Adapted from Solomon DH, Kavanaugh AJ, Schur PH, American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines: Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum 47(4):434–444, 2002.

The ANA panel that is available in most clinical laboratories includes ANAs of defined specificity: anti–double stranded DNA (anti-dsDNA), anti-Smith (anti-Sm), anti–U1 ribonucleoprotein (anti–U1-RNP), anti–Sjögren syndrome antigen A (anti-SSA/Ro), anti–Sjögren syndrome antigen B (anti-SSB/La), anticentromere, antiscleroderma 70 kD (anti–Scl-70) (also known as antitopoisomerase I), and anti–tRNA synthetase (anti–Jo-1). Other laboratories offer tests for antinucleosome, antihistone, anti–ribosomal P, and anti–single stranded DNA (anti-ssDNA) antibodies.

When the immunofluorescent ANA test is positive in a patient suspected of having SLE, an ANA panel should be obtained. Anti-dsDNA and anti-Sm antibodies are considered highly diagnostic, and the presence of either or both antibodies confirms the clinical diagnosis of SLE. However, a negative test for either or both does not exclude the diagnosis because anti-dsDNA antibodies are seen in up to 60% of patients, whereas anti-Sm antibodies are present in approximately 30% of the patients with SLE. The other specific types of ANAs in the panel have a lesser value as a diagnostic marker for SLE except in special situations such as positive anti-SSA/Ro antibodies in a patient with subacute cutaneous lupus or neonatal lupus syndrome.

A positive test for APLAs measured as anticardiolipin antibodies, lupus anticoagulant, or a biologic false-positive VDRL is included in the American College of Rheumatology (ACR) criteria for the classification of SLE. These are helpful in delineating a subset of patients with SLE and secondary antiphospholipid syndrome. Moderate to high titers of immunoglobulin G (IgG) and/or immunoglobulin M (IgM) anticardiolipin antibodies on two separate occasions, 12 weeks apart, are the criteria for antiphospholipid syndrome. The clinical significance of low-titered anticardiolipin antibodies is not known.

Serum complement levels are measured as concentration of C3 and/or C4 or as CH50 hemolytic units. Although most commonly used clinically to monitor disease activity, the presence of both hypocomplementemia and elevated titers of anti-dsDNA is highly associated with the diagnosis of SLE. Additionally, genetic deficiencies of early components of classical complement (C1) pathway are associated with increased risk for SLE or lupus-like syndrome. Genetic deficiencies of C1q and C1r/C1s have the highest risk, whereas deficiencies of C4 and C2 have a lower risk. A combination of normal serum C3 and low CH50 should raise the possibility of genetic complement deficiency. In patients with fewer than four of the ACR criteria, the presence of low C4 levels was predictive of subsequent evolution into SLE.⁴

Monitoring Disease Activity in Systemic Lupus Erythematosus

Serologic tests are widely used for assessing disease activity and predicting exacerbations. Determinations of the serum titer of anti-dsDNA and of serum complement are the most common and probably the most useful serologic tests that are readily available to the clinician.

Although applicable to most patients, both tests have important clinical limitations. Elevated titers of anti-dsDNA and hypocomplementemia do not occur in every patient with active SLE, and their correlation with the disease activity is not absolute. A subset of these patients test positive for anti-dsDNA antibodies (i.e., “serologically active”) but without evidence of clinical disease activity, even when followed for several months.^5,⁶ Box 43-2 provides recommendations by the European League Against Rheumatism (EULAR) on laboratory assessment for monitoring SLE in clinical practice.⁶

Box 43-2

EULAR Recommendations on Laboratory Assessment for Monitoring Systemic Lupus Erythematosus in Clinical Practice

1. Changes in anti–double stranded DNA (anti-dsDNA) antibody titers sometimes correlate with disease activity and active renal disease and may be useful in monitoring disease activity.

2. Treating patients with anti-dsDNA antibodies in the absence of clinical activity is not recommended.

3. Anti–Sjögren syndrome antigen A (anti-SSA/Ro), anti–Sjögren syndrome antigen B (anti-SSB/La), and antiribonucleoprotein (anti-RNP) may have prognostic value in systemic lupus erythematosus (SLE).

4. Complement levels are sometimes associated with active disease, although no predictive value for the development of disease flares is available.

5. Antiphospholipid antibodies are associated with general disease activity, thrombotic manifestations, damage development, and pregnancy complications.

EULAR, European League Against Rheumatism.

Adapted from Mosca M, Tani C, Aringer M, et al: European League Against Rheumatism recommendations for monitoring patients with systemic lupus erythematosus in clinical practice and in observational studies. Ann Rheum 69:1269–1274, 2010.

Clinical Significance of Anti–Double Stranded DNA Antibodies

Diagnostic Value

Anti-dsDNA should be tested if the screening test for ANAs is positive in a patient suspected of having SLE.⁷ The presence of anti-dsDNA is highly characteristic of SLE and is rarely seen in other rheumatic conditions except for drug-induced lupus secondary to anti–tumor necrosis factor agents used for rheumatoid arthritis (RA) and seronegative spondyloarthropathies.⁸

Anti-dsDNA antibodies are listed as an immunologic criterion for the classification of SLE by the ACR. In a large prospective study, the combination of an elevated titer of anti-dsDNA and low serum C3 has a high positive predictive value for the diagnosis of SLE.⁹

Clinical Tests for Anti–Double Stranded DNA

The most commonly available tests for anti-dsDNA in clinical practice are the radioimmunoassays using the Farr or Millipore filter binding technique, Crithidia luciliae immunofluorescent test, and ELISA. The Farr technique is a sensitive, highly reproducible method; it provides greater sensitivity for diagnosis and is helpful in monitoring disease activity but may miss low-avidity anti-dsDNA antibodies. Approximately 60% to 70% of patients with SLE will test positive by this method some time along the course of their illness. The immunofluorescent test uses fixed smears of C. luciliae, a nonpathogenic hemoflagellate containing a cytoplasmic organelle—called kinetoplast—that consists of pure circular dsDNA. The test is simple, has relatively good sensitivity and specificity, and measures both high- and intermediate-avidity anti-dsDNA antibodies. However, a precise serum titer cannot be easily determined. The ELISA test for anti-dsDNA is technically easier to perform, can be automated, and is thus less labor intensive and rapid, as well as avoids the use of radioactive reagents. The serum titer can be readily quantified, and both low- and high-avidity anti-dsDNA antibodies can be detected. False-positive test results can be observed when impure DNA is used as a substrate.

The qualitative properties of anti-dsDNA, including avidity, complement-fixing property, and immunoglobulin class, may affect their pathogenicity. The various clinical tests for anti-dsDNA preferentially measure antibodies of different properties and thus do not necessarily provide identical information on an individual patient.

In general, the highly specific Farr technique or the C. luciliae immunofluorescent test is best used for the diagnosis of SLE. The ELISA test can also be used, but using it later to confirm a positive result from either the Farr technique or the C. luciliae immunofluorescent test may be preferable. Box 43-3 shows guidelines for anti-dsDNA testing in the rheumatic diseases.⁷

Box 43-3

Guidelines for Anti–Double Stranded DNA Testing in the Rheumatic Diseases

1. Anti–double stranded DNA (anti-dsDNA) antibodies provide strong support for the diagnosis of systemic lupus erythematosus (SLE) in the correct clinical setting. Patients who are positive for antinuclear antibodies (ANAs) should be tested.

2. A positive anti-dsDNA test result does not necessarily make a diagnosis of SLE because anti-dsDNA antibodies may be found in a small number of patients with other conditions.

3. A negative test result for anti-dsDNA antibodies does not exclude the diagnosis of SLE.

4. Testing for anti-dsDNA antibodies is not useful in establishing the diagnosis of systemic sclerosis, rheumatoid arthritis, and other rheumatic diseases.

5. Anti-dsDNA antibodies correlate with overall disease activity, but the results must be interpreted in the overall clinical context.

6. Anti-dsDNA antibodies correlate with disease activity of lupus nephritis but only to a limited extent.

7. Increasing titers of anti-dsDNA antibodies may antedate or be associated with lupus disease flares.

Adapted from Kavanaugh AF, Solomon DH, American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines: Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests. Arthritis Rheum 47(5):546–555, 2002.

For monitoring the disease course, especially lupus nephritis, quantitative measurement by ELISA or the Farr technique and expressing the results in international units per milliliter (IU/mL) are recommended.¹⁰

Preemptive Treatment of Serologically Active Systemic Lupus Erythematosus

Prospective controlled studies have examined whether increasing the daily dose of corticosteroids soon after a rise in serum titer of anti-dsDNA antibodies and/or the elevation in serum C3a can prevent clinical relapse. Bootsma and associates ¹¹ reported that early treatment with prednisone as soon as a 25% rise in anti-dsDNA was measured by the Farr technique prevented a clinical relapse in most but not all patients. Tseng and colleagues¹² used a more stringent criterion for a serologic relapse—an elevation in anti-dsDNA level by 25% and an elevated level of serum C3a—and reported that a short-term, moderate dose of prednisone in clinically stable patients with SLE may have averted a severe disease flare. The results of this preliminary study, however, cannot be generalized and recommended to all patients with SLE. Certain limitations in the study design and an estimated positive-predictive value of 40% for the serologic change to predict flares indicated that these were not strong biomarkers for disease flares.¹³ Moreover, disease flares can occur in patients without a rise in anti-dsDNA and/or a lowering of serum C3 or C4 levels. The use of medications other than systemic corticosteroids as preemptive treatments has not been investigated.

Summary

The quantitative determination of anti-dsDNA antibodies does not adequately predict disease flares in every patient, which is not unexpected considering the heterogeneity of the clinical disease and the anti-dsDNA antibodies. The qualitative properties of the anti-dsDNA antibodies, such as the complement-fixing property, avidity, dissociation constant, and immunoglobulin class, as well as the total antibody content are important determinants in the pathogenicity and correlation with disease activity. Most of these measurements, however, are not readily available to the practicing clinician. Meanwhile, the anti-dsDNA antibody titer continues to be widely used as a serologic parameter for assessing disease activity. Combined with serum complement and other renal laboratory parameters, anti-dsDNA antibody titer is a valuable parameter in patients with lupus nephritis. It is especially useful if the patient in question has had a high anti-dsDNA and a low serum complement in past exacerbations of the disease. Anti-dsDNA should be measured at frequent intervals when following the clinical course of the patient.

Anti-Smith Antibodies

Anti-Sm antibodies react to multiple antigens in small ribonucleoprotein particles that function in the splicing of precursor messenger RNA. Different methods and antigen preparations are used in the clinical laboratories for measuring anti-Sm antibodies, including immunodiffusion, ELISA, counterimmunoelectrophoresis (CIE), multiplex bead assays, and hemagglutination. The ELISA test, using purified antigens, is more sensitive than immunodiffusion or CIE but less specific; however, it is superior in quantifying the serum antibody titer.

Anti-Sm antibodies are present in only 30% of patients with SLE, but these autoantibodies have considerable diagnostic value because they are rarely found in other rheumatic diseases, such as mixed connective tissue disease (MCTD), systemic sclerosis, and RA. Anti-Sm is included in the ACR criteria for the classification of SLE, and, as an immunologic parameter, it carries the same weight as anti-dsDNA antibodies and APLAs.

As a diagnostic test, anti-Sm has a relatively low sensitivity but a high specificity; thus a positive test result is useful in confirming a diagnosis. However, a negative test result does not exclude the diagnosis of SLE. When patients with SLE were compared with healthy control patients, anti-Sm had a weighted mean sensitivity of 24% and a specificity of 98%. On the other hand, when SLE was compared with other rheumatic conditions, anti-Sm had a mean sensitivity of 30% and a specificity of 96%.¹⁴

Prevalence

The prevalence of anti-Sm antibodies in SLE varies among the racially different population groups in the world, ranging from 10% to 44%. Both anti-Sm and anti–U1-RNP antibodies are more prevalent in African Americans and Afro-Caribbeans when compared with Caucasians.¹⁴ The test system, antigen used, and selection of patients and controls are different in these studies, which may suggest that the results may not be comparable. In the United States, Arnett and colleagues¹⁵ found anti-Sm and anti-U1-RNP antibodies to be more common in African Americans (25% and 40%, respectively) than in Caucasians (10% and 24%, respectively). Antibodies to SSA/Ro and SSB/La, however, occurred with equal frequencies in the two racial groups.

Anti-Smith Antibody Association with Organ Involvement

Whether the presence of anti-Sm antibodies defines a clinical subset of patients with SLE or carries a prognostic value in SLE remains uncertain. Cross-sectional studies have reported an association with nephritis, neuropsychiatric lupus, serositis, pulmonary fibrosis, and peripheral neuropathy. These associations have not been consistently confirmed by other investigators, and often the purported association has been based on a single serum specimen rather than on a sequential determination in a prospective study.

Anti-Smith Antibodies and Disease Activity

Few longitudinal studies have been performed on the usefulness of anti-Sm antibody titers in monitoring disease activity in patients with SLE. A recent analysis of published investigations concluded that an anti-Sm–positive result is not useful in the diagnosis of nephritis or in predicting renal flares in patients with SLE. Although the analysis found no strong evidence for its utility in predicting neuropsychiatric lupus or other organ system involvement, very few superior evidenced-based medicine articles have been published on the topic.

A prospective quantitative study, conducted over a period of 2 years, of anti–extractable nuclear antigen (anti-ENA) antibodies that included anti–Sm antibodies provided no useful additional information in assessing overall lupus disease activity.¹⁶

Anti-U1 Ribonucleoprotein

Anti–U1-RNP antibodies react to antigens in small nuclear ribonucleoprotein particles distinct from anti-Sm specificities. Both autoantibodies are measured together using the same test system. Different laboratory methods are available; however, the ELISA is probably most commonly used by clinical laboratories.

Arnett and colleagues ¹⁵ found that the prevalence of anti–U1-RNP antibodies measured by the immunodiffusion and CIE tests is higher in African-American patients (40%) than it is in Caucasian patients with SLE (23%). The ELISA test for anti–U1-RNP has a higher sensitivity for SLE and MCTD.¹⁴

Unlike anti-Sm antibodies, anti–U1-RNP antibodies are not considered specific for SLE. Anti–U1-RNP antibodies can be found in MCTD, RA, Sjögren syndrome, systemic sclerosis, and inflammatory myositis.

Clinical Association of Anti–U1 Ribonucleoprotein Antibodies

The presence of high titers of anti–U1-RNP antibodies is associated with MCTD, a clinical entity that is characterized by overlapping clinical features of SLE, scleroderma, and polymyositis. The diagnosis of MCTD requires the presence of anti–U1-RNP antibodies and the absence of anti-Sm and anti-dsDNA antibodies. Multiple types of ANAs in an individual patient suggest an alternative diagnosis such as SLE instead of MCTD. The issue of whether MCTD is a distinct rheumatic disease or a clinical syndrome that may occur during the course of SLE, systemic sclerosis, or another systemic rheumatic disease remains controversial.

In analyzing published data, Benito-Garcia and associates ¹⁴ concluded that a positive anti–U1-RNP test result supports a diagnosis of MCTD in the appropriate clinical setting. On the other hand, a negative anti–U1-RNP result will exclude MCTD, and an alternative diagnosis should be considered.

As a diagnostic test for SLE, anti–U1-RNP has a low sensitivity and moderate specificity. Unlike the anti-Sm test, the anti–U1-RNP test is not useful in supporting a diagnosis of SLE in the appropriate clinical setting. Among patients with SLE, the presence of anti–U1-RNP antibodies does not predict the occurrence of neuropsychiatric manifestations or lupus nephritis.

Serum Antibody Titer

Published data on the utility of serial quantitative testing of anti–U1-RNP antibodies as a measure of SLE disease activity have yielded inconclusive results. The serum titer of anti–U1-RNP antibodies fluctuated with disease activity in some but not all patients, whereas other investigators have reported no correlation either with specific organ involvement or with disease activity. In current clinical practice, a rising serum titer of anti–U1-RNP and/or anti-Sm is not used independently of clinical assessment and other laboratory parameters to predict disease exacerbation or to make changes in drug therapy.

The presence of anti–U1-RNP and/or anti-Sm antibodies does not appear to affect survival in SLE. Patients with undifferentiated connective tissue disease (UCTD) have signs and symptoms suggestive of a systemic autoimmune disease but do not fulfill the classification criteria for SLE, RA, systemic sclerosis, and other disorders. A large proportion of patients with UCTD, who tested positive for anti–U1-RNP antibodies, subsequently developed MCTD.¹⁷ Box 43-4 lists guidelines for anti-Sm and anti-U1-RNP testing in the rheumatic diseases.

Box 43-4

Guidelines for Anti-Smith and Antiribonucleoprotein Testing in Rheumatic Diseases

1. Anti-Smith (anti-Sm) antibodies are very useful for confirming the diagnosis of systemic lupus erythematosus (SLE). A positive test result strongly supports the diagnosis; however, a negative test result cannot exclude the diagnosis.

2. Antiribonucleoprotein (anti-RNP) antibodies are useful in the diagnosis of mixed connective tissue disease but not in the diagnosis of SLE.

3. Neither anti-Sm nor anti-RNP antibodies are useful in establishing the diagnosis of dermatomyositis or polymyositis, rheumatoid arthritis, systemic sclerosis, drug-induced lupus erythematosus, or Sjögren syndrome.

4. Anti-Sm and anti-RNP antibodies are not useful in predicting lupus nephritis or in diagnosing neuropsychiatric lupus or other systemic manifestations of SLE.

Adapted from Benito-Garcia E, Schur PH, Lahita R, American College of Rheumatology Ad Hoc committee on Immunologic Testing Guidelines: Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-Sm and anti-RNP antibody tests. Arthritis Rheum 51:1030–1044, 2004.

Anti–Sjögren Syndrome Antigen A

Anti-SSA/Ro antibodies are the most common specific ANA type encountered in the clinical laboratory. Anti-SSA/Ro antibodies are generally associated with Sjögren syndrome and SLE; however, these autoantibodies may also be seen in RA, polymyositis, systemic sclerosis, and other conditions.

Anti-SSA/Ro antibodies are detected by different methods including ELISA and bead immunoassays, which are the tests most commonly used by clinical laboratories. Different preparations of purified antigen are used by manufacturers. An indirect IFA test using transfected HEp-2 cells that overexpress the human necrosis factor receptor 1 (60-kDa) SSA/Ro antigen is highly sensitive, screening a ring-shaped RNA-binding protein test.

Anti-SSA/Ro antibodies are of two distinct types reacting with different antigens from the ribonucleoprotein complex: 60-kDa and 52-kDa. The 52-kDa autoantigen is a ubiquitin ligase involved in the proteasomal degradation of a variety of proteins, whereas the 60-kDa autoantigen may function in noncoding RNA quality control. Although most patients have both types of autoantibodies, some patients may have a single type of anti-SSA/Ro antibody. In most clinical laboratories, anti-SSA/Ro60 and anti-SSA/Ro52 are not tested separately on a routine basis.

Diagnostic Specificity and Associations

Anti-SSA/Ro antibodies are present in 30% to 40% of patients with SLE and in 60% to 90% of patients with primary Sjögren syndrome, depending on the test method. Anti-SSA/Ro antibodies do not carry a high diagnostic specificity for SLE; however, their presence is associated with photosensitivity and certain clinical subsets including subacute cutaneous lupus, neonatal lupus syndrome, secondary Sjögren syndrome in patients with SLE, homozygous C2 and C4 deficiency with lupus-like disease, and interstitial pneumonitis.

Anti-SSA/Ro antibodies are found in 60% to 90% of patients with subacute cutaneous lupus erythematosus (SCLE), depending on the assay system, and are primarily directed to the 60-kDa Ro antigen, although anti-SSA/Ro 50-kDa antibodies may be concomitantly present. SCLE is a distinct clinical subtype of SLE characterized by recurrent, erythematous, photosensitive, widespread, and nonscarring skin lesions in a typical distribution involving the face, trunk, and arms and by mild systemic disease.¹⁸

Neonatal lupus syndrome is a rare condition in infants born of mothers with SLE. It is characterized by photosensitive, annular, discoid, or erythematous skin lesions of the face and trunk, which appear at or before 2 months of age and disappear by 6 to 12 months of age. Congenital heart block with or without structural cardiac defects is observed in 50% of patients. Almost all afflicted infants and their mothers have anti-SSA/Ro and/or anti-SSB/La antibodies. Buyon and colleagues ¹⁹ found that women with both antibodies, especially if the anti-SSA/Ro antibodies identify the 52-kDA component, have an increased risk of giving birth to an infant with neonatal lupus syndrome. Most of the commercially available tests for anti-SSA/Ro antibodies do not distinguish between antibodies to the 52-kDA and the 60-kDA components.

Genetic deficiencies of the early components of classical pathway C1q, C2, and C4 can clinically exhibit a lupus-like illness. Anti-dsDNA antibodies are absent in affected patients, but a high frequency of anti-SSA/Ro and other anti-ENA antibodies are present. The patients exhibit symptoms of fever, rash, arthritis, and sometimes glomerulonephritis.²⁰

ANA-negative SLE refers to the rare patient with clinical features of SLE or SCLE with a negative ANA result by the immunofluorescent test using rodent kidney or liver as substrate. With a sensitive ELISA, these patients uniformly have anti-SSA/Ro and, in addition, some have anti-SSB/La and/or anti–U1-RNP antibodies.²¹

Among Caucasian patients with SLE, photosensitivity is associated with anti-SSA/Ro antibodies. In contrast, the presence of anti-SSA/Ro antibodies in South African black patients has been reported to be negatively correlated with photosensitivity.²²

Both anti-SSA/Ro and anti-SSB/La are strongly associated with sicca symptoms in patients with SLE. Other features of SLE reported to have a probable association with anti-SSA/Ro antibodies include interstitial pneumonitis, shrinking lung syndrome, and a deforming arthropathy.

Cavazzana and associates ²³ reported that 24% of patients with UCTD who test positive for anti-SSA/Ro antibodies progressed within a short period to either SLE or primary Sjögren syndrome.