Laboratory Evaluation of Connective Tissue Disease

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).

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.

C Reactive Protein (CRP)

It is one of the acute phase proteins whose value is raised in inflammatory arthritic condition such as RA, JCA and patients with seronegative spondyloarthopathy (SSA). Interestingly, in SLE though the ESR rises the CRP is normal in active disease but in presence of infection it CRP rises. This is measured by latex agglutination and also by turbidimetry and, therefore, more expensive than ESR.

HLA (Human Leukocyte Antigen)

Despite the strong association of HLA B27 with SSA group of disease, this does not have diagnostic significance. Therefore, in conclusion I have attempted to describe briefly about the various investigations that are currently being used for management of patients with connective tissue disease. They are very useful in making a diagnosis and following up a patient provided the limitations are kept in mind.

Table I: Prevalence of ANA positivity in various disorders

Table II: Spectrum of antibodies associated with Systemic lupus erythematosus

Table III: Staining Patterns of antinuclear antibodies on Indirect Immunoflourescence

Table IV: Diversity of anti-ENA antibodies and their clinical relevance

Dr. Ajay Wanchu, M.D., D.M
Department of Internal Medicine, PGIMER, Chandigarh

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