Cite this page
FeedbackConnective tissue or systemic autoimmune rheumatic diseases (SARDs) are characterized by autoantibodies that can affect tissues and organs throughout the body. Laboratory testing for antinuclear antibodies (ANAs) and specific autoantibodies associated with the presence of SARDs may be useful in the evaluation of these diseases. The SARDS include systemic lupus erythematosus (SLE), Sjögren syndrome, mixed connective tissue disease/undifferentiated connective tissue disease, systemic sclerosis (also referred to as scleroderma), idiopathic inflammatory myopathies (including polymyositis, dermatomyositis, and necrotizing autoimmune), and overlap syndromes (OSs).
Quick Answers for Clinicians
Patients with connective tissue or systemic autoimmune rheumatic diseases (SARDs) may present with very different signs and symptoms, depending on disease type and which tissues and organ systems are affected. For example, patients with systemic lupus erythematosus (SLE) may present with rash or photosensitivity, whereas those with Sjögren syndrome may report dry mouth and dry eyes. See Indications for Testing for common indications of various SARDs.
Testing is particularly important in children with suspected SARDs because of the growth and development issues associated with these diseases.
Testing for antinuclear antibodies (ANAs) and autoantibodies associated with ANA presence may assist in the diagnosis of a systemic autoimmune rheumatic disease (SARD); these tests are best used in individuals with a high pretest probability of having a SARD. The gold standard ANA test is the indirect fluorescent antibody (IFA) assay. Although labor intensive and subjective, it offers high sensitivity and is recommended by the American College of Rheumatology (ACR) as important for the evaluation of a number of SARDs. Methods other than IFA for the detection of ANAs (eg, immunoassays) are generally less sensitive for SARDs but offer greater specificities, faster turnaround times, and more objectivity. (See the Comparison of ANA Testing Methods table.) If an ANA evaluation results in a negative immunoassay and a positive IFA assay, the reported ANA pattern and titer, along with the patient’s clinical history and presentation, may be useful in determining which autoantibody test(s) to perform for disease confirmation. If an ANA evaluation results in a positive immunoassay and a negative IFA assay, the positive predictive value for SARDs is generally low.
Interpretation of antinuclear antibody (ANA) tests is based on common ANA patterns that have shown consistent correlation with systemic autoimmune rheumatic diseases (SARDs). The International Consensus on ANA Patterns has comprehensive information on patterns.
Indications for Testing
The following SARDs may manifest with these indications:
- SLE: rash, photosensitivity, oral ulcers, polyarticular arthritis, proteinurea or cellular casts, pleuritis, pericarditis, interstitial nephritis, fatigue, cytopenia, seizures or psychosis
- Sjögren syndrome: dry mouth, dry eyes
- Mixed or undifferentiated connective tissue disease: Raynaud syndrome, joint aches, puffy fingers, low-grade myositis
- Systemic sclerosis: Raynaud syndrome, sclerodactyly, interstitial lung disease/pulmonary hypertension, gastroesophageal reflux
- Inflammatory myopathies (including polymyositis, dermatomyositis, and necrotizing autoimmune): progressive muscle weakness (usually proximal) with or without rashes
Laboratory Testing
Diagnosis
Antinuclear Antibody Testing
ANA testing should be performed in the initial evaluation of SARDs. Antibodies in SARDs can target the nucleus as well as any intracellular components of the cell, most notably the cytoplasm. ANA tests are best used for individuals with a high pretest probability of having a SARD because ANA reactivity may be present in healthy patients as well as those with certain infections, cancer, and other autoimmune diseases.
The gold standard for ANA testing is the indirect fluorescent antibody (IFA) assay, performed in a lab experienced with ANA testing. ANA test interpretation is based on common ANA patterns that have shown consistent correlation with SARDs. Nuclear patterns reported by ARUP Laboratories include homogeneous, speckled, centromere, nucleolar, and nuclear dots; cytoplasmic patterns reported by ARUP Laboratories include reticular/antimichondrial antibody (AMA), speckled, discrete dots/GW body-like, golgi/polar, and rods/rings (see table below). The International Consensus on ANA Patterns has comprehensive information on patterns, targets, and clinical associations. ANA IFA reports should include the method, type of substrate, results obtained (including qualitative response [positive or negative]), pattern(s) observed, and their titers.
In cases of high clinical suspicion for SARDs, specific autoantibody testing should be performed, even if ANA testing is negative.
| Patterns | Primary Autoantibody Targets | Clinical Associations |
|---|---|---|
| Nuclear Patterns | ||
| Homogeneous | dsDNA, chromatin, Scl-70 (in some cases) | SLE, SSc, AIH, JIA |
| Speckled | Smith, Sm/RNP, Scl-70, SSA-52 (Ro52), SSA-60 (Ro60), SSB (La), RNA polymerase III, Ku | SjS, SLE, DM, SSc, PM/SSc OS, MCTD, UCTD |
| Centromere | CENP-Aa, CENP-B | SSc, PBC/SSc OS |
| Nucleolar | Fibrillarin, RNA polymerase III, PM/Scl-100, Th/To,a Scl-70 (in some cases) | SSc, PM/SSc OS, Raynaud phenomenon, SjS, cancer, other SARD |
| Nuclear dots | Sp-100, PML,a NXP-2 (MORC3 or MJ) | PBC, DM, other inflammatory conditions |
| Cytoplasmic Patterns | ||
| Reticular/AMA | AMA | PBC, SSc, PBC/SSc OS, PBC/SjS OS |
| Speckledb | EJ, Jo-1, OJ, PL-7, PL-12, MDA5, ribosomal P, SRP | SLE, SjS, CTD-ILD, IIM, Raynaud phenomenon |
| Discrete dots/GW body-like | Anti-GW182,b anti-Su/Agob | Neurologic conditions, SjS, SLE, RA, PBC, UCTD |
| Golgi/polar | Giantin/macrogolgin,b golgin-95/GM130,b golgin-160,b golgin-97,b golgin-245c | SLE, SjS, RA, OSs, cerebellar ataxia |
| Rods/rings | IMPDH2b | HCV treated with pegylated interferon-α/ribavirin combination therapy |
|
aIncludes fine speckled and dense fine speckled cytoplasmic patterns. bNo confirmatory tests are performed at ARUP Laboratories. AIH, autoimmune hepatitis; CTD, connective tissue disease; DM, dermatomyositis; HCV, hepatitis C virus; IIM, idiopathic inflammatory myopathies; ILD, interstitial lung disease; JIA, juvenile idiopathic arthritis; MCTD, mixed connective tissue disease; PBC, primary biliary cholangitis; RA, rheumatoid arthritis; PM, polymyositis; OS, overlap syndrome; RA, rheumatoid arthritis; SjS, Sjögren syndrome; SRP, signal recognition particle; SSc, systemic sclerosis; UCTD, undifferentiated connective tissue disease |
||
In addition to IFA ANA testing, other testing options are available. See table below for comparison of ANA testing methods.
| Method | Advantages | Disadvantages |
|---|---|---|
| IFA |
Preferred method Strong screening tool; resulting patterns can guide confirmatory testing for specific diagnosis or prognosis |
Labor intensive Subjective (in titer and pattern recognition) Reagents not well standardized Has limited diagnostic specificity Requires significant expertise to interpret Appropriate cutoff value for positivity not established |
| ELISA |
Commonly available Specificities for SARDs may be affected by limited antigenic targets Offers faster TAT More objective than IFA High throughput |
Has lower sensitivity than IFA Clinical performances of commercial ELISAs vary May not be useful in assessing ALDs or SARDs that target complex autoantigens |
| CLIA |
More sensitive than ELISA Easier to automate than ELISA Wide analytical measurement range (vs ELISA) Offers faster TAT More objective than IFA High throughput |
May not be useful in assessing ALDs or SARDs that target complex autoantigens Has lower sensitivity than IFA |
| Multiplex assay (LIA, MBA) |
Can detect multiple independent antibodies associated with ANAs Can be automated Can provide quantitative or qualitative results Faster TAT More objective than IFA High throughput Useful in analyzing autoantibody clusters and evaluating diseases with multiple autoantibody specificities (vs. ELISA) |
Has lower sensitivity than IFA Diagnostic accuracies differ among immunoassays LIA is not as technologically sophisticated as MBA |
|
ALDs, autoimmune liver diseases; CLIA, chemiluminescence immunoassay; ELISA, enzyme-linked immunosorbent assay; LIA, line immunoassay; MBA, multiplexed bead assay; TAT, turnaround time |
||
Monitoring
Once a SARD has been diagnosed, monitoring tests should be based on organ involvement. If cytopenias are present, the patient should be monitored with sequential CBCs. Certain drugs used for treatment require testing for liver function. In addition, patients should be monitored for malignancy. Dermatomyositis and mixed connective tissue disease have shown increased risk for malignancy, and Sjögren syndrome is specifically associated with an increased risk for hematologic malignancy.
ARUP Laboratory Tests
Semi-Quantitative Indirect Fluorescent Antibody
Semi-Quantitative Indirect Fluorescent Antibody/Qualitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Multiplex Bead Assay/Qualitative Immunoblot
Confirmatory for specific SARD
Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Semi-Quantitative Multiplex Bead Assay
First-line testing for connective tissue disease
Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody (IFA)/Semi-Quantitative Multiplex Bead Assay
Panel includes dsDNA, IgG; Smith/RNP, IgG; Smith (ENA), IgG; SSA 52 and 60, IgG; SSB, IgG; Jo-1, IgG; Scl-70, IgG
Useful in the differential diagnosis of SARDs with or without myopathy
Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Recommended to differentiate SLE and Sjögren syndrome
Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Semi-Quantitative Multiplex Bead Assay
Recommended first-line test to evaluate polymyositis or inflammatory myopathies
Semi-Quantitative Multiplex Bead Assay
Secondary screen based on ANA test results or if ANA IFA is negative but Sjögren syndrome, SLE, systemic sclerosis, or myositis is strongly suspected
Semi-Quantitative Multiplex Bead Assay
May be useful when evaluating for systemic sclerosis or SARD associated with overlapping features of systemic sclerosis and/or myositis
Qualitative Immunoblot
May be useful in differential diagnosis of SLE and Sjögren syndrome
Semi-Quantitative Multiplex Bead Assay
Secondary screen for SLE based on ANA results
Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody
Confirmatory test for SLE based on ANA results
Semi-Quantitative Multiplex Bead Assay
May be useful in detecting central nervous system SLE (somewhat rare) or renal involvement in SLE
Semi-Quantitative Multiplex Bead Assay
Aids in diagnosis of systemic sclerosis; preferred test is Comprehensive Systemic Sclerosis Panel
Semi-Quantitative Multiplex Bead Assay
Semi-Quantitative Multiplex Bead Assay
Recommended for diagnosis of systemic sclerosis in patients negative for centromere, SCl-70, or RNA polymerase antibodies
Qualitative Immunoblot
Qualitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody/Semi-Quantitative Multiplex Bead Assay/Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Quantitative Immunoturbidimetry/Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Semi-Quantitative Indirect Fluorescent Antibody/Quantitative Chemiluminescent Immunoassay/Semi-Quantitative Multiplex Bead Assay
Semi-Quantitative Indirect Fluorescent Antibody/Semi-Quantitative Multiplex Bead Assay/Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Qualitative Immunoblot/Semi-Quantitative Indirect Fluorescent Antibody/Semi-Quantitative Multiplex Bead Assay/Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Qualitative Immunoprecipitation/Semi-Quantitative Multiplex Bead Assay/Qualitative Immunoblot
Qualitative Immunoprecipitation/Semi-Quantitative Multiplex Bead Assay
Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Qualitative Immunoprecipitation/Semi-Quantitative Multiplex Bead Assay/Qualitative Immunoblot
Qualitative Immunoprecipitation/Qualitative Immunoblot
Qualitative Immunoprecipitation/Semi-Quantitative Multiplex Bead Assay/Qualitative Immunoblot/Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Quantitative Immunoturbidimetry
References
-
29021301
Tebo AE. Recent approaches to optimize laboratory assessment of antinuclear antibodies. Clin Vaccine Immunol. 2017;24(12):e00270-17.
-
22553077
Petri M, Orbai A, Alarcón GS, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012;64(8):2677-2686.
-
ACR - Position statement: methodology of testing for antinuclear antibodies
American College of Rheumatology. Position statement: methodology of testing for antinuclear antibodies. [Accessed: Mar 2021]
-
Nuclear Patterns ICAP
International Consensus on Antinuclear Antibody Patterns. Nuclear patterns. International Consensus on ANA Patterns. [Updated: 2019; Accessed: Mar 2021]
-
27548593
Lam NV, Ghetu MV, Bieniek ML. Systemic lupus erythematosus: primary care approach to diagnosis and management. Am Fam Physician. 2016;94(4):284-294.
-
29514034
Mariette X, Criswell LA. Primary Sjögren's syndrome. N Engl J Med. 2018;378(10)931-939.
-
27421219
Gunnarsson R, Hetlevik SO, Lilleby V, et al. Mixed connective tissue disease. Best Pract Res Clin Rheumatol. 2016;30(1):95-111.
-
24122180
van den Hoogen F, Khanna D, Fransen J, et al. 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthritis Rheum. 2013;65(11):2737-2747.
-
26210125
Pope JE, Johnson SR. New classification criteria for systemic sclerosis (scleroderma). Rheum Dis Clin North Am. 2015;41(3):383-398.
-
29079590
Lundberg IE, Tjärnlund A, Bottai M, et al. 2017 European League Against Rheumatism/American College of Rheumatology classification criteria for adult and juvenile idiopathic inflammatory myopathies and their major subgroups. Ann Rheum Dis. 2017;76(12):1955-1964.
-
24126457
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.
-
28541299
Pisetsky DS. Antinuclear antibody testing - misunderstood or misbegotten? Nat Rev Rheumatol. 2017;13(8):495-502.
-
22237992
Satoh M, Chan EKL, Ho LA, et al. Prevalence and sociodemographic correlates of antinuclear antibodies in the United States. Arthritis Rheum. 2012;64(7):2319-2327.
-
30862649
Damoiseaux J, Andrade LEC, Carballo OG, et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis. 2019;78(7):879-889.
26347739
Chan EK, Damoiseaux J, Carballo OG. Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015. Front Immunol. 2015;6:412.
32271157
Peterson LK, Tebo AE, Wener MH, et al. Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists. Clin Chem Lab Med. 2020;58(9):1489-1497.
For more information, see the Antinuclear Antibody (ANA) with HEp-2 Substrate Test Fact Sheet.