Autoantibody testing is available for the diagnosis of many different autoimmune conditions. In the last decade, many new more specific and sensitive assays have been developed. Multiplexing and array tests are replacing the traditional enzyme-linked (ELISA) tests for antinuclear antibodies (ANA). Genetic profiles or haplotypes, along with antibody tests, are also being used to determine the risk of autoimmune disease in type 1 diabetes.
Autoimmune Diseases
Autoimmune diseases are on the rise, with more than 80 diseases considered autoimmune. Three of the most common autoimmune diseases are hypothyroidism (Hashimoto’s thyroiditis and autoimmune atrophic thyroiditis), rheumatoid arthritis, and Sjogren’s syndrome. According to Steven Binder, Director of Bio-Rad Laboratories, because symptoms vary in severity in these common disorders, mild cases of these three disorders are frequently not diagnosed.
Binder explains that less common but more severe diseases such as systemic lupus, multiple sclerosis, and type 1 diabetes tend to present with more acute symptoms. Consequently, these conditions are more likely to be encountered in laboratory testing.
Autoantibodies
Autoantibodies are antibodies produced by the immune system that target the body’s own organs and cells. Specific tests for thyroid antibodies are used to diagnose autoimmune thyroid disorders whereas tests for rheumatoid factor are used to diagnose rheumatoid arthritis. Other specific autoantibody tests include tests for platelet antibodies to diagnose idiopathic thrombocytopenic purpura and the Direct Coombs test used for autoimmune hemolytic anemia. ANA tests are used to rule out or help with the diagnosis of connective tissue disorders, such as systemic lupus erythematosus.
ANA Testing
In many regions, ELISA tests have been the mainstay for detecting ANAs. However, HEp-2 extracts used in these tests offer reduced specificity. Indirect immunflourescent (IIF) methods have also been used and offer the detection of a large number of autoantibodies that bind to a variety of nuclear antigens from nearly 100 different protein antigens.
Because of test interferences, immunfluorescent (IFA) tests were introduced in the 1990s. However, they’re no longer commonly used because they can be difficult to interpret and are hard tests to automate. Today’s ANA tests typically rely on recombinant protein antigens, which react with antibodies in the patient’s serum. Some immunologists question the use of recombinant proteins since they differ from human antigens. In these tests, a positive ANA screen and its pattern lead to further laboratory investigations such as tests for anti-dsDNA antibodies or anti-extractable nuclear antigens (anti-ENA) antibodies.
Modern ANA tests include multiplex testing, which detects reactions against up to 16 different protein antigens. Multiplex tests have been available for about 5 years. Here, distinct protein targets are combined and antibodies to these proteins are detected in the patient’s serum.
Multiplex systems utilize a new approach for the simultaneous measurement of autoantibodies based on multiple-dyed (fluorescent) beads coated with specific antigens and flow cytometry detection. Multiplex techniques enhance the testing process and are able to measure multiple antibodies. Multiplex ANA tests typically detect: dsDNA, Chromatin, Ribosomal protein, SS-A (52 and 60), SS-B, Sm, Sm/RNP, RNP (A and 68), Scl-70, Jo-1 and Centromere B antibodies.
With this method ANA screens are reported as positive if any of the 13 autoantibodies investigated is positive (above cut-off level), and this report will show the actual levels of individual analytes. Further confirmation can be made using the IFA method.
Comparing ANA Methods
In one small study of pediatric patients, the overall concordance between multiplex and ELISA for dsDNA was 89%. The researchers concluded that multiplex ANA testing is efficient and can provide more information than IFA method and individual ENA analysis. In addition, multiplex ANA decreases the false-positive rate of IFA ANA screening.
The American College of Rheumatology recognizes that the new multiplex procedures are more cost effective but still considers the IFA test for ANA the gold standard.
Insurance Reimbursement Problems
A problem lies in the restrictive reimbursement of multiple tests mandated by insurance providers. Various insurers are reluctant to pay for tests that may not be needed. Originating with Medicare Services, insurance providers do not typically pay for panels. Their reasoning is that this is a form of screening, rather than definitive diagnostic testing or running one test at a time.
Genetic Tests
Dr. Noel Rose, Directory of Immunology at Johns Hopkins, predicts that tests for HLA haplotype will be used along with antibody tests as a diagnostic aid. This has been the case for years with HLA B27 tests performed in suspected cases of ankylosing spondylitis and Reiter’s disease. Rose predicts that testing for genetic haplotypes associated with type1 diabetes along with tests for insulin antibodies will be regularly used to identify potential cases of childhood diabetes before symptoms occur.
Resources:
Todd Smith, Developments in Autoantibody Detection, Advance for Medical Laboratory Professionals, June 15, 2009: 11-15.
Barbara Roberts, Min Xu, Barbara Busby, Evaluation of Multiplex Antinuclear Antibody Assay in Pediatric Patients, Medscape Today, Jan 2008.
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