The recommendations for detection of autoantibodies to cellular components commonly referred to as ANA
| Recommendation | Delphi score (mean±SD) | |
|---|---|---|
| 1 | The diagnosis of SARD requires a panel of specific laboratory tests (ie, ANA, anti-dsDNA and anti-ENA antibodies) | 9.6±0.9 |
| 2 | ANA, anti-dsDNA and anti-ENA testing should be included in the autoantibodies detection as part of the diagnostic work-up of SARD as well as some other autoimmune diseases (table 2) | 9.9±0.2 |
| 3 | The detection of ANA is the first level test for laboratory diagnosis of SARD | 9.8±0.4 |
| 4 | ANA testing is primarily intended for diagnostic purposes, and not for monitoring disease progression | 9.6±0.6 |
| 5 | The IIFA* is the reference method for ANA screening. Alternative assays can be used while keeping in mind that false negative and false positive ratio of these methods may be different. Thus, if the clinical suspicion is strong and the alternative method is negative, it is mandatory to perform IIFA | 9.7±0.6 |
| 6 | Diagnostic laboratories should specify the methods used for detecting ANA when reporting their results | 9.4±0.9 |
| 7 | Tests based on a (restricted) mixture of defined nuclear antigens should not be referred to as ANA test or ANA screen | 9.7±0.5 |
| 8 | Laboratories using in-house assays for detecting ANA, as well as anti-dsDNA and specific anti-ENA antibodies, should standardise each assay according to international standards (eg, WHO, CDC/IUIS) | 9.7±0.5 |
| 9 | For ANA screening by IIFA the conjugate should consist of fluorochrome-labelled anti-human IgG-specific secondary antibodies | 9.2±1.2 |
| 10 | A proper ANA–IIFA is dependent on reagents, equipment and other local factors, thus the screening dilution should be defined locally. An abnormal ANA should be the titre above the 95th percentile of a healthy control population. In general, a screening dilution of 1 : 160 on conventional HEp-2(000) substrates is often suitable for the detection of ANA in adult populations being evaluated for SARD | 9.0±1.4 |
| 11 | In the case of a positive ANA test, it is recommended that the pattern and the highest dilution to demonstrate reactivity be reported | 9.4±0.9 |
| 12 | ANA–IIFA patterns should be reported according to standardised terminology (table 2) | 9.7±0.4 |
| 13 | Besides nuclear patterns also cytoplasmic and mitotic apparatus patterns should be reported and specified when possible (table 2) | 9.5±1.0 |
| 14 | If ANA result is positive, testing for anti-dsDNA antibodies is advised when there is clinical suspicion of SLE | 9.7±0.4 |
| 15 | For anti-dsDNA antibody determination, the Farr assay and the CLIFT offer high clinical specificity. Alternative methods may yield lower specificity and, if so, it is recommended that positive results obtained by these methods be confirmed by CLIFT or Farr assay—and be reported separately | 8.0±2.5 |
| 16 | The method used for anti-dsDNA antibody detection should be included in the test result | 9.4±0.7 |
| 17 | Results of anti-dsDNA antibody detection should be reported quantitatively (or semiquantitatively for CLIFT) | 9.7±0.4 |
| 18 | For monitoring of SLE disease activity by quantitative determination of anti-dsDNA antibodies the same method should be used | 9.9±0.3 |
| 19 | In case of a positive ANA test during the diagnostic work-up (depending on pattern, titre and/or clinical setting), it is recommended to perform specific tests for anti-ENA antibodies (table 2) | 9.8±0.3 |
| 20 | For anti-ENA antibodies detection the method used should be reported. In the case of discrepancy with IIFA or with clinical suspicion, the use of an additional method should be considered | 9.5±0.5 |
| 21 | Results of assays for antibodies to specific ENA should be reported separately (including negative results); if the result of a screening assay is reported as negative, it is sufficient to communicate which ENA are present in that assay | 9.8±0.4 |
| 22 | Quantitative determination of positive anti-RNP antibodies is recommended in case of a clinical suspicion of mixed connective tissue disease | 8.2±2.7 |
| 23 | In case of high clinical suspicion the physician request for determination of antibodies to specific ENA should be granted, irrespective of the result of the ANA test. For instance, anti-Jo-1 antibodies for clinically suspected IM, anti-ribosomal P for SLE or anti-SS-A/Ro antibodies for congenital heart block/neonatal lupus/Sjögren's syndrome/subacute cutaneous lupus | 9.9±0.1 |
| 24 | Each laboratory should verify the recommended cut-off for kits used to determine ANA. It is recommended to use age and gender matched sera from healthy subjects from the general local population; cut-offs should be defined as the 95th percentile | 8.4±2.8 |
| 25 | Each laboratory should verify the recommended cut-off for kits used to determine anti-dsDNA and anti-ENA antibodies. It is recommended to use an adequate number of samples from patients with the appropriate autoimmune diseases, disease controls and healthy controls; cut-offs should be defined using ROC curve analysis | 7.6±2.9 |
*See supplementary materials (available online only) for further information on the methodology of immunoassays.
ANA, anti-nuclear antibodies; CDC, Centers for Disease Control and Prevention; CLIFT, Crithidia luciliae immunofluorescence test; dsDNA, double-stranded DNA; ENA, extractable nuclear antigens; IIFA, indirect immunofluorescent assay; IM, inflammatory myopathies; IUIS, International Union of Immunologic Societies; RNP, ribonucleoproteins; ROC, receiver operating characteristic; SARD, systemic autoimmune rheumatic diseases; SLE, systemic lupus erythematosus; WHO, World Health Organization.