|GUIDELINES FOR ANTINUCLEAR ANTIBODY TESTING
*See text about the various ANA.
It is assumed that the ANA found in a patient with any of these diseases somehow reflect genetic predisposition and lesional pathology in a particular individual. Since involvement and severity of different organs is directly related to disease prognosis the revelation of a particular ANA in a patient can guide the clinician in the follow-up and surveillance of incipient organ manifestations so that rational therapy can be instituted early.
A specially illustrating example of such relationships is SSc, where anti-centromere antibodies mostly are associated with a slowly developing form of limited SSc which has a good long-term prognosis, while anti-topoisomerase I (anti-Scl-70) antibodies relate to a more rapidly progressing form of diffuse SSc commonly complicated by fibrosing alveolitis and a more cumbersome prognosis. Anti-RNA polymerase I antibodies have been found to be associated with a particularly severe form of rapidly progressing diffuse SSc, that commonly involves the kidneys and manifests with malignant hypertension, cardiovascular disease and cerebral infarctions. In SSc patients that harbour anti-U1RNP antibodies the disease is practically always overlapping with another immuno-inflammatory rheumatic disease e.g. SLE, PM/DM or MCTD, and the prognosis may be very different from case to case. Similar clinical subsyndromes have been found in SLE, primary SjS, PM/DM, JCA. Each subsyndrome is thus associated with presence of a particular specific ANA, and the nature of that ANA should be revealed if at all possible.
Some ANA can be difficult to reveal with certainty, probably due to different properties or different epitope specificities seen in relationship to a njumber of diseases. Nevertheless, credible results are absolutely necessary in order to allow a meaningful use of the serologic information. A typical example is that of anti-double stranded DNA (-dsDNA), where independent studies have reached the same conclusion, i.e. anti-dsDNA that are characteristic for SLE can only be disclosed by using at least two different methods for their demonstration.
This may be explained by the fact that production of some types of anti-dsDNA can be a normal response to certain infections or to any type of long-standing tissue injury. In our laboratory we have reached at a strategy where we start screening for anti-dsDNA using an EIA that is known to be broadly reacting and thus has a low specificity for SLE, but then a positive result is followed up by use of a Crithidia luciliae IF test which is highly specific for SLE if found positive. Only a if a positive IF test is found we report anti-dsDNA to be present. A positive result of the EIA only is not reported as positive. With that approach we have shown that the sensitivity in SLE patients is around 45-50 % and the diagnostic specificity around 97 %.
12.5 How can we judge the clinical utility of results from solid phase assays?
It is clear from many reports that a positive test for ANA using EIA or some other solid phase based technology does not correspond well to what is found by the classical double immuno-diffusion or counter-immuno-electrophoresis techniques which rely on presence of precipitating antibodies. Before one can use results from such solid phase assays in clinical work-up it is necessary to explore the diagnostic potential by studying sera from local populations of immuno-inflammatory diseases. Healthy donor controls cannot be used for a clinically meaningful cut-off setting. Results derived from the study of a prototype disease must be compared with those of inflammatory disease controls that manifest features somewhat similar to the prototype disease ("critical controls"). By constructing receiver-operation curves and choosing a preferred high level of specificity cut-off can be set.
It is very important to prove the value of a test for early diagnostics. In early disease a positive ANA result has a relatively higher impact on clinical decision-making than later in follow-up. The most informative ANA results are those that are unique to one diagnostic entity (disease-specific).
12.6 What should be done to establish serologic positivity in borderline cases?
Since the early start of the European consensus studies the recommendation has been to confirm or refute doubtful borderline results ("grey area" results) by e.g. performing two different techniques. Alternatively one can agree with clinicians to report such results with a written "caveat notice" that the result cannot be used with confidence for differential diagnostics. Another possibility is to agree on calling all such results "negative". This has to be discussed between laboratory personnel and clinicians, so the policy is always the same.
12.7 Use of algorithms
Collaboration between clinicians and laboratory scientists may also lead to agreement on the use of practical algorithms for test ordering, for rational stepwise exploration of a preliminary result at screening, and for interpretation of a positive final result. As an alternative to an algorithm for test ordering it may be practical to set up the order form in such a way that the doctor can tick either a tentative diagnosis or one or more single tests (Fig 1). Thereby the flexibility of test ordering is maximal and people who may be uncertain about which tests will be rational to do can learn from the form.
Figure 1. Choice of test packages or single specified orders on test order form.
12.8 Use of international serum standards in the laboratory
The IUIS/WHO/CDC/AF International Committee on Standardization of Autoantibodies in Rheumatic and Related Diseases have established a repository of well-characterized freeze-dried sera ampoules into glass vials as standards or alignment tools for producing national and local serum standards. These standards can be ordered free of charge from Center for Disease Control in Atlanta, GA. Until now 14 different standards are available and in the coming year more standards will be made available.
12.9 Efforts to harmonize clinical/laboratory collaborative work
For five years annual meetings have been organized in the Nordic countries to make clinicians aware of the importance of collaborating with clinical immunology laboratories to optimize diagnostics and make the diagnostic process more appropriate for differential diagnostic use.
Clinicians and laboratory scientists have discussed a number of items that are handled very differently in different centres with the aim to harmonize such activities to the benefit of the end user, the patient. This led to the formation of a European steering group of leading scientists in rheumatology, called EASI (European Autoimmunity Standardization Initiative). Senior people from rheumatology and clinical immunology are now being recruited from hopefully every European country with the task to plan national discussions of the items laid forward as suggestions from the steering committee, hoping that controversies between different country policies can be bridged and the suggested measures amended in such a way that all nations get a unified concept of how to interact across each country. The final plan is to have European open meetings where these plans and strategies are presented by all national delegates for fruitful discussions. The 5th International Autoimmunity Congress in Sorrento next year will set up such a general session for interested parties.
12.10 Modern technical platforms and new assays.
Many new assay platforms and new technologies to detect and quantify specific ANA have been introduced by the industry, now is the time to find out what should be their role in future autoimmune serodiagnostics using the strategy outlined above. There is no doubt that precision and speed of testing can be made much better with automation, but that is just a small part of rationalizing laboratory work and may not contribute to better diagnostics. We need to know the clinical implications of getting positive results that are not substantiated by IF methodology or precipitation techniques. We need to have many more autoantigens ideally expressed on solid phases (addressable laser bead assays, multiplex assays etc.) so that true pathological ANA are binding but polyreactive low affinity (diagnostically unimportant) ANA are not. We also need to look at the possible value of quantitating various ANA as part of disease surveillance, an area of research that has been much neglected until now. Hopefully we can also start to look at pathobiological effects of certain ANA (e.g. the complement-fixing properties of lupus-related ANA) as compared to the same ANA specificity in other diseases. We know very little about the ANA found in inflamed tissues and fluids compared to the corresponding serum ANA. Until now there are no indications that high quality detection of ANA using a solid phase principle can take the place of HEp-2 cell ANA demonstration by IF, and there are multiple reasons for that.
12.11 Important issues in health cost estimation
Many scientists have wondered how to handle the increasing complexity and demand for autoimmune serodiagnostics. Many have switched from manpower-dependent to automated technical platforms trying to keep short-term costs low. One needs to realize that health costs are very low in the early phase of chronic diseases, total laboratory costs mounting to 2-3% of the patient-related costs in Sweden, whereas the heavy costs arrive during the later phases of such diseases. These long-term costs are dependent on many factors some of which are number of visits to clinics, length of stay and cost of stay in hospital, readmission rate, working days lost for the patient and family, productive years gained, economic compensation for inability to work etc. The best way to cut these long-term costs is to set an early diagnosis through the use of optimal clinical/serological diagnostics, making decisions about interventions as rational as possible and thus effect ultimate outcome.
ANA most likely reflect tissue lesion mechanisms, genetic predisposition and perhaps etiology, are associated to diagnosis, subsyndrome categorization, and prognosis, may help planning of clinical follow-up and therapeutic strategies, are of particular value in early diseases cases, can best be revealed by IF using HEp-2 cells, and can be credibly interpreted by non-medical personnel. Modern testing platforms are perhaps easier in use but not better. To arrive at optimal clinical diagnostics patients need to donate blood for testing purposes, and clinicians and laboratory scientists need to collaborate closely.
1. Wiik A. Anti-nuclear autoantibodies: clinical utility for diagnosis, prognosis, monitoring, and planning of treatment strategy in systemic immunoinflammatory diseases. Scand J Rheumatol 2005; 34:260-8.
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