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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter

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Volume 57, Issue 7


Harmonizing by reducing inter-run variability: performance evaluation of a quality assurance program for antinuclear antibody detection by indirect immunofluorescence

Laura Bogaert
  • Department of Laboratory Medicine, OLV Hospital Aalst, Aalst, Belgium
  • Department of Laboratory Medicine, GZA Hospitals, Antwerp, Belgium
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Stefanie Van den Bremt / Sofie Schouwers / Xavier Bossuyt
  • Department of Laboratory Medicine, University Hospital Leuven, Leuven, Belgium
  • Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Lieve Van Hoovels
  • Corresponding author
  • Department of Laboratory Medicine, OLV Hospital Aalst, Moorselbaan 164, 9300 Aalst, Belgium, Phone: +32 (0)53/72 42 91, Fax: +32 (0)53/72 45 88
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2019-01-07 | DOI: https://doi.org/10.1515/cclm-2018-0933



The introduction of automated anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) analysis may allow for more harmonized ANA IIF reporting, provided that a thorough quality assurance program controls this process. The aim of this study was to evaluate various quality indicators used for ANA IIF analysis with the final goal of optimizing the iQC program.


In an experimental setup, we introduced artificial errors, mimicking plausible problems during routine practice on a QUANTA-Lyser-NOVA View® system (Inova Diagnostics, San Diego, CA, USA). Predetermined quality indicators were evaluated against predefined acceptance criteria. In addition, we retrospectively investigated the applicability of the selected quality indicators in the daily routine practice during three pre-defined periods.


Both the experimental as the retrospective study revealed that pre-analytical, analytical and post-analytical errors were not highlighted by company internal quality control (iQC) materials. The use of patient derived iQC samples, median fluorescence intensity results per run and the percentage of positive ANA IIF results as additional quality indicators ensured a more adequate ANA IIF quality assurance. Furthermore, negative and moderate positive sample iQC materials merit clinical validation, as titer changes of >1 correspond to clinically important shifts. Traditional Westgard rules, including a clinically defined stop limit, revealed to be useful in monitoring of the supplemental quality indicators.


A thorough ANA IIF quality assurance for daily routine practice necessitates the addition of supplemental quality indicators in combination with well-defined acceptance criteria.

This article offers supplementary material which is provided at the end of the article.

Keywords: antinuclear antibodies; automation; indirect immunofluorescence; quality control


  • 1.

    Solomon DH, Kavanaugh AJ, Schur PH, American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum 2002;47:434–44.CrossrefGoogle Scholar

  • 2.

    Fritzler MJ. Choosing wisely: review and commentary on anti-nuclear antibody (ANA) testing. Autoimmun Rev 2016;15: 272–80.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 3.

    Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010;69:1420–2.Web of SciencePubMedCrossrefGoogle Scholar

  • 4.

    Rigon A, Infantino M, Merone M, Iannello G, Tincani A, Cavazzana I, et al. The inter-observer reading variability in anti-nuclear antibodies indirect (ANA) immunofluorescence test: a multicenter evaluation and a review of the literature. Autoimmun Rev 2017;16:1224–9.CrossrefWeb of ScienceGoogle Scholar

  • 5.

    Infantino M, Meacci F, Grossi V, Manfredi M, Benucci M, Merone M, et al. The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescence test. Immunol Res 2017;65:345–54.PubMedCrossrefWeb of ScienceGoogle Scholar

  • 6.

    Meroni PL, Chan EK, Damoiseaux J, Andrade LE, Bossuyt X, Conrad K, et al. Unending story of the indirect immunofluorescence assay on HEp-2 cells: old problems and new solutions? Ann Rheum Dis 2018 Apr 17; doi: 10.1136/annrheumdis-2018-213440.Web of ScienceGoogle Scholar

  • 7.

    Agmon-Levin N, Damoiseaux J, Kallenberg C, Sack U, Witte T, Herold M, et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis 2014;73:17–23.PubMedCrossrefWeb of ScienceGoogle Scholar

  • 8.

    CLSI I/LA02-A2. Quality assurance of laboratory tests for autoantibodies to nuclear antigens: (1) Indirect Fluorescence Assay for Microscopy and (2) Microtiter Enzyme Immunoassay Methods; Approved guideline – Second edition. Wayne, PA: Clinical and Laboratory Standards Institute, 2006.Google Scholar

  • 9.

    Mahler M. Lack of standardisation of ANA and implications for drug development and precision medicine. Ann Rheum Dis 2018 Mar 24; doi: 10.1136/annrheumdis-2018-213374.Web of ScienceGoogle Scholar

  • 10.

    Van Hoovels L, Schouwers S, Van den Bremt S, Bossuyt X. Variation in antinuclear antibody detection by automated indirect immunofluorescence analysis. Ann Rheum Dis 2018 Apr 20; doi: 10.1136/annrheumdis-2018-213543.Web of ScienceGoogle Scholar

  • 11.

    Maenhout TM, Bonroy C, Verfaillie C, Stove V, Devreese K. Automated indirect immunofluorescence microscopy enables the implementation of a quantitative internal quality control system for anti-nuclear antibody analysis. Clin Chem Lab Med 2014;52:989–98.PubMedWeb of ScienceGoogle Scholar

  • 12.

    Mulliez SM, Maenhout TM, Bonroy C. Impact of the routine implementation of automated indirect immunofluorescence antinuclear antibody analysis: 1 year of experience. Clin Chem Lab Med 2016;54:e183–6.PubMedWeb of ScienceGoogle Scholar

  • 13.

    Van den Bremt S, Schouwers S, Van Blerk M, Van Hoovels L. ANA IIF Automation: moving towards harmonization? Results of a multicenter study. J Immunol Res 2017;2017:6038137.PubMedWeb of ScienceGoogle Scholar

  • 14.

    Andrade LE, Klotz W, Herold M, Conrad K, Rönnelid J, Fritzler MJ, et al. International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I. Clin Chem Lab Med 2018;56:1783–88.PubMedCrossrefWeb of ScienceGoogle Scholar

  • 15.

    CLSI EP5-A2. Evaluation of precision performance of quantitative measurement methods; Approved guideline – Second edition. Wayne, PA: Clinical and Laboratory Standards Institute, 2004.Google Scholar

  • 16.

    Willems P, De Langhe E, Claessens J, Westhovens R, Van Hoeyveld E, Poesen K, et al. Screening for connective tissue disease-associated antibodies by automated immunoassay. Clin Chem Lab Med 2018;56:909–18.CrossrefWeb of SciencePubMedGoogle Scholar

  • 17.

    Bossuyt X, Fieuws S. Detection of antinuclear antibodies: added value of solid phase assay? [Letter] Ann Rheum Dis 2014;73:e10.CrossrefGoogle Scholar

  • 18.

    Pérez D, Gilburd B, Azoulay D, Shovman O, Bizzaro N, Shoenfeld Y. Antinuclear antibodies: is the indirect immunofluorescence still the gold standard or should be replaced by solid phase assays? Autoimmun Rev 2018;17:548–52.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 19.

    Bizzaro N, Brusca I, Previtali G, Alessio MG, Daves M, Platzgummer S, et al. The association of solid-phase assays to immunofluorescence increases the diagnostic accuracy for ANA screening in patients with autoimmune rheumatic diseases. Autoimmun Rev 2018;17:541–7.CrossrefPubMedWeb of ScienceGoogle Scholar

  • 20.

    Claessens J, Belmondo T, De Langhe E, Westhovens R, Poesen K, Hüe S, et al. Solid phase assays versus automated indirect immunofluorescence for detection of antinuclear antibodies. Autoimmun Rev 2018;17:533–40.Web of ScienceCrossrefPubMedGoogle Scholar

  • 21.

    Willems P, De Langhe E, Westhovens R, Vanderschueren S, Blockmans D, Bossuyt X. Antinuclear antibody as entry criterion for classification of systemic lupus erythematosus: pitfalls and opportunities. Ann Rheum Dis 2018 Jun 23; doi: 10.1136/annrheumdis-2018-213821.Web of ScienceGoogle Scholar

  • 22.

    Meroni PL, Bizzaro N, Cavazzana I, Borghi MO, Tincani A. Automated tests of ANA immunofluorescence as throughput autoantibody detection technology: strengths and limitations. BMC 2014;12:38.Web of ScienceGoogle Scholar

  • 23.

    Tozzoli R, Villalta D, Bizzaro N. Challenges in the standardization of autoantibody testing: a comprehensive review. Clin Rev Allergy Immunol 2017;53:68–77.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 24.

    Van Hoovels L, Schouwers S, Van den Bremt S, Bogaert L, Vandeputte N, Vercammen M, et al. Analytical performance of the single well titer function of NOVA View®: good enough to omit ANA IIF titer analysis? [Letter] Clin Chem Lab Med 2018;56:e258–61.Web of ScienceCrossrefGoogle Scholar

About the article

Received: 2018-08-27

Accepted: 2018-12-05

Published Online: 2019-01-07

Published in Print: 2019-06-26

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: None declared.

Employment or leadership: XB has been consultant for Inova Diagnostics.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 57, Issue 7, Pages 990–998, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2018-0933.

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