From ANA to ENA: How to proceed?
Introduction
Anti-nuclear antibodies (ANA) are directed to distinct constituents of the nucleus and ANA are traditionally identified by the indirect immuno-fluorescent technique (IFT) [1]. In daily practice, also antibodies to cytoplasmic antigens, like the aminoacyl-tRNA-synthetases, are referred to as ANA. Salt extraction of nuclear proteins was originally used for identifying the antigens that are recognized by ANA, hence the name extractable nuclear antigens (ENA). Although several antigens, like topoisomerase-I (Topo-I), are not extractable in this way, in this review we will refer to these antigens as ENA in order to cover all of the most relevant anti-ENA antibodies that are tested for in a routine laboratory.
The presence of ANA is associated with autoimmune connective tissue diseases. Their detection is even one of the diagnostic criteria for systemic lupus erythematosus (SLE) [2]. Further analysis of the reactivity to extractable nuclear antigens (ENA) may be of help to discriminate between the different types of autoimmune connective tissue diseases. For instance, antibodies to the Smith (Sm) antigen are specific for SLE [2], while the presence of anti-Sjögren's Syndrome (SS)A and/or-SSB antibodies is a hallmark for Sjögren's Syndrome [3]. Besides having diagnostic potential, the detection of anti-ENA antibodies is also of prognostic importance. The presence of anti-SSA in the circulation of the mother may cause neonatal lupus erythematosus and/or congenital heart block [4], [5], while the presence of anti-Topo-I antibodies predict a more severe course of disease in systemic sclerosis (SSc) [6].
Originally, most of these anti-ENA antibody specificities and clinical associations were determined by immuno-assays based on immuno-diffusion [7], [8]. However, further knowledge on the structure of the autoantigens, the identification of other specificities, and recombinant technology has enabled the launch of many different anti-ENA antibody detection systems [9], [10], [11]. These systems give a lot more information than the immuno-diffusion assays, but the clinical relevance of this information may be ill-defined. In this review we will discuss the main logistic aspects of ENA testing. This is especially relevant since the detection of these antibodies is more and more done by using new methodologies.
Section snippets
Follow-up of ANA by ENA
The first question to be asked is if all positive ANA tests should be followed by anti-ENA antibody testing. ANA testing was traditionally performed on liver tissue sections, but the substrate has now been replaced by HEp-2 cells. These cells have large nuclei and contain a considerable number of dividing cells, displaying mitotic figures, which enables the detection of antigens that are differentially expressed during the cell cycle. The HEp-2 cells may be transfected with additional antigens,
New methods for anti-ENA antibody detection
Anti-ENA antibody testing was originally performed by immuno-diffusion based techniques using thymic and/or spleen extracts [7], [8]. Also immuno-blotting of cell extracts from for instance HeLa-cells (nuclear and cytoplasmic) was often used for the detection of antibodies to ENA [13]. Although these techniques are quite laborious and results are difficult to read, the use of cell extracts enabled the identification of new antigenic entities. In addition, these studies revealed that some ENA
Clinical relevance of anti-ENA antibody results
The second question about anti-ENA antibody testing is: what is the proper choice of anti-ENA antibody assay to be used? For making a good choice between the distinct anti-ENA antibody assays that are available, it is important to have information on the clinical relevance of the obtained results. Items to be evaluated include test characteristics for diagnosis and prognosis of a certain disease. For instance, while anti-Sm antibodies are part of the diagnostic criteria for SLE, antibodies to
Procedure and interpretation of anti-ENA antibody detection
There is a consensus statement that a combination of two or more techniques should be used to detect all specificities with an adequate efficiency [17], the final question is how to report the data obtained to the clinician. Although many countries do not allow multiple test systems for economical reasons, the need for additional tests is based on the fact that some techniques as well as some commercial assays may be sub-optimal for detecting a certain (conventional) anti-ENA antibody
Conclusions
In the discussion on how to proceed from a positive ANA test to anti-ENA antibody testing more questions than answers are generated. With respect to the first question if a positive ANA should always be followed by anti-ENA antibody testing our own data reveal that both the titre and pattern give a clue to which samples could be excluded from further testing for antibodies to the conventional ENA. For the proper appreciation of the clinical value of the new anti-ENA antibody detection systems
Acknowledgements
We thank Professor Allan Wiik (Statens Serum Institut, Copenhagen, Denmark) for helpful comments during the preparation of the manuscript.
Take-home messages
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ANA titre and pattern give information on the necessity to proceed with anti-ENA antibody testing.
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High through-put anti-ENA antibody assays enabling detection of multiple antigenic specificities, including novel antigens and split antigens, are available nowadays, but evaluation of its clinical relevance is at present lacking.
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There is a high
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