Elsevier

Clinica Chimica Acta

Volume 415, 16 January 2013, Pages 101-106
Clinica Chimica Acta

Detection of antinuclear antibodies by automated indirect immunofluorescence analysis

https://doi.org/10.1016/j.cca.2012.09.021Get rights and content

Abstract

Background

Testing for antinuclear antibodies is useful for the diagnosis of systemic rheumatic diseases. Automated systems for image acquisition and interpretation of indirect immunofluorescence-based tests are increasingly used. The diagnostic performance of such automated approach in untreated patients has not been reported.

Methods

Antinuclear antibodies were measured by automated indirect immunofluorescence using Zenit G. Sight on HEp2 and HEp2000 substrate in 268 consecutive samples submitted to the laboratory for antinuclear antibody testing, and in 231 patients with a systemic rheumatic disease at the time of diagnosis, 143 blood donors, 134 patients with chronic fatigue syndrome, and 133 diseased controls.

Results

Image acquisition by G-Sight was of high quality. The accuracy of pattern assignment was limited. There was a significant correlation between automated estimation of fluorescence intensity (probability index of positivity) and end-point titer. Probability index interval specific likelihood ratios for systemic rheumatic disease increased with increasing level of positivity probability. With the HEp-2 substrate, the likelihood ratio for systemic lupus erythematosus was 0.06, 0.4, 6.8, 12.1, and 43.9 for a probability measure of positivity of ≤ 10, 11–≤ 30, 31–≤ 50, 51–≤ 85, and > 85, respectively.

Conclusion

Quantitative data generated by automated image acquisition facilitates standardized interpretation.

Highlights

► Image acquisition by automated indirect immunofluorescence (G-Sight) is of high quality. ► Fluorescence intensity estimated by automated indirect immunofluoresce correlate with antibody titer. ► Quantitative data generated by automated image acquisition facilitates standardized interpretation.

Introduction

Antinuclear and anti-cytoplasmic antibodies are important diagnostic markers for systemic rheumatic diseases and autoimmune hepatitis [1], [2], [3]. Traditionally, indirect immunofluorescence on Hep-2 cells is used to screen for antinuclear antibodies. More specific, second line tests are performed to identify the target antigen of the antibodies (e.g. dsDNA or extractable nuclear antigens). Although quantitative (multiplexed) solid phase immunoassays for specific antibodies can be automated, they cannot fully replace indirect immunofluorescence for antinuclear antibody testing, because of the lower sensitivity [4], [5]. Therefore, recent recommendations state that immunofluorescence antinuclear antibody testing should remain the gold standard [6].

Indirect immunofluorescence, however, suffers from low-throughput and intra- and inter-laboratory variance. Visual evaluation is time consuming, subjective and requires considerable expertise of the technicians [7]. To overcome these shortcomings, automated approaches for indirect immunofluorescence analysis are being developed [8]. The Aklides system (Medipan, Germany) was the first automated system for indirect immunofluorescence analysis of antinuclear antibodies. Good agreement between Aklides reading and visual reading has been reported [7], [9], [10]. Other systems are currently being developed and/or introduced in autoimmune laboratories. Few studies are available on the performance of automated indirect immunofluorescence analysis and none of these studies have assessed the clinical performance characteristics of such systems in patients who are presenting for diagnosis of a systemic rheumatic disorder.

In the present study we evaluated detection of antinuclear antibodies by G-Sight (Menarini), an automated system for image acquisition and interpretation of indirect immunofluorescence-based tests. We evaluated the ability of the system (i) to estimate the fluorescence intensity and (ii) to correctly classify fluorescence patterns. A major objective of the study was to determine the diagnostic performance of G-Sight for systemic lupus erythematosus and other connective tissue diseases in a cohort of untreated patients presenting for diagnosis and controls.

Section snippets

Study population

A first cohort consisted of samples with mono-specific antibodies, including antibodies [i] to centriole (n = 3), centromere (n = 37), the cytoplasm (fibrillar pattern) (n = 7), nuclear membrane (n = 14), midbody (n = 8), proliferating cell nuclear antigen (n = 8), mitotic spindle (n = 16) [detected by indirect immunofluorescence], and [ii] Scl-70 (n = 39), RNP (n = 26), and SSA (n = 48) [detected by EliA (Thermo-Fisher).

A second cohort consisted of 268 consecutive samples submitted to the laboratory for analysis

Image acquisition by G-Sight

Automated antinuclear antibody analysis by G-Sight was performed on 268 consecutive samples submitted to the laboratory. The analysis was done on HEp-2 cells as well as on HEp-2000 cells (which contain SSA-transfected cells). Each sample was visually checked for positivity and a pattern assigned in case of positivity. Cytoplasmic staining was also considered antinuclear antibody positive. Overall, there was good agreement between the two substrates (for cutoff at dilution 1:80, agreement for

Discussion

Various systems for automated analysis of indirect immunofluorescence are becoming available. These systems include Aklides (Medipan, Berlin, Germany), G-Sight (Menarini, Florence, Italy), NovaView (Instrumentation Laboratory, Inova, Barcelona, Spain), EuroPattern (Euroimmun, Lübeck), Helios (Aesku, Wendelsheim, Germany), and Image Navigator (Immuno-Concepts, Sacramento, United States). The systems differ from each other with respect to the use of counterstain (DAPI staining for focusing is

Conflict of interest

Heidi de Baere is an employee of A. Menarini.

The following are the supplementary data related to this article.

. Automated antinuclear antibody analysis by G-Sight was performed on 268 consecutive samples submitted to the laboratory. The analysis was done on HEp-2 cells as well as on HEp-2000 (which contain SSA-transfected cells). For each sample, a visual inspection was done and a pattern assigned (in case of positivity). An overview of the different patterns obtained is given.

Acknowledgments

We thank F. Nencini, A. Foggi and M. Donnini for helpful discussions. X. Bossuyt is a senior clinical investigator of the Fund for Scientific Research-Flanders.

Financial support or other benefits were from commercial sources.

A. Menarini provided the G-Sight instrument and the reagents to perform the study. The study was financially supported by A. Menarini.

XB has been funded by A. Menarini for participation in an international meeting on autoimmunity.

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