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Abstract
The impact of autoimmune diseases is growing from both a clinical and a laboratory point of view. Diagnostic assays are now being transferred from dedicated specialised laboratories into high-throughput service laboratories. The increasing number of available methods has raised the variability among the laboratories, making their reproducibility a critical problem. On the other hand, reliable tests are needed for early diagnosis and prompt treatment, and the cost of repeated confirmatory tests should be reduced and unnecessary further investigations avoided. New methodologies, particularly for antinuclear antibodies (ANAs), have been applied to autoantibody detection in order to screen and process larger volumes of clinical specimens more quickly and at less cost than the traditional methods. However, because of the lack of their sensitivity to detect all ANAs and standardisation, inaccuracies (including false positives and negatives) in the results have been reported. A committee of the American College of Rheumatology was formed to address this issue. Specific recommendations have been suggested that represent a realistic first step in the standardisation of diagnostic tests for autoimmune diseases.
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Background
Detection and quantitation of autoantibodies are vital tests in the diagnosis and management of patients with autoimmune diseases (AIDs).1 2 Manual, qualitative or semiquantitative assays were initially used for the detection of these autoantibodies but have now been largely replaced by quantitative automated high-volume solid phase methods. The assays now available have improved our diagnostic power and consequently increased the impact of AIDs in daily practice, both from a clinical and a laboratory point of view. The current tests are mainly based on ELISA or other new solid phase methods, including the micro array systems.1 2 These assays are now widely available on automated analysers and are being moved from dedicated specialised laboratories into highthroughput routine service laboratories.2
The increasing number of available methods and the spread of autoimmunity testing make the problem of quality control (reproducibility, sensitivity and specificity) and clinical interpretation of particular concern. The accuracy of any test is also critical for making an early diagnosis, a prerequisite for the successful treatment of many AIDs. In addition to the problem of a delay in treatment, a wrong diagnosis—either through false positive or false negative tests—may also be responsible for additional costs due to the repetition of confirmatory tests and/or to consequent unnecessary diagnostic investigations.1 2 The last point is a further reason why an ethical aspect of the issue has recently been raised.3
In addition, the use of new automated screening systems for autoantibodies raises the problem of their clinical significance in comparison with those detectable by ‘historical’ methods. No well-planned studies comparing the sensitivity, specificity, positive and negative predictive values of the old and new methods have been undertaken, so there are no specific guidelines regarding how to use and interpret these new assays.
This problem is currently being addressed by international ad hoc committees such as the IUIS/WHO/AF/CDC Committee for the Standardization of Autoantibodies in Rheumatic and Related Diseases and by a Task Force of the American College of Rheumatology (ACR) relating to antinuclear antibodies (ANAs), as well as others.3,–,6
ANA screening: a paradigmatic example of the problem
Members of the ACR recently informed the College regarding inaccurate results for ANA tests. In response, the ACR created an ANA Task Force in 2007 to evaluate the extent of the problem and to recommend solutions.
The Task Force reported that many large commercial laboratories as well as some smaller hospital laboratories are not using the most accepted ANA screening by immunofluorescence (IFA) on HEp-2 (or other) cells. In fact, many of these laboratories have been testing for ANAs by ELISA or coated beads as a cost-saving method. This has resulted in a significant increase in false negative tests, as many patients with systemic lupus erythematosus (SLE) and other connective tissue diseases may have autoantibodies detectable by IFA but not by solid phase substrates that employ a limited number of autoantigens. This issue is well illuminated in a recent case report from the Massachusetts General Hospital where the diagnosis of SLE was significantly delayed because of a false negative ANA test determined using a non-IFA method.7
The Task Force has reviewed the relevant literature published to date and has concluded that solid phase immunoassays may not be appropriate at present for replacing IFA as a screening test for the detection of ANA. The number of patients with AIDs failing to be diagnosed because of a negative ANA ‘screen’ using ANA ELISA or multiplex assay cannot be adequately ascertained from the available literature, but may be as high as 35% (table 1).8,–,33 Accordingly, the Committee prepared a position report addressing this problem and suggesting specific recommendations (box 1).
Antinuclear antibodies (ANA) in systemic lupus erythematosus (SLE) by immunofluorescence (IFA) and solid phase immunoassay
Box 1 Recommendations of the American College of Rheumatology (ACR) Antinuclear Antibody (ANA) Task Force
▶. Immunofluorescence ANA test should remain the gold standard for ANA testing.
▶. Hospital and commercial laboratories using bead-based multiplex platforms or other solid phase assays for detecting ANA must provide data to ordering physicians on request that their assay has the same or improved sensitivity and specificity as the immunofluorescence ANA.
▶. In-house assays for detecting ANA as well as anti-DNA, anti-Sm, anti-RNP, anti-Ro/SS-A, anti-La/SS-B, etc, should be standardised according to national (eg, CDC) and/or international (eg, WHO, IUIS) standards.
▶. Laboratories should specify the methods used for detecting ANA when reporting their results.
Members of the ACR ANA Task Force: Peter Schur (Chair), Donald Bloch, Joe Craft, John A Goldman, Pier L Meroni, Eileen Moynihan, Morris Reichlin, Westley Reeves, Eng Tan, Dan Wallace and Mark Wener.
Rheumatologists should be kept informed of such problems and of the possible solutions to avoid misdiagnosis.
Efforts in standardisation of diagnostic tests for AIDs are a necessary task whose goals include the validation of new tests in comparison with the historical ones, the evaluation of their diagnostic/prognostic value, the identification/interpretation of useful algorithms and the standardisation of reference material.
Acknowledgments
The authors thank Dr Donald Bloch for reviewing the literature and Drs Eng Tan and Edward K L Chan for their help in the preparation of this manuscript.
References
Footnotes
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Competing interests None.
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Funding This paper was supported by Minister of Health, Italy, Ricerca Corrente IRCCS Istituto Auxologico Italiano to PLM.
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Provenance and peer review Not commissioned; externally peer reviewed.