editor’s Pick: “ANA’s are some of the most commonly ordered tests in rheumatology today, so understanding what they are and why they’re so important is vital for rheumatologists.” —Physician Editor Bharat Kumar, MD, MME, FACP, FAAAAI, RhMSUS

Rheumatic disease labs have advanced since our 2004 article by Dr. Peter Schur (updated in 2009).^1-3 However, universally available laboratory advancements are not keeping pace with advancements in rheumatic disease pharmaceutical drugs.⁴ Diagnosing and managing rheumatic diseases is similar to putting the pieces of a puzzle together. Being the experts in ordering and evaluating the meaning of serologies, such as autoantibodies, complements and other immune system biomarkers, is a fun, challenging part of our profession. It is important to know the strengths and weaknesses of these tests and how they can change one’s pre-test probabilities.

In this series, we explore the fascinating world of autoantibodies.

Anti-Cell vs. Anti-Nuclear Antibodies

Patients positive for anti-nuclear antibodies (ANA) are among the most common reasons for rheumatology referrals.

ANAs are antibodies that target any part of the nucleus. However, a positive ANA by indirect immunofluorescence assay (IFA) can also be positive due to autoantibodies targeting extra-nuclear structures, such as in the cytoplasm, or other organelles, such as the mitochondria. Anti-Jo-1 antibody, an anti-synthetase syndrome autoantibody, is one example.

The International Consensus on ANA Patterns proposes using the term anti-cell antibodies instead of ANAs.⁵ We agree. However, because the term has not been widely adopted, we use ANA throughout this article.

The Expanding Identification

Millions of different autoantibodies can theoretically be directed toward cellular (and extracellular) contents. The list of autoantibodies that play a role in autoimmune diseases continues to grow. More than 200 autoantibodies have been identified to have a role in systemic lupus erythematosus (SLE), more than 60 in systemic sclerosis (SSc) and more than 25 in idiopathic inflammatory myopathies.⁵

ANAs in Healthy People

Depending upon methodology and the demographic study population, 10–20% of healthy individuals are ANA positive. A positive ANA in a healthy person could be a normal finding or could herald the possibility of an autoimmune disease developing in the future.⁶ Other possible causes include drugs, genetics and most inflammatory conditions (e.g., infections and cancer).

More than 75 common autoantibodies have been identified in healthy people.⁷ The most prevalent of these occur in over 25% of healthy individuals. These may occur due to such mechanisms as loss of tolerance and molecular mimicry after infections (e.g., adeno- and herpes viruses).

Several theories exist for why common autoantibodies in healthy people are nonpathogenic. Some are produced by immature B cells that are not programmed for affinity maturation by antigen stimulation or somatic mutation. Another theory is that the autoantigen is not in a location accessible to antigen-processing cells and autoreactive B cells. One method of this immune system sequestration is that many autoantigens are intracellular. Healthy apoptosis prevents immune system exposure.

ANA IFA

The ACR recommends ordering ANA by IFA methodology using human epidermoid tumor cell-line #2 (HEp-2) cells. Part of the reason is that ANA by IFA identifies many more possible nuclear antigen targets than the cheaper and easier to perform solid-phase immunoassays, including the multiplex immunoassay and the enzyme-linked immunosorbent assay (ELISA).⁸

However, several studies have demonstrated wide variations in ANA IFA results, even between instruments used by the same manufacturer.⁹ ANA may be positive in one laboratory, yet negative in another. Due to this, there is a push for improved ANA IFA standards among commercial laboratories.

ANA IFA Titer

The IFA ANA test applies the person’s serum to HEp-2 cells on a slide. ANAs from the patient’s blood then bind to antigens in the HEp-2 cells. The technician adds fluorescent-tagged immunoglobulins that bind to the antibodies.

Under the microscope, fluorescent-tagged ANAs appear as a yellowish-green glow. If ANAs are present, dilution tests are performed. When the technician adds an equal part of the diluting solution to the serum, the result is a 1:1 mix (or titer). When the lab technician adds a second equal amount of diluting solution, the result is a 1:2 titer and so on. This is repeated until the serum no longer fluoresces. The last titer that fluoresced is noted. Higher ANA concentrations result in higher titers.

ANA results are reported with the pattern noted by the technician as well as the titer. The titer result is the most helpful clinically. Most labs consider a titer of 1:40 or 1:80 and higher to be a positive ANA.

The greater the titer, the greater the likelihood for a systemic autoimmune rheumatic disease (SARD). An ANA of 1:80 has a positive predictive value of only 2% for a SARD, but it is around 50% with an ANA of 1:640 or higher.⁹

The likelihood, or post-test probability, that someone with a positive ANA has a SARD depends on the pre-test probability and the likelihood ratio of the ANA result. In clinical practice, we rarely calculate actual numbers. However, it is important to at least note subjective approximations.

For example, if someone has an ANA of 1:80 in the setting of mild fatigue and arthralgias without arthritis, the post-test probability for a SARD is very low—but not impossible. However, a patient with severe fatigue, inflammatory polyarthritis and thrombocytopenia with an ANA of 1:1,280 would have a very high likelihood. A negative ANA for the former patient would essentially rule out the possibility of SLE. Yet, a negative ANA by IFA for the latter patient makes SLE less likely but should be followed by further testing (e.g., by ordering an ANA by solid phase assay, anti-SSA, anti-dsDNA and assessing for cellular-bound complement activation products, such as EC4d and BC4d).¹¹

The EULAR/ACR 2019 SLE classification criteria requires a positive ANA on HEp-2 cells by IFA of at least 1:80, which has a high sensitivity of 97.8%.¹⁰ However, the sensitivity is not 100%. Some labs define a positive ANA as being at least 1:40, and there are SLE patients whose ANA is 1:40.

It is important to exercise caution when considering SLE with an ANA of 1:40 to 1:80. Thirteen percent of healthy people are positive at this level and higher.⁵ Noting the patient’s phenotype (clinical manifestations) to drive the workup and final diagnosis is of utmost importance.

ANA Patterns

The International Consensus on ANA Patterns working group describes 29 different patterns on ANA IFA testing. These include nuclear, cytoplasmic and mitotic-phase (e.g., centromere pattern) antibodies. Due to high intraobserver variation in pattern identification and the non-specificity of the patterns, pattern identification is rarely useful in clinical practice.

Centromere, followed closely by nucleolar pattern, has the highest predictive value for a SARD. These two patterns are, therefore, most helpful.⁹ Most patients with centromere titers of 1:640 and higher have SSc, SLE or Sjögren’s disease, while nucleolar pattern is more highly associated with SSc, SLE and inflammatory myositis.

Anti-diffuse fine-speckled (anti-DFS) patterns must be interpreted cautiously because there is more than a 50% variation in inter-technician interpretation. However, if subsequent anti-DFS70 testing by immunoblotting is positive, it may help rule out a SARD if specific autoantibodies are negative.¹²

The future of ANA IFA pattern assessment appears more promising. Machine learning may help identify specific autoantibodies through pattern assessment.

When to Order ANA

ANA positivity is essential in making a diagnosis of SLE, SSc, mixed connective tissue disease, drug-induced lupus and autoimmune hepatitis.⁸ Related SARDs, such as Sjögren’s disease and idiopathic inflammatory myositis are also usually ANA positive, but ANA positivity is not essential. However, because the SARDs can cause similar manifestations (e.g., musculoskeletal pain, Raynaud’s, rash, fatigue, cytopenias, and neurologic and cardiopulmonary disease), ordering an ANA when considering any SARD is reasonable.

ANA can also help with prognosis in juvenile idiopathic arthritis (JIA). ANA-positive JIA patients are at increased risk for uveitis. Ophthalmologic monitoring in asymptomatic ANA-positive patients helps identify and treat this devastating complication.

Are Too Many ANAs Ordered?

It is a common complaint that doctors order too many inappropriate ANA tests. However, until we improve diagnostic biomarkers, doctors may not be ordering enough ANAs in appropriate clinical settings.⁴ The lack of better tests is a major cause of delayed diagnoses—four to six years on average in SLE—leading to worse outcomes and prolonged suffering.⁴

For example, some rheumatologists feel ANAs should not be ordered in the setting of peripheral neuropathy. However, Sjögren’s disease is a commonly missed diagnosis in this setting. Therefore, the Sjögren’s Foundation Neurologic Disease Guidelines (publication pending) recommend ordering ANA in this setting. Also, autoantibodies, such as ANA and anti-SSA, often appear over a decade before SARDs, SLE for example, become symptomatic.¹³ Patient education and continued follow-up can lead to early diagnosis and treatment if an autoimmune disease develops in this setting.

There Is No False-Positive ANA

The finding of a positive ANA should be documented, along with its quantitative result and methodology. This should be enough to convey a sense of whether the patient could potentially have a disorder related to it. The term false-positive ANA should not be used because ANA positivity can occur during the subclinical phase of disease, many years before disease is evident.⁶ We cannot confidently state that an ANA is not due to a pathologic process.

ANA-Negative SLE

Around 6% of newly diagnosed SLE patients are negative for anti-cellular antibodies.¹⁴ Therefore, it is imperative not to dismiss a diagnosis of SLE in a suspected individual based on one negative ANA result.

Two separate, large, multi-center studies demonstrated the superiority of ordering ANA by both IFA and solid-phase assay to improve SLE diagnosis sensitivity.¹⁵ Patients with a SARD, including SLE, can have a positive solid-phase ANA and be negative by IFA. Therefore, clinicians should check a solid-phase ANA in IFA-negative patients suspected of SARD.

Because solid-phase assays are inexpensive, laboratories often run them as the screening test of choice. Unfortunately, up to 35% of SLE patients are ANA negative using solid-phase assays. Patients suspected of a SARD who are negative for solid-phase ANA should have it repeated by IFA.

Anti-SSA should also be ordered in ANA-negative patients suspected of having a SARD because anti-SSA can be present.¹⁶

Up to 30% of treated SLE patients become ANA negative over time.^17,18 When we see a new patient with a previous diagnosis of SLE who is ANA negative during the evaluation, it is imperative you obtain previous records and review the initial results on which the diagnosis of SLE was based. Assuming the patient does not have SLE because the ANA is negative and stopping therapy without exercising due diligence and executing a previous record review could be catastrophic.

Donald Thomas, MD, FACP, FACR, RhMSUS, is a rheumatologist who specializes in caring for patients with lupus and Sjögren’s disease at Arthritis and Pain Associates of P.G. County, Greenbelt, Md. He is the author of The Lupus Encyclopedia: A Comprehensive Guide for Patients and Health Care Providers. He also enjoys teaching at the Walter Reed National Military Medical Center as a clinical associate professor of medicine at the Uniformed Services University, Bethesda, Md.

Jason Liebowitz, MD, is an assistant professor of medicine in the Division of Rheumatology at Columbia University Vagelos College of Physicians and Surgeons, New York.

Disclosures

Dr. Thomas has spoken for Exagen Diagnostics Inc. and is a scientific advisor for Progentec Diagnostics.

References

1. Schur PH. Know your labs. The Rheumatologist. 2009 Feb. https://www.the-rheumatologist.org/article/know-your-labs.
2. Schur PH. Know your labs, Part 2. The Rheumatologist. 2009 Apr. https://www.therheumatologist.org/article/know-your-labs-part-2.
3. Schur PH. Laboratory testing for diagnosis, management of patients with rheumatic disease. The Rheumatologist. 2014 Dec.
4. Thomas DE, Liebowitz J. What’s holding back biomarker innovation, & how can we solve it? The Rheumatologist. 2023 Oct.
5. Irure-Ventura J, López-Hoyos M. The past, present, and future in antinuclear antibodies (ANA). Diagnostics (Basel). 2022 Mar 7;12(3):647.
6. Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. 2003 Oct 16;349(16):1526–1533.
7. Shome M, Chung Y, Chavan R, et al. Serum autoantibodyome reveals that healthy individuals share common autoantibodies. Cell Rep. 2022 May 31;39(9):110873.
8. Solomon DH, Kavanaugh AJ, Schur PH, et al. Evidence-based guidelines for the use of immunologic tests: Antinuclear antibody testing. Arthritis Rheum. 2002 Aug;47(4):434–444.
9. Vulsteke JB, Van Hoovels L, Willems P, et al. Titre-specific positive predictive value of antinuclear antibody patterns. Ann Rheum Dis. 2021 Aug;80(8):e128.
10. Aringer M, Costenbader K, Daikh D, et al. 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Arthritis Rheumatol. 2019 Sep;71(9):1400–1412.
11. Qin S, Wang X, Wang J, et al. Complement C4d as a biomarker for systemic lupus erythematosus and lupus nephritis. Lupus. 2024 Feb;33(2):111–120.
12. Bossuyt X. DFS70 Autoantibodies: Clinical utility in antinuclear antibody testing. Clin Chem. 2024 Feb 7;70(2):374–381.
13. Eriksson C, Kokkonen H, Johansson M, et al. Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden. Arthritis Res Ther. 2011 Feb 22;13(1):R30.
14. Choi MY, Clarke AE, St Pierre Y, et al. Antinuclear antibody-negative systemic lupus erythematosus in an international inception cohort. Arthritis Care Res (Hoboken). 2019 Jul;71(7):893–902.
15. Pérez D, Gilburd B, Azoulay D, et al. Antinuclear antibodies: Is the indirect immunofluorescence still the gold standard or should be replaced by solid phase assays? Autoimmun Rev. 2018 Jun;17(6):548–552.
16. Blomberg S, Ronnblom L, Wallgren AC, et al. Anti-SSA/Ro antibody determination by enzyme-linked immunosorbent assay as a supplement to standard immunofluorescence in antinuclear antibody screening. Scand J Immunol. 2000 Jun;51(6):612–617.
17. Wallace DJ, Silverman SL, Conklin J, et al. Systemic lupus erythematosus and primary fibromyalgia can be distinguished by testing for cell-bound complement activation products. Lupus Sci Med. 2016 Feb 1;3(1):e000127.
18. Wallace DJ, Stohl W, Furie RA, et al. A phase II, randomized, double-blind, placebo-controlled, dose-ranging study of belimumab in patients with active systemic lupus erythematosus. Arthritis Rheum. 2009 Sep 15;61(9):1168–1178.