The variability in serological testing for antinuclear antibodies (ANA) is under investigation after unexpected findings were reported from clinical trials of new agents to treat systemic lupus erythematosus (SLE).
In “Assay Variation in the Detection of Antinuclear Antibodies in the Sera of Patients with Established SLE,” David S. Pisetsky, MD, PhD, and his colleagues demonstrate variability in assays for identifying ANAs in patients with established SLE. First published online on March 27, 2018, in Annals of the Rheumatic Diseases, this study provides evidence that the performance characteristics of test kits can lead to assay variability.
The tests investigated included three immunofluorescence assays (IFA), an enzyme-linked immunosorbent assay (ELISA), and a bead-based multiplex assay. Study results may prompt some clinicians to consider how they interpret results of ANA assays and to adjust their testing protocols for patients with lupus.
The Research Rationale
“We had a very specific motivation for doing this study,” says lead author Dr. Pisetsky, professor of medicine and immunology at Duke University in Durham, N.C. “ANA testing has been around for a long time. The IFA for ANA determination has been identified as the so-called gold standard. But the reason to look in detail at the results from different ANA kits was the experience in clinical trials.”
One study in particular, a phase 2 trial of belimumab, failed to demonstrate statistically significant therapeutic effects of the drug in patients with established SLE. The unexpected results led the investigators to examine whether any subpopulations showed responses. The laboratory results they looked at included ANA determinations. Although ANA assays are believed to be positive in 95–99% of patients with lupus, surprisingly, they found 30% of the patients were serologically negative.
“The company went back, reanalyzed the data and saw efficacy in the people who were ANA positive,” Dr. Pisetsky explains. “In addition to ANA, anti-dsDNA [anti-double-stranded DNA] results were tested. When the company went forward in the phase 3 trial, it was for those patients who were ANA or anti-dsDNA positive. Other companies have followed this approach, and it’s not uncommon that they experience the same problem [with patients who have SLE demonstrating negative ANA results],” he says.
Because of the developments from the belimumab study and other clinical trials, “serological testing is being used as a companion diagnostic or theranostic biomarker although existing tests have not been validated for this purpose,” Dr. Pisetsky and colleagues wrote in their study. This nature of the enrollment criteria for clinical trials led them to investigate the variability of ANA test kits in patients with established SLE.
“The focus of our work was to figure out what this [variability] was about, because the general belief is that almost everyone with lupus is ANA positive,” Dr. Pisetsky says.
Study Results & Implications
Dr. Pisetsky and colleagues studied sera from 103 patients from an Ohio State University cohort with established SLE who were historically ANA positive. The investigators screened the patients’ sera using three commercially available kits for IFA ANA determinations, as well as with an ELISA and a bead-based multiplex assay.
Results indicated marked variation in ANA positivity depending on the assay platform and kit used. Of the three IFA kits, 4.9–22.3% of the samples tested negative. Similarly, 12 samples tested negative with the ELISA assay, and 14 tested negative with the multiplex assays.
A couple of years ago Dr. Pisetsky and a different group of researchers studied 181 patients enrolled in a clinical trial for a new agent to treat SLE and found a wide variation in ANA negativity using five kits (0.6–27.6%). They presented their results at the 2016 ACR/ARHP Annual Meeting.
These results have implications for the clinical trial setting because of how researchers screen and enroll patients in trials of new therapeutic agents. “But the study has implications beyond the clinical trial setting,” Dr. Pisetsky says. “One of the important messages is that the so-called gold standard [IFA test] really has limitations, and there are significant differences in the detection of antibodies by kits produced by different manufacturers. So, the frequency of ANA negativity can vary.”
Beyond that, two implications are significant for practicing rheumatologists. “First, increasingly, the ANA is being used as an important criterion for classification of lupus,” Dr. Pisetsky says, which assumes nearly everyone with lupus is ANA positive. However, if the kits used for ANA testing are limited in their detection, it’s possible some patients who have lupus will test negative and potentially be misdiagnosed. Practicing rheumatologists may consider exploring different ANA assays used for diagnosis and possibly adapting other protocols.
“Second, in general, people are tested for ANA only at diagnosis—on initial presentation—and they’re not tested subsequently because of the belief that the information is already there,” Dr. Pisetsky says. Some patients may be retested if they change providers or care settings, but routinely, retesting for ANA is not usual in patients with lupus. “This study suggests that there may be reasons to retest.”
Retesting could become important as clinicians and researchers continue to investigate how patients respond to different treatments. The current approach of using ANA results to determine eligibility for clinical trials suggests differences exist in the responsiveness of patients with lupus to treatment depending on ANA status, although this depends on which kit is used for the ANA assay, Dr. Pisetsky says.
Study results mean “you can’t make broad statements about ANA results because the kits are behaving so differently,” he says. “There are other assay formats available, including ELISA and multiplex, and, in the context of a clinical trial, if the presence of ANA is a determinant of eligibility, the company doing the study may want to do more than one test to screen patients. In the regular world of practice, there’s a balancing act that has to go on.”
Clinicians must balance their knowledge of testing protocols with their need to properly diagnose and treat their patients. IFAs are not simple tests—they are time consuming, results can be subjective, reproducibility can be limited, and they aren’t easily automated, Dr. Pisetsky says. In comparison, the ELISA and the multiplex assays are easier to perform in the laboratory. Still, no test is perfect, and it’s up to the provider to determine which test is the most appropriate given the particular patient’s situation. The IFA is still considered the gold-standard for ANA testing, despite the variability in the test kits.
“There are two approaches,” Dr. Pisetsky says. “One is to use the ELISA or multiplex for screening and then confirm it with IFA. Other people recommend the other direction: IFA first, and then do the multiplex to figure out which antibodies are present.”
Kimberly J. Retzlaff is a freelance medical journalist based in Denver.
- Pisetsky DS, Spencer DM, Lipsky PE, Rovin BH. Assay variation in the detection of antinuclear antibodies in the sera of patients with established SLE. Ann Rheum Dis. 2018 Jun;77(6):911–913.