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Tolerance, ANA and systemic autoimmunity

16 min

  • Distinguish central from peripheral mechanisms of self-tolerance
  • Interpret an ANA pattern and titer without assigning an unmeasured specificity
  • Match systemic autoantibodies such as ANA, anti-dsDNA, and anti-Sm to their diseases

Read the full reference

Try first

Try first

An antinuclear antibody (ANA) screen by indirect immunofluorescence on HEp-2 cells shows a coarse speckled nuclear pattern with an endpoint titer of 1:320. No antigen-specific antibody test has been run on the serum. Which report fits?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

How tolerance fails

Central tolerance acts during lymphocyte development in the thymus and bone marrow, where many self-reactive cells are deleted or edited. Some self-reactive cells still reach the periphery. There, inadequate costimulation leaves them anergic, and inhibitory receptors and regulatory T cells restrain them. Autoimmune disease can follow a failure at one or more of these checkpoints.1

Reading an ANA

Indirect immunofluorescence on HEp-2 cells is the reference ANA screen. Patient serum is incubated on fixed HEp-2 cells, developed with fluorescein-labeled anti-human immunoglobulin and read at 400×, usually at a screening dilution of 1:80 to 1:160.1,2 The report states the endpoint titer and the pattern, named by the International Consensus on ANA Patterns (ICAP) with its AC code.2

A pattern narrows the possible antibodies. It does not identify one, and each specific antibody is reported from its own assay.3,4

PatternLook in interphase nucleiSpecificities it suggests
Homogeneous (AC-1)Even, diffuse stainingdsDNA, histones, nucleosomes
Coarse speckled (AC-5)Coarse granulesSm, U1 RNP
Fine speckled (AC-4)Fine granulesSS-A/Ro, SS-B/La
Centromere (AC-3)About 40 to 80 discrete dotsCentromere proteins A, B and C
Dense fine speckled (AC-2)Dense fine speckles, with bright chromosomes in dividing cellsDFS70

A confirmed anti-DFS70 with negative disease-specific antibodies is associated with a lower likelihood of systemic autoimmune rheumatic disease when clinical suspicion is low.6

Matching antibodies to systemic disease

ANA is present in most people with systemic lupus erythematosus (SLE), so it is a sensitive screen. It also occurs in other autoimmune diseases, infections, some cancers, pregnancy and healthy people, more often with age and at lower screening dilutions.3 A positive ANA alone therefore does not mean SLE. The 2019 EULAR/ACR classification criteria use an ANA of at least 1:80 on HEp-2 cells only as the entry point.5

Specific antibodies add weight:3,4

  • Anti-dsDNA and anti-Sm support SLE.
  • Anti-SS-A/Ro supports Sjögren disease and also occurs in SLE.
  • Anti-centromere fits limited cutaneous systemic sclerosis, and anti-Scl-70 fits systemic sclerosis.
  • Anti-histone fits drug-induced lupus.

Complement, renal findings and blood counts show organ involvement.5

References
  1. Abbas AK, Lichtman AH, Pillai S, Henrickson S. Cellular and Molecular Immunology. 11th ed. Elsevier; 2025.
  2. Damoiseaux J. The International Consensus on ANA Patterns (ICAP): from conception to implementation. Clin Chem Lab Med. 2024;62(5):789-792. doi:10.1515/cclm-2023-1211
  3. Castro C, Gourley M. Diagnostic testing and interpretation of tests for autoimmunity. J Allergy Clin Immunol. 2010;125(2 suppl 2):S238-S247. doi:10.1016/j.jaci.2009.09.041
  4. ARUP Laboratories. Connective tissue diseases. ARUP Consult. Accessed September 27, 2026. https://arupconsult.com/content/connective-tissue-diseases
  5. 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. doi:10.1002/art.40930
  6. International Consensus on ANA Patterns. AC-2: nuclear dense fine speckled. Updated August 2025. Accessed September 27, 2026. https://www.anapatterns.org/view_pattern.php?pattern=2

Watch one

Serum is screened for ANA by indirect immunofluorescence on HEp-2 cells and titrated to its endpoint. The positive control shows its expected pattern and titer, and the negative control is dark.

How do you report this ANA?

TestResultPreviousReference intervalFlag
ANA screen, 1:80PositiveNegative
ANA, 1:160 to 1:1,280Positive at each dilutionNegative
ANA, 1:2,560NegativeNegative
Interphase nucleiAbout 50 discrete bright dots per nucleus
Dividing cellsDots lined up along the condensed chromosomes

Specimen: H 2, L 3, I 1. Serum, HEp-2 indirect immunofluorescence

  1. Check the controls: the positive control shows its expected pattern and titer, and the negative control is dark.

    A patient's pattern and titer count only on a run whose controls behaved.

  2. Read the screen: nuclear fluorescence at 1:80, so the ANA is positive.

    The screening dilution decides positive or negative.

  3. Look at the interphase nuclei: about 50 discrete bright dots in each nucleus.

    Discrete dots numbering about 40 to 80 set the centromere pattern apart from speckled patterns.

  4. Look at the dividing cells: the dots line up along the condensed chromosomes.

    The centromere pattern shows its dots aligned on the chromosomes of dividing cells.

  5. Find the endpoint: positive through 1:1,280 and negative at 1:2,560.

    The report gives the highest dilution that still shows the pattern.

  6. Check what else was run: no antigen-specific assay.

    The pattern suggests anti-centromere antibody, and only an antigen-specific assay can report it.

Report ANA positive at 1:1,280, centromere pattern (AC-3). Anti-centromere antibody is reported only from its own assay.

Your turn

Problem 1 of 3

HEp-2 indirect immunofluorescence shows a homogeneous nuclear pattern with an endpoint titer of 1:640. No antigen-specific test has been performed. Which report is appropriate?

Incorrect. A homogeneous pattern is associated with anti-dsDNA, anti-histone, and anti-nucleosome antibodies, so only an antigen-specific assay can report anti-dsDNA.

Correct. The report states the endpoint titer and the pattern, named with ICAP nomenclature. The specificity behind the pattern needs a separate antigen-specific assay.

Incorrect. The report states the endpoint titer and the pattern, because both carry interpretive information that a bare positive loses.

Hint
  1. Ask which assays were run on this serum.
  2. One pattern points to several possible antibodies.

Review Antinuclear antibodies

Problem 2 of 3

Selection in the thymus deletes many self-reactive T cells. What keeps the self-reactive T cells that reach the periphery in check?

Central selection removes many self-reactive cells and misses some. The peripheral controls exist for the ones that escape.

Assumed all self-reactive cells are removed in development

Central selection removes many self-reactive cells. Some reach the periphery, where anergy, inhibitory signals, and regulatory T cells keep them in check. Assuming deletion is complete leaves out these peripheral controls, whose failure lets autoimmunity develop.

Class switching changes the antibody class a B cell makes. It does not restrain a self-reactive T cell.

Positive selection happens in the thymus. Lymph nodes are where mature T cells meet antigen.

These peripheral controls act on mature T cells. Recognition without costimulation leaves a T cell anergic, and regulatory T cells and inhibitory signals restrain the rest.

Hint
  1. Some self-reactive cells do leave the thymus.
  2. Ask what else a T cell meets when it is first activated.

Review Autoimmunity and loss of tolerance

Problem 3 of 3

A serum shows an ANA of 1:160 with a fine speckled pattern. The antigen-specific assays give anti-SS-A/Ro positive, anti-SS-B/La negative, anti-dsDNA negative and anti-Sm negative. C3 and C4 are within their reference intervals. Which disease association does this profile fit best?

ANA is sensitive for SLE and occurs in many other conditions. The antibodies that support SLE, anti-dsDNA and anti-Sm, are negative here.

Read an isolated antibody as proof of systemic disease

Many autoantibodies occur in more than one disease and in some healthy people. ANA is sensitive for SLE with limited specificity. Anti-dsDNA and anti-Sm add specificity, and complement, renal, and blood-count findings define organ involvement. A positive ANA alone does not mean SLE.

Drug-induced lupus is associated with anti-histone antibody, which was not measured, and usually with a homogeneous pattern.

Anti-SS-A/Ro is the stronger serologic association of Sjögren disease. It also occurs in SLE, so the clinical picture completes the interpretation.

That association belongs to anti-centromere antibody, which gives a centromere pattern of discrete dots.

Review Systemic autoimmune patterns

Use it

  • Serum from Marguerite Vail, 63, MRN 6120457, comes in for an ANA ordered for fatigue. Nothing on the requisition suggests a connective tissue disease.
  • HEp-2 indirect immunofluorescence is positive at the 1:80 screen, with an endpoint of 1:320. Interphase nuclei show dense fine speckles, and the chromosomes of dividing cells stain brightly.
  • The laboratory's algorithm sends this pattern for an anti-DFS70 assay, which is positive.
  • The anti-dsDNA and extractable nuclear antigen panel ordered with the ANA are negative.
Decision 1 of 2

How is the ANA reported?

The screen is positive, and the report says so. The confirmed anti-DFS70 is reported beside it from its own assay.

Anti-SS-A/Ro was measured on the panel and is negative. A speckled look never reports a specificity, and this one is confirmed as DFS70.

Reported an antigen-specific antibody from pattern alone

A homogeneous pattern is associated with anti-dsDNA, anti-histone, and anti-nucleosome antibodies, and other patterns likewise match several specificities. Reporting anti-dsDNA from the pattern gives a result for an assay never performed. The ANA report states the titer and pattern, and a specific antibody is reported from its own assay.

The ANA report gives the titer and the ICAP pattern. The confirmed specificity is reported separately from the assay that measured it.

Review Antinuclear antibodies

Decision 2 of 2

What does this antibody profile support?

An ANA of 1:80 or higher is only the entry point for SLE classification. The anti-dsDNA and extractable nuclear antigen results are negative.

Read an isolated antibody as proof of systemic disease

Many autoantibodies occur in more than one disease and in some healthy people. ANA is sensitive for SLE with limited specificity. Anti-dsDNA and anti-Sm add specificity, and complement, renal, and blood-count findings define organ involvement. A positive ANA alone does not mean SLE.

A confirmed anti-DFS70 with negative disease-specific antibodies is associated with a lower likelihood of systemic autoimmune rheumatic disease when clinical suspicion is low, as it is here.

Sjögren disease is supported by anti-SS-A/Ro, which is negative. The pattern here is dense fine speckled, confirmed as DFS70.

Review Systemic autoimmune patterns

The clue that settled it is the confirmed anti-DFS70 beside negative disease-specific antibodies:

  • Dense fine speckled pattern, confirmed as anti-DFS70
  • Anti-dsDNA and the extractable nuclear antigen panel negative
  • Nothing on the requisition suggests a connective tissue disease

The report gives the ANA titer and pattern and the anti-DFS70 result, each from its own assay. With strong clinical suspicion, the clinician can still order further antigen-specific tests.

Keep

Sources checked