INTRODUCTION
Rheumatic diseases encompass a vast spectrum of disorders that range from localised to complex systemic illnesses affecting joints and connective tissues. They are often associated with pain and can be extremely debilitating to the sufferer. Many non-specific presentations may make diagnosis problematic. Key tests used to facilitate this are the serological autoantibody tests that measure antibodies against self antigens.
AUTOANTIBODY TESTS
When should they be ordered?
Within the rheumatology framework, some clues in the history and examination that may prompt the GP to order autoantibody tests include: Raynaud’s phenomenon; thinning of the skin; vasculitic-type rashes; keratoconjunctival sicca and xerostomia; uveitis; isolated photophobia; unexplainable myalgia, arthralgia, or fatigue; or evidence of unexplained acute kidney injury. Obviously, the suspected disease entity will dictate which autoantibody test to order, as will be reviewed below. There are also other times when other non-rheumatic clinical situations warrant autoantibody tests, for example, organ-specific autoimmune conditions, and these should be considered as well.
Healthy individuals, particularly older people, may also have positive autoantibodies in low concentrations. Although these are not perfect as markers for specific diseases, with reasonable pretest probability, they are useful for ruling in and out the possibility of certain diseases. As autoantibodies are rarely sufficient alone to do this, other appropriate tests to assist, including basic blood workups, should be ordered as well.
How are they reported?
Most autoantibodies are measured using immunoassays (for example enzyme-linked immunosorbent assay [ELISA]), and are read as a titre (for example 1:80) for the patient’s serum to react to or display positive antibodies. Results are reported as a titre or in standardised international units (IU)/mL. Positivity of certain autoantibodies in low titres (for example 1:40) in the absence of clinical features of rheumatism have little consequence. In general, the higher the reported titre, the more likely that there is an associated rheumatic illness with definite positivity ≥ 1:640.
Anti-nuclear antibodies
The archetypal autoantibody, anti-nuclear antibodies (ANA), are targeted against conserved intranuclear antigens. The test is reported in titres (often performed by ELISA) as well as staining pattern when immunofluorescence microscopy is performed using the patient’s serum. Four main staining patterns are recognised: homogeneous, speckled, nucleolar, and centromere. These are often associated with certain disease entities (Table 1). As there are overlapping disease entities and more specific autoantibodies available, the emphasis on staining pattern in diagnoses has diminished.
ANA testing and interpretation are quite complicated. As ANA may be positive in chronic infections, malignancy, other autoimmune conditions, and healthy people (up to 5%), it has only moderate specificity at best for diagnosing rheumatic conditions. Consequently, reasonable pretest probability for the latter is advised when ordering. The issue of false-positives and false-negatives should be considered as well, and ANA test results should not be taken as definitive. One factor that affects these rates is the testing method. Although the American College of Rheumatology asserts that the indirect fluorescent antibody (IFA) method of ANA detection is the gold standard and is more sensitive than ELISAs,1 some laboratories still utilise the latter since it is cheaper and faster.
If ANA is positive and is reported as homogeneous in staining pattern, then a possible follow-up test with anti-double-stranded DNA (dsDNA) is appropriate. This autoantibody to DNA is particularly useful for the diagnosis of systemic lupus erythematosus (SLE) since it has very high specificity for this disease (∼99%) and low prevalence in healthy patients (1%). Thus, a patient who is positive for both ANA and anti-dsDNA has a high probability of SLE. Anti-dsDNA levels are also generally parallel with SLE disease activity so it is useful for monitoring progression and response to treatment.2
Extractable nuclear antigen antibodies
Extractable nuclear antigens (ENA) are so-named because they were originally isolatable from the soluble saline fraction of disrupted cells. ENA consists of a large number of antigens, but only autoantibodies to selected antigens are tested for (Table 2). If ANA is ‘positive’, it should be followed with ENA testing to ascertain the precise antigen ANA is targeting, as this will assist with a diagnosis. A speckled ANA pattern will usually produce positive anti-ENAs. However, it is possible for ENA to be positive in light of a negative ANA result, and anti-ENA as a group may be seen in 1% of apparently healthy patients. If clinical suspicion is strong for a particular disease entity detectable by ENA testing then this test is warranted. A suggested clinical workup using ANA and ENA is depicted in Figure 1.
Rheumatoid factor and anti-CCP
Rheumatoid factor (RF) is an autoantibody directed to the Fc region of self immunoglobin G (IgG). The most commonly measured autoantibody is the IgM isotype. RF complexes activate complement, promote inflammation, and therefore create local tissue damage. When combined with the anti-cyclic citrullinated peptide antibody (anti-CCP/ACPA), the two become particularly useful in the laboratory diagnosis of rheumatoid arthritis (RA). IgM RF and anti-CCP have sensitivities of 69% and 67%; and specificities of 85% and 95% for RA respectively.3 RF, especially anti-CCP, has prognostic value, as high titres tend to correlate with a more severe form of RA. It should be noted, nonetheless, that RF can be also found in other rheumatic conditions such as Sjögren’s syndrome and SLE. Positivity may also be found in other conditions including hepatitis C, sarcoidosis, and infective endocarditis. Again, healthy individuals can display positivity to these autoantibodies (5% for RF and 2% for anti-CCP); hence, interpretation within the clinical context is vital for these to be of value.
Anti-neutrophil cytoplasmic antibodies
Anti-neutrophil cytoplasmic antibodies (ANCA) are antibodies directed against cytoplasmic components of neutrophils. The primary component of this test involves subjecting the patient’s serum to ethanol-fixed neutrophils and observing the staining pattern of these antibodies under indirect immunofluorescence microscopy. Two staining patterns can be appreciated: first, cytoplasmic (c)-ANCA (diffuse staining in cytoplasm); and, second, perinuclear (p)-ANCA (peripheral staining around nuclei). ELISA is used to ascertain the antigen that the autoantibodies are directed against (commonly PR3 for c-ANCA, and MPO for p-ANCA). ANCA are useful as markers for when small-vessel vasculitides (for example Churg-Strauss syndrome) are suspected. Positive p-ANCA (and the closely related ‘atypical p-ANCA’) titres may be seen also in drug-induced vasculitis, glomerulonephritis, ulcerative colitis, and autoimmune hepatitis.4 ANCA levels generally do not reflect disease activity and can fluctuate.
CONCLUSION
The sheer variety and amount of rheumatological autoantibody tests available can be somewhat confusing; however, with a basic understanding for the indications and limitations of each test, the diagnostic workup and monitoring for suspected rheumatological diseases can be strategically performed. These tests are not screening tests, and therefore, clinical acumen and reasonable pretest probability are still required before they are ordered by the clinician.
Notes
Provenance
Freely submitted; not externally peer reviewed.
Competing interests
The authors have declared no competing interests.
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- Received January 4, 2014.
- Accepted January 30, 2014.
- © British Journal of General Practice 2014