October 2009: Volume 34, Number 10
2009 Annual Meeting Highlights
Challenges and Controversies in Anti-Nuclear Antibody Testing
Are Multiplex Assays the New Gold Standard?
By Genna Rollins
New methods have emerged for testing anti-nuclear antibodies (ANA), leading some labs to transition from traditional immunofluorescence (FANA) to EIA or solid phase multiplex assays based on flow cytometry. However, all the assays have pros and cons, making it imperative that labs know their methods well and educate physicians about the limitations of the various tests in diagnosing systemic autoimmune diseases such as systemic lupus erythematosus, Sjögren syndrome, and scleroderma. Particularly when the tests are used in the initial work-up of patients with suspected autoimmune disorders, “automated assays may be the way to go, but with educational comments that alert physicians that there might be situations where they want to reflex to other, more sensitive, less specific assays,” noted John L. Carey, MD, vice chair of clinical pathology at Henry Ford Health System in Detroit. Carey spoke as part of a three-person panel at the symposium, “Issues in Immunodiagnostics: Are ELISA ANA and Traditional Immunofluorescent ANA Results Comparable?”
Donald Bloch, MD, a rheumatologist and assistant professor of medicine at Harvard Medical School, agreed that close collaboration between labs and physicians is needed for physicians to make an accurate diagnosis of systemic autoimmune diseases. “Anyone may order this test—surgical interns, nurse practitioners, OB-Gyns, rheumatologists—and they all have different reasons for ordering it and different levels of expertise to deal with the result.” Bloch emphasized that an ANA test alone does not diagnose lupus or other systemic autoimmune disorders, although a positive result is one of 11 criteria used to determine whether a patient is eligible for participation in clinical trials. “In the beginning [of a work-up] all autoimmune diseases look the same. The ANA test is not a screening test for lupus, but the healthcare provider is looking for a little help,” he noted.
Even when providers understand the role of an ANA test in screening, there often is confusion about how to interpret results, according to Carey. “There is no perfect ANA screening test. You will have false negatives, even with FANA, but there have been issues with primary care providers running off with false positive and ill-defined results and in treating patients who don’t have autoimmune disease,” he said. Approximately 20% to 25% of normal, healthy adults will have positive ANA results, but not an autoimmune disease. Given the relatively rare prevalence of autoimmune disease, this false-positive rate translates into a poor positive-predictive value in a general population undergoing initial testing. “It would be nice if we had a test that predicted the entire range of autoimmune disorders, but what has developed is that ANA testing has a good negative-predictive value and therefore a rule-out role,” Carey observed.
What’s Behind the Result?
Just as clinicians need to understand the interpretation nuances of ANA testing, labs need to know the ins-and-outs of the tests they offer. The Hep-2 cells used as substrate in FANA assays contain more than 100 auto-antigens, but the various staining patterns, such as homogenous, speckled, and centromeric, can look similar in different diseases, posing challenges for labs in providing reliable results. “Many labs perform IFA very well, and they’re expert at reading the patterns and have more than one lab tech who can do it. They take steps to ensure consistency, but that’s not the case across the country,” according to Steven Binder, director of technology development at Bio-Rad Laboratories in Hercules, Calif. The relatively high percentage of positives found in healthy individuals is the greatest limitation of the method, he said. Labs can modify this somewhat by changing the dilution titers. “I prefer a 1/80 dilution, particularly in a mixed population where you’ll have a lot of clinically irrelevant positives,” Carey said. “It reduces by about half the number of positives you’ll see on non-autoimmune patients and has essentially the same clinical sensitivity.”
FANA is not alone in its limitations. Initial reports based on relatively modest sample sizes suggested that multiplex assays might be more sensitive than FANA, leading some labs to use multiplex tests as the frontline diagnostic, with FANA as a confirmatory test for positive results. This testing algorithm troubles Bloch. “Why are we doing this? Why change from Hep-2 cells with more than 100 antigens to solid phase assays with 13 or fewer antigens?” he asked. At least one other study produced contradictory findings that multiplex assays performed poorly in comparison to EIA. “Does this confirm that we should go back to FANA? No, but it does raise some concerns about the sensitivity of this assay format,” said Carey. The speakers agreed that multiplex assays need further validation in larger, more extensive clinical trials.
While EIA and multiplex assays offer the advantage of high-throughput and ease of operation, the different kits use different auto-antigens, which impact each test’s ability to detect particular antibodies. As an example, Binder pointed to anti-Ro/SSA 52 and SSA 60. “Sometimes the manufacturers use both, others use one or the other, so even a report of SSA(Ro) can be situational, and up until 2 years ago, the FDA didn’t allow SSA 52 and 60 to be reported separately,” he explained.
However, Carey suggested that using EIA as a screening test and FANA to confirm positive results by EIA is the dominant approach in use today. Carey and Binder agreed that when EIA ANA tests were first introduced they were not particularly robust, but the assays have improved over time.
Still, EIA kits “are not uniform in their performance,” and two sold in the U.S. do not contain Hep-2, according to Binder. “Have the labs that use those kits communicated that to their physicians?” he asked. Carey agreed that the variability of auto-antigens used in both EIA and multiplex assays require clear feedback to physicians. “Non-FANA tests with high through-put and reasonable sensitivity are a good choice. However, there should be educational material explaining that false negatives can occur, and there should be a list of specific antigens,” he suggested.
As the industry keeps an eye on improvements in both EIA and multiplex assays, Binder cautioned that FANA is not about to be put out to pasture. “I would never use the term ‘replacement’ for FANA. Just as some specimens will only be positive using a multiplex method, others will only be positive by FANA. I expect the two methods to co-exist for quite a long time."