There is often a correlation between the ANA pattern and the presence of anti-DNA and autoantibodies to other intracellular autoantigens. Identification of the staining pattern is useful for the laboratory because it may influence the search for the most appropriate autoantibodies by disease specific autoantibody profiles or other more specific tests. For example, in the presence of a cytoplasmic or nuclear dot type of fluorescence, an immunoassay that includes the cytoplasmic antigens Jo-1, M2/PDC (mitochondria), ribosomal P, EEA1, GW Bodies or the Sp-100 autoantigens, may be indicated (3). In the presence of a homogenous pattern, a search for dsDNA, histone or chromatin antibodies may be indicated. Anti-nucleolar patterns remain one of the main challenges for the clinical laboratory because it is difficult using current technologies to identify the target antigens (fibrillarin, B23, PM/Scl, Pol I/III, Th/To and others) (4). However, when an anti-centromere pattern is present, confirmation is usually not necessary. More recently, there has been attention to the dense fine speckled (DFS) pattern and evidence indicating that patients with ‘monospecific’ anti-DFS antibodies DO NOT have an autoantibody associated rheumatic disease (5).
In general, the screening HEp-2 IFA titer does not correlate with clinical characteristics such as disease activity or flares, and therefore is not a particularly useful parameter for following the course of the disease or estimating the efficacy of therapy (10;11). It should be emphasized that this conclusion is not based on careful prospective laboratory studies using standardized tests on advanced diagnostic platforms or in defined indices of clinical disease (e.g. SLEDAI, SLAM). In general, titers of Hep-2 IFA may fluctuate over time, and the antibodies tend to be detectable in phases both of disease activity and remission (12;13), although there are reported exceptions. One exception may be the presence of high levels of anti-U1-RNP antibodies that are characteristic of mixed connective tissue disease (14).
Another exception is related to evidence that anti-dsDNA antibody levels often correlate with certain clinical features, e.g. lupus nephritis, and its determination is obligatory in the diagnostic work-up of SLE patients and the follow-up of nephritic cases (15;16). However, it is appreciated that some assays for anti-dsDNA detection are better than others in measuring clinically important shifts in antibody levels.
The HEp-2 IFA is the preferred screening method for detecting autoantibodies in human systemic autoimmune rheumatic diseases (SARD) and several other autoimmune conditions (i.e. primary biliary cirrhosis, autoimmune liver diseases, juvenile arthritis at risk of uveitis). In SARD the ANA has been referred to as the ‘gold standard’ for ANA screening (1). However, given the apparent low levels of some autoantigens in the HEp-2 cell substrates, such as the SSA/Ro, Jo-1 and ribosomal P, the test may have a negative result even when these autoantibodies are present (2). Therefore, if clinical findings are highly suggestive of Systemic Lupus, Polymyositis (Autoimmune Inflammatory Myopathy), Sjögren’s syndrome, Scleroderma or other systemic autoimmune conditions, the search for specific autoantibodies is most efficiently done by ordering a disease-specific profile, especially if the ANA result is negative.
Yes, when possible, it is important to try and identify specific antibody targets. There is a wide variety of known autoantibody targets in systemic rheumatic diseases and this is expanding at a rapid pace (7-9). The HEp-2 IFA screening test is able to reveal more than 100 different types of autoantibodies (1), only a portion of which have a validated clinical association and only about 30-40 of these can be revealed by routine laboratory assays. From a cost-benefit point of view, therefore, it is not possible to detect the target specificity in all positive ANA cases. There is international consensus that to bring clinical value to HEp-2 IFA it should be reflexed testing to a disease-specific solid-phase assay.
The presence of high titer IgG autoantibodies and their persistence over time is characteristic of several autoimmune rheumatic diseases, such as SLE, scleroderma, Sjögren’s syndrome, mixed connective tissue disease and autoimmune liver disease (PBC, autoimmune hepatitis). High titer autoantibodies should not be regarded as epiphenomenona of infection or inflammation. However, autoantibodies at low titers (<1:80) may be present in patients with various non-autoimmune diseases (viral and bacterial infections, neoplasia, etc.), in relatives of patients with autoimmune diseases and in apparently healthy subjects MitogenDx has set a fixed cut-off for positivity at a titer of 1:80 to decrease the percentage of false positives. A large multicenter study has shown that ANA without any clinical significance may be found in 30% of healthy subjects at a titer of 1:40 and in 5% at a titer of 1: 160 (6).
Both tests use basically the same technology and assay – an indirect immunofluoresence assay (IFA) on HEp-2 cell substrates. The main difference is that many labs that perform the anti-nuclear antibody (ANA) test only report antibodies that react with the cell nucleus, while ignoring a wide spectrum of clinically-relevant autoantibodies that react with the cytoplasm and mitotic or cell cycle targets. Hence, the term ANA is restrictive, and the term anti-cell/cellular antibodies is more comprehensive and complete.
MitogenDx does not recommend repeating a Hep-2 IFA or ENA test until at least 6 months has passed from a previous blood draw and subsequent test. However, if there is a compelling clinical reason an additional ENA or HEp-2 IFA test may be of insightful. Repeated ANA and autoantibody profile tests are most useful in the diagnostic phase of patient evaluations (i.e. sera with initially negative or low titer positive ANA from a patient with a clinically defined systemic autoimmune disease). A repeat ANA or disease specific autoantibody profile is not indicated unless a change in the clinical picture raises the suspicion of a change in the underlying disease presentation or the appearance of another associated rheumatic disease (e.g. secondary Sjögren’s syndrome, secondary anti-phospholipid syndrome or an overlap syndrome, vasculitis, sudden appearance of Raynaud’s phenomenon).
This Q&A includes extractions from articles by Bizzaro and Wiik (21) and Fritzler, et al. (22).