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Home , Lupus anticoagulant, Rheumatoid factor

LaboratoryLaboratory investigation investigation of rheumatic of and rheumatic connective tissue diseasesand connective tissue

Introduction Table 1 At the present time, tests for CCP 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 reported in numeric terms. The magnitude diseases has always complicated the task of of the level has not been reliably • Rheumatoid segregating disease entities. Some recent associated with clinical data. developments in immunopathology have • Systemic 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 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 organ damage – for example, the synovial • Fibrosing alveolitis This test was superseded by the ANA membrane in rheumatoid disease.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 • 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 • active SLE. Many different patterns of diagnosis and monitoring of disease activity • cellular staining can be seen using this – for example, CRP levels in rheumatoid • technique and may indicate the specificity of arthritis (RA), though they are less useful in the antibody (Table 3). Tests for antibodies systemic (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 (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 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 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 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 found in SLE, and rarely in polymyositis/ Anti-PM/Scl antibodies are not detected • Lymphoma and other malignancies 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 associated myositis. Anti-Jo-1 rarely occurs in most connective tissue diseases, but in patients with lung or skin disease without Penicillamine myositis. particularly in SLE and Sjögren’s syndrome. Moderate Anticonvulsants Sjögren’s syndrome – Anti-SSA is Antihistone Antibodies Hydantoins: phenytoin found in 25% of patients with secondary Antihistone antibodies are detected by line Succinimides: sicca syndrome, and in 80% with the immuno-assay and are useful in evaluating ethosuximide primary disease, often in association with patients who have SLE-like syndromes Low Antithyroid drugs hypergammaglobulinaemia and high-titre induced by various drugs (Table 4). Greater IgM RF. than 90% of such patients have antihistone Thiourea derivatives antibodies. As antihistone antibodies are Psychotropic drugs ANA-negative SLE – occasionally patients present with varying frequencies in other Phenothiazine with clinical features typical of SLE have autoimmune diseases, the test is only useful derivatives a negative ANA by HEp2. Now HEp2000 in excluding drug-induced SLE. cells are used, all active SLE patients will Undefined Oral contraceptive have a positive ANA screen. A hallmark of Anti-Proliferating Cell Nuclear Antigen agents (PCNA) Antibodies the previously called ANA negative SLE Oestrogen-progestin These antibodies are directed against the condition appears to be a photosensitive combination rash. Anti-SSA is found in this subset of SLE cyclin complex, which is involved in the Sulfonamides patients. regulation of cell division. They are found in Laboratory investigation of rheumatic and connective tissue diseases

Table 3 Specificity of defined ANA in various disorders

C – Centromere, CCD – Cell cycle dependent, Cyto – Cytoplasmic, H – Homogeneous, N – Nucleolar, R – Rim, S – Speckled * ANA – Anti-nuclear antibody ** Anti-ds DNA performed by Farr assay. *** > 90% of patients with drug-induced SLE have these antibodies. SLE – Systemic lupus erythematosus; RA – ; 1oSS – Primary Sjögren’s syndrome; MCTD – Mixed connective tissue disease; PSS – Progressive systemic sclerosis (scleroderma); CREST – Calcinosis, Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia; DM – Dermatomyositis; PM – Polymyositis.

OTHER USEFUL TESTS 3. Lupus and anticardiolipin activate complement pathways and cause and Anticardiolipin antibodies have been detected in a low inflammatory reactions. SLE patients with renal Antibodies percentage (< 15%) of otherwise healthy disease show consumption and glomerular Lupus anticoagulant and anticardiolipin women with a history of unexplained deposition of early complement components antibodies are closely related auto- recurrent abortion. Aspirin has been of the classical pathways, hence reduction antibodies that recognise similar shown to improve foetal outcome and of C3, C4 and CH50 levels and increased phospholipid antigenic determinants. Lupus thus all pregnant women with a history of levels of immune complexes. RA patients with anticoagulant acts as an inhibitor of the these antibodies should be referred for systemic vasculitis may show a similar pattern. specialist management, regardless of the prothrombinase complex which is required Anti-Neutrophil Cytoplasmic Antibodies presence of SLE. for conversion of prothrombin to . Multiple patterns of anti-neutrophil This inhibition prolongs in vitro clotting tests 4. Prospective studies of a series of patients cytoplasmic antibodies (ANCAs) for both intrinsic and extrinsic pathways. with SLE show those who have lupus are distinguishable by indirect Patients with these antibodies are artificially anticoagulant and anticardiolipin immunofluorescence techniques using split into 2 groups: antibodies, and a history of multiple patient serum. Classical (c-ANCA) and failed pregnancies, are at high risk of 1. Primary antiphospholipid syndrome: perinuclear (p-ANCA) antibodies differ in experiencing subsequent foetal loss. The Antibodies plus clinical events without their disease specificities and sensitivities. prognostic value of these antibodies in SLE and other related syndromes. Atypical ANCAs have many associations patients without a history of foetal loss has including sclerosing cholangitis, auto- 2. Secondary antiphospholipid syndrome: only recently begun to be assessed. immune liver disease and cystic fibrosis. Antibodies plus clinical events with SLE or HLA B27 related syndromes. All patients with vasculitis diagnosed with There are many reported associations Wegener’s granulomatosis (WG) (a variant of There is very little evidence that the between certain HLA antigens, especially necrotising vasculitis) will have ANCA, and in management of primary and secondary HLA B27, which is found in 8% of Caucasians, greater than 95% it will be c-ANCA. However, disease should be any different. and a number of diseases. 90% of patients to complicate matters, not all patients with Current opinions as to the significance of suffering from carry WG have vasculitis, and are often termed these auto-antibodies are as follows: the HLA B27 antigen. Although the chance of limited stage disease. In some patients, this HLA B27- positive individuals developing the 1. Approximately one-third of patients with may progress to systemic disease, though disease is 100 times that of HLA B27-negative SLE have one or both antibodies. Patients in many the disease remains limited to the individuals, the vast majority of positive with non-SLE disorders probably account initial organ. During acute presentation individuals will never develop ankylosing for more than half of lupus anticoagulant with c-ANCA, antibodies are directed at the spondylitis. HLA B27 is also associated with and anticardiolipin antibody positive proteinase 3 antigen (Anti-PR3). The level more prolonged reactive arthritis and recurrent persons. of antibody is dependent upon disease iritis. HLA B27 is associated with one of the activity, such that the degree of positivity 2. In patients with or without SLE or closely variants of psoriatic arthropathy. falls to < 70% in patients with only limited related disorders, a significant association Complement disease, and to 30 – 40% in those patients exists between lupus anticoagulant, The interaction of antibodies with specific who are in clinical remission. Presenting anticardiolipin antibodies and a history antigens to form immune complexes is a features include non-specific signs of of , neurologic disease and significant factor in the development of organ systemic illness with evidence of respiratory thrombocytopaenia. damage in rheumatic diseases. In excess, tract involvement. The latter is a consistent these complexes localise in blood vessels, feature of this particular illness, which may Laboratory investigation of rheumatic and connective tissue diseases

involve the entire respiratory tract from nose autoimmune disorders, including SLE and disease. In combination, the ANA Anti-ds to lungs. Other manifestations of systemic its variants. Other auto-antibodies involved DNA and anti-ENA tests are extremely disease are often present, particularly renal in the p-ANCA response include anti- helpful in the diagnosis of many of the involvement presenting as a necrotising elastase and antilactoferrin antibodies, and connective tissue diseases. A negative glomerulonephritis, inflammatory arthritis, these often give an atypical appearance. ANA, anti-ds DNA and anti-ENA excludes and hearing loss. The disease can be The atypical ANCA pattern is now used the diagnosis of SLE. Tests for antihistone rapidly progressive at any time, so early for the detection of auto-immune liver and antibodies are useful in excluding drug- referral to a specialist is advisable. In some biliary duct disease. Patients with classic induced SLE. Tests for lupus anticoagulant patients, fluctuating titres of c-ANCA may Wegener’s granulomatosis rarely have and anticardiolipin antibodies identify be related to the degree of disease activity p-ANCA. patients with thrombosis and recurrent and therefore may be useful in monitoring abortion. Presence of the HLA B27 antigen Testing for ANCA by immunofluorescence therapy. A rise in titre from a previous is important, but not essential, in the and ELISA (proteinase 3 and negative test almost always precedes a diagnosis of ankylosing spondylitis. Immune myeloperoxidase) remains a valuable disease flare. complexes and serum complement levels tool for the investigation of patients with are useful in the monitoring of subsets of PR3 antibodies can be seen in bacterial suspected necrotising vasculitis. As with SLE and RA patients with vasculitis. Testing endocarditis, possibly in association with all auto-antibody investigations, the clinical for ANCA is useful in the investigation renal failure. Immune suppression is not picture needs to be taken into consideration of patients with suggested necrotising required and is contraindicated in this when interpreting the results. vasculitis. illness. Conclusion p-ANCAs are less specific than c-ANCAs. Despite the lack of definitive tests for the This is, in part, due to the fact that p-ANCAs diagnosis of rheumatic and connective are not a homogeneous group of antibodies. tissue diseases, there are many pointers to The major antibody involved in p-ANCA their diagnosis. Rheumatoid factor assays, reactions is an antimyeloperoxidase; although moderately sensitive for RA, lack approximately 90% of p-ANCAs possess specificity, being found in many illnesses such antimyeloperoxidase antibody and normal subjects, though the recent activity. These antibodies are classically addition of CCP antibodies is a valuable tool. associated with a microscopic polyarteritis The ANA test remains a useful non-specific which may present with a necrotising screening test for many connective tissue glomerulonephritis. These antibodies are diseases. Elevated anti-ds DNA levels have one of the few proven to be pathogenic a high association with SLE and its disease to small blood vessels. They can also be activity. Anti-ENAs are useful in identifying found less frequently in patients with other subsets of patients with connective tissue

Dr Daman Langguth FRACP FRCPA Dr Daman Langguth trained in and immunology in Perth and Brisbane. Daman has particular expertise in the investigation of auto-immune disease, allergy, and immune deficiency. He is also involved in maintaining professional standards in immunopathology and general pathology through his membership of various committees. Dr Langguth is available for consultation. T: (07) 3377 8698 E: [email protected]

Dr Carl Kennedy FRACP FRCPA A graduate of the University of Queensland, Carl trained in Clinical Immunology and Allergy and Immunopathology at the Royal Brisbane Hospital and Princess Alexandra Hospital. In addition to his work in pathology, Carl practises as a consultant immunologist and allergist, which gives him personal insight into difficulties faced by his clinical colleagues in the diagnosis and management of allergy and autoimmune disorders. As a medical student and later a registrar under a Queensland Health rural scholarship Carl worked in many remote locations as far afield as Mt Isa, Roma, Childers, Bundaberg, Gympie and Gin Gin, which gave him an appreciation of the challenges faced by rural and remote practitioners. Dr Kennedy is available for consultation. T: (07) 3377 8698 E: [email protected]

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