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Laboratory Evaluation of Connective Tissue Disease

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

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Laboratory investigations are helpful in the management of patients with connective tissues diseases as they help in establishing the diagnosis, assessing disease activity, organ damage and evaluate progression of disease. Apart from routine haemogram, blood chemistry and urinalysis several immunological tests are employed. Some of the latter tests are not routinely available and require an immunological set-up to perform. As awareness of these diseases grows it is hoped that such investigations will eventually be more freely available. It is important to know the indications and also the limitation of each investigation that will result in an appropriate request on the part of the treating physicians. Unnecessary requests for such investigations only defeat the purpose of getting the tests done. Besides there is cost implication too as some of the investigations are expensive.

In the practice of modern medicine, laboratory investigations are increasingly becoming an important factor in the diagnosis and management of diseases, particularly so in the field of rheumatology and clinical immunology. This unfortunately undermines the importance of physical examination and undue reliance is placed on slightly abnormal but more sophisticated investigation results. All immunological investigations, even the most recently available ones should be interpreted in the light of clinical picture and sole reliance on a particular abnormal result without proper clinical examination adds more confusion in sorting out a clinical problem. Moreover, abnormal investigation results alone should not be an indication of treatment.

Various investigations are needed for evaluating a patient with connective tissue diseases that include serum and other tissue fluid (synovial, pleural) investigations, imaging (radionuclide studies, radiography, CT and magnetic resonance imaging) and histological studies. I shall restrict to the investigations that are carried out in the immunological laboratory for connective tissue diseases. Thus synovial fluid, analysis, nuclear medicine and radiological investigations shall not be covered.

Rheumatoid Factor (RF)

This is one of the most frequently requested immunological investigations for managing patients with connective tissue diseases. This is based on the principle of agglutination of latex particle coated with human immunoglobulin G (IgG) with serum containing IgM antibodies directed against the Fc portion of IgG. It is available from commercial sources as a kit and does not need any expensive equipment. The result is expressed as the highest dilution of serum showing a positive agglutination (also called the titre). High titre positivity is suggestive of rheumatoid arthritis (RA) and primary Sjogren’s syndrome (SS). Also sera from patients with polyarticular type of juvenile chronic arthritis (JCA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc) are positive. A negative RF does not rule out RA, as up to 20-30% of patients is persistently seronegative. It is mostly negative in JCA (except in 20% of children with polyarticular type of disease) and always negative in the group of patients with seronegative spondyloarthopathy (ankylosing spondylitis, Reiter’s syndrome or reactive arthritis, psoriatic arthritis, enteropathic arthritis, etc.) Certain disease such as infective endocarditis, tuberculosis and leprosy can give rise to false positive rheumatoid factor.

A more sensitive assay for RF by enzyme linked immunosorbent assay (ELISA) is also becoming available. The results can be expressed in quantitative terms as well as the three isotypes of RF, namely IgM, IgG and IgA. There is evidence to suggest that the level of isotype of RF could be helpful as a prognostic parameter in assessing joint damage.

Antinuclear Antibodies (ANA)

ANA is one of the most important serological tests in the diagnosis of SLE. The LE cell test, which was earlier used, for the diagnosis of SLE is no longer required to be done, as ANA is far more sensitive. The latter test is done by immunofluorescence technique using rat tissues (liver, kidney or stomach) or cells lines such the HEp2 cells as substrate. Antibodies to nuclear antigens are allowed to react with the animal nuclei and anti-human Ig conjugated with a fluorescein compound and observed probes bound immunoglobulins by a fluorescence microscope. This is also a quantitative test and the results expressed in titre of sera dilution. I would not recommend the ANA testing by ELISA which is often promoted as more sensitive than the classical immunofluorscent assay.

It is 99% sensitive for the diagnosis of SLE and more than 90 out of 100 SLE patients will be positive for ANA. Patients with SLE who are on long term steroid treatment or with chronic renal failure can be negative. However, a negative test with a clinical diagnosis of SLE needs reassessment of the patient. ANA is positive in primary SS (990%), SSc (70%), Dermato/ polymyositis (20%) and a small subset of patients with RA and JCA. It is uniformly negative in all primary vasculitis (WG, PAN and hypersensitivity vasculitis) as well as in SSA (Table I).

Department of Internal Medicine, PGIMER, Chandigarh

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