Detection of antinuclear antibodies by automated indirect immunofluorescence analysis
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.
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.
References (18)
- et al.
Detection of antinuclear antibodies by indirect immunofluorescence and by solid phase assay
Autoimmun Rev
(2011) - et al.
Challenges of automated screening and differentiation of non-organ specific autoantibodies on HEp-2 cells
Autoimmun Rev
(2009) - et al.
Novel opportunities in automated classification of antinuclear antibodies on HEp-2 cells
Autoimmun Rev
(2011) Clinical performance characteristics of a laboratory test. A practical approach in the autoimmune laboratory
Autoimmun Rev
(2009)- et al.
American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing
Arthritis Rheum
(2002) - et al.
Appropriateness in anti-nuclear antibody testing: from clinical request to strategic laboratory practice
Clin Exp Rheumatol
(2004) - et al.
Autoimmune hepatitis
Nat Rev Gastroenterol Hepatol
(2011) - et al.
Antinuclear antibody detection by automated multiplex immunoassay in untreated patients at the time of diagnosis
Autoimmun Rev
(Feb 23 2012) - et al.
ANA screening: an old test with new recommendations
Ann Rheum Dis
(2010)