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.
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.
The Sm and nuclear ribonucleoprotein (RNP) antigens are a particulate complex composed of small nuclear RNAs (U1-RNAs) and proteins. This complex has also been referred to as extractable nuclear antigens (ENA), since it is soluble in saline. Autoantibodies to these antigens occur in systemic lupus erythematosis and mixed connective tissue disease.
The Sm (Smith) and related nuclear ribonucleoproteins (nRNPs) are targets for autoantibodies in SLE. These antigens are present in sub-cellular 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 (60%) 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. High titers of Sm and RNP antibodies have been reported in patients with less renal and central nervous system disease, though others have refuted these findings. Sm antibodies may disappear with treatment, while RNP antibodies persist.
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.
Results are reported as positive or negative. The reference value is negative.
Specimen requirement is one plain red top tube of blood.
References
Cohen ML, et al Clinical significance of antibodies to ribonucleoprotein, Ann Rhem Dis 1979;38(1):74-78.
Migliorini P, et al, Anti-SM and anti-RNP antibodies, Autoimmunity, 2005; 28(1):47-54.
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