With more than 80 autoimmune disorders identified so far, the scope of this diagnostic field is vast. Unfortunately, many parts of it are correspondingly vague, with multiple tests and many subjective interpretations often required before a diagnosis is reached. As a result, the cost of diagnosis may climb while patient satisfaction drops. Research-and-development efforts in this area are consistently productive, however, so more precise tests and instruments reach the market with some regularity. In addition, recent discoveries concerning the genes underlying autoimmune diseases and the cascade of events leading to signs and symptoms are fueling a strong push to develop whole new classes of autoimmune diagnostics for the future.

Autoimmune Disorders

Autoimmune disorders fall into two basic families, systemic and tissue specific, although a single patient may be subject to multiple disease entities within one family or in both. The patient’s course of illness also may proceed from a single set of signs and symptoms to another, either sequentially or, in overlap syndromes, simultaneously. Because of this broad variation in how an autoimmune problem is expressed in an individual over time, the most complete clinical picture possible always must be taken into consideration along with the results of laboratory testing.

The best-known members of the family of systemic autoimmune disorders are rheumatoid arthritis, systemic lupus erythematosus, Sjogren syndrome, autoimmune vasculitis, scleroderma, and mixed connective-tissue disease. Overlap syndromes also are described as systemic, since they typically involve rheumatoid arthritis or lupus.

Although any organ can theoretically be attacked by the body’s own immune defenses, the most common tissue-specific autoimmune disorders are myasthenia gravis, autoimmune thyroid disease (Graves and Hashimoto diseases), and skin disorders such as bullous pemphigoid. The endocrine, cardiovascular, and gastrointestinal systems also may be affected with some regularity, as can the blood, liver, kidney, lung, and brain.

Current Technologies

The Binding Site Ltd, Birmingham, United Kingdom, produces a broad range of enzyme immunoassay (EIA) and immunofluorescence assay (IFA) tests for the investigation of autoimmune vasculitides. These diseases have widely varying clinical presentations and are diagnostic challenges. They include the small-vessel vasculitides of Wegener granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome; these often were fatal before today’s diagnostic testing made the early administration of immunosuppressive agents possible.

Enzyme-linked immunosorbent assay (ELISA) kits for detecting antiphospholipid syndrome are among the clinical diagnostic products for autoimmune disease available from Corgenix Inc, Broomfield, Colo. The company also offers test kits for autoimmune-related vascular problems, such as bleeding and clotting disorders.

Kronus, Boise, Idaho, serves both clinical and research laboratories by offering test kits for autoimmune disorders, particularly type 1 diabetes, Addison disease, thyroid autoimmunity, and myasthenia gravis/Lambert-Eaton myasthenic syndrome. The company also provides detailed, complimentary technical education on autoimmune aspects of diabetes.

Incubation times that are complete as soon as the sample is dry are offered in a series of autoimmune screening panels requiring only very small amounts of whole blood or serum (International Immuno-Diagnostics, Foster City, Calif.

The EliA™ system, Phadia, Uppsala, Sweden, is a completely automated instrument for autoimmune testing that accepts bar-code, mainframe, or manual data input. Calibration is needed only monthly. Efficiency and flexibility are increased because various autoimmune tests can be part of the same run, and the instrument also can perform allergy testing. Primary or secondary tubes can be used, and serum and plasma are both acceptable.

Diamedix, Miami is the source of a diagnostic test for Wegener granulomatosis. This serum test, which can be performed either manually or using the Mago® Plus/APTUS® automated EIA processor, provides semiquantitative and qualitative detection of antibodies against the proteinase-3 antigen; these antibodies are found in 84% to 100% of patients with Wegener granulomatosis.

The PhD™ system from Bio-Rad Laboratories, Hercules, Calif, is designed to provide flexibility in autoimmune testing. Each workstation can be changed from EIA to IFA in 30 seconds, and a single computer can control one to 10 workstations, whether each is running EIA or IFA and using microplates or slides. One worklist, programmed manually or via bar-code scanner, can accommodate eight EIAs or four IFAs.

Biochip Mosaic™ human epithelial cells/primate liver from Euroimmun, Lübeck, Germany, are used in antinuclear antibody (ANA) screening for rheumatic diseases. The company notes that these cells permit the detection of many types of antibodies against cell nuclei. The liver component allows verification of results between substrates and helps in the establishment of the titer levels that are important in distinguishing between diagnostic and background findings.

Recent Innovations

Autoimmune gastritis, which can lead to pernicious anemia, involves serum autoantibodies against gastric parietal cells and a cobalamin-absorbing protein, intrinsic factor. As many as 50% to 70% of patients with pernicious anemia will exhibit antibodies to intrinsic factor, but many complications of the condition can be prevented or delayed through prompt diagnosis and treatment. For this reason, Immco Diagnostics, Buffalo, NY, introduced an improved intrinsic-factor antibody ELISA and a new gastric parietal-cell antibody ELISA in 2006.

The Access® line of immunoassay systems from Beckman Coulter, Fullerton, Calif, now has a complete, automated thyroid panel available due to the addition of a new immunoassay that identifies thyroperoxidase antibodies. The assay yields a quantitative determination of thyroperoxidase antibodies in serum and heparinized plasma. This helps clinicians determine whether thyroid dysfunction is autoimmune (Graves or Hashimoto disease) or not (goiter or other thyroid problems). A small sample can give results in as few as 30 minutes. The assay also can be performed using the company’s UniCel DxC 600i Synchron® Access clinical system, its newest integrated chemistry/immunoassay instrument.

Inova Diagnostics Inc, San Diego specializes in diagnostics for autoimmune disease. It now offers anticardiolipin kits with a proprietary wash buffer formulation that the company says will prevent the front-to-back drift that can occur when testing procedures are automated. The quality-control processes for the kits incorporate the Sapporo standards.

Wampole Laboratories, Princeton, NJ, a division of Inverness Medical, suggests a newer alternative to the IFA and ELISA for ANA testing in autoimmune diseases. The AtheNA Multi-Lyte® is a multiplexed, bead-based ANA test system that the company says can provide more consistent results than IFA or ELISA testing while reducing both direct costs and staff time.

DiaSorin (Saluggia, Italy) specializes in high-performance autoimmune diagnostics with a long shelf life. What the company describes as a complete line of IFA and EIA products emphasizes ready-to-use reagents and simple test procedures involving short incubation times. The EIA line is particularly broad in the areas of autoimmune gastroenterology, rheumatology, hematology, and vascular disorders. IFA autoimmunity training is available using the Multimedia Autoimmune Reference Image Assistant (MARIA™) software package, which incorporates a complete library of reference images accessed using a floating menu. Recently, DiaSorin introduced chemiluminescence assays for the diagnosis of autoimmune diseases using LIAISON® technology.

Laboratories helping to further the autoimmunity investigations now in progress in many academic and private-sector settings can take advantage of an extensive array of antibodies. For research purposes (not diagnostic use), Biodesign International®, Saco, Me, makes more than 4,000 antibodies and antigens available, including an expanded range of human autoimmune-related products. Animal and human crude cell extracts also are produced by the company.

On the Horizon

A huge research effort spanning decades has been dedicated to determining why T cells sometimes fail to recognize the self, creating autoimmune disease by attacking tissues instead of defending them. Investigators1 from the Dana-Farber Cancer Institute and the Whitehead Institute announced, in late January 2007, a series of discoveries that may change both diagnostic methods and therapies for autoimmune disorders. A set of genes associated with autoimmune disease has been identified; eventually, it could be possible to test for the presence of the genetic signature of autoimmunity instead of the many autoantibodies involved.

The regulatory T cells that prevent other T cells from attacking healthy tissue are no longer produced when a master gene regulator called Foxp3 does not function properly. The result is autoimmune disease. Using a newly developed DNA microarray technology that is not yet available commercially, the researchers located the 30 genes affected by Foxp3 after scanning the entire T-cell genome. Of these genes, one not previously suspected of affecting regulatory T cells, Ptpn22, was strongly associated with Graves disease, systemic lupus erythematosus, rheumatoid arthritis, and type 1 diabetes.

A few months earlier, researchers2 had found another piece of the autoimmunity puzzle by detecting the stepwise process through which the body begins to damage itself in a murine model. The distribution of immunoglobulin G (IgG) subclasses appears, based on this work, to have a profound effect on autoimmune disease. Each IgG subclass binds to a particular Fc receptor on the T cell’s surface, and the strength of an IgG subclass varies according to which Fc receptors it engages. Various autoimmune diseases, particularly systemic lupus erythematosus and Goodpasture disease, are identifiable because a specific distortion of the IgG subclass ratio is present.

Once immune cells, early in their lives, start reacting to DNA, a three-step process is set in motion. The chemical pathway called toll-like receptor 9 causes signaling by an adaptor protein (MyD88) and transcription factor (T-bet); this forces antibodies to switch to the more harmful IgG subclasses. Among the worst is IgG2b, which binds to an Fc receptor that activates T cells and causes inflammation. In mice, administering the IgG subclasses in their healthy ratio, in blood, halts autoimmune disease.

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Eventually, this work may lead to a new form of diagnostic testing for autoimmune disorders: detection of the proportions of IgG subclasses represented in a sample. Refinements of such a technique could make it possible to determine not only that autoimmune disease is present, but which organs are most likely to be affected. If the ratio of Fc receptors on T cells can be changed, inflammatory symptoms could even be prevented. For laboratorians, clinicians, and patients, the ability to move directly from firm diagnosis to definitive treatment of autoimmune disorders in this way will be welcome news.

Kris Kyes is technical editor of  CLP. For more information, contact .

References

  1. EurekAlert. Cracking open the black box of autoimmune disease. Available at: www.eurekalert.org/pub_releases/2007-01/wifb-cot011607.php. Accessed January 22, 2007.
  2. Kaneko Y, Nimmerjahn F, Madaio MP, Ravetch JV. Pathology and protection in nephrotoxic nephritis is determined by selective engagement of specific Fc receptors. J Exp Med. 2006;203:789-797.