BIDMC Recommended Approach to Diagnostic Laboratory Testing for Connective Tissue Diseases

Transcription

1 BIDMC Recommended Approach to Diagnostic Laboratory Testing for Connective Tissue Diseases The following discussion is provided by Anders Berg, who is the assistant medical director of the Clinical Chemistry laboratory at Beth Israel Deaconess Medical Center. This discussion is meant to aid our physicians when they are considering when to order autoantibody tests, which tests to order, and how the results will impact their patients diagnoses. This discussion is by no means complete; for complex cases, we always recommend consultation with an appropriate specialist (e. g. rheumatology, gastroenterology) as the case requires. These recommendations are based upon multiple sources which are referenced throughout, relying primarily upon consensus guidelines published by the American College of Rheumatology. I. Initial evaluation of a patient with suspected connective tissue disease Diagnosis of connective tissue disease (CTD) is best founded upon a strong clinical suspicion based upon symptomatology. Serological tests for CTD-associated autoantibodies can provide helpful corroborating evidence for specific diseases, but they should never be used on their own as a basis for a diagnosis. The often-cited positive and negative predictive values of these tests only pertain to patients with symptoms consistent with the disease in question, and their results are uninterpretable when the test is used to screen patients with nonspecific symptoms. The ANA screen by indirect immunofluorescence is the standard-of-care for screening for connective tissue disease autoantibodies, it is sensitive for the detection of the most clinically relevant specific nuclear autoantibodies (SNAs) including antibodies directed against double-stranded DNA (dsdna), Ro (SSA), La (SSB), Scl-70, Smith, centromeric, and nucleosomal antigens (1). If there is a clinical suspicion that a patient has systemic lupus erythematosus or other diseases associated with anti-nuclear antibodies (including Sjogren s Syndrome, Drug-induced lupus, Scleroderma or Mixed Connective Tissue Disease), the approach we advocate is to first order an antinuclear antibody screen (ANA), and, if the ANA is positive,follow-up with more specific antibody tests if necessary. We do not advocate ordering panels of SNA tests in the absence of a positive ANA result, and we also do not advise testing for specific antibodies unless the patient s symptoms suggest a specific disorder. The reasoning behind these recommendations is that, with a negative ANA, in the absence of specific clinical suspicion the results of any SNA assay are likely to confuse the diagnosis instead of resolve it. If clinical symptoms do provide a reasonable clinical suspicion for a specific disease, e. g. a young woman with a fatigue, hematuria, and malar rash, it is reasonable to initially order both an ANA and anti-dsdna antibodies tests because of the high degree of suspicion for SLE. In patients whose symptoms include a polyarticular inflammatory arthritis, it is advisable to also order an autoantibody test for rheumatoid arthritis. This may be achieved by testing for either rheumatoid factor (RF) or antibodies against cyclic citrullinated polypeptide (anti-ccp antibodies) (2, 3). Anti-CCP testing is a newer test for rheumatoid arthritis with a growing number of advocates, and clinical trials indicate that this

2 marker is as good or perhaps better for the detection and diagnosis of RA. However, assays for this disease marker are not yet standardized, and thus valid assay comparisons are problematic. Advocates for this test would suggest that the advantage of the anti-ccp antibody is its apparent high specificity for rheumatoid arthritis, although whether this assay has a clear advantage over tests for rheumatoid factor activity is still a subject of dispute. In patients with symptoms which are consistent with aspects of a connective tissue disease but are nonspecific and provide no clear diagnostic picture, it is perhaps reasonable to order an ANA screen, erythrocyte sedimentation rate or C-reactive protein measurement to evaluate for an inflammatory or rheumatologic disease (infectious causes of the patient s symptoms, which are potentially fatal and usually treatable, should always be considered and evaluated). Even when the ANA and subsequent specific nuclear antibody tests are positive, physicians should be cautious about finalizing any diagnosis. Antinuclear antibodies are found in up to 25%-30% of healthy controls (1), they are associated with a long list of non-connective tissue disorders, and our own internal quality assurance studies have demonstrated that 20% of our patients have low ANA titers in the absence of any apparent autoimmune disease. This relatively poor specificity raises the possibility of a misdiagnosis, and in these cases the physician should strongly consider deferring diagnosis until the patient s clinical picture becomes clearer. II. Methodology and ANA test interpretation(4) The anti-nuclear antibody test is an indirect immunofluorescence assay. The substrate used is a standardized cultured cell line called Hep2 cells. The cells are grown, plated, and fixed on glass slides, incubated with patient serum, washed, and stained with fluorescent anti-human IgG antibodies. According to recommended guidelines, the standard protocol is to screen using patient serum diluted 1:40, and if any nuclear staining is observed the result is reported as positive. When the ANA is positive, the nuclear staining pattern is also reported and in many cases the pattern correlates with the specific antibodies present (i.e. a peripheral pattern is often seen in patients with anti-dsdna antibodies). However, staining patterns are not perfectly specific, and they should not be used on their own to interpret antibody specificity and diagnosis, but instead the specificity of the autoantibodies should be determined, when necessary, by immunoassay testing for specific nuclear autoantibodies. In specimens that are positive at a dilution of 1:40, the serum is serially diluted (1:80, 1:160, 1:320, etc.) until the fluorescence is barely detectable, and the last dilution to produce visible staining is reported as the antibody titer. ANA titering is performed largely because it was once the only way to quantitate the patient s autoantibody. However, this titer is irreproducible; less than a 4-fold change in the titer could simply be experimental error, and the correlation between titer and disease flares is irreproducible between individuals. As a consequence of this, the antibody titer does not necessarily correlate with disease severity, and in most cases we strongly advise against monitoring ANA titers as a method of following

3 disease activity (when applicable, monitor anti-dsdna as an alternative see below). Although the ANA titer is not helpful for monitoring disease in patients with established disease, it may be helpful in strengthening the diagnosis: untreated connective tissue diseases are usually associated with higher ANA titers (1:160 and above). In our experience, low titers of 1:40 and 1:80 are more often false positives with no clear explanation. There is a large list of diseases and disorders unrelated to specific CTDs that are associated with low titer ANA antibodies, and as already mentioned above, 20% of patients have low positive ANA results in the absence of autoimmune disease. Therefore, finalizing a diagnosis based on low positive ANA test results can be problematic. Nonspecific symptoms like chronic fatigue combined with a low titer ANA and no positive specific nuclear antigen tests should prompt the treating physician to search for alternative diagnoses, perform additional testing and follow-up with additional tests later instead of finalizing the diagnosis. The best specific nuclear antigen antibody test available is the test for double-stranded DNA antibodies (anti-dsdna) (1). This test differs from the ANA screen in several regards. Specific antibodies to double stranded DNA (dsdna) are often found in systemic lupus erythematosus (up to 70% sensitivity for SLE), and are 95% specific for this disease). The anti-dsdna antibody test is analogous to an ELISA test performed in a microtiter plate with immobilized dsdna as a substrate. Titers measured by this method are more reproducible and quantitative than the titer associated with the ANA screen. Furthermore, there is evidence that a rising anti-dsdna antibody titer (along with falling serum complement concentrations) may be associated with worsening disease activity and development of lupus nephritis (although there is no standardized method for the interpretation of specific values). Tests for antibodies to other specific nuclear antigens are generally less robust in their predictive values than anti-dsdna testing. Other tests have either lower sensitivity, lower specificity, or both. This is largely the reason why we discourage clinicians sending indiscriminate panels for these antibodies and we emphasize that final diagnosis should rather be based largely upon clinical presentation over time. (It is important to note that delaying final diagnosis in this manner should not necessarily delay empiric therapy for symptoms, however). Furthermore, instead of using simple testing algorithms which we think oversimplify the clinical gestalt that should go into a diagnosis, we prefer to present our recommendations as discussions of the most common connective tissue diseases and which tests can be most contributory to their diagnosis. Here s a listing of the specific nuclear antibodies associated with the various CTDs. Again, these tests should only be ordered when the ANA screen is found to be positive and there is symptomatic evidence of a specific autoimmune disease.

4 Systemic Lupus Erythematosus Rheumatoid Arthritis Scleroderma (Systemic Sclerosis) Mixed Connective Tissue Disease Polymyositis/Dermatomyositis Drug-induced Lupus Neonatal Lupus Sjogren s Syndrome anti-dsdna Rheumatoid Factor and/or anti-ccp anti-scl-70 anti-rnp anti-pm-1 and/or anti-jo-1 anti-histone anti-ro/la anti-ro/la III. Discussion of testing for suspected specific connective tissue diseases Systemic Lupus Erythematosus The best laboratory approach to diagnosis of systemic lupus erythematosus is the combination of screening for anti-nuclear antibodies by our in-house indirect immunofluorescence assay followed by ELISA testing for specific anti-dsdna antibodies (1). By one published estimate, amongst a mixture of healthy patients, patients with SLE, and patients with other CTDs, the ANA has an overall sensitivity of 93% and specificity of 57% (1). When combined with a positive anti-dsdna, result, the specificity approaches 88%-100% (5). Antibodies to dsdna most often produce a peripheral nuclear staining pattern. Other antibodies that may be found in patients with SLE include anti-smith (Sm), anti-ro/la (SS-A and SS- B), anti-histone H1/H2B, and anti-ribonucleoprotein (RNP) antibodies, however none of these are both as sensitive and specific as anti-dsdna testing for this disease. Anti-Sm is relatively specific for SLE, but is not present in most patients with SLE (6). Anti-Ro/La may be positive but are more often associated with Sjogren s syndrome. Anti-RNP may also be found in patients with SLE but they may also be consistent with a diagnosis of mixed connective tissue disease (MCTD) (1). Antibodies to histones H1 and H2B may be found in SLE, whereas anti-h2a-h2b and anti-h3-h4 antibodies are found in drug-induced lupus. Antichromatin antibodies (anti-h2a-h2b-dna nucleosome complex) are commonly found (69%) in patients with SLE, they are relatively specific, and they are associated with renal complications in patients with SLE. Antibodies against single-stranded DNA (ssdna) are often present but are very nonspecific and this test is no longer recommended.

5 Sjogren s Syndrome Anti-nuclear antibodies are present in approximately 48% of patients with Sjogren s Syndrome; the most common specific antibodies in these patients are directed against Ro and La antigens (formerly known as SS-A and SS-B). These antibodies classically produce a speckled nuclear immunofluorescence staining pattern. Although ANA and Ro/La antibodies may be positive in these patients, because of their low sensitivity they are not part of any diagnostic criteria or recommendations (1). Scleroderma/Systemic Sclerosis) Cases of systemic sclerosis are commonly associated (85% sensitive) with ANA antibodies of several specificities which produce speckled, centromeric, and nucleolar patterns by immunofluorescence (1, 7). These antibodies may be directed against centromere (CENP-A and -B), Scl-70 (topoisomerase-i), RNA polymerase, and U3-RNP antigens. Antibodies directed against any of these is relatively specific for systemic sclerosis, and there is some evidence to indicate that the specific antibodies present may have prognostic value in differentiating between systemic sclerosis and the milder forms of scleroderma; the clinical significance of specific nuclear antibodies in cases of suspected scleroderma is a developing field of investigation. Rheumatoid Arthritis Rheumatoid arthritis is most commonly associated with rheumatoid factor (RF) antibodies and/or antibodies against cyclic citrullinated polypeptide (CCP). RF is an IgM autoantibody directed against patients endogenous IgGs. Rheumatoid arthritis is also associated with antibodies directed again cyclic citrullinated polypeptide (CCP).. CCP is a peptide component of the extracellular matrix protein filaggrin that has been post-translationally modified by arginine peptidyl deiminase (3). Citrullination of this protein may play a part in its recognition by the immune system as an autoantigen. Recent metaanalyses have demonstrated that both RF and anti-ccp assays are 67-69% sensitive for RA, but anti-ccp antibodies are much more specific (2, 3). Rheumatoid factors may also be found in SLE and Sjogren s syndrome, as well as chronic hepatitis C infection (especially when associated with cryoglobulins). Trials indicated that both of these tests may someday provide some indication of the patients risk of developing joint disease, however how specific assay values can be translated into absolute risk for individual patients is at this time unclear. Mixed Connective Tissue Disease (MCTD) MCTD may be a subtype of SLE that is typified by symptoms that combine aspects of Lupus along with other connective tissue diseases, especially scleroderma and inflammatory myositis. MCTD is the only connective tissue disease defined by a serologic test; a dignosis of MCTD requires demonstration of the presence of antibodies against RNP ribonucleoprotein antigen (also sometimes called U1-RNP). Accordingly, these patients will also be positive for the ANA screen) (8).

6 Neonatal Lupus Neonatal lupus is a syndrome of intrauterine fetal heart block caused by autoantibodies in the mother s serum which cross the placenta and affect the fetal myocardial conduction system (9). The autoantibodies associated with this syndrome are directed against Ro or La antigens, and thus this syndrome may occur in women with pre-existing autoimmune disease or in any woman with these circulating autoantibodies during the pregnancy. Note that the mother may be asymptomatic and unaffected and the antibodies may only affect the fetus. Accordingly, anti-ro/la testing is sometimes used to test women with histories of unexplained late gestational spontaneous abortions although the utility of such testing is unclear. Polymyositis/Dermatomyositis Positive ANA screen results in up to 80% of patients with polymyositis and dermatomyositis syndrome, but because of the overlap of symptoms with other CTDs, other diagnoses must also be considered when the ANA is positive (1). Specific nuclear antigen tests also have poor utility. Anti-Jo-1 and anti-pm-1 antibodies are relatively specific for these diseases, but they are only found in 30% of the patients with these syndromes. Autoimmune myositis may also be found in mixed connective tissue disease, although these patients will instead be positive for ANA and anti-rnp antibodies. GI inflammatory diseases Recent progress into the understanding of autoimmune diseases of the gastrointestinal system has demonstrated useful applications for autoantibody tests in these diseases. Type I autoimmune hepatitis is highly associated with autoantibodies; positive ANA screens are found in 61-90% of cases and anti-smooth muscle antibodies are highly specific for the disease (10, 11). P -ANCA antibodies and anti-liver kidney microsomal (LKM) antibodies may also be present. These antibodies may also be found in patients with chronic hepatitis C, although the titers are usually lower. Note that the current literature on the subject indicates that chronic hepatitis C may be coexistent with autoimmune hepatitis, and so differentiating between the two based on serologic tests can be difficult. Furthermore, the distinction between AIH and chronic hepatitis C may have an impact upon the decision to treat the autoimmune hepatitis with long-term immunosuppressive therapy. Note that a positive ANA may also be associated with non-alcoholic steatohepatitis (NASH), making this disease difficult to differentiate from AIH without a liver biopsy. Primary biliary cirrhosis is almost always (95%) associated with anti-mitochondrial antibodies; ANA screening is also usually positive, although the prevalence of this depend upon the study. Antimitochondrial antibodies are very specific for this disease, particularly in patients in higher risk categories (e.g. Caucasian women of childbearing age). Ulcerative colitis, with or without associated primary sclerosing cholangitis, may be associated with either a positive ANA screen, or more frequently with p-anca autoantibodies (30-80% incidence,

7 depending upon the study) without specificity for myeloperoxidase (as found with pauci-immune glomerulonephritis or vasculitic disorders). These antibodies are less frequently associated with Crohn s disease, making these tests somewhat helpful for differentiating inflammatory bowel diseases. IV. References 1. Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum Aug;47(4): Newsome G. Guidelines for the management of rheumatoid arthritis: 2002 update. J Am Acad Nurse Pract Oct;14(10): Nishimura K, Sugiyama D, Kogata Y, Tsuji G, Nakazawa T, Kawano S, et al. Meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann Intern Med Jun 5;146(11): Laboratories B-R. Kallestad HEp-2 Cell Line Substrate (ANA Screening test package insert) Gutierrez-Adrianzen OA, Koutouzov S, Mota RM, das Chagas Medeiros MM, Bach JF, de Holanda Campos H. Diagnostic value of anti-nucleosome antibodies in the assessment of disease activity of systemic lupus erythematosus: a prospective study comparing anti-nucleosome with anti-dsdna antibodies. J Rheumatol Aug;33(8): Benito-Garcia E, Schur PH, Lahita R. Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-sm and anti-rnp antibody tests. Arthritis Rheum Dec 15;51(6): Reveille JD, Solomon DH. Evidence-based guidelines for the use of immunologic tests: anticentromere, Scl-70, and nucleolar antibodies. Arthritis Rheum Jun 15;49(3): Venables PJ. Mixed connective tissue disease. Lupus. 2006;15(3): Buyon JP, Clancy RM, Friedman DM. Cardiac manifestations of neonatal lupus erythematosus: guidelines to management, integrating clues from the bench and bedside. Nat Clin Pract Rheumatol Mar;5(3): Vergani D, Alvarez F, Bianchi FB, Cancado EL, Mackay IR, Manns MP, et al. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. J Hepatol Oct;41(4): Alvarez F, Berg PA, Bianchi FB, Bianchi L, Burroughs AK, Cancado EL, et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol Nov;31(5):