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Laboratory Evaluation of Connective Tissue Disease

Dr. Ajay Wanchu, M.D., D.M

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Antibodies to DNA

Antibodies to double- stranded (ds) DNA are highly specific to SLE and correlate well with renal activity whereas antibodies to single stranded (ss) DNA are not specific to SLE and can occur in other connective tissue diseases too. Anti-dsDNA antibodies are useful in monitoring disease activity and optimizing immunosuppressive treatment. These are done by indirect immunofluorscent method using Crithidia lucillae, a non-pathogenic haemoflagellate, as substrate by a fluid phase radioimmunoassay (Farr assay) or by an ELISA method. The latter is conveniently done and there are several commercial kits available. Both by the radioimmunoassay and ELISA the results are expressed quantitatively, the upper limit of normal varying from kit to kit. A moderate to high level is seen characteristically in SLE and hence is of diagnosis value. The only other disease it maybe elevated is chronic active hepatitis, but in the latter the tests by C. lucillae is often negative. Various antibodies seen in patients with SLE are shown in Table II.

Extractable Nuclear Antigens (ENA)

There have been significant advances in our understanding of the nature of nuclear antigens to which ANA are reacting. The diversity of antigens and their reactivity to autoantibodies have been also proved useful in the clinical setting. The initial clue that ANA are heterogeneous group of antibodies came from variance of patients of ANA observed. The common patterns observed are the homogenous, peripheral or rim, speckled and nucleolar (Table III). Homogenous pattern is given by sera containing antibodies to DNA and histones, the rim pattern is caused by antibodies to DNA and the speckled pattern due to reactivity with ribonuclear proteins antigens (RNP). The latter are small polypeptides associated with ribonucleic acid (RNA) distributed both in the nucleus as well as the cytoplasm but can be extracted by saline treatment of rabbit thymus and human spleens, hence called extractable nuclear antigens (ENA). Antibodies to ENA are heterogeneous too and are detected by immunoprecipitation in gels, Western blot and ELISA using pure antigens. The various anti-ENA that have been useful in clinical setting are shown in Table IV.

Anticardiolipin Antibodies

Cardiolipin is a phospholipid and an increase in IgG or IgM anticardiolipin antibodies occurs in up to 40% of patients with SLE where it is associated with the presence of recurrent mid term foetal loss, venous/arterial thrombosis and thrombocytopenia. It often occurs in young and middle aged females in the absence of features of SLE, when it is called primary antiphospholipid antibody syndrome. These autoantibodies have also been associated with a variety of clinical manifestations including migraine, focal neurological deficits, livedo reticularis, renal and portal vein thrombosis both in the setting of SLE or without it. Though its pathogenic role is still questionable its presence in high level requires consideration for anticoagulation therapy along with immunosuppressive measures.

Antineutrophil Cytoplasmic Antibodies (ANCA)

These have received a lot of attention in recent years because of the association with systemic vasculitic disorders such as Wegener’s granulomatosus, microscopic polyarthritis nodosa and crescentic glomerulonephritis. There are mainly two types of antibodies: those that give a diffuse cytoplasmic stain, c-ANCA (antigens is seine protease) and the perinuclear pattern, p-ANCA. Antibodies to c-ANCA are associated with WG (90% of sera positive) and polyarteritis nodosa (50%) whereas antibodies directed against p-ANCA are seen in patients with microscopic polyarteritis nodosa (50%), idiopathic crescentic glomerulonephritis (95%) and drug induced renal vasculitis (100%) and less commonly in RA (12%) and WG (5%). The target antigens are myeloperoxidase, lactoferrin elastase and other granule proteins. Monitoring their level also is useful in assessing disease activity and can be done quantitatively using an ELISA assay.

Complement Component C3 and C4

Complement component C3 and C4 measurement is done by single radial immunodiffusion method of Mancini and more accurately by turbidimetry. In disease like SLE level these are lowered and correlate with disease activity. Elevation of C3 levels is associated with infection.

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