|New Detection Tools for Autoimmune Disease|
|Friday, 02 July 2010 10:44|
More than 50 million people suffer from at least one autoimmune disease, and that number is expected to grow as the baby boomers creep into their sixties and beyond; autoimmunity is more prevalent in an aging population. The laboratory's role is becoming increasingly important in diagnosing these diseases featuring immunity systems attacking the body's own cells.
Autoimmune disease may be classified as organ-specific (e.g., thyroid, adrenals, pancreas, etc.) or systemic, which produce autoantibodies routinely guiding diagnosis and clinical decision making. In systemic lupus erythematosus (SLE), Sjögren's syndrome, rheumatoid arthritis and mixed connective tissue disease, detection of autoantibodies is key to diagnosis.
"The role of autoantibody testing today has become important in many autoimmune diseases," said Marvin J. Fritzler, MD, PhD, professor of Medicine, chair of the Alberta Research and Innovation Authority, department of Biochemistry and Molecular Biology, Faculty of Medicine, Calgary, Canada. "Historically, it has been used in only a few diseases but the list is expanding quickly."
In the past, diagnosis relied heavily on indirect immunofluorescence assays utilizing a HEp-2 cell line in which cells are incubated with patient samples and stained with immunofluorescent markers to detect nuclear and cytoplasmic autoantibodies, said Toni Clinton, PhD, vice president, Laboratory Operations, American Esoteric Laboratories (a division of Sonic Healthcare USA). She said the technique is time consuming, requires a high level of technologist training and yields sensitive but less specific results.
Newer methods include multiplexing-mixing color-coded autoantigen-coated beads with a patient's sample and fluorescent dye in a flow cytometer to analyze patterns. For about 7 years, Steven L. Mendelsohn, MD, PhD, president of Mountain Regional Arthritis Center, Asheville, NC, has been using the AtheNA Multi-Lyte Test System from Inverness Medical Innovations Inc., enabling him to look at 10 antibodies on one sample. He checks for rheumatoid arthritis and vasculitis.
Enzyme immunoassays (EIAs)--also called enzyme-linked immunosorbent assays (ELISA)--screen for antibodies to double-stranded DNA. Though a higher throughput method, EIA uses a limited number of antigens and produces more false positives and negatives, according to Hal Scofield, MD, associate member, Arthritis and Immunology Program, Oklahoma Medical Research Foundation, professor, department of Medicine, University of Oklahoma Health Sciences Center, and dean of Clinical and Translational Research, University of Oklahoma College of Medicine, Oklahoma City.
Dr. Scofield explained many people with SLE, for example, have antibodies that don't react with traditional antigens: "You've got to know that. And you'll miss [the disease] if you don't do immunofluorescence."
Antibody-specific assays now diagnose the most prevalent autoimmune disorders. For rheumatoid arthritis, antibody to cyclic citrullinated peptides (CCP) is widely used and offers high sensitivity and specificity. For SLE, double-stranded DNA antibody to Smith antigen as well as ribosomal-P are highly specific. (Dr. Fritzler pointed out that double-stranded DNA antibody tests are more complex and difficult to perform because nucleic acid, as opposed to protein, serves as the analyte.) For scleroderma, antibody to centromere proteins, topoisomerase 1 and RNA polymerase III identify patients with emerging or full-blown scleroderma.
In deciding what test method to use, Dr. Clinton relies on physician input. For a quick screen to determine if a patient is at risk for autoimmune disease, her lab defaults to EIA.
"If it's positive, we'll report that, and often the physician will call back and say, 'OK, I want to test for whatever the specific antigens are that would correspond with that patient's symptoms and other clinical criteria.'"
When selecting assay kits, Dr. Fritzler looks for manufacturers consistently producing accurate results. He also checks to make sure analytes perform reliably in terms of sensitivity and specificity or are "at least operating in the range in which you would expect them to."
He mentioned INOVA Diagnostics Inc. (part of the Werfen Group), Bio-Rad Laboratories and Immuno Concepts as companies with significant market share. Dr. Clinton added Inverness to the list.
"We use Immuno Concepts, INOVA and products from two companies in Europe--one called EUROIMMUN US and Mikrogen," Dr. Fritzler said. "And we're having a serious look at Bio-Rad and Phadia."
Additionally, KRONUS Inc. provides a number of antibody-specific kits for diagnosing autoimmune diseases. And HYCOR offers a broad menu of FDA-cleared ELISA assays for autoantibodies involved in systemic and organ-specific diseases.
Dr. Mendelsohn looks forward to one day being able to do a single panel for as many as 100 autoantibodies. "As we learn more of these diseases, we're realizing there were more than we ever thought," he said.
He's also excited about the possibility for fewer false-positive results and less patient anxiety. "As this testing becomes more available, particularly with this kind of multichannel testing all at one time, we'll be able to do that," Dr. Mendelsohn added. "I think that's better patient care, it's more cost effective and more accurate for the doctor to be more definitive about the diagnosis."
In emerging technology, novel autoantigens have led to new diagnostic assays not commercially available at this time. Dr. Fritzler's lab offers autoantibodies to GW182 and other components of structures known as processing bodies (P-bodies)--sites of mRNA degradation and post-transcriptional gene silencing.
The autoantibodies are related to a condition called Sjögren's syndrome-a chronic autoimmune disease characterized by an immune attack on moisture-producing glands. "So that's the newest kid on the block," he said. "I believe we're the only lab in the world offering that diagnostic assay."
Promise of Biomarkers
As the number of patients with autoimmune diseases increases, the most promising studies may be those showing that biomarkers--including autoantibodies--can predict diseases before symptoms are present.
A landmark study published in 2003 by a group at the University of Oklahoma tested archived sera from U.S. armed services recruits over time and found autoantibody markers in SLE predicted individuals who would develop the disease.
More studies need to be done, but Dr. Fritzler said, "slowly, the literature is adding more evidence to the idea that autoantibodies can actually predict the onset of disease." Taking this notion a step further, Dr. Scofield, who coauthored the 2003 study, said, "If you study [autoantibodies] in large numbers of people, you might be able to say, 'Hey, we found some people who are going to get lupus. Can we prevent it?'"
Source: Hoffman, J. (2010), "New Detection Tools for Autoimmune Disease: New testing assays and biomarkers are improving diagnosis for patients", Advance. Can be viewed online here.