Laboratory Evaluation of Rheumatic Diseases
ACUTE-PHASE REACTANTS
Acute-phase reactants are proteins whose plasma concentration increases (positive acute-phase proteins) or decreases (negative acute-phase proteins) by at least 25% during inflammatory states.1 Box 1 lists positive and negative acute-phase reactants. The effect of inflammatory molecules such as interleukin (IL)-6, IL-1, tumor necrosis factor α (TNF-α), interferon gamma (IFN-γ), and transforming growth factor β (TGF-β) causes a change in hepatic protein synthesis collectively known as acute-phase response. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are the most widely measured acute-phase reactants in clinical practice.
Both ESR and CRP levels can be elevated in a wide variety of conditions including trauma, infection, infarction, neoplasms, and inflammatory arthritis. Usually ESR and CRP levels correlate well, but in some patients levels may be discordant for reasons that are unclear. They are very useful in monitoring disease activity in rheumatologic conditions such as rheumatoid arthritis, polymyalgia rheumatica,2 and giant cell arteritis. Some studies have shown that the pretreatment ESR value is of some prognostic value in polymyalgia rheumatica. Most patients with active lupus have normal or minimally elevated CRP levels, and markedly elevated concentrations of CRP in SLE should raise a suspicion of bacterial infection. Other causes for elevated CRP in SLE patients include serositis, synovitis, and vasculitis.
ANTINUCLEAR ANTIBODIES
Immunofluorescent microscopy performed on human epithelial-2 (Hep-2) cells is widely used for initial screening. It is a highly sensitive test and is often abnormal in patients with ANA-associated diseases, but the specificity is low and the test has many false-positive results. It is reported as positive or negative and includes a titer. ANA testing performed using ELISA technology is very sensitive and has a high incidence of false-positive results (Fig. 1).
Figure 1 Antinuclear antibody pattern in mouse liver.
A, Peripheral (rim) pattern. B, Homogenous (diffuse) pattern. C, Speckled pattern. D, Nucleolar.
(From Schur PH, Shmerling RH: Laboratory Tests in Rheumatic Diseases. In Hochberg MC, Silman AJ, Smolen JS, et al (eds): Practical Rheumatology. St. Louis, Mosby, 2004, pp 60. Courtesy of Dr. Peter Schur and UpToDate.)
Clinical Applications
ANA testing is very useful in establishing a diagnosis of systemic lupus erythematosus (SLE). Nearly all patients with SLE have a positive ANA test, with a sensitivity of 93% to 95% and a specificity of 57%.3 However, even healthy persons can have a positive ANA test at lower titers. About 25% to 30% of healthy persons have a positive test with a titer of 1 : 40, 10% to 15% at a titer of 1 : 80, and 5% at a titer of 1 : 160 or greater. The frequency increases with age, particularly in women. ANA titer of 1 : 40 is seen in 25% to 30% of relatives of patients with rheumatologic disorders.3
In addition to lupus, ANA testing is helpful in diagnosing other rheumatic diseases such as systemic sclerosis and Sjögren’s syndrome (Table 1). The sensitivity of ANA in diagnosing systemic sclerosis is 85% and the specificity is 54%.3 Although ANA is not included in the 2002 classification criteria for Sjögren’s syndrome, it is found in 80% of patients with primary Sjögren’s syndrome and at high titers (>1 : 320) in nearly one half of the patients.4 Patients presenting with Raynaud’s phenomenon should also have ANA testing because a positive ANA test indicates an increased risk of developing an associated systemic rheumatic disease from 19% to 30%, whereas a negative test indicates a risk of 7%.5 Additionally, ANA testing helps to stratify the risk of uveitis in patients with juvenile idiopathic arthritis.
Table 1 Sensitivity and Specificity of Antinuclear Antibody in Various Connective Tissue Diseases
| Disease | Sensitivity (%) | Specificity (%) |
|---|---|---|
| Systemic lupus erythematosus | 93-95 | 57 |
| Scleroderma | 85 | 54 |
| Polymyositis, dermatomyositis | 61 | 63 |
| Rheumatoid arthritis | 41 | 56 |
| Sjögren’s syndrome | 48 | 52 |
| Raynaud’s phenomenon | 64 | 41 |
| Juvenile chronic arthritis | 57 | 39 |
| Juvenile chronic arthritis with uveitis | 80 | 53 |
Types
There are different types of ANAs based on their target antigen, including single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), nuclear histone and nonhistone nuclear proteins, and RNA protein complexes. The staining pattern seen on indirect immunofluorescence (IIF) gives some indication of the specificity of the antibodies in the sample (Table 2 and see Fig. 1). Identification of the specificity for extractable nuclear antigens (ENA) is warranted because this can further differentiate between the distinct types of autoimmune connective tissue diseases. Hence, a positive ANA test should be followed by an anti-DNA antibodies assay.
Table 2 Identifying Antinuclear Antibodies
| Antigen | Disease Association |
|---|---|
| Homogenous and Diffuse | |
| DNA-histone complex (nucleosome) | |
| Peripheral Rim | |
| dsDNA | SLE |
| Speckled | |
| RNA polymerase types II and III | Systemic sclerosis |
| RNP | MCTD (100%) |
| Scl-70 | Systemic sclerosis (15%-70%) |
| Sm | SLE (25%-30%) |
| SS-A | |
| SS-B | |
| Nucleolar | |
| Nucleolar RNA, RNA polymerase 1 | Systemic sclerosis |
| Pm-scl | Polymyositis |
| Centromere | |
| CENP | Limited scleroderma |
MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus.
Anti-DNA Antibodies
The sensitivity of anti-dsDNA antibody for diagnosis of SLE is 57.3% and the specificity is 97.4%.6 These antibodies are present at some time in the course of the disease as the levels fluctuate and may be absent at times. Anti-DNA antibodies have been reported in patients with a variety of other rheumatologic and nonrheumatologic diseases including rheumatoid arthritis, Sjögren’s syndrome, scleroderma, drug-induced lupus, Raynaud’s phenomenon, mixed connective tissue disease, discoid lupus, myositis, chronic active hepatitis, uveitis, Graves’ disease, and anticardiolipin antibody syndrome and in women with silicone breast implants. Not all patients with SLE have positive anti-dsDNA antibodies; therefore, a negative test does not exclude the diagnosis of SLE. The prevalence of patients with a positive anti-DNA assay despite a negative ANA has been reported to be 0% to 0.8%. Therefore, unless there is a reasonable suspicion that the ANA is falsely negative, anti-DNA antibody testing is not generally indicated in ANA-negative patients.
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