INTRODUCTION — The detection of antinuclear antibodies (ANA) in serum facilitates the diagnosis of patients with systemic lupus erythematosus (SLE) and related autoimmune diseases. Conversely, the absence of ANA 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 indirect immunofluorescence test for ANA
●The significance of common ANA staining patterns and titer of ANA
●Additional techniques to detect ANA
●Advantages and disadvantages of methods to detect ANA
●Clinical limitations of ANA testing
Additional information concerning antibodies directed against specific autoantigens and cellular structures are presented separately. (See "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP" and "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems" and "Antiribosomal P protein antibodies" and "Drug-induced lupus" and "Clinical manifestations and diagnosis of systemic sclerosis (scleroderma) in adults" and "Clinical manifestations of dermatomyositis and polymyositis in adults" and "Overview of autoimmune hepatitis" and "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis (primary biliary cirrhosis)" and "Clinical significance of antinuclear antibody staining patterns and associated autoantibodies".)
TECHNIQUES TO DETECT ANA — Several tests have been developed to detect antinuclear antibodies (ANA). In the past, immunodiffusion and indirect immunofluorescence techniques using rodent liver sections as substrate were used to detect autoantibodies. These techniques have largely been replaced by indirect immunofluorescence using a human neoplastic cell line and by solid phase assays. Both indirect immunofluorescence with human epidermoid carcinoma cell line (HEp-2) cell substrate and solid phase assays have technical limitations that decrease their sensitivity for the detection of some antibodies. It is critical that the clinician have an understanding of the advantages and disadvantages of these assays for ANA.
Indirect immunofluorescence test for ANA — The indirect immunofluorescence test is the most widely used assay for the detection of ANA and remains the reference method of choice for the detection of these antibodies [1,2]. The test 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.
Significance of ANA staining patterns — Most clinical laboratories report ANA results as the endpoint titer and the staining pattern (or patterns) produced by the patient's serum. Nuclear staining patterns include: homogeneous, speckled, centromere, and nucleolar (picture 1).
●In the homogeneous staining pattern, the entire nucleus is diffusely stained. Antibodies that produce this staining pattern include those directed against histone proteins, deoxyribonucleic acid (DNA), and DNA-histone complexes.
●In the speckled staining pattern, fine or coarse speckles are seen throughout the nucleus. Many different antibodies may produce the speckled pattern, including those directed against U1 RNP, Sm, and La antigens.
●The centromere pattern refers to the presence of 30 to 60 uniform speckles distributed throughout the nucleus of resting cells. In mitotic cells, the speckles localize to the chromosomes at the metaphase plate.
●The nucleolar pattern refers to homogeneous or speckled staining of the nucleolus; it is produced by autoantibodies directed against fibrillarin, ribonucleic acid (RNA) polymerase I and III, Th, PM-Scl, and RNA helicase.
ANA staining patterns are loosely associated with underlying autoimmune diseases. As an example, the most common ANA pattern in patients with mixed connective tissue disease is nuclear speckled, which is produced by antibodies directed against U1 RNP. In patients with limited systemic sclerosis, centromere staining is the predominant pattern; serum from patients with diffuse systemic sclerosis may produce speckled nuclear staining or nucleolar staining. Antibodies in the serum of Sjögren's syndrome patients are likely to produce speckled or homogenous staining. In patients with systemic lupus erythematosus (SLE), homogeneous, speckled, or nucleolar staining patterns may be observed. Because ANA patterns are not specific for individual autoimmune disorders, a positive test for ANA by indirect immunofluorescence often leads to additional testing using solid phase assays (see 'Solid phase assays' below) to detect specific, disease-associated autoantibodies. The clinical significance of the different ANA staining patterns is discussed in more detail separately. (See "Clinical significance of antinuclear antibody staining patterns and associated autoantibodies".)
Significance of ANA titer — There is considerable controversy concerning the ideal initial dilution to be used to screen for the presence of ANA. 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 SLE, systemic sclerosis, and Sjögren's syndrome was determined. ANA were detected at a dilution of 1:40 in 97, 100, and 84 percent of patients with SLE, systemic sclerosis, and Sjögren's syndrome, 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. The 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 the majority of individuals with ANA detected at a dilution of 1:40 (approximately 30 percent of the normal population) have a false positive result.
Because the indirect immunofluorescence 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 [3]. A dilution that produces a positive result in 5 percent of normal controls should be considered the optimal screening dilution. This recommendation has not yet been universally accepted.
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 (ds)DNA may assist in monitoring disease activity in patients with SLE. (See "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP".)
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 enzyme-linked immunoabsorbant assays (ELISA), fluorescent microsphere assays, and immunoline assays. In these 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, La, Sm, U1 RNP, Scl-70, PM-Scl, Jo-1, centromere, histone, ribosomal P, and DNA. Diluted human serum is incubated with the immobilized antigen and, as with the indirect immunofluorescence assay, a secondary antibody is used to detect bound autoantibodies.
ADVANTAGES AND DISADVANTAGES OF METHODS TO DETECT ANA
Indirect immunofluorescence test — The major advantage of indirect immunofluorescence using the human epidermoid carcinoma cell line (HEp-2) cell substrate to detect antinuclear antibodies (ANA) is the large number of autoantibodies that can be detected using the HEp-2 cells [3]. Disadvantages of the indirect immunofluorescence test include the labor intensity of the assay and the requirement for well-trained technicians to read and interpret the results. In addition, because the staining pattern usually does not identify the responsible autoantibody, additional testing using solid phase assays may be required.
Some autoantigens may not be present in the HEp-2 cell substrate. The Ro60 antigen, for example, may be lost from the HEp-2 cell substrate during the cell membrane permeabilization step. A patient with systemic lupus erythematosus (SLE) or Sjögren's syndrome may therefore be ANA-negative by indirect immunofluorescence if anti-Ro60 is the only autoantibody present in serum [2] (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 [4]. Anti-ribosomal P antibodies have low sensitivity, but high specificity for the diagnosis of SLE. (See "Antiribosomal P protein antibodies".)
Solid phase assays — The major advantages of solid phase assays are their suitability for high-throughput testing and the semi-quantification of test results. The efficiency offered by automation is especially important for laboratories that conduct high-volume ANA testing. It has been estimated that solid phase assays may decrease the labor cost of ANA testing by as much as 95 percent [5,6]. An additional advantage of solid phase assays is that a positive test also provides identification of the responsible autoantibody.
Concerns have been raised about the use of solid phase assays as the initial test for the detection of ANA because of the potential lack of sensitivity compared with indirect immunofluorescence 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 indirect immunofluorescence for the diagnosis of systemic autoimmune diseases, including SLE [7,8]. Because of concerns about the lack of sensitivity of solid phase assays, the American College of Rheumatology (ACR) recommended that indirect immunofluorescence, using the HEp-2 cell substrate, should remain the initial test to detect ANA [9,10]. Many, but not all, clinical laboratories have adopted this recommendation. It is critical that each clinician be aware of the method used by his or her laboratory to detect ANA.
CLINICAL LIMITATIONS OF ANA TESTING — The higher the prior probability that a patient has a systemic autoimmune disease, the more likely the results of an antinuclear antibody (ANA) test will assist in establishing the diagnosis. As an example, if there is clinical evidence of systemic lupus erythematosus (SLE) (eg, photosensitivity, pleurisy), systemic sclerosis (eg, Raynaud phenomenon, skin changes), or Sjögren's syndrome (eg, unexplained dry eyes and dry mouth), the ANA results are likely to be helpful. In contrast, if the ANA test is ordered less discriminately, the majority of positive results will likely represent false positive results and may potentially distract the clinician from the correct diagnosis. A list of diseases in which ANA are detected at higher frequency than in normal individuals is presented in the table (table 1).
In 2002, a committee appointed by the American College of Rheumatology (ACR) published evidence-based guidelines for testing for ANA [11]. The committee concluded that a positive ANA result was very useful for the diagnosis of SLE and systemic sclerosis, and somewhat useful for the diagnosis of Sjögren's syndrome and polymyositis/dermatomyositis. ANA testing was useful for identifying patients with juvenile idiopathic arthritis at risk for asymptomatic uveitis and for distinguishing patients with primary Raynaud's phenomenon from those with Raynaud's phenomenon associated with an underlying systemic autoimmune disease. A positive ANA result is also a component of the diagnostic criteria for drug-induced lupus, mixed connective tissue disease, and autoimmune hepatitis.
Although these guidelines are helpful, they do not address the problem most frequently faced by clinicians: Patients with a wide range of underlying diseases may initially present with similar, vague symptoms, including musculoskeletal pain and fatigue. Many clinicians use the test for ANA as an adjunct to their history and physical examination to distinguish patients who may have an underlying autoimmune disease from those who have other illnesses. Whether this is an appropriate use of the ANA test is controversial. If a test for ANA is ordered indiscriminately, 5 percent will have a positive result at the predetermined screening dilution (usually 1:160). If one assumes that the prevalence of ANA-associated diseases in the population is 1 percent, the majority of these patients will have a false positive result. (See "Clinical manifestations and diagnosis of systemic lupus erythematosus in adults".)
INDICATIONS FOR ORDERING ADDITIONAL, SPECIFIC TESTS FOR AUTOANTIBODIES — If a test for antinuclear antibodies (ANA) by indirect immunofluorescence is positive, the reported staining pattern may provide a clue regarding the underlying autoantibodies. However, as indicated above (see 'Significance of ANA staining patterns' above), the association between staining patterns and disease-associated autoantibodies is relatively weak. Additional testing for specific autoantibodies, using solid phase assays, may be indicated.
If a test for ANA by indirect immunofluorescence is negative, but the clinical suspicion of systemic lupus erythematosus (SLE) or other ANA-associated autoimmune disease is high, additional testing may still be appropriate. Some autoantigens, including Ro-60, Ro-52, ribosomal P, and inflammatory myositis-associated antigens may be absent, or present in low concentration, in the human epidermoid carcinoma cell line (HEp-2) cell substrate (see "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems" and "Antiribosomal P protein antibodies" and "Clinical manifestations of dermatomyositis and polymyositis in adults"). Depending on the clinical setting, solid phase assays may be ordered to detect these antibodies, independent of the results of ANA testing by indirect immunofluorescence.
THE SIGNIFICANCE OF A POSITIVE TEST FOR ANA IN THE AS-YET UNDIAGNOSED PATIENT WITH MUSCULOSKELETAL SYMPTOMS — Having obtained a positive test for antinuclear antibodies (ANA) in a patient with symptoms suggesting a rheumatologic autoimmune disease, what are the diagnostic possibilities to be considered? A retrospective study published in 1989 reported the diagnostic associations of 276 patients with a positive test for ANA who were referred to a community rheumatology practice for further evaluation [12]. Of the 276 patients, 126 were diagnosed with a systemic autoimmune disease, including systemic lupus erythematosus (SLE) (52), drug-induced LE (12), undefined connective tissue disease (18), rheumatoid arthritis (11), limited systemic sclerosis (11), polymyalgia rheumatica (4), and systemic vasculitis (3). Drug-induced ANA, without apparent drug-induced LE, was diagnosed in 18 patients. Organ-specific autoimmune diseases were identified in 44 patients of whom the majority had autoimmune thyroid disease. No diagnosis was established at the time of chart review in 37 patients. The results of this study suggest that, in the general community, a positive test for ANA is very helpful in identifying patients with underlying systemic or organ-specific autoimmune diseases.
A second retrospective study of 153 patients with a positive test for ANA was performed at a large teaching hospital [13]. Systemic autoimmune disease was identified in 39 patients: SLE (17), rheumatoid arthritis (8), drug-induced LE (4), Sjögren's syndrome (3), undifferentiated connective tissue disease (3), and systemic sclerosis (2). The prevalence of patients with organ-specific autoimmune diseases or other ANA-associated disorders was not reported.
The results of these retrospective studies provide some guidance to the clinician caring for an undiagnosed patient with a positive test for ANA: It would seem prudent to consider the full spectrum of ANA-associated diseases (table 1). ANA may also be detected in patients who have first-degree relatives with autoimmune diseases, even though the patient him- or herself need not have an autoimmune disease [14]. ANA may also develop in patients who are taking certain medications, even though the patient does not have evidence of drug-induced autoimmune disease at the time of examination. Finally, studies have shown that a patient may have ANA for years, if not decades, before developing the symptoms of an autoimmune disease [15].
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
●Techniques to detect ANA
•Indirect immunofluorescence – The indirect immunofluorescence test is the most widely used assay for the detection of antinuclear antibodies (ANA), in part because of the large number of autoantigens that are present in the human epidermoid carcinoma cell line (HEp-2). (See 'Indirect immunofluorescence test for ANA' 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 ANA staining patterns' above.)
-Significance of ANA titer – The optimal serum dilution to be used to screen for ANA by indirect immunofluorescence should be defined by each laboratory and should be chosen so as to detect autoantibodies in approximately 5 percent of a normal population. (See 'Significance of ANA titer' above.)
Changes in ANA titer are not helpful as a way to monitor disease activity in patients diagnosed with an ANA-associated autoimmune disease. Once a positive test has been obtained, repeat determinations of ANA are not indicated. (See 'Significance of ANA titer' above.)
•Solid phase assays – Additional tests for ANA, including different types of solid phase assays, are suitable for high-throughput testing and semi-quantification of results. However, these assays use a limited number of autoantigens and are therefore less sensitive than indirect immunofluorescence for the detection of autoantibodies. (See 'Advantages and disadvantages of methods to detect ANA' above.)
●Clinical limitations of ANA testing – 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 systemic lupus erythematosus (SLE) (eg, photosensitivity, pleurisy), systemic sclerosis (eg, Raynaud phenomenon, skin changes), or Sjögren's syndrome (eg, unexplained dry eyes, dry mouth), 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 'Clinical limitations of ANA testing' above.)
ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Morris Reichlin, MD, who contributed to an earlier version of this topic review.
