Antinuclear Antibody (ANA) Dx

Synonyms: ANA, Fluorescent ANA (FANA), LE preparation.

CPT Code: ANA 86038;  ANA titer 86039;  Positive ANA

ICD-9 Code: 795.79

ICD-10 Code: Raised antibody titer R76.0; Abnormal immunological finding in serum, unspecified R76.9

Description: Autoantibodies that react with various components of the cell nucleus are called ANAs. ANA is the characteristic laboratory finding of SLE.  However, ANA may be found in patients with a variety of other autoimmune conditions as well as in normal persons. The ANA test defines a population of autoantibodies that react with specific intracellular constituents (Table). This is clinically relevant because the presence of specific autoantibodies may correlate with particular organ involvement and prognosis. Some manifestations are more typical of patients possessing particular autoantibodies, such as the presence of renal disease in patients with anti-dsDNA antibodies.

Method: The ANA test was preceded by the discovery of the LE cell (a leukocyte that phagocytizes a nucleus) in 1948 as the first diagnostic test for SLE. When it was later realized that the LE cell phenomenon was mediated by high titers of antinuclear antibodies, the LE cell test was replaced by more sensitive immunofluorescent tests specific for antibodies capable of binding various nuclear constituents. Hence, the LE cell preparation is seldom performed any more, but LE cells may be coincidentally found in body fluids (e.g., pleural fluid). LE cells are suggestive, but not proof, of a lupus diagnosis.

Initially, ANA tests were performed on rodent tissue sections. Of note, some nuclear antigens (e.g., Ro) are absent in rodents, and some organelles (e.g., nucleoli, centromeres) are present in limited numbers in normal tissue. Thus, before the mid-1980s, there were patients who had clinical manifestations characteristic of SLE and anti-Ro antibodies but a negative ANA test. With replacement of rodent tissues by the human HEp2 tumor cell as the standard substrate for ANAs, the concept of ANA-negative lupus has largely disappeared. The generic ANA test is now universally performed by IIF, using an HEp2 substrate.

Tests for the antigens to which specific ANAs react (e.g., Sm, RNP, Ro, La) can be performed by several methods, including ELISA.

Interpretation: In addition to being positive or negative, the ANA test is quantitatively reported as a titer. The clinical significance of the ANA test often parallels the strength of the titer reported. Typically, positive ANA results are reported in terms of both titer and pattern. Higher titers are more consistent with, but not diagnostic of, SLE. Typically, titers of >1:160 are considered positive, whereas titers of <1:80 are equivocal and often nonspecific.  Low titer results (<1:320) are more likely to be seen in normal individuals, the elderly or be related to comorbidities that may also show ANA positivity (e.g., thyroid disease, chronic renal, liver or lung disease).

The ANA test is also interpreted according to the pattern of nuclear staining observed. These patterns may correlate with different antigen reactivity (Table).  A speckled pattern of immunofluorescence is most commonly seen with the HEp2 substrate but is perhaps least specific. A speckled ANA result is associated with various so-called extractable nuclear antigens (ENA; so named because they can be extracted from the nucleus by saline). These include Ro (SS-A), La (SS-B), Sm (anti-Smith), RNP, Scl-70, Jo-1, and many others. Anti-Ro and anti-La antibodies are also observed in patients with Sjögren’s syndrome (hence the designations SS-A and SS-B). Anti-Ro antibodies may also be seen in neonatal lupus, subacute cutaneous lupus erythematosus or lupus arising in the elderly. Anti-Sm is relatively specific for the diagnosis of SLE because it is infrequent in other diseases or in normal persons. Along with anti-RNP antibodies, patients with SLE with anti-Sm may be more prone to develop interstitial lung disease. Anti-RNP antibodies were also previously associated with mixed connective tissue disease (now better termed undifferentiated connective tissue disease). Anti–Scl-70 antibodies are associated with the diffuse form of systemic sclerosis.

A nucleolar pattern of the ANA test is also seen in systemic sclerosis, SLE, and inflammatory myositis. A centromere pattern is associated with the limited form of systemic sclerosis (previously referred to as CREST syndrome). The homogeneous (or diffuse) ANA is also very nonspecific and often associated with antibodies to histones. Such antibodies are seen in SLE, and reactivity to specific histone proteins is characteristic of drug-induced lupus. A rim (or peripheral) pattern of immunofluorescence is associated with antibodies to “native” or double-stranded DNA (dsDNA). Anti-dsDNA antibodies are useful for the diagnosis of SLE because they are uncommon in other diseases. In addition, patients with high titers of anti-dsDNA antibodies are more prone to develop proliferative lupus nephritis. The titer of anti-dsDNA antibodies may vary with the activity of disease, particularly lupus nephritis, and sequential anti-DNA determination is sometimes used to follow the activity of SLE. Anti-DNA antibodies may be specifically determined by several assays, including the Crithidia luciliae assay and the Farr test. Results from these various tests are reported in different units, and it is important to be familiar with the laboratory performing these tests.

Normal Values: Local laboratories should establish positive and negative titers such that <5% of normal individuals have a positive result. For most clinical laboratories, an ANA result is said to be negative when the titer is <1:160.

Increased in: ANAs are found in patients with SLE and a variety of autoimmune diseases (e.g., Hashimoto thyroiditis, inflammatory myositis). A positive ANA result may be seen in patients with chronic liver disease (e.g., chronic active hepatitis, primary biliary cirrhosis), chronic renal disease, chronic interstitial lung disease, and drug-induced lupus and among intravenous drug abusers. The incidence of positive ANA results is threefold higher (15%) in the elderly. In addition, first-degree relatives of patients with autoimmune disease and normal persons (particularly women and older persons) may have a positive ANA result with no associated autoimmune disease.

ANA titers do not generally correlate with disease activity, and there is little value in repeating an ANA test in a patient known to be positive.

Indications: The ANA test is most commonly used in the diagnosis of SLE. It is almost 100% sensitive because virtually all patients with SLE have a positive ANA result. However, ANAs are not specific at all because they may be seen in other connective tissue diseases such as drug-induced lupus (>95% of patients are ANA positive), scleroderma (70%–90%), inflammatory myositis (40%–60%), and Sjögren’s syndrome (75%–90%). Moreover, even some healthy persons have positive ANA results, particularly at low titer. The ANA test should not be used to screen patients with joint pain or presumed systemic illness.

Cost: Performed by indirect immunofluorescence on an HEp2 substrate, but are also available by ELISA. ANA tests are widely available and typically cost $50–75.

BIBLIOGRAPHY

Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol 2000;53:424–432. PMID: 10911799

Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. American College of Pathologists. Arch Pathol Lab Med. 2000;124:71-81. PMID:10629135

Tan EM. Autoantibodies, autoimmune disease, and the birth of immune diagnostics. J Clin Invest. 2012;122:3835-6.PMID: 23154275