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Autoantibody testing is becoming increasingly important in rheumatological diagnostics. It delivers crucial information for differential diagnostics and sometimes even allows prediction of the disease progression or helps in the choice of therapy. Before diagnostic use, all serological tests undergo thorough investigation. They are only approved when they meet the respective criteria. However, a result obtained from these serological tests should never be the sole basis of a diagnosis as discrepancies may occur due to the very nature of autoimmunity.
Discrepancies between the ANA screening test and the monospecific confirmation test
Indirect immunofluorescence tests (IFA) with human epithelial cells (HEp-2 cells) are the gold standard in the screening for antibodies against nuclear antigens (ANA).1 IFA is a very sensitive but not very specific method and ANA can also be detected in up to 20% of the healthy population.2 A positive ANA screening test result could therefore also occur without relation to the suspected disease.
To confirm a positive ANA screening test using a monospecific test, the selection of antigens to be tested must also contain the target antigen. It is important that all antibodies tested negative in the confirmatory test are documented in the patient file. It might be necessary to test further antibodies. EUROIMMUN offers different EUROLINE ANA profiles for parallel monospecific detection of up to 23 antibodies or of only the most prevalent ANA. However, especially for autoantibodies with a very low prevalence, a monospecific test might not yet have been developed or the target antigen might not even be identified yet. In these cases, EUROIMMUN actively contributes to research by discovering target antigens and developing confirmatory tests. For example, systemic sclerosis diagnostics could be optimised with the newly discovered target antigen NVL (nuclear valosin-containing protein-like), which is offered exclusively on the EUROLINE Systemic Sclerosis Profile 2.
A negative ANA screening test, on the other hand, does not exclude the presence of autoantibodies relevant for rheumatology. For some anti-cytoplasmic antibodies, IFA on HEp-2 cells is not the appropriate method. This is the case for many myositis-specific antibodies (MSA). They (often) do not produce a recognisable fluorescence pattern in the ANA screening test as the target antigens only occur in small amounts and are distributed throughout the cytoplasm. Examples include anti-cN1A antibodies and antibodies against tRNA synthetases such as anti-Jo-1. In suspected cases of myositis, monospecific testing for MSA is therefore recommended in parallel to the ANA screening test. The EUROLINE myositis profiles are a particularly good choice as they allow to test for 20 myositis-relevant autoantibodies with just one blot strip.
Finally, discrepancies can also occur due to different antigen presentations. In HEp-2 IFA results, antigens are present in situ while in monospecific confirmatory tests the antigens are purified and immobilised. This means that they undergo modification, which may influence antibody binding.
Discrepancies between different monospecific tests
Discrepancies between different monospecific tests are also usually caused by the different antigen presentations. It not only matters which detection method is used (e.g. ELISA, ChLIA or immunoblot), but also the origin, type, purification and immobilisation of the antigens. Depending on the test and manufacturer, proteins as antigens may be recombinant (e.g. produced in bacteria) or native purified proteins (e.g. from tissue) and may thus contain different posttranslational modifications. Recombinant peptides may differ in the sequence selected. Last but not least, the process of purification, the immobilisation and the coating surface may cause changes to the antigen. Any modification of an antigen can affect the binding affinity of the antibody. The autoantibodies to be detected are also not identical but individually formed polyclonal autoantibodies. This means they are different from patient to patient even when they are directed against the same antigen. Due to the described differences, the different antibody populations can be detected differently well by the different tests. Thorough knowledge of antibody binding and corresponding advancement of a test system can increase the analytical sensitivity and specificity. For example, by using highly purified nucleosomes for coupling of double-stranded DNA (dsDNA) to the solid phase in the Anti-dsDNS-NcX ELISA (IgG), its diagnostic detection rate for anti-dsDNA antibodies is superior to that of the Anti-dsDNA RIA (Farr assay). However, there are some sera in which anti-dsDNA antibodies could only be detected using the Farr assay or the Crithidia luciliae immunofluorescence test (CLIFT).3 The use of different tests can therefore significantly increase the detection rate in antibody testing.
However, even a perfect antibody test would not be sufficient to make or exclude a diagnosis. The clinical picture of the patient should always be given priority in rheumatological diagnostics. Depending on the indication, other examinations such as histological analyses or imaging techniques should be used in addition to serological tests to confirm a diagnosis. We are happy to advise you on our antibody diagnostic test systems as an important contribution to rheumatological diagnostics. Our experienced specialists are also available to support you in the evaluation of especially challenging serological results.
1 International Consensus on ANA Patterns
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