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Measurement and clinical significance of antinuclear antibodies

Measurement and clinical significance of antinuclear antibodies
Literature review current through: May 2024.
This topic last updated: Apr 04, 2024.

INTRODUCTION — The detection of antinuclear antibodies (ANAs) in serum facilitates the diagnosis of patients with systemic lupus erythematosus (SLE) and related autoimmune diseases. Conversely, the absence of ANAs in the serum of a patient with suspected SLE also provides important information in that it makes the diagnosis much less likely. This topic will review the clinical utility and limitations of ANA testing, techniques for detecting ANAs, and interpretation of ANA testing, including significance of staining patterns and associated autoantibodies.

Additional information concerning antibodies directed against specific autoantigens and cellular structures or antibodies that may be detected in specific rheumatic diseases are presented separately:

Double-stranded deoxyribonucleic acid (dsDNA), Smith, and U1 ribonucleoprotein (RNP) antibodies (see "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP")

Ro/SSA and La/SSB antibodies (see "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems")

Antiribosomal P protein antibodies (see "Antiribosomal P protein antibodies")

Drug-induced lupus (see "Drug-induced lupus", section on 'Laboratory tests and characteristic autoantibodies')

Systemic sclerosis (SSc) (see "Clinical manifestations and diagnosis of systemic sclerosis (scleroderma) in adults", section on 'Laboratory testing')

Dermatomyositis (DM) and polymyositis (PM) (see "Clinical manifestations of dermatomyositis and polymyositis in adults", section on 'Laboratory findings')

Autoimmune hepatitis (see "Overview of autoimmune hepatitis", section on 'Autoantibodies')

Primary biliary cholangitis (PBC) (see "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis", section on 'Laboratory tests')

CLINICAL UTILITY AND LIMITATIONS OF ANA TESTING

Background — Antinuclear antibody (ANA) testing is an important tool to facilitate the diagnosis of certain autoimmune diseases. ANAs are detected in patients with a variety of systemic as well as organ-specific autoimmune conditions, as presented in the table (table 1). The threshold for a positive test was chosen to favor sensitivity for conditions like systemic lupus erythematosus (SLE).

There are important limitations to consider when deciding to test a patient for ANAs. In general, the indirect immunofluorescent test for ANAs is positive, at a dilution of 1:160, in 5 percent of the normal adult population [1]. The ANA test was intended to have this degree of sensitivity to assist in the diagnosis of patients with SLE. Because the prevalence of ANA-associated diseases in the general population is only approximately 1 percent, four out of five individuals will have false-positive results. Due to the sensitivity of the ANA test for the detection of autoantibodies, the test cannot be used to screen a healthy population for the presence of autoimmune diseases. Of note, the prevalence of ANAs in the healthy adult population increases with age, and ANAs are twice as likely to be detected in healthy women compared with men [2].

The importance of pretest probability — ANA testing is most likely to be clinically useful in establishing a diagnosis when there is moderate to high pretest probability that a patient has a systemic autoimmune disease. For example, if there is clinical evidence of SLE (eg, photosensitivity, pleurisy), systemic sclerosis (SSc; Raynaud phenomenon, skin changes) or Sjögren's disease (SjD; unexplained dry eyes and dry mouth), the ANA results are likely to be helpful. By contrast, if the ANA test is ordered less discriminately, the majority of positive results will likely reflect false positives and may potentially distract the clinician from the correct diagnosis.

When to test for ANAs — ANA testing is helpful when a patient’s symptoms, physical findings, and laboratory results suggest a moderate to high suspicion of one or more of the following diagnoses:

SLE (see "Clinical manifestations and diagnosis of systemic lupus erythematosus in adults", section on 'Laboratory testing')

Drug-induced lupus (see "Drug-induced lupus", section on 'Laboratory tests and characteristic autoantibodies')

Mixed connective tissue disease (MCTD) (see "Mixed connective tissue disease", section on 'Laboratory testing')

SSc (see "Clinical manifestations and diagnosis of systemic sclerosis (scleroderma) in adults", section on 'Laboratory testing')

SjD (see "Diagnosis and classification of Sjögren's disease", section on 'Serologic and other laboratory testing')

Dermatomyositis (DM) and polymyositis (PM) (see "Diagnosis and differential diagnosis of dermatomyositis and polymyositis in adults", section on 'Laboratory testing and imaging')

Raynaud phenomenon (see "Clinical manifestations and diagnosis of Raynaud phenomenon", section on 'Laboratory testing for secondary Raynaud phenomenon')

Autoimmune hepatitis (see "Overview of autoimmune hepatitis", section on 'Autoantibodies')

Primary biliary cholangitis (PBC) (see "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis", section on 'Laboratory tests')

In 2002, a committee appointed by the American College of Rheumatology (ACR) issued guidelines about when to test for ANAs in considering the diagnosis and prognosis of specific conditions [3]. Although these guidelines are helpful when the clinician knows the patient’s diagnosis, the guidelines do not address the problem most frequently faced by clinicians: Patients who have vague musculoskeletal symptoms and an unknown diagnosis. For example, a patient might experience musculoskeletal pain and fatigue. These symptoms are common in both autoimmune diseases and many other disorders. Some clinicians use the ANA test, somewhat indiscriminately, to try to identify patients who have an underlying autoimmune disease. Whether this is an appropriate use of the ANA test is controversial, as it will lead to more false-positive ANA results. Guidelines developed by experts in some countries advise against ordering an ANA test at the initial evaluation of patients with vague symptoms (see "Society guideline links: Antinuclear antibodies"). However, there have not been well-designed studies to inform these guidelines.

Rheumatologic causes of a positive ANA — Many systemic autoimmune conditions are associated with a positive ANA, including SLE, drug-induced SLE, MCTD, SSc, SjD, rheumatoid arthritis (RA), DM, PM, juvenile DM (JDM), and juvenile idiopathic arthritis (JIA) (table 1). ANAs are also frequently detected in patients with Raynaud phenomenon and people who have given birth to a child with neonatal lupus syndrome (NLS). In addition, a patient may have ANAs for years, if not decades, before developing the symptoms of an autoimmune disease [4].

Several studies have described the types of diagnoses that were ultimately made in adults with a positive test for ANA. The reported percent of patients with a positive ANA who are diagnosed with an ANA-related autoimmune disease varies from 9 to 51 percent, likely reflecting differences in the clinical reasons for ordering an ANA test and therefore the pretest probability [5-7]. Across studies, the most commonly diagnosed ANA-related autoimmune disease was SLE, followed by drug-induced SLE, undefined or mixed connective tissue disease, and SSc [5-7].

Nonrheumatologic causes of a positive ANA — ANAs are detected in the serum of patients with other conditions, including organ-specific autoimmune diseases, infections, and malignancy. ANAs may also be detected in healthy people taking certain medications and in individuals who have a family history of autoimmunity (table 1).

Organ-specific autoimmune diseases

Autoimmune thyroid disease – Hashimoto's thyroiditis is the most common organ-specific autoimmune disease associated with a positive test for ANA [5,7]. The rate of developing hypothyroidism in patients with Hashimoto's thyroiditis is approximately 5 percent per year (see "Pathogenesis of Hashimoto's thyroiditis (chronic autoimmune thyroiditis)"). Hypothyroidism develops very gradually and patients and their caregivers may not recognize the subtle progression of symptoms; patients may therefore be quite symptomatic before the correct diagnosis is made. The presence of ANAs and antithyroperoxidase antibodies may identify patients at risk for hypothyroidism and thereby facilitate diagnosis. Graves' disease is also associated with positive ANAs [8].

Other – Other organ-specific autoimmune diseases with increased prevalence of ANAs include autoimmune hepatitis, PBC [9], inflammatory bowel disease [10], and interstitial lung disease. (See "Overview of autoimmune hepatitis", section on 'Autoantibodies' and "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis", section on 'Laboratory tests'.)

Infection – ANAs have been detected in patients with various types of viral infections including Epstein-Barr virus, human immunodeficiency virus (HIV), hepatitis C virus, and parvovirus B19 [11].

Malignancy – ANAs have been described in patients with malignancies including lymphoma [12] and hepatocellular carcinoma [13]. ANAs have also been detected in patients with paraneoplastic syndromes [14,15].

Medications – Certain medications are associated with a positive ANA test in the absence of drug-induced lupus, such as tumor necrosis factor (TNF) inhibitors and minocycline [16-18].

Family history of autoimmune disease – ANAs may be detected in an asymptomatic person who has a first-degree relative with autoimmune disease [19-21].

TECHNIQUES TO DETECT ANA AND NOMENCLATURE

Methodologies — The two most commonly used methods to detect antinuclear antibodies (ANAs) are:

Indirect immunofluorescence (IIF) using the human epidermoid carcinoma (HEp-2) cell line as substrate; and

Solid-phase assays, such as enzyme-linked immunosorbent assays (ELISA), fluorescent microsphere assay, and immunoline assays

Both methods have technical limitations that decrease their sensitivity for the detection of some specific antibodies. It is critical that clinicians understand the advantages and disadvantages of these ANA assays and be aware of the method that is used by their laboratory. Because of concerns about the lack of sensitivity of solid-phase assays [22], in 2009, the American College of Rheumatology (ACR) recommended that the initial test to detect ANAs should be IIF with the HEp-2 cell substrate. This recommendation has been supported by the European Federation of Laboratory Medicine, the European Autoimmune Standardization Initiative, and the International Consensus on Antinuclear Antibody Patterns [23]. Despite these recommendations, some clinical laboratories use solid-phase assays as the initial test for ANAs for reasons that include the efficiency of automated testing and lower labor costs.

Indirect immunofluorescence test for ANA (preferred) — The IIF test using the HEp-2 cell substrate is the most widely used assay for the detection of ANA and remains the reference method of choice for the detection of these antibodies [1,24]. When positive, the IIF ANA test report will include both the endpoint titer and staining pattern or patterns.

Technique – IIF takes advantage of a HEp-2 cell line, which has cells with large nuclei (making staining patterns easier to see). In addition, HEp-2 cells contain nearly all of the clinically important autoantigens, making these cells ideal for the detection of the corresponding autoantibodies. The cells are grown on and subsequently fixed to glass slides, permeabilized with a solvent, and then overlaid with diluted patient serum. After an initial incubation, the slides are washed to remove nonadherent immunoglobulins and other serum proteins, and the cells are then incubated with a fluorescein-conjugated antibody directed against human immunoglobulin. The fluorescein-conjugated secondary antibodies bind to human antibodies, which have reacted with antigens present in the Hep-2 cell substrate. After washing to remove unbound fluoresceinated antibodies, slides are examined using an ultraviolet microscope. If fluorescence is detected at one or more screening dilutions (often 1:40 and 1:160), the serum is serially diluted and retested. An endpoint is reached when fewer than half of the cells on the slide show detectable fluorescence. The ANA titer is reported as the dilution prior to this endpoint, or the highest dilution at which fluorescence is detectable in at least than half of the Hep-2 cells. In some patients with very high titer ANAs, the titer is reported to be greater than the highest dilution tested. Some laboratories stop diluting the sample after reaching a titer of 1:2560.

Preparation of the HEp-2 cell substrate, including fixative and permeabilization techniques, can affect which autoantibodies might be detected by IIF.

Advantages – The major advantage of this method is that a large number of autoantibodies can be detected using the HEp-2 cells [25]. IIF using the HEp-2 cell substrate is also more sensitive for the diagnosis of systemic autoimmune diseases such as systemic lupus erythematosus (SLE) [26,27].

Disadvantages – Disadvantages of the IIF technique include the labor-intensive nature of the assay and the requirement for well-trained technicians to read and interpret the results. Because the staining pattern usually does not identify the responsible autoantibody, additional testing using solid-phase assays is often required. In addition, some autoantigens may not be present in the HEp-2 cell substrate or may be difficult to detect due to variations in the techniques used to prepare the HEp-2 cell-containing slides. These limitations may lead to a false-negative ANA test. The Ro60 antigen, for example, may be lost from the HEp-2 cell substrate during the cell membrane permeabilization step and may not be detected in HEp-2 cell substrate if the cells are fixed with methanol (as opposed to acetone) [28]. A patient with SLE or Sjögren's disease (SjD) may therefore be ANA negative by IIF if anti-Ro60 is the only autoantibody present in serum [24] (see "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems"). Similarly, antibodies directed against ribosomal P antigens may be difficult to detect using the HEp-2 cell substrate [29]. (See "Antiribosomal P protein antibodies".).

Solid-phase assays — Several techniques have been introduced to make the process of detecting autoantibodies more efficient. These techniques, collectively referred to here as "solid-phase assays," include ELISA, immunoline assays, and addressable laser bead immunoassay (ALBIA). Solid-phase assays provide semiquantitative results that may be reported as being within a "positive," "indeterminate," or "negative" range for each specific autoantibody.

Technique – In solid-phase assays, a panel of purified native or recombinant autoantigens is prepared and each antigen is immobilized on a solid surface (microtiter plate, fluorescent microsphere, or membrane). The panel of antigens used in solid-phase assays may include all or some of the following: Ro/SSA, La/SSB, Sm, U1 ribonucleoprotein (RNP), Scl-70, PM-Scl, Jo-1, centromere, histone, ribosomal P, and double-stranded DNA (dsDNA). Diluted human serum is incubated with the immobilized antigen and, as with the IIF assay, a secondary antibody is used to detect bound autoantibodies. Fluorescein, or an enzyme that can catalyze a colorimetric assay, is conjugated to the secondary antibody. Quantification of the amount of autoantibody in the patient’s serum can be achieved by measuring the amount of fluorescence (fluorescent microsphere assay) or the extent of colorimetric change (as in the ELISA and immunoline assays).

Advantages – The major advantages of solid-phase assays are their suitability for high-throughput testing and the semiquantification of test results. The efficiency that automation offers is especially important for laboratories that conduct high-volume ANA testing. Solid-phase assays may significantly decrease the labor cost of ANA testing [30]. An additional advantage of solid-phase assays is that a positive test also provides identification of the responsible autoantibody.

Disadvantages – A serious concern about using the solid-phase assay as the initial assay for ANA is its potential lack of sensitivity compared with IIF using the HEp-2 cell substrate. The number of autoantigens that are included in solid-phase assays is limited compared with the number that are present in the HEp-2 cell substrate. As an example, most solid-phase assays do not contain antigens found in the nucleolus; patients with autoantibodies directed against these structures will have a falsely negative solid-phase ANA result. In addition, some studies suggest that solid-phase assays are less sensitive than IIF for the diagnosis of systemic autoimmune diseases, including SLE [26,27].

INTERPRETING A POSITIVE TEST FOR ANA USING INDIRECT IMMUNOFLUORESCENCE — Indirect immunofluorescence (IIF) testing for antinuclear antibody (ANA) will report a titer, representing the highest dilution at which fluorescence is observed in at least than half of the human epidermoid carcinoma (HEp-2) cells.

Definition of a positive result — There is considerable controversy concerning the ideal dilution considered to indicate a "positive" ANA test. The optimal serum dilution to be used to screen for ANA by IIF should be defined by each laboratory and should be chosen so as to detect autoantibodies in approximately 5 percent of a normal population.

It is helpful to understand how the threshold for a positive test was developed. In a large multicenter study of healthy volunteers 20 to 60 years of age, ANA were detected in 32 and 5 percent of the sera at dilutions of 1:40 and 1:160, respectively [1]. In this same study, the prevalence of ANA in patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjögren's disease (SjD) was determined. ANA were detected at a dilution of 1:40 in 97, 100, and 84 percent of patients with SLE, SSc, and SjD, respectively. At the 1:160 dilution, the sensitivity of the ANA test decreased to 95, 87, and 74 percent, respectively. Based on these results, the authors suggested that positive ANA results at the 1:40 dilution should be reported so as to permit detection of as many patients with ANA-associated autoimmune diseases as possible. Unfortunately, this suggestion has caused confusion among clinicians, because a high prevalence of low-titer ANAs in healthy individuals is an inherent feature of the assay. If one estimates that the prevalence of ANA-associated diseases in the general population is 1 percent, it is evident that most individuals with ANA detected at a dilution of 1:40 (approximately 30 percent of the normal population) [1] have a false-positive result.

Because the IIF test for ANA has the potential to result in a large number of false-positive results, an international group of experts recommended that the initial screening dilution should be defined by each individual laboratory, based on testing serum from an adequate number of normal individuals [25]. A dilution that produces a positive result in 5 percent of normal controls should be considered the optimal screening dilution. We therefore consider an ANA titer of greater than or equal to 1:160 as positive. Of note, the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) classification criteria for entry of patients into clinical studies of SLE require the presence of ANA at a dilution of 1:80 [31].

Significance of titer — As noted above, the threshold at which an ANA is considered positive may differ by laboratory. A high titer is more suggestive of an underlying autoimmune disease such as SLE [32]. However, a high-titer ANA in isolation is not diagnostic of any particular autoimmune disease. Some ANA may be present in very high titer and yet not indicate that an autoimmune disease is present. As an example, if anti-DF70 antibodies are the only cause of the positive ANA, the presence of these antibodies suggests the absence of a systemic autoimmune disease (see below).

After ANA have been detected in a patient with autoimmune disease, the test does not need to be repeated because there is no evidence that changes in ANA titer serve as a marker of disease activity.

Significance of staining patterns — The results of IIF testing for ANA includes the staining pattern (or patterns) produced by the patient's serum, in addition to the antibody titer (picture 1). Although the term anti-"nuclear" antibody would imply that only nuclear staining patterns are important, the international consensus is that all staining patterns observed in HEp-2 cells, including nuclear, cytoplasmic, and cell-cycle related patterns, should be reported [25]. ANA staining patterns are somewhat helpful in identifying the autoantibodies that are present in a patient’s serum and are loosely associated with the underlying autoimmune disease. As an example, the most common ANA pattern in patients with mixed connective tissue disease (MCTD) is nuclear speckled, which is produced by antibodies directed against U1 ribonucleoprotein (RNP). However, many different ANA patterns, including homogeneous, speckled, or nucleolar, may be detected in patients with SLE. Because ANA patterns are not specific for autoantibodies or individual autoimmune disorders, a positive test for ANA by IIF often leads to additional testing using solid-phase assays (see 'Solid-phase assays' above) to detect specific, disease-associated autoantibodies.

Nomenclature — An International Consensus on Antinuclear Antibody Patterns (ICAP) workshop was held in São Paolo, Brazil in 2014. The goal of the first and subsequent meetings was to "promote harmonization and understanding of autoantibody nomenclature, thereby optimizing ANA usage in patient care." Members of ICAP reached consensus on the definition and clinical relevance of 29 distinct HEp-2 cell patterns [33,34]. Images of the HEp-2 cell patterns, labeled AC-1 to AC-29, are available at the ICAP website.

ICAP workshop participants suggested that there are 11 staining patterns (six nuclear, five cytoplasmic) that "must" be reported by all "competent-level" laboratories. An additional 18 ANA staining patterns "should" be reported by "expert-level" laboratories [33,34]. Results of a survey of members of the Association of Medical Laboratory Immunologists and American Society for Clinical Pathology suggest increasing awareness and acceptance of the ICAP guidelines [35].

ANA staining patterns involving the nucleus — These include homogeneous, nuclear speckled, nucleolar, nuclear dot staining, and nuclear envelope patterns.

Homogeneous

Definition – The homogeneous ANA pattern refers to diffuse staining of the nucleus in resting cells and of the chromosome region in dividing cells (picture 1). The ICAP code is AC-1.

Prevalence – The homogeneous staining pattern is the most commonly reported and was detected in 36 percent of more than 9200 ANA-positive serum samples tested at the University Hospitals in Leuven, Belgium [36].

Disease associations – This pattern is associated with systemic autoimmune diseases including SLE, drug-induced lupus, SjD, SSc, and rheumatoid arthritis (RA). It is also seen in organ-specific autoimmune diseases including Hashimoto's thyroiditis, primary biliary cholangitis (PBC), and autoimmune hepatitis.

Antibody targets – The homogeneous ANA pattern may obscure other staining patterns, such as nuclear speckled. Therefore, additional testing for specific autoantibodies is required. Antibodies that produce this staining pattern include those directed against:

Double-stranded DNA – Anti-double-stranded DNA (dsDNA) antibodies are present in approximately 40 to 60 percent of patients with SLE and are highly specific for the disease (75 to 99 percent, depending on the assay used to detect the antibodies) [37]. In addition, anti-dsDNA antibodies may develop in patients who are treated with inhibitors of tumor necrosis factor (TNF) alpha [38]. These patients may or may not have symptoms of drug-induced lupus. (See "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP", section on 'Anti-DNA antibodies'.)

Single-stranded DNA – Anti-single-stranded DNA (ssDNA) antibodies can be seen in SLE but have low specificity. (See "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP", section on 'Anti-DNA antibodies'.)

Histone proteins – The presence of antihistone antibodies support a diagnosis of drug-induced SLE. (See "Drug-induced lupus", section on 'Laboratory tests and characteristic autoantibodies'.)

Nuclear speckled — Three types of nuclear speckled patterns ("fine," "coarse," and "fine dense") have been described. Individual laboratories may or may not distinguish between these different patterns.

Fine speckled

Definition – Fine speckled staining refers to hundreds to thousands of dots present throughout the nucleus. Nucleoli may or may not be stained, depending on the specific autoantibody. Except for antibodies directed against Scl70 (topoisomerase I), there is no staining of the chromosome region in dividing cells. The ICAP code is AC-4.

Prevalence – The fine nuclear speckled pattern was reported in 20 percent of more than 9200 ANA-positive serum samples [36].

Disease associations – This pattern is seen in systemic autoimmune diseases including SLE, subacute cutaneous SLE, SjD, SSc, dermatomyositis (DM), polymyositis (PM), and RA.

Antibody targets – Antibodies that produce this staining pattern include those directed against:

-Ro60 – Anti-Ro60 antibodies are seen in subacute cutaneous SLE, SjD, SLE, PBC, PM, and RA. They are also present in nearly all people who give birth to a child with neonatal lupus syndrome (NLS). Note that the ability to detect anti-Ro60 antibodies depends on the source of the HEp-2 cell substrate used in the IIF assay. (See "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems" and "Neonatal lupus: Epidemiology, pathogenesis, clinical manifestations, and diagnosis", section on 'Autoantibodies'.)

-La – Anti-La antibodies are relatively specific for SjD but are also seen in patients with SLE and in people who give birth to a child with NLS. (See "Diagnosis and classification of Sjögren's disease", section on 'Antibodies to Ro/SSA and La/SSB' and "Clinical manifestations and diagnosis of systemic lupus erythematosus in adults", section on 'Laboratory testing' and "Neonatal lupus: Epidemiology, pathogenesis, clinical manifestations, and diagnosis", section on 'Autoantibodies'.)

-Topoisomerase I (Scl70) – Anti-Scl70 antibodies are associated with diffuse SSc, where they are associated with the presence of interstitial pulmonary fibrosis [39]. Anti-Scl70 antibodies are rarely detected in patients with other autoimmune diseases or in healthy individuals. (See "Clinical manifestations and diagnosis of systemic sclerosis (scleroderma) in adults", section on 'Laboratory testing'.)

-Ku – Anti-Ku antibodies are rare, with an estimated prevalence of 0.5 percent of all ANA-positive sera. They may be seen in SSc, where they are associated with the presence of myositis and arthritis [40], and in SLE and PM. (See "Neuromuscular manifestations of systemic sclerosis (scleroderma)", section on 'Laboratory testing' and "Overview of and approach to the idiopathic inflammatory myopathies", section on 'Myositis-associated autoantibodies'.)

-Mi-2 – Anti-Mi-2 antibodies can be seen in DM [41]. In general, patients with these antibodies have skin findings characteristic of DM, including heliotrope rash and Gottron papules, but do not develop lung disease and respond well to corticosteroids [42]. (See "Overview of and approach to the idiopathic inflammatory myopathies", section on 'Myositis-specific autoantibodies'.)

-Transcriptional intermediary factor 1gamma – Antibodies directed against transcriptional intermediary factor (TIF) 1gamma may be present in 15 to 25 percent of patients with DM and may be associated with the presence of an underlying malignancy [43]. (See "Malignancy in dermatomyositis and polymyositis", section on 'Serum autoantibodies'.)

-Melanoma differentiation-associated protein-5 – Anti-melanoma differentiation-associated protein-5 (MDA5; also known as CADM-140 and ribonucleic acid [RNA] helicase) antibodies may produce speckled staining in either the nucleus or cytoplasm of HEp-2 cells. Some HEp-2 cell preparations have little or no detectable MDA5, resulting in a negative ANA test. Anti-MDA5 antibodies can be seen in DM, where their presence is associated with amyopathic disease and rapidly progressive interstitial lung disease [42,44]. (See "Overview of and approach to the idiopathic inflammatory myopathies", section on 'Myositis-specific autoantibodies'.)

Coarse speckled

Definition – The coarse nuclear speckled pattern refers to hundreds to thousands of nuclear dots of variable sizes but generally larger than the dots seen in the fine speckled pattern. There is no staining of nucleoli or dividing chromosomes (picture 1). The ICAP code is AC-5.

Prevalence – The coarse nuclear speckled pattern was reported in 1.5 percent of more than 9200 ANA-positive serum samples [36].

Disease associations – This pattern is seen in SLE and MCTD.

Antibody targets – Antibodies that produce this staining pattern include those directed against:

-Sm – Anti-Sm antibodies are relatively specific for the diagnosis of SLE but may be seen in MCTD. They are rare in other autoimmune diseases. (See "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP", section on 'Anti-Sm antibodies'.)

-U1 RNP – Anti-U1 RNP antibodies are present in all patients with MCTD because the antibodies are a critical component of the diagnosis of this disease. They may also be present in SLE, SjD, SSc, PM, and RA. (See "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP", section on 'Anti-U1 RNP antibodies'.)

Dense fine speckled pattern

Definition – In the dense fine speckled pattern, the speckles are distributed throughout the nucleus of interphase cells, excluding nucleoli. This pattern differs from the fine and coarse speckled patterns in that the speckles associate with chromosomes in dividing cells. The ICAP code is AC-2.

Prevalence – Estimates of the prevalence range from 0.8 to 37 percent [45,46].

Disease associations – The dense fine speckled pattern is common in healthy people with a positive ANA test and is uncommon in people with SLE. This pattern was noted in over half of healthy hospital workers who had a positive ANA test [47]. Other studies have confirmed that patients with antibodies producing this staining pattern have a low prevalence of underlying autoimmune disease [48-50]. Anti-DFS70 antibodies were found to be the only detectable autoantibody in only 1.1 percent of patients with SLE in a large, international cohort. These results further suggest that anti-DFS70 autoantibodies are not associated with systemic rheumatic disease [51]. In a patient population with a low prior probability of having an autoimmune disease, the presence of antibodies producing the DFS70 pattern may be considered a reassuring result, even when these autoantibodies are present in high (>1:640) titer [50]. However, the DFS70 staining pattern may obscure patterns produced by co-occurring autoantibodies. In patients with a high prior probability of autoimmune disease (as determined by history, physical examination, and other laboratory findings), additional testing using solid-phase assays may be required to detect disease-associated autoantibodies, including those directed against Ro/SSA, La/SSB, Sm, U1 RNP, Scl70, and histone proteins.

Antibody target – Antibodies that produce this staining pattern are directed against dense fine speckled, 70 kD (DFS70; also known as lens epithelium-derived growth factor protein 75 kD [LEDGFp75]).

Nucleolar

Definition – A nucleolar patter shows homogeneous or speckled staining of the nucleolus. Three distinct types of nucleolar staining patterns have been described: "homogeneous," "clumped," and "speckled" or "punctate." However, most clinical laboratories do not distinguish between these subtypes (picture 1). The ICAP codes are AC-8 ("homogeneous"), AC-9 ("clumped"), and AC-10 ("speckled" or "punctate").

Prevalence – The prevalence of antinucleolar antibodies in serum samples varies widely, from 1.4 percent of all samples submitted for ANA testing to a hospital in the United Kingdom [52] to 17 percent of more than 9200 ANA-positive sera [36].

Disease associations – Antinucleolar antibodies may be detected in patients with SLE, RA, SjD, SSc, PM, DM, MCTD, and the Raynaud phenomenon. Antibodies producing the nucleolar staining pattern have been reported in 15 to 40 percent of patients with SSc [39] and the presence of these antibodies has important implications for diagnosis and prognosis. Therefore, in the appropriate clinical setting, additional testing using solid-phase or immunoprecipitation assays may be required to identify the autoantibodies that are responsible for the nucleolar staining pattern.

Antibody targets – Antibodies that produce this staining pattern include those directed against:

PM/Scl complex (PM-Scl-75, PM-Scl-100) – The PM/Scl complex, also known as the exosome, is a multiprotein structure that degrades RNA. Antibodies directed against one or both of two components of the exosome, PM-Scl-75 and PM-Scl-100, are present in 5 to 10 percent of SSc patients and are associated with limited skin disease and decreased risk of pulmonary and kidney disease but increased risk of associated inflammatory myositis [39]. (See "Overview of and approach to the idiopathic inflammatory myopathies", section on 'Myositis-associated autoantibodies'.)

U3-RNP – U3-RNP (fibrillarin) is involved in the first step of processing pre-ribosomal RNA. Anti-U3-RNP antibodies are present in 5 to 10 percent of patients with SSc and are associated with diffuse skin disease, pulmonary artery hypertension, pulmonary fibrosis, and myositis [53]. (See "Clinical manifestations, evaluation, and diagnosis of interstitial lung disease in systemic sclerosis (scleroderma)", section on 'Laboratory findings'.)

Th/To complex (7-2RNP) – The Th/To complex functions as a ribonuclease that removes the 5' leader sequences from precursor tRNA molecules. Antibodies directed against components of the Th/To complex are present in 2 to 5 percent of patients with SSc and are associated with limited skin disease but increased risk of pulmonary fibrosis and renal crisis [53]. (See "Clinical manifestations, evaluation, and diagnosis of interstitial lung disease in systemic sclerosis (scleroderma)", section on 'Laboratory findings'.)

RNA polymerases I, II, and III – Antibodies directed against RNA polymerases are detected in approximately 20 percent of patients with SSc and have a high degree of specificity for this disease. The presence of anti-RNA polymerase antibodies is associated with diffuse skin involvement and increased risk of renal crisis. Pulmonary fibrosis is uncommon in patients with anti-RNA polymerase antibodies [54]. (See "Clinical manifestations and diagnosis of systemic sclerosis (scleroderma) in adults", section on 'Laboratory testing'.)

Topoisomerase I (Scl70) – Antibodies directed against Scl70 (topoisomerase I) produce nuclear speckled as well as nucleolar staining patterns.

Nuclear dot staining patterns — Three types of nuclear dot staining patterns have been described: centromere, promyelocytic leukemia protein (PML) speckled-100 kD (Sp100) nuclear body, and Cajal body.

Centromere

Definition – The centromere staining pattern is characterized by the presence of 30 to 60 discrete, large speckles in the nucleus of resting cells that align with the chromosomes at the metaphase plate in mitotic cells (picture 1). The speckles are larger and fewer in number than those seen in the fine and coarse speckled patterns. The ICAP code is AC-3.

Prevalence – Antibodies directed against centromeres were detected in 3 percent of more than 9200 ANA-positive sera [36].

Disease associations – Anticentromere antibodies are associated with limited SSc, PBC, SjD, SLE, and isolated Raynaud phenomenon. In limited SSc, they are present in approximately 30 percent of patients and are associated with calcinosis and pulmonary hypertension. Among patients with PBC, approximately 15 percent will have anticentromere antibodies that are associated with a worse outcome than anticentromere antibody-negative patients [9]. Anticentromere antibodies are also seen in 4 to 15 percent of SjD patients [55,56] and approximately 4 percent of SLE patients [57]. They may be also detected in patients with isolated Raynaud phenomenon who do not have evidence of a systemic autoimmune disease [58]. (See "Clinical manifestations and diagnosis of systemic sclerosis (scleroderma) in adults", section on 'Laboratory testing' and "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis", section on 'Laboratory tests' and "Diagnosis and classification of Sjögren's disease", section on 'Diagnostic testing' and "Clinical manifestations and diagnosis of Raynaud phenomenon", section on 'Diagnosing secondary versus primary Raynaud phenomenon'.)

Antibody targets – Antibodies that produce this staining pattern are directed against centromere proteins A, B, and C.

PML-Sp100 nuclear body

Definition – The PML-Sp100 nuclear body staining pattern is characterized by the presence of 5 to 20 discrete, large speckles present in the nucleus of resting cells. The pattern can be distinguished from the centromere pattern because there are fewer dots in each cell and there is no staining of chromosomes in dividing cells. The ICAP code is AC-6.

Prevalence – Antibodies directed against PML-Sp100 nuclear bodies were detected in 0.2 percent of more than 9200 ANA-positive sera [36].

Disease associations – This pattern is associated with PBC seen with or without antimitochondrial antibody (AMA). Antibodies directed against components of the PML-Sp100 nuclear body are present in 20 percent of patients with AMA-positive PBC [9]. Anti-PML-Sp100 nuclear body antibodies are also present in approximately 40 percent of PBC patients who do not have AMA as detected by IIF [59] and may be the only antibody marker in some PBC patients. Anti-PML-Sp100 nuclear body antibodies are rarely detected in patients with other autoimmune diseases. Because the PML-Sp100 nuclear body staining pattern is similar to that of the Cajal body staining pattern and because anti-PML-Sp100 nuclear body antibodies are specific for the diagnosis of PBC, it is critical to confirm the presence of these antibodies using a solid-phase assay. (See "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis".)

Antibody targets – The PML-Sp100 nuclear body is a multiprotein cellular structure that is involved in a variety of cellular functions including gene transcription, cellular apoptosis, cell-cycle control, and DNA repair. Antibodies may be formed against Sp100 or PML, or both proteins together.

Cajal body

Definition – The pattern is characterized by the presence of a variable number of large speckles (zero to eight) in the nucleus of resting cells. There is no staining of chromosomes in dividing cells. The ICAP code is AC-7.

Prevalence – Antibodies directed against Cajal bodies were present in 1.3 percent of more than 9200 ANA-positive sera [36].

Disease associations – Autoantibodies directed against Cajal bodies are not disease-specific, making the clinical significance uncertain. They are rarely seen in patients with SjD, PBC, SLE, SSc, and PM [9,60-62] or in healthy individuals.

Antibody targets – Antibodies that produce this staining pattern are directed against p80-coilin.

Nuclear envelope

Definition – Two types of nuclear envelope staining patterns have been reported. The first is characterized by smooth, continuous, linear staining of the nuclear envelope; the second by discontinuous linear staining of the nuclear envelope. Not all clinical laboratories distinguish between the two nuclear envelope staining patterns. The ICAP codes for the continuous and discontinuous nuclear envelope patterns are AC-11 and AC-12, respectively.

Prevalence – Autoantibodies directed against components of the nuclear envelope were detected in 0.5 percent of more than 9200 ANA-positive sera [36].

Smooth, continuous nuclear envelope pattern

Disease associations – The clinical significance of this pattern is uncertain given unclear sensitivity and lack of specificity for any one condition. The pattern has been described in patients with SLE, SjD, antiphospholipid syndrome, and autoimmune liver disease [63].

Antibody targets – Antibodies that produce this staining pattern are directed against nuclear lamin and lamin-associated proteins.

Discontinuous, nuclear pore complex pattern

Disease associations – This pattern is seen in PBC.

Antibody target – The target of the antibody that produces the nuclear pore complex pattern is glycoprotein 210 (Gp210) (picture 1). Anti-Gp210 antibodies are detected in 25 percent of PBC patients and are more than 95 percent specific for this diagnosis [9]. Because of the specificity of anti-Gp210 antibodies for the diagnosis of PBC, it is important to confirm the presence of these antibodies by enzyme-linked immunosorbent assays (ELISA). (See "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis", section on 'Laboratory tests'.)

Cell cycle-associated staining patterns

Proliferating cell nuclear antigen

Definition – Autoantibodies producing the proliferating cell nuclear antigen (PCNA) pattern produce nuclear speckled staining during the G1 phase of the cell cycle, when cells are increasing in size, and dense homogeneous nuclear staining during S phase, when DNA replication occurs. There is no staining in dividing cells. The ICAP code is AC-13.

Prevalence – Autoantibodies producing the PCNA staining pattern were detected in 0.3 percent of more than 9200 ANA-positive sera [36].

Disease associations – The clinical significance of antibodies producing the PCNA staining pattern is uncertain. They were originally identified in a small percentage of patients with SLE and subsequently noted in patients with other autoimmune diseases, viral hepatitis, and malignancies [64,65]. In a retrospective study, only 1 of 28 patients with antibodies producing the PCNA staining pattern was diagnosed with SLE, while six patients were diagnosed with autoimmune thyroid disease [36].

Antibody targets – Antibodies that produce this staining pattern are directed against the delta chain of DNA polymerase.

Centromere protein-F

Definition – The centromere protein-F (CENP-F) staining pattern is characterized by fine speckled staining of the nucleus in resting cells; staining spares the nucleolus. The strongest staining cells are in the G2 phase of cell division, during the "gap" between DNA synthesis and mitosis. There is centromere-associated staining of dividing cells. In addition, during the late stage of cell division, there is staining of the middle region of separating spindles, known as the spindle "midzone." In cells that have progressed further in cell division, there may be staining of a small structure between the two dividing cells known as the mid-body. The ICAP code is AC-14.

Prevalence – The CENP-F staining pattern was detected in 0.3 percent of more than 9200 ANA-positive sera [36].

Disease associations – Approximately half of the patients with autoantibodies producing the CENP-F staining pattern have an underlying malignancy. Associated malignancies include breast, lung, and prostate cancer, as well as lymphoma [66].

Antibody targets – Antibodies that produce this staining pattern are directed against CENP-F.

Mitotic spindle apparatus — Two types of nuclear mitotic apparatus staining patterns have been described: nuclear mitotic apparatus-1 (NuMA1) and NuMa2.

NuMA1 staining pattern

Definition – The NuMA1 staining pattern is characterized by speckled nuclear staining in resting cells. In dividing cells, there is staining of the spindles near the centrosomes, but the staining does not extend to the centromeres. There is no staining of spindles between the dividing chromosomes. The ICAP code is AC-26.

Prevalence – The NuMA1 staining pattern was detected in 0.7 percent of more than 9200 ANA-positive sera [36].

Disease association – In a review of the diagnoses in patients whose serum produced the NuMA1 staining pattern, 20 percent had SjD, 12 percent had SLE, and 40 percent had other autoimmune diseases [67]. (See "Diagnosis and classification of Sjögren's disease".)

Antibody targets – Antibodies that produce this staining pattern are directed against NuMA1.

NuMA2 staining pattern

Definition – Antibodies producing the NuMA2 staining pattern do not stain the nuclei of resting cells. In dividing cells, there is staining of the spindle fibers that extends from the centrosomes to the centromeres. Later in cell division, there is also staining of spindles between the centromeres. The ICAP code is AC-25.

Prevalence – The NuMA2 staining pattern was detected in 0.06 percent of 9200 ANA-positive sera [36].

Disease associations – Antibodies that produce the NuMA2 staining pattern are not associated with any specific autoimmune disease [67].

Antibody targets – Antibodies that produce this staining pattern are directed against human kinesin-like protein (HsEg5; also known as kinesin family member 11 [KIF11]).

Cytoplasmic staining patterns

Antimitochondrial antibody

Definition – The AMA staining pattern is characterized by the presence of granular and filamentous staining throughout the cytoplasm when using HEp-2 cells. The ICAP code is AC-21. The presence of AMA may be confirmed by using a solid-phase assay or a triple tissue substrate consisting of sections of rat kidney, liver, and stomach.

Prevalence – The AMA staining pattern was detected in less than 1 percent of 8300 serum samples submitted for testing to the Clinical Immunology Laboratory at the Massachusetts General Hospital in 2012 [68].

Disease association – Several antibodies causing this staining pattern are highly specific for PBC. However, this pattern can also be seen with other autoimmune diseases like SLE and in non-autoimmune conditions like infection. If the AMA staining pattern is detected by IIF, then solid-phase assays should be used to confirm the presence of autoantibodies seen in PBC [69]. (See "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis".)

Antibody targets – Antibodies that produce this staining pattern include those directed against:

Mitochondrial alpha-ketoacid dehydrogenase complex family of mitochondrial proteins – These include the E2 component of pyruvate dehydrogenase complex (also known as dihydrolipoamide S-acetyltransferase [DLAT]), 2-oxo-glutarate dehydrogenase (dihydrolipoamide dehydrogenase [DLD]), and branched-chain 2-oxo-acid dehydrogenase (BCKDC). These autoantibodies are highly specific for PBC.

Other mitochondrial components – Autoantibodies directed against different components of mitochondria have been described in drug-induced hepatitis, SLE, myocarditis, and infections [70].

Cytoplasmic fine speckled

Definition – In this staining pattern, fine speckles are distributed throughout the cytoplasm of resting HEp-2 cells. The ICAP codes are AC-19 and AC-20.

Prevalence – Antibodies producing the cytoplasmic fine speckled staining pattern were present in 1.2 percent of more than 9200 ANA-positive sera [36].

Disease associations – This pattern can be caused by multiple autoantibodies, as outlined below, that are seen in SLE, SjD, SSc, DM, PM, and other inflammatory myopathies, as well as in people who give birth to children with NLS.

Antibody targets – Antibodies that produce this staining pattern include those directed against:

Jo-1 (histidine tRNA synthetase) and other tRNA synthetases – Antibodies directed against aminoacyl-tRNA synthetases are present in 25 to 35 percent of patients with DM or PM and are rarely detected in patients with other autoimmune diseases. In general, anti-tRNA synthetase antibodies are markers of a subtype of DM or PM characterized by interstitial lung disease, myositis, nonerosive arthritis, Raynaud phenomenon, and "mechanic's hands." Antibodies directed against Jo-1 (histidyl or histidine tRNA synthetase) are the most common anti-tRNA synthetase autoantibody, found in 20 to 30 percent of patients with PM and in 60 to 70 percent of PM patients with interstitial lung disease [43]. (See "Overview of and approach to the idiopathic inflammatory myopathies", section on 'Myositis-specific autoantibodies'.)

Ribosomal P proteins – Antibodies directed against ribosomal P antigens are found in 10 to 20 percent of patients with SLE and are rare in other autoimmune diseases. (See "Antiribosomal P protein antibodies".)

Ro52 – Anti-Ro52 antibodies are present in patients with SLE, SjD, SSc, and PM [71]. Anti-Ro52 and/or anti-Ro60 antibodies are present in nearly all people who have children with NLS. (See "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems", section on 'Ro52 (also known as TRIM21)'.)

Signal recognition particle – Anti-signal recognition particle (SRP) antibodies are detected in approximately 10 percent of patients with inflammatory muscle disease and identify a subset of patients with rapidly progressive, necrotizing disease [72,73]. (See "Overview of and approach to the idiopathic inflammatory myopathies", section on 'Myositis-specific autoantibodies'.)

Cytoskeletal staining

Definition – The cytoskeletal staining pattern is characterized by staining of a network of fibers in the cytoplasm. The ICAP codes are AC-15, AC-16, and AC-17.

Disease associations – This pattern can be seen in type I autoimmune hepatitis or viral hepatitis when caused by anti-actin antibodies [74]. However, it can also be caused by antibodies directed against other components of the cytoskeleton that are not disease-specific. Therefore, additional testing for anti-actin antibodies should be sent when available in patients with suspected autoimmune hepatitis. (See "Overview of autoimmune hepatitis", section on 'Autoantibodies'.)

Antibody targets – Antibodies that produce this staining pattern are directed against actin, actin-associated proteins, cytokeratin, tropomyosin, and vimentin.

Golgi apparatus

Definition – The Golgi apparatus pattern is characterized by irregular staining of the cytoplasm adjacent to the nucleus. The ICAP code is AC-22.

Prevalence – The Golgi apparatus staining pattern was detected in 0.4 percent of more than 9200 ANA-positive sera [36].

Disease associations – The clinical significance of these antibodies is uncertain because of their rarity and lack of consistent disease associations. Anti-Golgi apparatus antibodies were initially identified in a patient with SjD and have subsequently been detected in patients with SLE, RA, sarcoidosis, idiopathic cerebellar ataxia, and viral infections [75-77].

Antibody targets – A large number of components of the Golgi apparatus have been found to be targets of autoantibodies, including giantin, golgin 245, golgin 110, and others.

Rods and rings

Definition – The rods and rings staining pattern is characterized by staining of one or a few circular and/or cylindrical structures in the cytoplasm of HEp-2 cells. This staining pattern can be detected using some, but not all, commercial HEp-2 cell slides [78]. The ICAP code is AC-23.

Disease associations – This cytoplasmic staining pattern was identified in 15 of 75 hepatitis C virus patients who were treated with pegylated interferon-alpha and ribavirin [79]. Antibodies directed against rods and rings are rarely detected in patients with autoimmune diseases [80].

Antibody targets – Antibodies that produce this staining pattern are directed against inosine monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthase 1 (CTPS1).

INTERPRETING A POSITIVE ANA WITH SOLID-PHASE ASSAYS — Solid-phase assays use a panel of autoantigens to detect the presence of specific autoantibodies in patient serum. If a patient has antibodies against one of the autoantigens in the panel, then the antinuclear antibody (ANA) test is considered positive. The panel of autoantigens may vary between laboratories and is more limited than the autoantigens present in human epidermoid carcinoma (HEp-2) cells.

WHEN TO REFER TO A SUBSPECIALIST — Patients with a positive ANA test should be referred to a rheumatologist whenever there is concern for systemic autoimmune disease and when assistance is needed for further patient evaluation. In the absence of other evidence of an ANA-related rheumatologic condition, there is no ANA titer or pattern that requires rheumatologic consultation.

FOLLOW-UP EVALUATION — The need for follow-up testing in patients with positive or negative antinuclear antibody (ANA) will depend on the degree of clinical suspicion of an ANA-related autoimmune disease, as well as the type of ANA test used.

When to send additional autoantibody testing — The decision as to whether additional antibody testing is needed will depend on the type of test that was used to detect the antibodies.

Patients with a positive ANA by indirect immunofluorescence — If a test for ANA by indirect immunofluorescence (IIF) is positive, the reported staining pattern may provide a clue regarding the underlying autoantibodies. However, as indicated above (see 'Significance of staining patterns' above), the association between staining patterns and disease-associated autoantibodies is relatively weak. Additional testing for specific autoantibodies with solid-phase assays is therefore indicated, as dictated by the patient’s clinical presentation.

For people who have a positive ANA test and may become pregnant, tests for anti-Ro/SSA and anti-La/SSB antibodies should be requested. The presence of these autoantibodies indicate increased risk during pregnancy for the development of neonatal lupus syndrome (NLS). (See "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems", section on 'Neonatal lupus syndrome'.)

Patients with a negative ANA by indirect immunofluorescence — Additional testing for specific autoantibodies, using solid-phase assays, may be appropriate if a test for ANA by IIF is negative, depending on the clinical presentation. For example, if the ANA is negative but clinical suspicion of systemic lupus erythematosus (SLE) is high, solid-phase assays to detect antibodies directed against Ro60, Ro52 and ribosomal P might be ordered, because these antigens may be absent from the human epidermoid carcinoma (HEp-2) cell substrate [81]. (See 'Indirect immunofluorescence test for ANA (preferred)' above and 'Solid-phase assays' above.)

Patients with an ANA by solid-phase assay — If the initial test for ANA was performed using a solid-phase assay, then a positive test identifies the specific autoantibody and additional testing may not be required. For example, the presence of antibodies directed against centromere protein or Scl70 would support the diagnosis of systemic sclerosis (SSc). Similarly, the presence of antibodies directed against Sm antigens would support a diagnosis of SLE. However, it should be noted that the number of autoantigens included in solid-phase assays is more limited than the number that is present in the HEp-2 cell substrate. Therefore, if the test for ANA by solid-phase assay is negative and the suspicion of a systemic autoimmune disease remains high, it may be necessary to order a test for ANA by indirect immunofluorescence using the HEp-2 cell substrate.

Solid-phase assays for ANA are not as sensitive as IIF and may produce false-negative results.

Repeating ANA testing — In patients with a negative ANA test, repeated testing for ANA usually is not indicated unless there are new signs or symptoms of a rheumatic disease [82].

In patients who have been diagnosed with an ANA-associated autoimmune disease, changes in ANA titer are not helpful as a way to monitor disease activity. Therefore, once a positive test has been obtained in a patient with ANA-associated autoimmune disease, repeat determinations of ANA are not indicated. In contrast with changes in ANA titer, changes in the level of antibodies directed against double-stranded DNA (dsDNA) may assist in monitoring disease activity in patients with SLE. (See "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP".)

Counseling patients with positive ANA and low suspicion of autoimmune disease — Sending an ANA test in patients with low suspicion of autoimmune disease is usually not indicated. However, if a patient with low suspicion for autoimmune disease has a positive ANA test, then the full differential diagnosis of a positive ANA test (table 1) should be considered, including the high prevalence of false-positive results in the healthy adult population. Due to how the IIF for ANA is designed, 5 percent of patients by definition will have a positive test at the predetermined screening dilution (usually 1:160). However, the prevalence of ANA-associated diseases in the adult population is only approximately 1 percent, so four out of five of those results will be falsely positive. False-positive rates are likewise higher when testing centers consider even lower ANA screening dilutions as positive, such as 1:40 or 1:80 (see 'Definition of a positive result' above). The prevalence of a positive ANA test among healthy patients increases nonlinearly with age and is also higher among females compared with males [2]. Consideration of nonrheumatologic conditions that can cause a positive ANA test is outlined above. (See 'Nonrheumatologic causes of a positive ANA' above.)

It is also important to note that ANAs may be present for years, if not decades, before a patient develops an autoimmune disease. Providers should counsel patients about symptoms of diseases related to a positive ANA. More information for patients is available elsewhere. (See "Patient education: Antinuclear antibodies (ANA) (Beyond the Basics)" and "Patient education: Antinuclear antibodies (The Basics)".)

CONSIDERATIONS IN PEDIATRIC PATIENTS — Testing for antinuclear antibody (ANA) in children has some notable differences when compared with testing in adults.

Prevalence of a positive ANA – The prevalence of a positive ANA in children is around 12 to 15 percent based on data from several cross-sectional studies, with higher rates among female versus male children [2,83,84]. Given the high prevalence of ANA in the pediatric population, testing for ANA should only be performed when there is suspicion of an autoimmune disease.

Clinical utility – In addition to being a useful diagnostic test for ANA-related autoimmune conditions, testing for ANA is also useful in juvenile idiopathic arthritis (JIA), since a positive ANA at any titer is associated with higher risk of uveitis in asymptomatic oligoarticular JIA, seronegative polyarticular JIA, and psoriatic JIA and therefore necessitates more frequent ophthalmological screening. (See "Oligoarticular juvenile idiopathic arthritis", section on 'Laboratory findings' and 'When to test for ANAs' above.)

Causes of a positive ANA – Children have a lower prevalence of ANA-associated autoimmune diseases than adults and higher rates of infections that can occasionally mimic symptoms of autoimmune disease and cause a false-positive ANA.

Several retrospective studies have described the diagnoses among pediatric patients with a positive ANA [85,86]. One study at a tertiary pediatric center found that of the 113 patients referred for a positive ANA (defined as a titer of 1:40 or higher), 72 had an autoimmune disease at the time of referral (including JIA in 44, systemic lupus erythematosus [SLE] in 16, dermatomyositis [DM] in 3, undifferentiated connective tissue disease in 2, uveitis in 2, and other in 5) [85]. The remaining 41 patients were not diagnosed with ANA-related autoimmune diseases; among those who were not lost to follow-up, diagnoses included musculoskeletal complaints, hypermobility, skin diseases, patellofemoral syndrome, viral syndromes, and fibromyalgia.

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Antinuclear antibodies".)

SUMMARY AND RECOMMENDATIONS

Clinical utility and limitations of ANA testing – Antinuclear antibody (ANA) testing is an important tool in diagnosis of certain autoimmune diseases. The threshold for a positive test was chosen to favor sensitivity for conditions like systemic lupus erythematosus (SLE). (See 'Clinical utility and limitations of ANA testing' above and 'Background' above.)

The importance of pretest probability – The higher the prior probability that a patient has a systemic autoimmune disease, the more likely the results of an ANA test will assist in establishing the diagnosis. As an example, if there is clinical evidence of SLE (eg, photosensitivity, pleurisy), the ANA results are likely to be helpful. By contrast, if the ANA test is ordered indiscriminately, the majority of positive results will be falsely positive and may potentially distract the clinician from the correct diagnosis. (See 'The importance of pretest probability' above.)

When to test for ANA – ANA testing is helpful when a patient’s symptoms, physical findings, and laboratory results suggest a moderate to high suspicion of an ANA-related autoimmune disease, including SLE, systemic sclerosis (SSc), and Sjögren's disease (SjD) (table 1). (See 'When to test for ANAs' above.)

Causes of a positive ANA – ANAs are detected in patients with a variety of systemic as well as organ-specific autoimmune conditions, infections, and malignancy, as presented in the table (table 1). ANA may also be detected in healthy people taking certain medications and in individuals who have a family history of autoimmunity. (See 'Rheumatologic causes of a positive ANA' above and 'Nonrheumatologic causes of a positive ANA' above.)

Techniques to detect ANA – Methods for testing ANA have different technical limitations that decrease their sensitivity for the detection of some specific antibodies. It is critical that clinicians understand the advantages and disadvantages of these ANA assays and be aware of the method that is used by their laboratory. (See 'Techniques to detect ANA and nomenclature' above and 'Methodologies' above.)

Interpreting a positive test for ANA using indirect immunofluorescence – When positive, the indirect immunofluorescence (IIF) ANA test report will include both the endpoint titer and staining pattern or patterns. (See 'Interpreting a positive test for ANA using indirect immunofluorescence' above.)

Significance of ANA titer – There is considerable controversy concerning the ideal dilution considered to indicate a "positive" ANA test. The optimal serum dilution to be used to screen for ANA by IIF should be defined by each laboratory and should be chosen so as to detect autoantibodies in approximately 5 percent of a normal population. (See 'Definition of a positive result' above and 'Significance of titer' above.)

Significance of ANA staining patterns – ANA staining patterns are loosely associated with underlying autoimmune diseases; however, additional testing is often required to identify specific, disease-associated autoantibodies. (See 'Significance of staining patterns' above.)

Interpreting a positive test for ANA using solid-phase assays – Solid-phase assays provide semiquantitative results that may be described as being within a "positive," "indeterminate," or "negative" range for each specific autoantibody. (See 'Interpreting a positive ANA with solid-phase assays' above.) A positive test for ANA by solid-phase assay identifies the target of autoantibodies.

Follow-up evaluation – The need for follow-up testing in patients with positive or negative ANA test will depend on the degree of clinical suspicion of an ANA-related autoimmune disease as well as the type of ANA test used. (See 'Follow-up evaluation' above.)

When to refer to a subspecialist – Patients with a positive ANA should be referred to a rheumatologist when there is concern for systemic autoimmune disease and when assistance is needed for further patient evaluation. In the absence of other evidence of an ANA-related rheumatologic condition, there is no specific ANA titer that requires rheumatologic consultation. (See 'When to refer to a subspecialist' above.)

Repeating ANA testing – In patients with a negative ANA test, repeated testing for ANA usually is not indicated unless there are new signs or symptoms of a rheumatic disease. In patients with a positive ANA test and ANA-related autoimmune disease, changes in ANA titer are not helpful as a way to monitor disease activity. (See 'Repeating ANA testing' above.)

Counseling patients with a positive ANA and low suspicion of autoimmune disease – If a patient with low suspicion for autoimmune disease has a positive ANA test, then the full differential diagnosis of a positive ANA test should be considered (table 1), including the high prevalence of false-positive results in the healthy adult population. (See 'Counseling patients with positive ANA and low suspicion of autoimmune disease' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Morris Reichlin, MD, who contributed to an earlier version of this topic review.

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Topic 1822 Version 32.0

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