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Diagnosing lupus: ANA testing comes under scrutiny

BY KERRY DOOLEY YOUNG

 

Researchers have found further evidence about how tests for systemic lupus erythematosus (SLE) can fail to detect certain cases, adding to a growing discussion about how to best diagnose this condition.

Authors of a new report say clinicians need to be aware of which approach their laboratory employs for routine antinuclear antibodies–indirect immunofluorescence assay (ANA-IIF) testing. Looking at results for 1,137 patients with lupus, they found shows that 1,049 (92.3%) were classified as positive for ANA, considered a hallmark for lupus diagnosis. But another 71 (6.2%) were anticellular antibody–negative, and 17 (1.5%) had isolated cytoplasmic and mitotic cell patterns (CMPs). In all, nearly one in five was “misclassified” as ANA-negative when they in fact have antibodies directed against a variety of CMP targets (Arthritis Care Res. https://doi.org/10.1002/acr.23712)

 
 

Marvin J. Fritzler, PhD, MD, one of the report’s authors, said that he expects a fairly rapid shift away from the current reliance on this approach to ANA testing for diagnosing lupus. He said its use could fade away within 10 years.

“I think it’s on its way out,” said Dr. Fritzler of University of Calgary (Alta.). “There are some people who think it isn’t even going to be 10 years.”

Dr. Fritzler and his colleagues, including lead author May Y. Choi, MD, also of University of Calgary, described their work as the first study of ANA-IIF in a large SLE inception cohort redefining negative ANA as the absence of any intracellular IIF staining, which they referred to as anticellular–antibody negative. 

This research involved data and patient sera collected by the Systemic Lupus International Collaborating Clinics (SLICC), a network of 53 investigators in 43 academic medical centers in 16 countries. During 1999-2011, SLICC investigators enrolled patients fulfilling the American College of Rheumatology’s classification criteria for definite SLE within 15 months of diagnosis.

IIF assays were first introduced more than 50 years ago and remain the most widely used conventional technique for detecting ANA, according to the American Association for Clinical Chemistry. In this test, a diagnostic dye, known as fluorescein, is used as a marker, the association said.

“I love the ANA test. I grew up on it. It’s a beautiful thing to look into the microscope and see this wonderful green, bright-green staining,” Dr. Fritzler said. “But now we know how it really performs in today’s diagnostic world; I think it’s on its way out.”

Dr. Fritzler said he expects diagnostic laboratories to drive a shift away from these tests once they see that they have a market advantage by offering a more reliable tests. Enzyme-linked immunosorbent assay (ELISA) and multiplex immunoassays also have been used in detecting lupus. Still, primary care physicians rely on traditional ANA screening to diagnose lupus, he said. Delays in obtaining the correct lupus diagnosis because of a misleading ANA may allow the condition to go untreated longer, perhaps putting patients at an otherwise avoidable risk for kidney damage.

“So it’s important to know what percentage of those are going to be negative and what other tests are needed then if there is still a reasonably high suspicion that the patient has lupus despite the tests being negative, what tested should replace it,” Dr. Fritzler said.

Lupus erythematosus cells (LE cells) on 100X light microscope stain
“I love the ANA test. I grew up on it. It’s a beautiful thing to look into the microscope and see this wonderful green, bright-green staining… But now we know how it really performs in today’s diagnostic world; I think it’s on its way out.” Marvin J. Fritzler, PhD, MD

In the paper, Dr. Fritzler and his colleagues reported that patients who were older, of white race/ethnicity, or on high-dose glucocorticoids at or prior to enrollment were more likely to be anticellular antibody–negative. Patients on immunosuppressants or with anti-SSA/Ro60 or anti-UI-RNP were less likely to be anticellular antibody–negative.

Wide variation in assay results 

Their work further highlights challenges with ANA testing raised by David S. Pisetsky, MD, PhD, of Duke University, Durham, N.C. Dr. Pisetsky and his colleagues showed wide variation in the results of commercially available assays. They found negative results that ranged from 5% to 22% of samples with three different commercially available immunofluorescence assays (IFAs), one ELISA assay, and one bead-based multiplex assay (Ann Rheum Dis. 2018;77:911-3).

The reliance on ANA tests as a gatekeeper in clinical trials can be frustrating for patients with lupus who are excluded from clinical trials because of a negative reading, Dr. Pisetsky said in an interview.

“What makes it hard is that, frequently, we don’t know what the kit was that led to the original ANA assessment,” Dr. Pisetsky said. “Most people don’t think there are differences, yet there are differences.”

Joan T. Merrill, MD, chief adviser of clinical development at the Lupus Foundation of America and a member of the Oklahoma Medical Research Foundation, said that the ANA test remain “very valuable.” Most patients with lupus have a positive ANA or at least one other autoantibody, such as SSA/Ro, at some point in their illness, said Dr. Merrill.

“What the recent publications have underscored is that, being positive for an ANA does not prove the diagnosis of lupus by itself and also being negative at one time with one testing method does not rule it out,” Dr. Merrill said. “Lupus is a spectrum of autoimmune/inflammatory features that need to be interpreted in context and in evaluation of the whole patient.”

The standard ANA test gives a broader view of antigens potentially involved in lupus than do the ELISA or other screens, according to Donald E. Thomas Jr., MD, a rheumatology specialist who teaches at Walter Reed National Military Medical Center in Bethesda, Md. 

“The antinuclear antibody test is essential and it’s not going away any time soon,” he said.

The American College of Rheumatology has repeatedly endorsed its support for the immunofluorescence ANA test, using human epithelial type 2 substrate, calling it the gold standard for ANA testing.

The American College of Rheumatology has repeatedly endorsed its support for the immunofluorescence ANA test, using human epithelial type 2 substrate, calling it the gold standard for ANA testing.

“The antinuclear antibody test is essential and it’s not going away any time soon.” Donald E. Thomas Jr., MD

“Hospital and commercial laboratories using alternative bead-based multiplex platforms or other solid phase assays for detecting ANAs must provide data to ordering health care providers on request that the alternative assay has the same or improved sensitivity compared to IF ANA,” the college said in a position statement,  which was approved in 2009, 2011, and 2015.

Dr. Thomas said that he has taken to ordering two tests from two different laboratories to try to prevent false-negatives results in screening for lupus, and he’s convinced his partners to do the same.

In his experience, he’s seen patients who tested negative for lupus on the immunofluorescence ANA test, but positive on an ELISA or multiarray test. Including the ELISA and multiarray test adds to the cost of care, but they are less expensive than the immunofluorescence ANA is. Still, the two-test approach can eliminate costs associated with false-negative results, he said.

In a case where a single ANA test fails to confirm lupus initially, a patient  “will be seeing more doctors, getting more tests done until the proper diagnosis is reached,” said Dr. Thomas.

 

Kerry Dooley Young is a freelance reporter for MDedge News.