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Immunology

Connective Tissue Disease

Current approach

Since there are many types of auto-immune (AI) disorders, some organ specific (for example, thyroid gland) and some generalised involving multiple organs (for example, systemic lupus erythromatosis [SLE]), early symptoms may be non-specific (fever, muscle aches and malaise). These variations in clinical presentation, along with patients seeking help from different types of doctors, leads to a non-standardised diagnostic approach and suboptimal use of laboratory resources.

At present, Lancet conducts an anti-nuclear factor (ANF) test as a connective tissue disease (CTD) screen. This is a manual test using an indirect fluorescent assay (IFA) technique with HEp-2 cells. Although the specificity of the IFA ANF test makes it a useful confirmatory test, it is not a suitable screening test. Reasons why an ANF test should not be used as a screening test include the following:

  • There are multiple auto-antibodies that may be detected by this method, many of which are not clinically significant. This leads to some uncertainty regarding interpretation as there may be low activity of auto-antibodies detected with the ANF that cannot be confirmed with an ENA ELISA screen that contains only 17 selected, clinically significant auto-antibodies.
  • Confusion often arises regarding the interpretation of ANF titres. Negative ANF's do not rule out auto-immune disease and only ANF titres > 1:160 are considered “positive”. Unfortunately almost 20 to 30% of healthy individuals will have low ANF levels, especially with increasing age, and further testing is not indicated.

From a laboratory perspective, IFA ANF tests are also not ideal for the following reasons:

  • They need to be interpreted by experienced operators.
  • Their interpretation is subjective.
  • Results are difficult to reproduce between different observers.
  • IFA slides fade quickly and cannot be stored for checks or audit purposes.

New approach

To rationalise the diagnosis of Connective Tissue Disease (CTD), a new laboratory algorithm will be introduced during May 2012. Using this approach, a sensitive screening ELISA test will be used to detect the clinically relevant autoantibodies to various intracellular antigens (including DNA, enzymes and ribo-nucleosomes) that may be selectively expressed in the serum of individuals with CTD. Individuals identified as positive for auto-antibodies at screening will then have confirmatory testing to obtain the correct diagnosis.

The new Lancet CTD laboratory diagnostic algorithm (Figure 1) will streamline CTD disease diagnostics by screening with an automated sensitive ELISA, the CTD screen, for the following auto-antibodies in one test: dsDNA, Sm, Rib-P, U1RNP, Ro, La, CENP, Scl-70, PM-Scl, Fibrillarin, RNA Pol III, Jo-1, Mi-2, PCNA. If positive, a confirmatory IFA ANF and a confirmatory dsDNA test will be done.

Only then will specific markers be identified using confirmatory ELISA's for individual auto-antibodies (see Table 1) that assist in localising the disease process. Lancet plans to introduce additional tests on this automated ELISA platform to signifcantly decrease turnaround time to results. Future tests include those for the diagnosis of rheumatoid arthritis (Rheumatoid factor IgA and IgM, anti-CCP); faecal calprotectin (inflammatory bowel disease); antiphospholipid syndrome (cardiolipins and β2 microglobins); ANCA vasculitis (PR3, MPO, GMB) and coeliac disease gliadin IgA and IgG.

Figure 1. new Lancet CtC algorithm

connective-tissue-disease.gif

Table 1. Interpretation of CtD eLISa tests: prevalence of auto-antibodies in various CtDs

AUTO-ANTIBODYASSOCIATED CTDAUTO-ANTIBODY PREVALENCE (%)
Sm SLE 20-30
U1RNP MCTD, SLE 100, 30-40
RNP70 MCTD, SLE 75-95, 10-15
SS-A (Ro) Sjögren's, SLE 60-90, 40-50
SS-B (La) Systemic sclerosis, SLE 50-95, 6-15
Scl-70 Systemic sclerosis, SLE 30-60
CENP Limited systemic sclerosis (CREST)
Primary biliary sclerosis (PBC)
70-80
10-20
Jo-1 Poly-/dermatomyositis 25-35

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