Deng et al. (1) compared the clinical utility of three antinuclear antibody (ANA)1 screening assays, i.e., an enzyme immunoassay (EIA), an immunofluorescence assay (IFA) on human epithelial (HEp)-2 cells, and a multiplex immunoassay (MIA). Among the conclusions from their data were the following statements: “the overall diagnostic performance of the IFA, EIA, and MIA were not statistically different….” Our primary focus will be on the above quoted words, with additional attention given to the propensity of HEp-2 cell lines to miss anti–Sjögren's Syndrome (SS)-A, which was also highlighted by Deng et al.
While we intend no criticism of the excellent research done by Deng et al. comparing 3 commonly used ANA screening methods, we do offer some words of caution. First, their research involved populations of patients, i.e., those suspected or not suspected of connective tissue disease (CTD). Clinical laboratories serve individual patients, not populations. Although Deng et al. went on to say that the clinical sensitivity and specificity of the 3 methods varied drastically, we are concerned that their finding of no statistical difference in overall diagnostic performance among the methods might be mistakenly taken to imply that there is equivalency of performance on individual patients; this is not the case, as evidenced by literature references too numerous to list here.
To illustrate the “individuality” of ANA methods, we present 2 cases that we have encountered in our own laboratory practices. One of us has recently published a clinical case study primarily concerning the diagnosis of congenital heart block, where ANA HEp-2 by IFA failed at detecting anti–SS-A in the mother's serum (2). The other 2 of us have had a similar experience with a case of lupus rash reported by a neonatologist when questioning the disparity between the negative ANA on his patient and his clinical findings. Fortunately, in both cases, the disparities were resolved by having available solid-phase assays that are more sensitive for the anti–SS-A case, and in the latter case of lupus rash, the resolution involved an ANA by an IFA that was transfected to overexpress the SS-A antigen (3).
Another illustrative case involved 2 false-negative ANA tests by a solid-phase assay (MIA) reported by a reference laboratory that initially misled a patient's physicians (4). This scenario resulted in additional needless physician visits and testing, in addition to the distress felt by the patient, who noted that she was “…virtually bedridden; it seemed like an eternity.” The case was resolved when a third ANA by IFA tested positive as a speckled pattern at high titer (1:1280) along with a positive, 1:40 titer, double-stranded DNA antibody. The anti-DNA antibody, as well as several other CTD autoantibodies, had also been negative on initial testing with the MIA. A subsequent sample referred to the commercial laboratory was again reported as ANA negative by MIA.
The move to solid-phase ANA screening by laboratories from IFA has been mainly occasioned by the labor-intensive and subjective nature of IFA. These objections are being overcome by ANA IFA automation; currently, at least 3 automated systems have been cleared by the U.S. Food and Drug Administration (5). This abandonment of ANA IFA has occurred despite recommendations of the American College of Rheumatology (6) that ANA by IFA is the gold standard for screening with greater sensitivity than solid-phase assays for connective tissue disease. Laboratories using solid-phase assays must provide data when requested showing the same or improved sensitivity and specificity compared to ANA IFA and specify the method used on laboratory reports.
The excellent paper of Deng et al., the many citations in the literature of ANA testing missteps, and the recommendations of the American College of Rheumatology on ANA testing prompt us to close with the following conclusion: ANA testing encompasses a variety of methodologies with differing sensitivities, applications, and implications for clinical practice. Knowledge by clinical scientists and clinicians of these differences and limitations (occurring even within a methodology class, e.g., the relatively low sensitivity of conventional HEp-2 substrates for anti–SS-A compared to transfected HEp-2 cells) is paramount to the efficient and accurate diagnosis of a variety of medical conditions served by ANA testing.
↵1 Nonstandard abbreviations:
- antinuclear antibody
- enzyme immunoassay
- immunofluorescence assay
- human epithelial
- multiplex immunoassay
- connective tissue disease
- Sjögren's Syndrome.
(see article on page 36 in the July 2016 issue)
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: None declared. Consultant or Advisory Role: F.F. Hall, Immuno Concepts. Stock Ownership: None declared. Honoraria: None declared. Research Funding: F.F. Hall, E.S. Hoy, Immuno Concepts. Expert Testimony: None declared. Patents: None declared.
- Received September 6, 2016.
- Accepted October 17, 2016.
- © 2016 American Association for Clinical Chemistry