Immunofluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systemic lupus erythematosus patients

doi:10.1016/j.clim.2007.04.010

Clinical Immunology

Volume 124, Issue 1, July 2007, Pages 18-21

https://doi.org/10.1016/j.clim.2007.04.010 Get rights and content

Abstract

Antinuclear antibodies (ANA) are a hallmark of systemic lupus erythematosus (SLE) and one of its key diagnostic criteria. Recently, new technologies based on antibody binding to fluorescence beads (FB) have been widely employed for ANA screening. We conducted a formal study in 385 consecutive patients who underwent both traditional immunofluorescence (IF) and FB testing and evaluated each patient for the diagnosis of SLE. The distribution of ANA test results was significantly different (χ² = 73.12; p < 0.0001) due to a marked discordance of double-negative and double-positive results. The concordance of the FB-negative and IF-negative test results was 240/256 (95.6%), while the concordance of double-positive results was 54/129 (41.9%). The sensitivity of IF was markedly higher (48/53; 90.6%) than that of the FB (26/53; 49.1%; p < 0.0001) for the diagnosis of SLE. IF had a lower specificity (76%) than FB (87%; p = 0.0002). The present data show that the IF assay has superior sensitivity for detection of ANA and should continue to be used as the primary screening test for the diagnosis of SLE.

Introduction

The presence of antinuclear antibodies (ANA) is a hallmark of SLE and one of its diagnostic criteria established by the American College of Rheumatology [1]. ANA are seen in 90–95% of patients with SLE. It is traditionally detected by indirect immunofluorescence (IF) assay in which the antibodies of the patients' sera that bind to the nucleus of Hep-2 human epipharynx carcinoma cells are detected by fluorescein isothiocyanate-conjugated anti-human IgG, using fluorescence microscopy [1]. The IF technique also provides information on the pattern of fluorescence, such as homogeneous, peripheral, nucleolar, or speckled [1]. Such patterns are relevant for antigen specificity and they have been associated with autoimmune disease subsets [1]. Notwithstanding, the detection of ANA by IF is laborious and requires an experienced technician. Flow cytometry with autoantigen-coated fluorescent beads (FB) has been gaining popularity for several years [2]. FB-based techniques, also commonly referred to as Reflex ANA, are claimed to have multiple advantages, such as simultaneous testing for recognition of several antigens, automation, cost effectiveness, and high sensitivity [2]. This ANA detection technique which was also used in our study, however, has not been systematically validated against IF ANA in patients with SLE and therefore its utility remains unproven. After replacement of the traditional Hep-2 cell-based IF with the FB assay at our Institution, we encountered 11 patients who met the diagnostic criteria for SLE, but tested ANA negative by FB assay. All of these patients were subsequently tested ANA-positive using the IF assay, which was retained by our laboratory for confirmatory testing. This was of concern because the FB technique was being offered as the primary screening method for the diagnosis of SLE. We therefore decided to conduct a retrospective study to analyze how these two methods of ANA detection correlated in SLE and non-SLE patients and compared their sensitivity and specificity for the diagnosis of SLE.

Section snippets

Patients and methods

Based on prior authorization by our Institutional Review Board for the studies of human subjects, we retrospectively analyzed all patients tested for ANA both by IF and FB in the time period of 1/1/03 through 4/30/06. Patients that had an antibody titer of more than 1:50 by IF were considered to have a positive test [1]. The Athena MultiLyte assay employing the Luminex microsphere technology (Zeus Scientific, Raritan, NJ), was used for flow cytometry-based (FB) ANA screening. We then studied

Results

FB-based ANA screening was done on 984 patients at our institution in the period from 1/1/03 to 4/30/06. Sera of 385 of these patients were also tested in parallel for the presence of ANA by IF. The electronic charts of these 385 patients were reviewed. The results of ANA testing are shown in Table 1. The distribution of ANA test results was significantly different (χ² = 73.12; p < 0.0001) due to a marked discordance of double-negative and double-positive results. The concordance of the FB-negative

Discussion

Antinuclear autoreactivity in patients with SLE was discovered in 1948 with the first detection of the LE (lupus erythematosus) cell [4]. Detection of ANA by indirect IF was introduced in 1957 [5]. In this assay, test sera are incubated with substrate cells, such as Hep-2 cells, at different dilutions and antibody binding is detected by fluorescein isothiocyanate-conjugated IgG by direct visualization under a fluorescent microscope. Because of high sensitivity, it is an excellent screening

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Rapid CommunicationImmunofluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systemic lupus erythematosus patients

Abstract

Introduction

Section snippets

Patients and methods

Results

Discussion

J. Autoimmun.

Clin. Immunol.

J. Immunol. Methods

J. Immunol. Methods

Range of antinuclear antibodies in “healthy” individuals

Arthritis Rheum.

Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases

Autoimmunity

Rapid Communication
Immunofluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systemic lupus erythematosus patients