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Antinuclear Antibodies

Serologic tests for autoantibodies, including antinuclear antibodies (ANAs) and antibodies to specific nuclear antigens such as double-stranded DNA (dsDNA), play an important role in the diagnosis of systemic rheumatic diseases. However, test results for autoantibodies alone are insufficient to establish the diagnosis of a systemic rheumatic disease. No tests for autoantibodies should be performed without a clinical evaluation that leads to a presumptive diagnosis.

The original discovery of ANA was based on indirect immunofluorescence. Immunofluorescent antibody (IFA) is still the most commonly utilized method for detection of antinuclear antibodies. In this method, diluted patient serum is incubated on a slide containing a monolayer of human epithelial cells. If antibody is present, it binds to cell nuclei. After washing, bound antibody is detected by adding fluorescent anti-human IgG. Positive cells demonstrate bright green nuclear fluorescence with a distinct staining pattern. Patient samples are initially tested at a dilution of 1:40 to 1:160. Positive samples are then diluted and both the fluorescent pattern and titer are reported. The titer is the highest dilution of serum that still shows immunofluorescent nuclear staining.

Hep-2 cells are the preferred cell line due to their human origin, high mitotic activity, and the ability to induce expression of clinically important antigens. HEp-2 cells have an estimated 100 to 150 antigens, the most of any method, allowing for detection of the greatest number of antibody specificities.

The four commonly recognized staining patterns are homogeneous, speckled, nucleolar, and centromere. Previously, attempts were made to correlate the fluorescent pattern with specific diseases. For example, the homogeneous pattern was believed to indicate SLE or drug induced SLE, nucleolar pattern scleroderma, centromere pattern the CREST syndrome, and speckled pattern a wide variety of diseases. During the last several years, however, the clinical importance of the staining pattern has diminished. Considerable overlap exists between the different fluorescent patterns and rheumatic diseases and more specific autoantibody tests have become available.

During the 1980s the ANA ELISA screen was developed. In this method, antigens are either extracted from cells or synthesized and then fixed to the surface of a microtiter plate well. Diluted patient serum is added to the microwell and allowed to incubate. If ANA are present, they bind to the solid phase antigens. After washing, bound antibodies are detected by the addition of an enzyme-conjugated antihuman IgG antibody. Colorless enzyme substrate is added, and if enzyme-conjugated antibody is present, it is converted to colored product. Color intensity is measured photometrically. Microwells with color intensities exceeding the established cut-off value are considered positive. ELISA offers several advantages over IFA including more automated sample preparation and more objective semi-quantitative measurement.

Disadvantages of ELISA include possible denaturation of nuclear antigens during extraction and adherence to the solid phase. Today, most commercial ELISA kits use HEp-2 cell extracts and/or recombinant antigens. Unfortunately, the number and quantity of antigens has not been standardized, resulting in manufacturer-to-manufacturer test variability. In general, ELISA kits based on HEp-2 cell extracts correlate best with IFA.

The newest analytical method is multiplexed immunoassay (MIA) that uses synthetic individual nuclear antigens covalently bound to fluorescent polystyrene beads possessing a distinct fluorescent signal. A mixture of antigen coated fluorescent beads is incubated with patient serum in a single tube. If ANA is present, it binds to the fluorescent bead coated with the complementary antigen. After washing, bound antibodies are detected using a fluorochrome conjugated, anti-human IgG antibody. The bead set is then analyzed by flow cytometry, which detects two fluorescent signals, from the polystyrene bead and from the labeled detection antibody. With this method, a single patient sample can be quickly analyzed for multiple different antibodies. This eliminates the multiple sample pipetting needed for ELISA and HEp-2 cell assay. Advantages of MIA include automation, faster turnaround time, and direct determination of antibody specificity.

MIA appears to be much less sensitive than IFA or ELISA. Comparative studies have suggested that if MIA was used alone as a ANA screen, it would miss a substantial number of ANA positive patients. Analysis of College of American Pathologist’s Proficiency Test (CAP PT) results since 2006 have demonstrated decreased sensitivity (higher false negative rate) for MIA, suggesting the inability of MIA to detect some ANA specificities that are detected by IFA. In fact, no nucleolar antigens are present in some MIA kits.

ANA screen is the sentinel laboratory test in the algorithm of CTD testing. Decreased sensitivity of MIA has raised concern among rheumatologists about the potential for missing cases of CTD. A case report of suspected delayed diagnosis of CTD due to false-negative solid-phase ANA screening was recently published (NEJM 2009;360:711-20). In February 2009, The American College of Rheumatology issued a position statement asserting that IFA should remain the standard for ANA testing because of its higher sensitivity. They suggested that hospitals and laboratories using ELISA or MIA should demonstrate that their assays have sensitivity and specificity equivalent to IFA.

An ANA test should be ordered if the physician feels there is a reasonable clinical suspicion of SLE or another systemic rheumatic disease based on the clinical history, physical findings and results of other laboratory tests. A large number of diverse conditions in addition to SLE are associated with a positive ANA including many chronic inflammatory and infectious diseases and some drug treatments.

Clinical Utility of ANA

Frequency (%) of Positive ANA

Diseases for which ANA is very useful for diagnosis




Systemic sclerosis (scleroderma)


Diseases for which ANA is somewhat useful for diagnosis


Sjogren syndrome


Idiopathic inflammatory myositis (dermatomyositis or polymyositis)


Diseases for which ANA is useful for monitoring or prognosis


Juvenile chronic oligoarticular arthritis w/ uveitis


Raynaud phenomenon


Conditions in which ANA is part of the diagnostic criteria


Drug induced SLE


Autoimmune hepatic disease


Mixed connective tissue disease (MCTD)


Diseases for which an ANA is not useful in diagnosis


Rheumatoid arthritis


Multiple sclerosis


Idiopathic thrombocytopenic purpura


Thyroid disease


Discoid lupus


Infectious diseases

Varies widely


Varies widely

Patients with silicone breast implants




Relatives of patients with autoimmune diseases


Normal persons with titer >1:40


Normal persons with titer >1:160



A substantial number of normal healthy persons have a positive ANA. The prevalence of ANAs among healthy individuals varies with sex and age; older persons, particularly women older than 65 years, more commonly have positive results. Titers are usually low; less than 5% of healthy people have a titer of 1:160 or higher. Due to the low prevalence of SLE (50 cases per 100,000 persons) in the general population, the majority of persons randomly discovered to have a positive ANA result do not have SLE.

ANAs and specific autoantibodies are an integral part of the American College of Rheumatology’s diagnostic criteria for systemic lupus erythematosis (SLE). Nearly all patients (95-100%) with active, untreated SLE have a positive ANA.

American College of Rheumatology Classification Criteria for SLE

Malar rash

Discoid rash


Oral or nasal ulcers

Arthritis of 2 or more peripheral joints

Pleuritis or pericarditis

Renal disease with persistent proteinuria or cellular casts

Unexplained seizures or psychosis

Hemolytic anemia, leukopenia or lymphopenia on 2 or more occasions

Positive ANA, anti-ds DNA antibodies, anti-Sm antibodies or antiphospholipid antibodies


Patients who have few signs or symptoms suggestive of SLE have a low pretest probability of having the disease. In these patients, a positive ANA test result does little to increase the probability that the patient has SLE. In fact, positive ANA results in cases such as this can be misleading and precipitate unnecessary testing, erroneous diagnosis, or even inappropriate therapy.

Patients with scleroderma (systemic sclerosis) usually present with a distinct set of clinical signs and symptoms. Sixty to 80% of patients have a positive result. A positive ANA is not required for diagnosis, but can be supportive. A negative result might lead the physician to consider other fibrosing illnesses such as local scleroderma, eosinophilic fasciitis, and scleredema.

Forty to 70% of patients with Sjogren syndrome have a positive ANA test result. This finding supports the diagnosis, but is not an absolute requirement. Testing is useful in patients with persistent sicca symptoms or in women who has given birth to a child with congenital heart block.

The ANA is positive in 30 to 80% of patients with polymyositis and dermatomyositis. A positive result is supportive, but a negative result does not rule out the diagnosis.

A positive ANA result is an integral component of the diagnosis of drug-induced lupus erythematosis, autoimmune hepatitis, and mixed connective tissue disease. Patients must have a positive ANA before these diagnoses can be made.All studies of drug-induced SLE have included a positive ANA result in the definition of the syndrome. Similarly, criteria for the diagnosis of certain types of autoimmune hepatitis and mixed connective tissue disease (MCTD) dictate that the ANA test result be positive for diagnosis.

Raynaud phenomenon is diagnosed either by physical examination or by eliciting a specific clinical history. An ANA test does not help establish the diagnosis, but may provide prognostic information. Raynaud phenomenon may be associated with several rheumatic diseases including SLE, rheumatoid arthritis and scleroderma. However, Raynaud phenomenon is also common among the general population and 80% of these patients never develop a systemic rheumatic disease. A positive ANA in a patient with Raynaud phenomenon increases the likelihood of development of a systemic rheumatic disease from 20% to 30%, while a negative result decreases the likelihood to about 7%.

The ANA is not useful for diagnosing juvenile chronic arthritis. However, in children with known disease, a positive ANA result may predict the development of uveitis, a serious complication. Approximately one third of patients with a positive ANA develop uveitis. Patients with a positive ANA should be screened for uveitis.

An ANA is not necessary for diagnosis of the antiphospholipid antibody syndrome. However, approximately 50% of patients with this syndrome have a positive ANA. The presence of ANA increases the likelihood that the syndrome is secondary to SLE.

Except for the diseases mentioned above, ANA testing does not provide useful information about prognosis. This is true even for those diseases in which the ANA is useful for diagnosis. The ANA is not useful in assessing disease activity. Since the ANA does not provide useful information about prognosis or disease activity, there is no indication for sequentially monitoring the ANA titer.

ANA Follow-up Tests

Specific follow-up tests for antibodies to the following antigens are available: dsDNA, Sm, RNP, SS-A (Ro), SS-B (La,) Scl-70, histones, and Jo-1. With rare exceptions, these tests should not be ordered if the ANA was negative or weakly positive, because less than 5% of patients with ANA titers <1:160 will have positive follow-up tests. With the exception of antibodies to double-stranded DNA, variation in the titers of these antibodies does not provide useful clinical information. Therefore, repeating tests other than anti-dsDNA, after a positive result is obtained, is not indicated. Because the diseases associated with these tests tend to be dynamic, negative findings might need to be rechecked if the clinical circumstances have changed considerably over time.

ANA Pattern


Disease Association



SLE & low level in other rheumatic disease


Drug induced lupus, SLE


Smith (Sm)


Ribonucleoprotein (RNP)


SS-A (Ro)

Sjogren’s, SLE

SS-B (La)






Scl-70 (DNA topoisomerase 1)







The ANA may be positive in a number of diseases, some of which have clinical features similar to SLE. Two additional tests can help establish the diagnosis of SLE: tests for autoantibodies to dsDNA (anti-dsDNA) and to Smith antigen (anti-Sm).

Anti-DNA antibody refers to antibodies that bind specifically to double stranded DNA. Anti-dsDNA antibodies are detected by a radioimmunoassay technique known as the Farr assay. The Farr assay is more specific for the diagnosis of SLE than the more commonly used enzyme immunoassays. Anti-dsDNA antibodies have specificity for SLE of nearly 100%. High antibody concentrations are more specific for SLE than are low concentrations just above the upper limit of normal (4 IU/mL). Between 50 and 85% of SLE patients have high titer antibodies. Weakly positive anti-dsDNA antibodies occur infrequently in other autoimmune diseases and in healthy persons.

In addition to diagnosis of SLE, measurement of anti-dsDNA antibodies is helpful in assessing prognosis and monitoring disease activity. Renal disease is more common in patients with high antibody levels. An increase in anti-dsDNA concentration often precedes flares of disease activity in SLE patients. Because of this association, many physicians measure anti-DNA levels serially. Appropriate testing intervals are every 1 to 3 months in patients with active disease and every 6 to 12 months in patients with less active disease. Serial tests should be performed in the same laboratory if disease activity is to be reliably monitored over time.

The Sm (Smith) and related nuclear ribonucleoproteins (nRNPs) are targets for autoantibodies in SLE. These antigens are present in subcellular organelles called spliceosomes that are composed of peptide containing small RNAs. Anti-Sm antibodies are only present in 15 to 30% of the patients with SLE, but they are highly specific for SLE. They occur more frequently in young black females with SLE. They almost never occur in healthy individuals or patients with other diseases. Anti-Sm antibodies should not be confused with anti-smooth muscle antibodies detected in autoimmune liver disease. Anti-RNP antibodies, which are commonly tested for in conjunction with anti-Sm, are present in 30 to 40% of SLE patients. However, anti-RNP antibodies are not specific for SLE and are not useful for establishing the diagnosis of SLE.

Antibodies to the ribonucleoprotein SS-A (Ro) are detected in 35 to 60% of SLE patients. Anti SS-A autoantibodies have been associated with photosensitivity, sicca symptoms, thrombocytopenia, and subacute cutaneous LE rash. Subacute cutaneous lupus erythematosis is a widespread, non-scarring, often photosensitive, form of cutaneous lupus with mild systemic manifestations and a low frequency of CNS and renal involvement. Anti SS-A (Ro) antibodies are strongly associated with neonatal lupus. Maternal IgG antibodies cross the placenta, causing disease in the neonate.The two major manifestations of neonatal lupus erythematosis are transient dermatitis and permanent heart block. Photosensitive dermatitis develops after the first few weeks of life and resolves within 6 months, coincident with the clearing of maternal antibodies from the infant’s circulation. Heart block is due to binding of SS-A antibodies in conducting system tissue.

Mixed Connective Tissue Disease

Many patients present with clinical signs and symptoms that are compatible with more than one systemic rheumatic disease. One such overlap syndrome is mixed connective tissue disease (MCTD). Patients with MCTD have overlapping features of SLE, scleroderma, and myositis. Arthritis, arthralgia, Raynaud phenomenon, esophageal dysfunction, and myositis are common, but renal involvement is rare. Detection of RNP antibody, in the absence of other antibodies, strongly suggests the diagnosis of MCTD. Two laboratory criteria are necessary to diagnose MCTD: (1) the presence of high titer RNP antibodies and (2) the absence of anti-DNA, anti-Sm, and histone antibodies.

Sjogren Syndrome

Lymphocytic infiltration of exocrine glands, particularly the salivary and lacrimal glands, and other organs characterize Sjogren syndrome. The most common clinical presentation is with sicca symptoms, xerophthalmia and xerostomia. The autoantibodies most closely associated with Sjogren syndrome are directed against the ribonucleoproteins SS-A (Ro) and SS-B (La). SS-A antibodies are detected in 40 to 60% of patients with Sjogren syndrome. SS-B (La) antibodies occur slightly less frequently and never occur in the absence of SS-A antibodies. The presence of SS-A and SS-B antibodies can be used to support the diagnosis of Sjogren syndrome. However, their presence must be interpreted in the proper clinical context because they are also found in patients with SLE and other diseases. In addition to diagnosis, these antibodies provide some prognostic information. Patients with these antibodies more often have extraglandular disease including vasculitis, purpura, lymphadenopathy, leukopenia, thrombocytopenia, hypergammaglobulinemia, and the presence of rheumatoid factor.


Patients with scleroderma are categorized primarily into two types of disease: limited and diffuse. Patients with limited disease (also known as CREST syndrome) tend to have a better prognosis than those with diffuse disease. Calcinosis, Raynaud phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia characterize the CREST syndrome. Autoantibodies may be useful in differentiating the two types of disease. Limited disease is most commonly associated with the anti-centromere pattern of ANA staining. Approximately 60% of patients with limited scleroderma have anti-centromere antibodies. They occur more frequently in Caucasians than in African Americans, Hispanics, or Asians. The specificity of anti-centromere antibodies for scleroderma is 95%.

Approximately 40% of patients with diffuse disease have autoantibodies to Scl-70. Scl-70 is an antigen present on DNA topoisomerase I, which is the nuclear enzyme responsible for twisting and untwisting the DNA helix during gene transcription. Antibodies directed against Scl-70 usually give a nucleolar ANA pattern. Scleroderma patients with anti-Scl-70 antibodies tend to have more widespread skin disease and internal organ damage, especially pulmonary fibrosis. Anti-Scl-70 and anti-centromere antibodies seldom coexist in the same individual.

Drug-Induced Lupus

The appropriate clinical setting for drug-induced lupus involves exposure to a causative drug, development of a high titer ANA, absence of anti-dsDNA antibodies, and presence of clinical features of SLE. A number of medications have been associated with the development of ANAs and clinical signs and symptoms suggestive of SLE. Common manifestations included arthritis, serositis, and rashes. Renal and CNS manifestations are usually absent. The most commonly associated medications are hydralazine, isoniazid, procainamide and several anticonvulsants. Drug-induced lupus is also associated with the development of autoantibodies to histones, which are DNA binding basic proteins. Antihistone antibodies are present in 90 to 100% of patients with drug-induced lupus. These antibodies are also detectable in 80% of patients with idiopathic SLE, as well as patients with rheumatoid arthritis, scleroderma, vasculitis and autoimmune hepatic diseases. The absence of antihistone antibodies makes the diagnosis of drug-induced SLE less likely, but their presence does not confirm that the case is drug induced. Thus, the presence of this antibody is occasionally less helpful than its absence.

Idiopathic Inflammatory Myositis

Patients with dermatomyositis and polymyositis have inflammatory infiltration and destruction of muscle and other organ systems. Autoantibodies to the antigen, Jo-1, have been associated with pulmonary involvement and arthritis. The Jo-1 antigen resides on the enzyme, histidyl-tRNA synthetase, which is usually located in the cytoplasm of cells, rather than the nucleus.Antibodies to the Jo-1 antigen are detected in approximately 25% of adult patients with myositis including; polymyositis, dermatomyositis, and overlap syndromes.

The following table summarizes the most useful follow-up tests for each of the systemic rheumatic diseases and their sensitivity.

Recommended Follow-up Tests for Patients with a Positive ANA

Suspected Disease

Follow-up Test(s)

Sensitivity (%)













Sjogren Syndrome






Limited Scleroderma

Centromere pattern


Diffuse Scleroderma



Drug induced Lupus



Inflammatory myositis




Specimen requirement is one SST tube of blood.

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