Antinuclear Antibodies, HEp-2 Substrate, IgG, Serum - Duke University Hospital (2023)

Specimen Required

Collection Container/Tube:

Preferred: Serum gel

Acceptable: Red top

Submission Container/Tube: Plastic vial

Specimen Volume: 0.5 mL

Collection Instructions: Centrifuge and aliquot serum into a plastic vial.

Useful For

Evaluation of patients suspected of having systemic autoimmune rheumatic disease (ANA-associated rheumatic diseases or connective tissue disease) or organ-specific autoimmune diseases such as autoimmune liver diseases

Method Name

Indirect Immunofluorescence

Reporting Name

Antinuclear Ab, HEp-2 Substrate, S

Specimen Minimum Volume

0.3 mL

Specimen Stability Information

Specimen TypeTemperatureTimeSpecial Container
SerumRefrigerated (preferred)21 days
Frozen28 days

Reject Due To

Gross hemolysisOK
Gross lipemiaOK
Gross icterusOK

Clinical Information

Autoantibodies targeting antigens in the nuclear region in the HEp-2 cell line substrate using the indirect immunofluorescence assay (IFA) have traditionally been called antinuclear antibody (ANA). ANA is the 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 (MCTD), Sjogren syndrome (Sjs), and systemic sclerosis (SSc) including CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) and inflammatory myopathies (IM) such as dermatomyositis (DM).(1-4) Testing for ANA may also be of diagnostic relevance in the differential evaluation of autoimmune liver diseases (ALD).(5-6)

The 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. SLE patients and those with 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 (reviewed in 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 anticellular antibody.(2) These changes are relevant as in addition 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 on anticellular antibody or specific associated antibodies may be components. Of all ANA-associated rheumatic diseases, the presence of anticellular antibody is considered 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 anticellular antibody-negative with 1.5% testing positive for isolated cytoplasmic or mitotic pattern.(11)

Although the anticellular antibody is a sensitive test, it 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 anticellular antibody test result may be guided by HEp-2 IFA patterns and/or titer, patient’s clinical presentation or in some cases the patient’s demographic.(12)

Reference Values

<1:80 (Negative)


Presence of anticellular antibody (formerly 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.

Anticellular 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.


Some patients without clinical evidence of systemic autoimmune rheumatic disease (SARD) maybe positive for anticellular antibody. 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.

Clinical Reference

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;73:17-23

2. Chan EK, Damoiseaux J, Gabriel Carballo O, 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;16:715-726

4. International Consensus on ANA Patterns. Nomenclature and Classification Tree. ICAP; 2021 Accessed August 13, 2021. Available at

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;67:145-172

6. Younossi ZM, Bernstein D, Shiffman ML, et al. Diagnosis and management of primary biliary cholangitis. Am J Gastroenterol. 2019;114:48-63

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;71:1400-1412

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;47:1768-1773

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;11:17

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;12:14

11. Choi MY, Clarke AE, ST Pierre Y, et al. Antinuclear antibody-negative systemic lupus erythematosus in an international inception cohort. Arthritis Care Res. 2019;71:893-902

12. Nandjwada 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;43:1816-1824

Method Description

Antibodies to nuclear antigens in a human epithelial type 2 (HEp-2) cell line by an indirect immunofluorescent technique. Commercial slides prepared from HEp-2 cells are used as a substrate. IgG antibodies in serum specimens are detected after incubation of serum with the commercial slides by the addition of a fluorescein isothiocyante (FITC)-labeled antihuman-IgG reagent. All patient specimens are initially screened at 1:80.(Package insert: NOVA Lite DAPI ANA. Inova Diagnostics; 05/2015)

Day(s) Performed

Monday through Saturday

Report Available

2 to 3 days

Specimen Retention Time

14 days

Performing Laboratory

Antinuclear Antibodies, HEp-2 Substrate, IgG, Serum -Duke University Hospital (1) Mayo Clinic Laboratories in Rochester

Test Classification

This test has been cleared, approved, or is exempt by the US Food and Drug Administration and is used per manufacturer's instructions. Performance characteristics were verified by Mayo Clinic in a manner consistent with CLIA requirements.

CPT Code Information


LOINC Code Information

Test IDTest Order NameOrder LOINC Value
NAIFAAntinuclear Ab, HEp-2 Substrate, S59069-5
Result IDTest Result NameResult LOINC Value
ANAHAntinuclear Ab, HEp-2 Substrate, S59069-5
1TANAANA Titer:33253-6
1PANAANA Pattern:49311-4
2TANAANA Titer 2:33253-6
2PANAANA Pattern 2:49311-4
CYTQLCytoplasmic Pattern:55171-3
LCOMLab Comment:77202-0


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