The Nuances of Antinuclear Antibodies Testing in Rheumatologic Diseases

Strong working knowledge of ANA testing in rheumatologic diseases increases effectiveness of detection and diagnosis

Tyler Radke, MLS(ASCP)CM
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Tyler is from Green Bay, WI, and graduated from the University of Wisconsin-Oshkosh in 2012 with a bachelor’s in medical technology. He is an ASCP-certified medical laboratory scientist and worked at Froedtert St. Joseph’s Hospital in West Bend before relocating back to Green Bay as the technical lead of microbiology at Bellin Health. In 2017, he became the laboratory manager at Bellin Memorial Hospital and Bellin Health Oconto Hospital in Wisconsin. He is also a member of the laboratory technical advisory group (LabTAG) for the Wisconsin Clinical Laboratories Network (WCLN), serving as the representative for Region 7.

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Kali Vincent, MLS(ASCP)CM
Photo portrait of Kali Vincent, MLS(ASCP)

Kali Vincent, MLS(ASCP)CM, is a ​microbiology bench tech, lead ANA testing, at Bellin Health in Wisconsin.

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Published:Jun 11, 2024
|8 min read
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Rheumatologic diseases are characterized by a wide variety of symptoms and occur in nearly 1 in 3 adults. Given the high prevalence, screening for rheumatologic disease typically occurs in a primary care setting as patients present with concerns and symptoms. Laboratory diagnosis can become complicated by the heterogeneous nature of antinuclear antibody detection among various rheumatologic diseases. Ultimately, definitive identification of a rheumatologic condition is not always indicated with a positive antinuclear antibody test, requiring referral to a rheumatologic specialist for further work-up.

Laboratory diagnostics help to define the etiologic cause of a suspected rheumatologic disease. However, many benefits and limitations exist among the two primary assay methods used in the clinical laboratory. A comprehensive  understanding of the diagnostic assays and markers in use today will help labs and providers succeed in providing appropriate test methods to detect, diagnose, and monitor rheumatologic disease.

Common rheumatologic diseases

Rheumatologic disease is quite common in the US, afflicting nearly 30 percent of the adult population, according to a recent study by the Centers for Disease Control and Prevention.1 When reviewing prevalence estimates from the American College of Rheumatology, many of the systemic rheumatologic diseases are less common, generally less than 2 percent for any of the disorders. These systemic diseases include systemic lupus erythematosus, Sjogren’s syndrome, scleroderma, rheumatoid arthritis, systemic vasculitis, and others.

Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is the most common type of lupus and is more likely to affect women than men. SLE is not organ specific and can affect several vital organs such as the kidneys and lungs. In SLE, the autoimmune  system generates B cells that produce a wide range of autoantibodies that can form immune complexes, cause inflammation, and may deposit in the kidneys causing irreversible tissue damage. At this time, there is no cure, so lifestyle changes and medical interventions are used to manage symptoms and progression of the disease.

Diagnosing systemic lupus erythematosus 

There are several factors that play into successfully diagnosing SLE. Considerations include symptom history and presentation, genetic predisposition to autoimmune diseases, physical examination for rash, tissue biopsy, and laboratory testing. A combination of both clinical and immunologic criteria are required for classification according to the American College of Rheumatology (ACR).

Laboratory testing for the immunologic presence of SLE may consist of any positive test result for various assays, such as antinuclear antibody (ANA), Ds-DNA, anti-Sm, antiphospholipid antibodies, low level complement, or direct Coombs positive without hemolytic anemia. Given the high prevalence of positive ANA findings in patients with SLE, the ANA test is the gold standard method for detecting SLE. Autoantibodies that may be detected in an ANA test that could indicate SLE include SSA/Ro, SSB/La, SmD, and RNP.

Sjogren’s syndrome

Sjogren’s syndrome (SS) is the second most common autoimmune rheumatologic disease and predominantly affects women. Clinically, symptoms are most often dry eyes and mouth. Other broad symptoms, affecting approximately 15 percent of patients, include fatigue, joint pain, swollen salivary glands, and skin dryness.2 

In SS, autoantibodies bind to excretory glands, causing inflammation and damage resulting in the loss of gland production and subsequent clinical dryness. Similar to SLE, though less common among SS, other organs like the kidneys can be damaged by lymphocytic infiltration or immune complex deposition.

Diagnosing Sjogren’s syndrome

There is no single diagnostic test for SS; instead, diagnosis involves a combination of clinical and immunological features. ACR & European League Against Rheumatism (EULAR) criteria place a heavy emphasis on testing performed in the clinical setting. This includes the Schrimer test that measures tear production, use of dyes to examine eyes for dry spots, and salivary flow to determine rate of salivary production. Although ANA testing is a key component of diagnosis, it is limited to the detection of the autoantibodies SSA/Ro and SSB/LA. Some 70 percent of patients have SSA/Ro, and 40 percent of patients have SSB/La.3


Scleroderma, also referred to as systemic sclerosis, is an autoimmune disease targeting skin and connective tissues. Though more commonly present in women than men, scleroderma is rare. As autoantibodies attack skin and connective tissue, associated symptoms are skin hardening and joint pain of the extremities, including fingers, hands, feet, and face.

Scleroderma can be classified into either diffuse cutaneous or limited cutaneous scleroderma. Diffuse cutaneous disease is more serious and can be rapidly progressing, causing more widespread tissue damage in other organs, such as the heart, lungs, digestive tract, and kidneys. In limited cutaneous scleroderma, disease is limited to the extremities and patients experience better clinical outcomes.

Diagnosing scleroderma

Scleroderma has some unique clinical characteristics that help to differentiate it from other rheumatologic diseases. With a predilection for affecting the skin and extremities, some presentations of scleroderma include shiny skin due to skin tightening and the development of small red spots on the hands and face (telangiectasia). Another unique feature affecting many patients is the presence of Raynaud’s phenomenon. Patients afflicted with Raynaud's phenomenon may experience a numbing sensation as their fingertips turn white in response to cold temperatures, restricting blood flow to the extremity. 

In addition to the various clinical features, laboratory testing is essential to help differentiate diffuse cutaneous from limited cutaneous disease. Again, testing for ANA is the primary laboratory test to assist with diagnosis and classification. Of the autoantibodies, those with the highest prevalence in scleroderma are Scl-70 followed by CENP and RNA Polymerase III. In patients with CREST, an acronym for a subset of limited cutaneous sclerodermas, several other autoantibodies can also be detected like RNP and SSA/Ro as well as the previously listed.

Common Autoantibodies and Potential Overlaps
AutoantibodiesSystemic Lupus ErythematosusSjoren's SyndromeSclerodermaMixed Connective Tissue Disease










What you need to know about ANA/ENA tests

      Illustrated diagram showing the difference in antinuclear antibody staining patterns on a blue background.

Main antinuclear antibody patterns on immunofluorescence.

Al-Mughales JA et al. Front Immunol. 2022;13:850759. doi:10.338fimmu.2022.850759

There are two primary methodologies used when testing a patient for ANAs:

Indirect immunofluorescence antibody

Indirect immunofluorescence antibody (IFA) has historically been the testing method of choice due to its high degree of sensitivity and large range of autoantibodies detected. In IFA, slides containing HEp-2 cells in various stages of mitosis are introduced to a dilution of patient serum. After an initial incubation step, the slide is washed with a buffer removing excess patient serum, a conjugate is added, and re-incubation occurs. Following a second wash with a buffer, a counterstain is applied, and the slide is ready to be coverslipped and viewed under a fluorescent microscope. If ANAs are present, samples are diluted further to give a final titration value and an ANA pattern (speckled, peripheral, etc.) is described. False positivity with either method can be encountered with increased patient age, cancer, infectious and inflammatory processes.

Automated enzyme immunoassays

In recent years, automated enzyme immunoassays (EIA) have gained popularity for detecting ANAs. Several manufacturers are available; our health system, Bellin Health, uses the Phadia™ ELiA platform. This test uses extractable nuclear antigens (ENA) in either a pooled format for ANA screening or as individual ENA markers for precise testing. In either case, antigen binds autoantibodies present in the patient serum, creating an enzyme labeled antibody-antigen sandwich complex. After a washing step, substrate is added that creates a fluorogenic molecule. Once that reaction is stopped, the amount of fluorescence is converted into a concentration of antibody present. 

Whether by IFA or EIA, the ANA test detects multiple autoantibodies to a wide range of nuclear antigens. It is therefore not recommended to test for individual ENAs without first having a positive ANA by IFA or automated EIA, as false negative results could occur.

Benefits and limitations of testing methodologies

Enzyme immunoassays (EIA)

Compared to IFA, EIA testing comes with several benefits. Though IFA and EIA have a comparable degree of sensitivity, EIA has a higher sensitivity to SSA/Ro and Jo-1 due to lower expression on IFA HEp-2 cells.4 EIA also has notably higher specificity from use of recombinant antigens and can help to reduce the number of false positives seen with IFA.5

EIA automated platforms have the capability to auto dilute and pipette, removing risk of human error. Though EIA automated platforms may come at a higher upfront cost, they provide a large catalog of options for other tests such as dsDNA, antiphospholipid antibodies, allergens, and more. 

Additionally, testing of the specific ENA marker  concentrations can be used for clinical decisions, such as medication modifications and detection of treatment failure. The notable drawback of EIA assays is the limited ability to detect only a subset of known markers associated with disease.

Indirect immunofluorescence antibody (IFA)

IFA is not selective to a specific autoantibody and can detect a very wide range of autoantibodies, improving test sensitivity. According to the International Consensus of ANA Patterns (ICAP), there are eight main nuclear patterns associated with IFA, with the ability to subcategorize certain patterns.6 Nuclear patterns can correlate with one or  multiple nuclear antigens. For example, a speckled pattern can be associated with SmD, RNP, SSA/Ro, SSB/La, Jo-1, and Scl-70. 

However, testing by IFA remains laborious with numerous manual steps, increasing chances of human protocol error. Additionally, IFA is susceptible to intradepartmental imprecision among performing labs. Some labs may use automated IFA platforms, which improve consistency and reproducibility of results. However, current automated IFA platforms do not offer other test options and are restricted to ANA. Unlike EIA, the IFA methodology uses doubling dilutions of titer values for clinical management decisions, subject to the same imprecisions already noted.

ANA overlap in rheumatologic diseases

The ANA test can detect and differentiate several different autoantibodies. However, autoantibody presence overlaps among several rheumatologic diseases, decreasing specificity of the ANA test. For example, consider SSA/Ro: though it has a high prevalence in SLE, it can also be detected in Sjogren’s syndrome and scleroderma. This overlap diminishes the ability to classify the various diseases by lab test. 

Some autoantibodies such as SmD are specific to SLE, but unfortunately, the prevalence of patient positivity with SmD is less than 30 percent.7

Provider test preference

Once rheumatologic symptoms arise, patients are likely to be seen initially by their primary care provider. Primary care is where the EIA test method has the most benefit given its high specificity. When testing is positive, primary care can feel confident their referral is not adding health care cost and waste. By contrast, when used for screening, the IFA method is prone to false positives,8 resulting in an unwarranted referral and health care waste.

Evidently, rheumatology still has a strong preference for IFA as some low prevalence rheumatology diseases are missed by EIA methods9 but detected by IFA. 

ANA testing takeaways

Tests for ANA play a key role in the successful diagnosis of several systemic rheumatologic diseases. However, labs need to consider several limitations associated with ANA tests, such as autoantibody overlaps, variable detection rates, and methodology differences that complicate result interpretation. 

The benefits/limitations of each ANA method have led to provider ordering preferences. As stated, specialties such as rheumatology favor traditional IFA despite its cumbersome performance. This is likely due to the paucity of markers that exist for the EIA method, diminishing reliable detection of all rheumatologic diseases. However, EIA benefits patients and specialists alike with its superior specificity, improving diagnostic acumen and removing health care waste in primary care.

Regardless of preference, test performance is nuanced, requiring a strong emphasis on other clinical features to assist the diagnosis of rheumatologic diseases.


  1. Duca LM et al. State-specific prevalence of inactivity, self-rated health status, and severe joint pain among adults with arthritis - United States, 2019. Prev Chronic Dis. 2022;19:E23. doi:10.5888/pcd19.210346
  2. Aiyegbusi O et al. Renal disease in primary Sjögren's syndrome. Rheumatol Ther. 2021;8(1):63-80. doi:10.1007/s40744-020-00264-x
  3. Diagnosis, Understanding Sjögren's. Sjögren's Foundation. Accessed May 2024.
  4. EliA Test Algorithms for Autoimmunity Diagnostics. Thermo Fisher Scientific.
  5. Connective Tissue Disease. Thermo Fisher Scientific.
  6. Nomenclature and Classification Tree. International Consensus on ANA Patterns. Updated September 2021.
  7. Fully Automated Solid-Phase Fluorescence Enzyme Immunoassays (FEIA). Thermo Fisher Scientific.
  8. Khalifah MJ et al. Comparison of indirect immunofluorescence and enzyme immunoassay for the detection of antinuclear antibodies. Cureus. 2022;14(11):e31049. doi:10.7759/cureus.31049
  9. Ostrov BE. Reliability and reproducibility of antinuclear antibody testing in pediatric rheumatology practice. Front Med (Lausanne). 2023;9:1071115. doi:10.3389/fmed.2022.1071115

Tyler Radke, MLS(ASCP)CM, Kali Vincent, MLS(ASCP)CM
Tyler Radke, MLS(ASCP)CM

Tyler is from Green Bay, WI, and graduated from the University of Wisconsin-Oshkosh in 2012 with a bachelor’s in medical technology. He is an ASCP-certified medical laboratory scientist and worked at Froedtert St. Joseph’s Hospital in West Bend before relocating back to Green Bay as the technical lead of microbiology at Bellin Health. In 2017, he became the laboratory manager at Bellin Memorial Hospital and Bellin Health Oconto Hospital in Wisconsin. He is also a member of the laboratory technical advisory group (LabTAG) for the Wisconsin Clinical Laboratories Network (WCLN), serving as the representative for Region 7.

Tyler Radke, MLS(ASCP)CM, Kali Vincent, MLS(ASCP)CM
Kali Vincent, MLS(ASCP)CM

Kali Vincent, MLS(ASCP)CM, graduated with a bachelor's degree in medical laboratory science from Michigan Technological University. Currently, she works for Bellin Health in Wisconsin as a bench tech in microbiology and is the lead tech for ANA testing.


DiagnosticsAntibodiesAutoimmune DiseasesLupusImmunoassays
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Laboratory diagnostics help to define the etiologic cause of a suspected rheumatologic disease.
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