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• Anti-nuclear antibodies for SLE classification versus diagnosis: titer-specific likelihood ratios to the rescue. Response to Aringer M et al. Ann Rheum Dis. 2021 Feb 10:annrheumdis-2020-219373
  Xavier Bossuyt, Walter Fierz and Pier Luigi Meroni
  Published on: 30 July 2021

• Published on: 30 July 2021

  Anti-nuclear antibodies for SLE classification versus diagnosis: titer-specific likelihood ratios to the rescue. Response to Aringer M et al. Ann Rheum Dis. 2021 Feb 10:annrheumdis-2020-219373

  • Xavier Bossuyt, MD, PhD, Clinical laboratory immunologist Laboratory medicine, University Hospitals Leuven, Belgium
  • Other Contributors:
    • Walter Fierz, MD, Clinical laboratory immunologist
    • Pier Luigi Meroni, MD, rheumatologist

  Anti-nuclear antibodies for SLE classification versus diagnosis: titer-specific likelihood ratios to the rescue. Response to Aringer M, Brinks R, Dörner T, et al. European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) SLE classification criteria item performance. Ann Rheum Dis. 2021 Feb 10:annrheumdis-2020-219373.

  Antinuclear antibodies (ANA) are important laboratory markers for the diagnosis and classification of systemic lupus erythematosus (SLE). In the 2019 European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) classification criteria for SLE, ANA with titer ≥1:80 are an entry criterion [1].
  Even though ANA 1:80 are highly sensitive for SLE, they have a low specificity. This has recently been reinforced by Aringer et al. [2] who analyzed the performance of the individual items included in the 2019 EULAR/ACR classification criteria for SLE on a large group of SLE patients (n=1197) and non-SLE disease controls (n=1074), including patients with other connective tissue diseases (two-thirds of the controls). In this study, ANA with titer ≥1:80 were highly sensitive (99.5%), but only 19.4% specific for SLE [2]. As ANA are an entry criterion for the 2019 EULAR/ACR classification criteria, the low specificity of ANA for SLE does not affect the specificity of the 2019 SLE classification criteria. An important item that conferred specificity to the 2019 SLE classification was the attribution rule [2]. The a...

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  Anti-nuclear antibodies for SLE classification versus diagnosis: titer-specific likelihood ratios to the rescue. Response to Aringer M, Brinks R, Dörner T, et al. European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) SLE classification criteria item performance. Ann Rheum Dis. 2021 Feb 10:annrheumdis-2020-219373.

  Antinuclear antibodies (ANA) are important laboratory markers for the diagnosis and classification of systemic lupus erythematosus (SLE). In the 2019 European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) classification criteria for SLE, ANA with titer ≥1:80 are an entry criterion [1].
  Even though ANA 1:80 are highly sensitive for SLE, they have a low specificity. This has recently been reinforced by Aringer et al. [2] who analyzed the performance of the individual items included in the 2019 EULAR/ACR classification criteria for SLE on a large group of SLE patients (n=1197) and non-SLE disease controls (n=1074), including patients with other connective tissue diseases (two-thirds of the controls). In this study, ANA with titer ≥1:80 were highly sensitive (99.5%), but only 19.4% specific for SLE [2]. As ANA are an entry criterion for the 2019 EULAR/ACR classification criteria, the low specificity of ANA for SLE does not affect the specificity of the 2019 SLE classification criteria. An important item that conferred specificity to the 2019 SLE classification was the attribution rule [2]. The authors stressed that ANA are a useful screening test, but not specific and that the classification criteria should not be used as diagnostic criteria [2].

  As ANA are employed in the context of both classification and diagnosis it is important to get better insights into the interpretation of ANA test results. We here argue that interpretation of ANA test results can be improved by assigning titer-specific likelihood ratios (LR)s. The LR is a good indicator of the clinical value of a test result. It is the ratio of the fraction of patients with a particular test results to the fraction of controls with the same test result. For example, a test result with a LR of 5 indicates that such result is 5 times more likely in patients than in controls.
  Positioning of ANA as an entry criterion in the 2019 EULAR/ACR classification was based on a literature review and meta-regression analysis on the performance of indirect immunofluorescence (IIF) on HEp-2 substrates [3]. In the paper that describes this meta-regression analysis (including 13080 SLE patients and 7539 controls), data on sensitivity, specificity and negative and positive likelihood ratio (LR) of ANA for SLE are given for different cutoffs (1:40, 1:80, 1:160 and 1:320) [3]. Figure 1 (left hand part) graphically recapitulates the results of the meta-regression analysis. ANA at a cutoff titer of 1:80 had a high sensitivity (97.8%) [3]. The specificity and positive likelihood ratio were 74.7 and 3.87, respectively [3]. Because of the high sensitivity, ANA at 1:80 cutoff by IIF was positioned as an entry criterion for the SLE classification [3].

  The LRs reported by Leuchten et al. [3] are for single cutoff values, implying that all values higher than the cutoff value are considered equal. For example, for a 1:80 cutoff, all patients with ANA ≥1:80 are grouped (i.e. 1:80, 1:160, 1:320, 1:640, …). Such approach does not take into account the fact that the likelihood for disease increases with increasing antibody levels. Conversely, titer-specific information gives more detailed information on the clinical value of a specific result. Based on the data provided by Leuchten et al. [3] we calculated the titer-specific LRs for a negative, 1:40, 1:80, and 1:160 test result (Figure 1, right-hand panel). The titer-specific LRs were 0.02, 0.08, 0.17, and 0.94, respectively. These titer-specific LRs are clearly lower than the LRs calculated using a dichotomous approach as reported by Leuchten et al. [3], the reason being that a dichotomous approach groups all values exceeding the cutoff. For example, the titer-specific LR for 1:80 was 0.17, whereas the positive LR associated with a single 1:80 cutoff was 3.87. Of note, the LR associated with a single 1:80 cutoff reported in the recent study by Aringer et al. [3] was 1.23.
  A test result with a LR of 0.17 (the 1:80 titer-specific LR) is 5.8 times more likely to be found in non-SLE than in SLE. Thus, even though an ANA with titer 1:80 is of use as entry criterion for classification, it is not supportive of a diagnosis of SLE (it rather makes this diagnosis less likely). According to the meta-analysis of Leuchten et al. [3], 2% of SLE patients (and 11.5% of controls) had a 1:80 ANA test result. The titer-specific LR increases with increasing titers and was 0.94 for a titer 1:160 and 25.29 for titers 1:≥320. Thus, the strength of ANA in supporting an SLE diagnosis increases with the increase in the antibody titer. Providing clinicians with such information will help them with the interpretation of ANA test results. However, there might be manufacturer-dependent differences between HEp-2 substrates [4]. Consequently, titer-specific LR may have to be established for each HEp-2 substrate.
  In addition to the titer, the IIF antibody pattern will likely provide additional information. Some patterns are associated with certain specific antibodies (and diseases) (e.g., a homogeneous pattern is found in SLE patients with antibodies to dsDNA, -histones or nucleosomes) [5-7]. The only unambiguous association is for anti-centromere antibodies with anti-centromere positive (limited cutaneous) systemic sclerosis.

  To conclude, we support the statement of Aringer er al. [1] that the classification criteria for SLE should not be used for diagnosis. A dichotomous interpretation of ANA test results overlooks the fact that the likelihood for SLE increases with increasing antibody level. Providing titer-specific LRs for ANA may be one way to help clinicians with the interpretation of ANA.

  Competing interests: none

  Contributorship: XB drafted the manuscript. All authors discussed the content and contributed to the final manuscript.
  Acknowledgements: we thank Martin Aringer for critically reading the manuscript

  Funding, grant/award info: not applicable

  Ethical approval information: not applicable

  Data sharing statement: not applicable

  Patient and Public Involvement: not applicable  

   
  References

  1. Aringer M, Costenbader K, Daikh D, et al. 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Ann Rheum Dis. 2019;78:1151-1159.
  2. Aringer M, Brinks R, Dörner T, et al. European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) SLE classification criteria item performance. Ann Rheum Dis. 2021 Feb 10:annrheumdis-2020-219373.
  3. Leuchten N, Hoyer A, Brinks R, Schoels M, Schneider M, Smolen J, Johnson SR, Daikh D, Dörner T, Aringer M, Bertsias G; Systemic Lupus Erythematosus Classification Criteria Steering Committee. Performance of Antinuclear Antibodies for Classifying Systemic Lupus Erythematosus: A Systematic Literature Review and Meta-Regression of Diagnostic Data. Arthritis Care Res (Hoboken). 2018;70:428-438.
  4. Pisetsky DS, Spencer DM, Lipsky PE, Rovin BH. Assay variation in the detection of antinuclear antibodies in the sera of patients with established SLE. Ann Rheum Dis. 2018;77:911-913.
  5. Willems P, De Langhe E, Westhovens R, et al. Antinuclear antibody as entry criterion for classification of systemic lupus erythematosus: pitfalls and opportunities. Ann Rheum Dis. 2019;78:e76.
  6. Vulsteke JB, Van Hoovels L, Willems P, et al. Titre-specific positive predictive value of antinuclear antibody patterns. Ann Rheum Dis. 2019 Oct 10:annrheumdis-2019-216245.
  7. Bossuyt X, De Langhe E, Borghi MO, Meroni PL. Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases. Nat Rev Rheumatol. 2020;16:715-726.

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  Conflict of Interest:
  None declared.

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