Test Id : IM_04
Antinuclear Antibodies, HEp-2 Substrate, with Reflex, Comment
Useful For
Suggests clinical disorders or settings where the test may be helpful
Interpretation for the evaluation of patients suspected of having systemic autoimmune rheumatic disease (antinuclear antibody-associated rheumatic diseases or connective tissue diseases), especially systemic lupus erythematosus, mixed connective tissue disease and Sjogren syndrome
Method Name
A short description of the method used to perform the test
Only orderable as part of a profile. For more information see RFLXP / Antinuclear Antibodies, HEp-2, Reflex Panel.
Technical Interpretation
NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.
Reporting Name
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Aliases
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Antinuclear antibody
Specimen Type
Describes the specimen type validated for testing
Serum
Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing
Only orderable as part of a profile. For more information see RFLXP / Antinuclear Antibodies, HEp-2, Reflex Panel.
Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Serum | Refrigerated (preferred) | 21 days | |
| Frozen | 28 days |
Useful For
Suggests clinical disorders or settings where the test may be helpful
Interpretation for the evaluation of patients suspected of having systemic autoimmune rheumatic disease (antinuclear antibody-associated rheumatic diseases or connective tissue diseases), especially systemic lupus erythematosus, mixed connective tissue disease and Sjogren syndrome
Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Autoantibodies targeting antigens in the nuclear region in the human epithelial type 2 (HEp-2) cell line substrate using the indirect immunofluorescence assay (IFA) have traditionally been called antinuclear antibodies (ANA). ANA is a commonly performed antibody test in the initial evaluation of patients with systemic autoimmune rheumatic diseases (also referred to as connective tissue disease). Classic ANA-associated rheumatic diseases include systemic lupus erythematosus (SLE), mixed connective tissue disease, Sjogren syndrome (Sjs), and systemic sclerosis (SSc) including CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia), and inflammatory myopathies (IM) such as dermatomyositis.(1-4) Testing for ANA may also be of diagnostic relevance in the differential evaluation of autoimmune liver diseases (ALD).(5-6)
Classical ANA patterns (antibodies targeting the nuclear region) include homogeneous, speckled, centromere, nuclear dots, and nucleolar. These patterns are routinely reported by most clinical laboratories. Patients with SLE, SSc, Sjs, IM (such as anti-synthetase syndrome and necrotizing autoimmune myopathy), or ALD have also been shown to have clinically significant antibodies that react with antigens in other cellular compartments such as the cytoplasm and structures associated mitosis or mitotic patterns with HEp-2 substrate.(1-3) Based on the increasing recognition of these non-nuclear antigenic targets and their documented clinical relevance, the First International Consensus on ANA Patterns established a classification tree for ANA with alpha-numeric anti-cell (AC) code for each pattern with a recommendation for a change in terminology from antinuclear antibody to anti-cellular antibody.(2) These changes are relevant as, in addition to the nuclear patterns, the classification includes cytoplasmic and mitotic patterns with descriptions for their interpretation, associated antibody targets, and clinical associations when available.(4)
The diagnosis of ANA-associated rheumatic diseases is usually based on a set of criteria of which the presence of anti-cellular antibody or specific associated antibodies may be components. Of all ANA-associated rheumatic diseases, the presence of anti-cellular antibodies is considered a mandatory entry criterion by the 2019 European League Against Rheumatism and the American College of Rheumatology classification criteria for SLE.(7) Since cytoplasmic staining patterns may be reported as "ANA negative" or as a comment with no quantitative or titer result, some patients with clinicopathological symptoms consistent with neuropsychiatric SLE would not qualify for entry based on where testing is performed.(8-10) This limitation may therefore exclude patients who may meet the clinical and other laboratory criteria for disease but are not reported as "ANA positive" due to the use of the current terminology. In an international inception cohort of newly diagnosed SLE patients, 6.2% were anti-cellular antibody-negative with 1.5% testing positive for isolated cytoplasmic or mitotic pattern.(11) In addition, a recent investigation of various HEp-2 IFA kits showed variabilities in the expression of specific patterns with high reproducibilities between tests for centromere, multiple nuclear dots, nuclear coarse speckled, nuclear homogeneous and cytoplasmic reticular AMA (antimitochondrial antibody) patterns.(12)
Overall, the anti-cellular antibody is a good screening test for ANA-associated rheumatic diseases with variable sensitivities in the different clinical subsets but lacks diagnostic specificity.(1-4) Therefore, positive results require confirmation with the use of specific ANA-associated antibody tests except for the centromere pattern, which is very characteristic for patients with limited diffuse SSc. Confirmation of a positive anti-cellular antibody test result may be guided by HEp-2 IFA patterns or titer, patient's clinical presentation, or, in some cases, the patient's demographic.(13)
Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
Only orderable as part of a profile. For more information see RFLXP / Antinuclear Antibodies, HEp-2, Reflex Panel.
An interpretation will be provided.
Interpretation
Provides information to assist in interpretation of the test results
Interpretive comments are provided.
Presence of anti-cellular antibody (also known as antinuclear antibody) is a feature of systemic autoimmune rheumatic diseases such as systemic lupus erythematosus, mixed connective tissue disease, Sjogren syndrome, and systemic sclerosis and some inflammatory myopathies (dermatomyositis, anti-synthetase syndrome and necrotizing autoimmune myopathy). It may also be of diagnostic relevance in patients with autoimmune liver diseases.
Patients' sera are screened at 1:80. The following nuclear patterns and their titers are reported: centromere, homogeneous, nuclear dots, nucleolar, speckled, fine dense speckled (also referred to as DFS70), and proliferating cell nuclear antigen (PCNA). If observed, the following cytoplasmic patterns are reported: reticular/AMA (antimitochondrial antibody), cytoplasmic speckled, fibrillar, polar/Golgi-like, or rods and rings. The spindle fiber and centrosome mitotic patterns are also reported if observed. Reported patterns may help guide differential diagnosis, although they may not be specific for individual antibodies or diseases. Negative results do not necessarily rule out systemic autoimmune rheumatic disease.
The anti-cellular antibody test lacks diagnostic specificity and is associated with some cancers, infectious, and inflammatory conditions, with variable prevalence in healthy individuals. The lack of diagnostic specificity requires confirmation of positive results using associated antibody tests such as those targeting extractable nuclear antigens.
Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Some patients without clinical evidence of systemic autoimmune rheumatic disease (SARD) may be positive for anti-cellular antibodies. This occurs at variable prevalence depending on the patient demographics. A positive result may also precede clinical manifestation of SARD or be associated with some viral or chronic infections, cancers, or use of certain medications. All results must be reported in the appropriate clinical context as the performance of the test can be variable.
Reflex testing is limited to specimens with three patterns namely, homogeneous, speckled or dense fine speckled. Not all patients with these three patterns will test positive in the confirmatory tests. Negative results do not rule out the presence of disease.
For individuals positive for other HEp-2 indirect immunofluorescence assay (IFA) patterns, additional testing may be available based on the pattern present, clinical suspicion, or availability of reliable antibody tests. In patients with certain autoimmune diseases such as myositis and Sjogren syndrome, testing for specific antibodies may be indicated in the absence of antinuclear antibody positivity using HEp-2 IFA.
Clinical Reference
Recommendations for in-depth reading of a clinical nature
1. 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
2. Chan EK, Damoiseaux J, Carballo OG, et al. Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015. Front Immunol. 2015;6:412
3. Bossuyt X, De Langhe E, Borghi MO, Meroni PL. Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases. Nat Rev Rheumatol. 2020;16(12):715-726
4. International Consensus on ANA Patterns. Nomenclature and Classification Tree. ICAP; 2021 Accessed April 21, 2026. Available at www.anapatterns.org/trees.php
5. European Association for the Study of the Liver. EASL Clinical Practice Guidelines: The diagnosis and management of patients with primary biliary cholangitis. J Hepatol. 2017;67(1):145-172
6. Younossi ZM, Bernstein D, Shiffman ML, et al. Diagnosis and management of primary biliary cholangitis. Am J Gastroenterol. 2019;114(1):48-63
7. 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;71(9):1400-1412
8. Naides SJ, Genzen JR, Abel G, Bashleben C, Ansari MQ. Antinuclear antibodies testing method variability: A survey of participants in the College of American Pathologists' Proficiency Testing Program. J Rheumatol. 2020;47(12):1768-1773
9. Van Hoovels L, Broeders S, Chan EKL, et al. Current laboratory and clinical practices in reporting and interpreting anti-nuclear antibody indirect immunofluorescence (ANA IIF) patterns: results of an international survey. Auto Immun Highlights. 2020;11(1):17
10. Tebo AE, Schmidt RL, Kadkhoda K, et al. The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation. Auto Immun Highlights. 2021;12(1):4
11. 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;71(7):893-902
12. Nandiwada SL, Peterson LK, Mayes MD, et al. Ethnic differences in autoantibody diversity and hierarchy: More clues from a US cohort of patients with systemic sclerosis. J Rheumatol. 2016;43(10):1816-1824
13. Silva MJ, Dellavance A, Baldo DC, et al. Interkit Reproducibility of the Indirect Immunofluorescence Assay on HEp-2 Cells Depends on the Immunofluorescence Reactivity Intensity and Pattern. Front Immunol. 2022;12:798322
Method Description
Describes how the test is performed and provides a method-specific reference
An interpretation based on the test results is generated by the laboratory information system.
PDF Report
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Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.
Monday through Saturday
Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.
Performing Laboratory Location
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Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.
Not Applicable
LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.
| Test Id | Test Order Name | Order LOINC Value |
|---|---|---|
| IM_04 | Antinuclear Ab,HEp-2,reflex Comment | No LOINC Needed |
| Result Id | Test Result Name |
Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
|
|---|---|---|
| IM_04 | Antinuclear Ab,HEp-2,reflex Comment | 77202-0 |