Laboratory Investigation of Rheumatic and Connective Tissue Diseases
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LaboratoryLaboratory investigation investigation of rheumatic of and rheumatic connective tissue diseasesand connective tissue diseases Introduction Table 1 At the present time, tests for CCP antibodies The overlap of clinical features in many Diseases commonly associated with RF are performed via an ELISA-based test and of the rheumatic and connective tissue Connective Tissue Disease reported in numeric terms. The magnitude diseases has always complicated the task of of the antibody level has not been reliably • Rheumatoid arthritis segregating disease entities. Some recent associated with clinical data. developments in immunopathology have • Systemic lupus erythematosus (SLE) Antinuclear antibodies, antibodies to extractable helped refine the process, though clinical • Sjögren’s syndrome nuclear antigens, and anti-double-stranded DNA assessment remains the cornerstone of • Scleroderma antibodies diagnosis. • Mixed connective tissue disease Auto-antibodies to a variety of cellular General Principles components are a frequent finding in Abnormal immunological activity forms Other Immunological Diseases (associated connective tissue diseases. Antinuclear the basis of the autoimmune disease with hyperglobulinaemia) antibodies (ANA) were first detected when process, the end result of which is target • Chronic active hepatitis the LE cell phenomenon was discovered. organ damage – for example, the synovial • Fibrosing alveolitis This test was superseded by the ANA membrane in rheumatoid disease. Most • Myelomatosis indirect immunofluorescence test (IIF). laboratory investigations are based on the Human epithelial cell line (HEp2000) cells detection of antibody, largely surrogate • Cryoglobulinaemia are used for screening for ANA. This is a markers of tissue damage, analogous • Sarcoidosis modification of the previous HEp2-based to high blood pressure and vascular ANA test so that SSA is also detected. Thus, disease. As well as antibody detection, Chronic Infections with this test, a negative result excludes basic investigations are also useful in the • Infective endocarditis active SLE. Many different patterns of diagnosis and monitoring of disease activity • Tuberculosis cellular staining can be seen using this – for example, CRP levels in rheumatoid • Syphilis technique and may indicate the specificity of arthritis (RA), though they are less useful in the antibody (Table 3). Tests for antibodies systemic lupus erythematosus (SLE). • Brucellosis to extractable nuclear antigen (anti-ENA) • Parasitic infections attempt to further identify these antibodies. INVESTIGATIONS Acute viral infections and post vaccination Seven anti-ENAs are routinely tested for Rheumatoid Factor (RF) by ELISA — anti-RNP, anti-SSA(Ro), anti- Neoplastic disease (especially after Rheumatoid factors are auto-antibodies Ro52, anti-SSB(La), anti-Sm, anti-Scl-70, and irradiation or chemotherapy). (usually IgM, though they can be IgG anti-Jo-1. A different method (Line immune- and IgA) directed against antigenic assay) is used to confirm results and to determinants on the Fc portion of IgG. Cyclic citrullinated peptide (CCP)antibodies detect anti-PM-Scl antibodies if specifically Agglutination tests using IgG-coated Over the past five years, the importance of requested. Anti-double-stranded DNA particles (latex or erythrocytes) form the antibodies directed against citrullinated antibodies (anti-ds DNA) are assayed by basis of most diagnostic tests. Sullivan peptides in the development of RA has FEIA and radioimmunoassay (Farr assay) if Nicolaides Pathology uses a quantitative allowed the subsequent production of requested. latex immunoturbidometric assay for the commercial assays. Citrulline is a non- detection of RF. standard amino acid formed during Interpretation Standard testing for ANA provides a rapid Interpretation cellular aging. CCP antibodies are present screening for SLE and related connective RF assays (particularly latex assays) are not in a significant number (up to 70%) of RA tissue disorders. A low titre (40-160) ANA specific for RA. RF may be found in a variety patients. The antibodies may be present in may have little or no clinical relevance, of acute and chronic inflammatory diseases those with a negative rheumatoid factor, occurring in a wide range of diseases (Table (Table 1), most of which are associated with and are useful in combination with the RF 2), as well as in a low percentage of normal a broadly elevated level of gamma globulin. assay. The antibodies are termed cyclic individuals, particularly the elderly and the RFs are also found in low titre (5%) in citrullinated as this is the form of the antigen pregnant. The ANA is positive, however, normal individuals, with titre and incidence used in the test; as the name suggests, these in 100% of patients with active SLE, in up increasing with age. Positive RF is, however, antibodies are directed against multiple to 90% of patients with RA, and in 50% of a highly sensitive diagnostic tool for RA, with citrullinated peptides. CCP antibodies are patients with scleroderma. 80% of RA patients having positive results, highly specific, though they are seen in though only 50% will be positive at time of psoriatic arthritis and in some acute viral diagnosis. Thus the frequency of positive seroconversions (EBV, CMV etc.). results increases with the duration of the disease. Laboratory investigation of rheumatic and connective tissue diseases Table 2 Neonatal SLE – Placental transfer of anti- SLE and in patients with chronic hepatitis B Causes of positive immunofluorescence for SSA from mother to foetus may result in a or C. In SLE, they have been associated with ANA transient lupus-like syndrome. This may glomerulonephritis. cause congenital heart block and thus • Connective tissue diseases all pregnant women with SSA antibodies Anti-PM/Scl Antibodies • Chronic liver diseases should be referred for specialist pregnancy These antibodies are directed against • Organ-specific autoimmune diseases: followup. a group of proteins collectively called the exosome, which is critical in RNA Pernicious anaemia Anti-RNP production. They are present in a subset of Hashimoto’s thyroiditis This antibody is present in many of the patients who have both scleroderma and Myasthenia gravis connective tissue diseases. The absence inflammatory myositis (polymyositis), and in Fibrosing alveolitis of anti-RNP virtually excludes the diagnosis around 5% of patients with either disease. of ‘classic’ MCTD. Anti-RNP is also • Chronic tuberculosis and leprosy found in SLE, and rarely in polymyositis/ Anti-PM/Scl antibodies are not detected • Lymphoma and other malignancies dermatomyositis and scleroderma. by the screening ELISA assay and must be • Old age (> 60 years) (low titre) specifically requested. They are present only Anticentromere Antibodies and Anti-Scl-70 if the ANA has a nucleolar pattern together • Pregnancy (low titre) Anticentromere antibodies are usually with a fine speckled or homogeneous associated with the CREST syndrome and nuclear pattern at high titre. Six distinct patterns of nuclear staining occur in low frequency in SLE, MCTD, may be seen using the ANA technique and scleroderma. Anti-Scl-70 is found DFS-70 Antibodies and may indicate the origin of the antigen in scleroderma and at low frequency in Dense fine speckled antibodies are involved. They are homogeneous, speckled, the CREST syndrome. Seen rarely in SLE identified based on their immunoflouresence nucleolar, rim and centromere and dense where it is associated with pulmonary and can be confirmed by ENA. The presence fine speckled. hypertension. of DFS-70 alone represents a low risk of an • Homogeneous and rim patterns are underlying connective tissue disease. usually associated with anti-ds DNA and Anti-Sm antihistone antibodies. Anti-Sm is a highly specific marker for SLE, Anti-Ribosomal Antibodies • Speckled patterns may be associated though it occurs in only 5% of SLE patients. Anti-ribosomal antibodies are present in with anti-RNP, anti-Sm, anti-SSA and There is no agreement on associations SLE, though they are not common (5% or anti-Scl-70. between anti-Sm and various disease less). Some have associated their presence features (e.g. CNS involvement and • Nucleolar antibodies may be associated with neuropsychiatric complications. They Raynaud’s phenomenon). with anti-Scl-70. are screened for by ANA with a cytoplasmic pattern combined with nucleolar pattern Anti-SSB • Dense fine speckled is associated with suggesting their presence. Confirmation is These antibodies occur with high frequency DFS-70. by line immuno-assay. in primary Sjögren’s syndrome, but can also COMMENTS be found in SLE and RA. Their presence Anti-ds DNA together with anti-SSA antibodies increases Table 4 Anti-ds DNA is moderately specific for SLE the risk of neonatal lupus syndromes. Drugs implicated in drug-induced SLE — 71% of patients with anti-ds DNA have Anti-Jo-1 Degree of Risk Agent been found to have SLE. The incidence rises Anti-Jo-1 are specific for a subset of High Antihypertensives to 82% five years after first detection of anti- patients with myositis and mild overlap Hydralazine ds DNA. Anti-ds DNA levels often correlate features (Raynaud’s phenomenon, sicca with disease activity. A rise in titre may syndrome), and who have the complications High Antiarrhythmics predict disease flare. of interstitial lung disease. It has also been Procainamide Anti-SSA described in patients with malignancy- High Chelating agents Anti-SSA is found with varying frequencies