Systemic Connective Tissue Diseases - Screening
If systemic autoimmune disease is being considered as a clinical diagnosis, screening for antinuclear antibodies (ANA), rheumatoid factor (RF), and raised immunoglobulins can be useful, although it should usually be possible to perform more directed investigations base on the clinical findings.
ANA are detected on the Bioplex 2200 analyser using a multiplex flow based immunoassay. This assay can simultaneously detect the presence of autoantibodies to the following autoantigen specificities: DNA, Ro (SS-A) 60, Ro (SS-A) 52, La (SS-B), Sm, SmRNP, RNP-68kD, Scl-70, Jo-1, centromere (CENP-B), chromatin and ribosomal P. Different ANA specificities can be associated with certain connective tissue diseases.
The majority of patients with rheumatoid arthritis will be positive for Rheumatoid Factor. High titre RF is indicative of a likely poor prognosis. Repeat testing of RF has no role in patient monitoring. Levels of complement components C3 and C4 are usually raised in RA, as is CRP, indicative of an acute phase response. Falling levels of C3 and C4 may indicate rheumatoid vasculitis. Although rheumatoid factor is often taken as an indicator of RA it is raised in 15% of the population without RA following chronic inflammation or infection and is not raised in 15% of adult RA, 95% of juvenile RA. In a patients where there is a strong suspicion of RA but negative for rheumatoid factor consider measuring anti-cyclic citrullinated peptide (CCP) antibodies.
Systemic Lupus Erythematosus
Where SLE is suspected, ANA, DNA binding, antibodies to ENAs, antibodies to Cardiolipin, C3, C4, CRP and immunoglobulin levels could be requested at presentation. When diagnosis is confirmed, monitoring should include measurement of C3, C4 and CRP at each visit with intermittent measurement of ANA and anti-dsDNA Ab. Since the half-life of antibodies is 3 weeks, serial measurement of antibodies at weekly or fortnightly intervals is unhelpful.
There may be considerable overlap of Sjogren’s syndrome with other autoimmune disorders, including SLE. Characteristically antinuclear antibodies and antibodies to extractable nuclear antigens (particularly antibodies to Ro or SSA, and antibodies to La or SSB) are found. Rheumatoid factor and raised immunoglobulins may be found.
Scleroderma / systemic sclerosis
The pattern of antinuclear antibody may help define this group (e.g. presence of anti-centromere antibody associated with the CREST syndrome). Other antibodies to extractable nuclear antigens (particularly antibodies to Scl-70) may be found.
Polymyositis / dermatomyositis
Antinuclear antibodies are common and antibodies to extractable nuclear antigens (particularly antibodies to Jo-1) are seen in >30% patients, especially those with pulmonary fibrosis. Other ENA antibodies may be found such as Pm-Scl found in those with overlap features with Scleroderma.
Mixed connective tissue disease
The presence of autoantibodies to antinuclear antibodies and to extractable nuclear antigens (particularly antibodies to RNP), without other lupus markers, may support this clinical diagnosis.
Primary anti-phospholipid antibody syndrome
Recurrent thrombosis (or fetal loss) may be associated with antibodies to phospholipids including cardiolipin and beta-2-glycoprotein I. Related anti-phospholipid antibodies include the lupus anticoagulant. Anti-cardiolipin antibodies may be found in other autoimmune disorders, particularly SLE. Repeat testing after a three monthly interval is recommended to confirm the diagnosis in appropriate clinical settings. Coagulation investigations (ordered from haematology) are also useful in diagnosis.
Measurement of ANA, ANCA, C3, C4, CRP, RF, Immunoglobulins and Cryoglobulins may be useful in establishing a diagnosis, in conjunction with a tissue diagnosis. In patients with active untreated Wegener's granulomatosis (granulomatous polyangitis (GPA), c-ANCA, specific for PR3, is present in 90% of cases. p-ANCA, specific for MPO occurs commonly in microscopic polyarteritis (MPA), idiopathic pauci-immune glomerulonephritis and in a few patients with Wegener's. Positive ANCA, NOT directed toward PR3 or MPO, are also present in a range of other autoimmune diseases as well e.g. SLE, RA, Ulcerative colitis. ANCA specific for MPO and PR3 provide good disease specificity. The ANCA and MPO/PR3 result should be interpreted together.
Investigation of Renal failure
The immunology laboratory can assist in establishing whether renal failure has an immunological cause, most commonly as part of a systemic vasculitis or in myeloma. The measurement of ANA, C3, C4, CRP, ANCA, Anti-GBM, Cryoglobulins, Serum immunoglobulins and electrophoresis, Urine electrophoresis may provide diagnostic information.
Autoimmne hepatitis AIH and Primary biliary Cirrhosis (PBC)
PBC and AIH are associated with characteristic autoantibodies that are helpful in classifying the hepatitis and separating autoimmune chronic active hepatitis from the other forms. Patterns may include antibodies to smooth muscle (actin), reported as SMA-t in type I AIH, liver/kidney microsome antibodies in LKM-positive autoimmune hepatitis (type II AIH) and antibodies to mitochondria in PBC. Presence of autoantibodies does not exclude a viral cause for the hepatitis. Because of the overlap between the various different forms of hepatitis it is usually best to test for all the types of autoantibody - AMA, SMA, LKM and ANA. The profound disturbance in immune regulation, and in the normal processing of gut derived antigens will lead to characteristic changes in the levels of IgG, IgA and IgM. Serum electrophoresis may reveal lack of alpha 1 antitrypsin if this is associated with the cirrhosis. Primary sclerosing cholangitis has no definitive serological markers, but may be associated with ANCA (anti-neutrophil cytoplasmic antibodies) or ANA or SMA.
Coeliac disease (Gluten sensitive enteropathy)
The immune response in coeliac disease is directed towards epitopes formed between tissue transglutaminase (tTG) and gliadin (the alcohol soluble fraction of gluten). IgA antibodies to tissue transglutaminase (antibodies to endomysium) are found in active disease, and can be used to monitor compliance with treatment. Similar antibodies are seen in dermatitis herpetiformis. Anti-tTG Aantibodies are now detected using the Bioplex 2200 analyser. This method includes an IgA verification bead. If this fails, IgA will be measured to ensure that IgA deficiency (particularly common in these patients) is not causing a false negative result. All positive anti-tTG Ab are confirmed by detecting anti-endomysial antibodies by indirect immunofluorescence. This method is more specific, but less sensitive, than the anti-tTG antibody assay.