Antinuclear antibody (ANA) testing for many years has been the primary laboratory screening approach for diagnosing connective tissue autoimmune diseases. The classic method for assessing ANA is an indirect immunofluorescent assay (IFA) using the HEp-2 cell line as a substrate. This assay is relatively simple to perform; however, reading the slides and interpreting the various patterns take the time of highly trained, experienced technologists. ANA testing volumes started to rise just as hospital laboratories were cutting back on personnel, which led manufacturers to develop more automated ANA testing methods, specifically enzyme-linked immunosorbent assays (ELISA) and multiplex technology. 

Aside from being automated, these methods offer several benefits, including economies of scale and ob-jective interpretations that do not require technologists’ readings. Multiplex methods may identify more pa-tients with multiple antibodies and may provide improved clinical disease association for specific antibod-ies. Meanwhile, ELISA testing’s automation lends itself particularly to high-volume screening. Still ELISA and multiplex methods are not without problems when compared with IFA, which the American College of Rheumatology has designated as the gold standard. Recently, manufacturers have combined the best of both worlds by developing IFA with more automated slide preparation and objectively interpreted image analysis systems. In this article I discuss the experience of Summa Health, a multihospital system in Akron, Ohio, as we consolidated our ANA testing operations.

The Summa Health Challenge

Summa had two laboratories performing ANA IFA, both with similar volumes of approximately 4,000–5,000 specimens per year. Lab A’s volume came mostly from inpatient testing and rheumatology referrals. This lab, which also trained medical technology students and pathology residents, performed ANA IFA 3 days per week, with both a technologist and a PhD immunologist reviewing slides. Meanwhile, Lab B’s clientele consisted mainly of outpatient specimens from physician offices. This site functioned like a reference laboratory, performing ANA IFA on weeknights, with slides reviewed by one technologist and results available the following morning right after physicians’ offices opened. While this two-lab system functioned well, Summa lab administrators decided to centralize ANA testing at one site.

Lab B staff were concerned that if we centralized testing at Lab A, Lab B’s turnaround time would suffer and it would lose business. Lab A staff were more concerned about continuing the lab’s training programs and providing consistent results with two readers per slide. This lab could perform ANA IFA daily, but with no immunology staffing at night, it could not make results available to physicians’ offices just as they were opening each morning. We wanted to keep ANA IFA as our principle screening method, possibly reserving multiplex or ELISA methods to determine specific antibodies—a decision with which our physicians concurred.

We decided that one of the newer automated IFA systems might address both labs’ concerns. This way, Lab B could run specimens at night, rendering results for negative or positive specimens first thing every morning. The instrument initially would detect negative specimens, which a technologist would confirm, thereby enabling two readings. Similarly, positive specimens could be signed out as “positive; titer and pattern to follow.”  The lab then would run dilutions on the instrument during the day, with patterns reviewed by a second technologist and/or the scientific director. Slides could be read online, either at the same lab or at another institution. Having the slides available digitally would also meet Lab A’s educational objectives. Overall, we believed that an automated IFA assay would facilitate excellent patient care and continued medical education, capitalizing on the best attributes of each laboratory and its personnel. 

An Imperfect Gold Standard

Despite our decision to go with IFA, we see it as an imperfect gold standard. It certainly detects more antibodies than any other method, but ELISA and multiplex techniques specifically identify which antibody a patient has as opposed to providing a pattern in the IFA procedure. Our data presented at the 2014 International Congress on Autoimmunity show that the same reader will obtain different results for some specimens on different manufacturers’ HEp-2 slides. These variations may be due to different HEp-2 fixation methods, different second antibody targets (IgG specific, heavy chain specific versus multi-isotype specific), the fluorescent:protein ratio of the second antibody, and even microscope characteristics. The bottom line is that IFA, while being consistent for a given manufacturer in a given lab, may yield different results from another laboratory using a different manufacturer’s slides and reagents. 

Another concern with IFA involves what to use as a screening dilution. False positive results have been reported at low titers, and most authors recommend determining the screening dilution for the population being tested at a particular location. Because most studies evaluating the performance of the ELISA, multiplex, and automated IFA systems use a manual IFA reading as the comparison point, their results may be dependent upon the slide manufacturer used as a reference.  

Current Status

Summa consolidated ANA testing at Lab A in September 2016. We informed clients of both laboratories of the consolidation and have not had any turnaround time complaints nor lost any clients. We currently are evaluating the possibility of acquiring an instrument to either automate the IFA procedure or perform follow-up specific antibody testing. 

Thomas Alexander, PhD, D(ABMLI), is an immunologist at Summa Health in Akron, Ohio.+Email: alexandt@summahealth.org