Frequently Asked Questions…
and Answers

Is the ANA test in indirect immunofluorescence (IIF) always positive in the workup of systemic rheumatic disease?

IIF on HEp-2 cells 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). In SARD it has been referred to as the ‘gold standard’ for ANA screening (1). However, given the apparent low levels of some autoantigens in the cell substrate, such as the SSA/Ro, Jo-1 and ribosomal P, the test may show a negative result even when these antibodies 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.

Does the ANA or anti-cytoplasmic antibody pattern have any diagnostic value?

There is often a correlation between the ANA pattern and the presence of anti-DNA and autoantibodies to related 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, it may be indicated to search for dsDNA, histone or chromatin antibodies. Anti-nucleolar patterns remain one of the main challenges for the clinical laboratory because it is difficult using current technologies to identify the target antigen (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).

Is it important to define one or several cut-off values for positivity?

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 healthy subjects, particularly women over 40 years of age and elderly persons. Mitogen has set a fixed cut-off for positivity at a titer of 1:160 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).

In the presence of a positive ANA screening test, is it always necessary to search for antibody specificity?

The variety of autoantibody targets in systemic rheumatic diseases is very wide and continues to grow almost weekly (7-9). The IIF-ANA 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.

Is it useful to repeat and monitor autoantibody levels in the follow-up of patients with systemic rheumatic disease?

In general, the screening IIF-ANA 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). Likewise, the quantification of specific autoantibodies has limited clinical value to monitor disease activity. In general, levels of ANA and related autoantibodies fluctuate over time, and the antibodies tend to be detectable in phases both of disease activity and remission (12;13), although there are some 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.

When is it useful to repeat an ANA test?

Unless there is a compelling clinical reason to do so, the Mitogen will not perform an ENA or ANA test if a requisition or sample is received on the same patient within a six month time period. 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).

How often should anti-dsDNA antibodies be checked in SLE patients?

The frequency of repeat evaluation depends on the activity of the disease and the clinical picture; in general, timing varies in the active forms depending on the diagnosis. Testing intervals of 8 to 12 months are advised in patients with the inactive disease. However, since an increase in autoantibody concentration may precede an episode of clinical exacerbation in subjects with lupus nephritis even by some months, and since in these cases early treatment can impede relapse or limit its severity (17-20), SLE patients with renal involvement ideally should be checked every 2-6 months, depending on the current estimate of disease activity.

When should an anti-ENA test be repeated?

Disease specific autoantibodies are normally present at the moment of diagnosis and generally do not become positive later on, so it is not usually important to repeat this test. However, in selected cases – i.e., if the clinical picture changes – a repeat test is indicated due to the possible evolution into a recognizable disease or into a different prognostic subgroup. It must be kept in mind that the analytical methods used to detect ANA and related autoantibodies have different performance characteristics leading to significant inter-laboratory sensitivity and specificity. A false negative test can be a problematic and if the physician is suspicious, the test should be repeated or confirmed with different methods.

This Q&A includes extractions from articles by Bizzaro and Wiik (21) and Fritzler, et al. (22).




       (1)    Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010;69:1420-2.

       (2)    Fritzler MJ. The antinuclear antibody (ANA) test: Last or lasting gasp? Arthritis Rheum 2011;16:19-22.

       (3)    Stinton LM, Eystathioy T, Selak S, Chan EKL, Fritzler MJ. Autoantibodies to protein transport and messenger RNA processing pathways: Endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes and GW Bodies. Clin Immunol 2004;110:30-44.

       (4)    Welting TJ, Raijmakers R, Pruijn GJ. Autoantigenicity of nucleolar complexes. Autoimmun Rev 2003;2:313-21.

       (5)    Mahler M, Hanly JG, Fritzler MJ. Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases. Autoimmun Rev 2012;11:642-5.

       (6)    Tan EM, Feltkamp TEW, Smolen JS, Butcher B, Dawkins R, Fritzler MJ, et al. Range of antinuclear antibodies in “healthy” individuals. Arthritis Rheum 1997;40:1601-11.

       (7)    Sherer Y, Gorstein A, Fritzler MJ, Shoenfeld Y. Autoantibody explosion in systemic lupus erythematosus. Semin Arthritis Rheum 2004;34:501-37.

       (8)    Mehra S, Walker J, Patterson K, Fritzler MJ. Autoantibodies in systemic sclerosis. Autoimmun Rev 2013;12:350-4.

       (9)    Bournia VK, Vlachoyiannopoulos PG. Subgroups of Sjogren syndrome patients according to serological profiles. J Autoimmun 2012;39:15-26.

    (10)    Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum 2002;47:434-44.

    (11)    Kavanaugh AF, Solomon DH. Guidelines for immunologic laboratory testing in the rheumatic diseases: Anti-DNA antibody tests. Arthritis Rheum 2002;47:546-55.

    (12)    Moder KG. Use and interpretation of rheumatologic tests: a guide for clinicians. Mayo Clin Proc 1996;71:391-6.

    (13)    Ward MM. Laboratory testing for systemic rheumatic diseases. Postgrad Med 1998;103:93-100.

    (14)    Amigues JM, Cantagrel A, Abbal M, Mazieres B. Comparative study of 4 diagnosis criteria sets for mixed connective tissue disease in patients with anti-RNP antibodies. Autoimmunity Group of the Hospitals of Toulouse. J Rheumatol 1996;23:2055-62.

    (15)    Swaak AJG, Groenwold J, Aarden LA, Statius Van Eps LW, Feltkamp EW. Prognostic value of anti-dsDNA in SLE. Ann Rheum Dis 1982;41:388-95.

    (16)    ter Borg EJ, Horst G, Hummel EJ, Limburg PC, Kallenberg CGM. Measurement of increases in anti-double-stranded DNA antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus. Arthritis & Rheumatism 1990;33:634-43.

    (17)    Swaak AJG, Aarden LA, van Epps LWS. Anti-dsDNA and complement profiles as prognostic guides in systemic lupus erythematosus. Arthritis Rheum 1979;22:226-35.

    (18)    Swaak AJ, Groenwold J, Bronsveld w. Predictive value of complement profiles and anti-dsDNA in systemic lupus erythematosus. Ann Rheum Dis 1986;45:359-66.

    (19)    Walz Leblanc BAE, Gladman DD, Urowitz MB. Serologically active clinically quiescent systemic lupus erythematosus – predictors of clinical flares. J Rheumatol 1994;21:2239-41.

    (20)    Bootsma H, Spronk P, Derksen R, De Boer G, Wolters-Dicke H, Hermans J, et al. Prevention of relapses in systemic lupus erythematosus. Lancet 1995;345:1595-9.

    (21)    Bizzaro N, Wiik A. Appropriateness in anti-nuclear antibody testing: from clinical request to strategic laboratory practice. Clin Exp Rheumatol 2004;22:349-55.

    (22)    Fritzler MJ, Wiik A, Fritzler ML, Barr SG. The use and abuse of commercial kits used to detect autoantibodies. Arthritis Res & Ther 2003;5:192-201.