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Clinical Chemistry and Laboratory Medicine (CCLM)

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

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Volume 56, Issue 1


Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting – strengths and weaknesses

Wymke Hormann / Melanie Hahn / Stefan Gerlach / Nicola Hochstrate / Kai Affeldt / Joyce Giesen
  • Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Kai Fechner / Jan G.M.C. Damoiseaux
  • Corresponding author
  • Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2017-06-23 | DOI: https://doi.org/10.1515/cclm-2017-0326



Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis.


We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months.


Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation.


Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

Keywords: autoantibodies; automation; Crithidia luciliae; dsDNA; indirect immunofluorescence; systemic lupus erythematosus (SLE)


  • 1.

    Tan EM, Cohen AS, Fries JF, Masi AT, Mcshane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7.PubMedCrossrefGoogle Scholar

  • 2.

    Hochberg MC. Updating the American college of rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725.CrossrefPubMedGoogle Scholar

  • 3.

    Petri M, Orbai AM, Alarcõn GS, Gordon C, Merrill JT, Fortin PR, et al. Derivation and validation of the systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012;64:2677–86.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 4.

    Ho A, Magder LS, Barr SG, Petri M. Decreases in anti-double-stranded DNA levels are associated with concurrent flares in patients with systemic lupus erythematosus. Arthritis Rheum 2001;44:2342–9.PubMedCrossrefGoogle Scholar

  • 5.

    Mastroianni-Kirsztajn G, Hornig N, Schlumberger W. Autoantibodies in renal diseases - clinical significance and recent developments in serological detection. Front Immunol 2015;6:221.PubMedWeb of ScienceGoogle Scholar

  • 6.

    Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol 2000;53:424–32.CrossrefGoogle Scholar

  • 7.

    Cozzani E, Drosera M, Gasparini G, Parodi A. Serology of lupus erythematosus: correlation between immunopathological features and clinical aspects. Autoimmune Dis 2014;2014:e321359.Google Scholar

  • 8.

    Kavanaugh AF, Solomon DH. Guidelines for immunologic laboratory testing in the rheumatic diseases: Anti-DNA antibody tests. Arthritis Rheum 2002;47:546–55.CrossrefPubMedGoogle Scholar

  • 9.

    Antico A, Platzgummer S, Bassetti D, Bizzaro N, Tozzoli R, Villalta D. Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsDNA antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test. Lupus 2010;19:906–12.Web of SciencePubMedCrossrefGoogle Scholar

  • 10.

    Rouquette AM, Desgruelles C. Detection of antibodies to dsDNA: an overview of laboratory assays. Lupus 2006;15:403–7.PubMedCrossrefGoogle Scholar

  • 11.

    Hughes R, Ui-Hassan S. Anti-dsDNA antibodies: their role in the detection and monitoring of SLE. Clin Lab Int 2006;7:12–7.Google Scholar

  • 12.

    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.Web of ScienceCrossrefPubMedGoogle Scholar

  • 13.

    Damoiseaux J, Agmon-Levin N, Van Blerk M, Chopyak V, Eriksson C, Heijnen I, et al. From ANA-screening to antigen-specificity: an EASI-survey on the daily practice in European countries. Clin Exp Rheumatol 2014;32:539–46.PubMedGoogle Scholar

  • 14.

    Haugbro K, Nossent JC, Winkler T, Figenschau Y, Rekvig OP. Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 2004;63:386–94.PubMedCrossrefGoogle Scholar

  • 15.

    Chiaro TR, Davis KW, Wilson A, Suh-Lailam B, Tebo AE. Significant differences in the analytic concordance between anti-dsDNA IgG antibody assays for the diagnosis of systemic lupus erythematosus-Implications for inter-laboratory testing. Clin Chim Acta 2011;412:1076–80.Web of ScienceCrossrefGoogle Scholar

  • 16.

    Biesen R, Dähnrich C, Rosemann A, Barkhudarova F, Rose T, Jakob O, et al. Anti-dsDNA-NcX ELISA: dsDNA-loaded nucleosomes improve diagnosis and monitoring of disease activity in systemic lupus erythematosus. Arthritis Res Ther BioMed Central Ltd 2011;13:R26.CrossrefGoogle Scholar

  • 17.

    Dieker J, Schlumberger W, McHugh N, Hamann P, Vlag J van der, Berden JH. Reactivity in ELISA with DNA-loaded nucleosomes in patients with proliferative lupus nephritis. Mol Immunol 2015;68:20–4.CrossrefPubMedWeb of ScienceGoogle Scholar

  • 18.

    Tozzoli R, Bizzaro N, Tonutti E, Villalta D, Bassetti D, Manoni F, et al. Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rhuematic diseases. Am J Clin Pathol 2002;117:316–24.CrossrefGoogle Scholar

  • 19.

    Aarden L, de Groot E, Feltkamp T. Crithidia luciliae, a simple substrate for the determination of anti-dsDNA with the immunofluorescence technique. Ann N Y Acad Sci 1975;254:505–15.PubMedCrossrefGoogle Scholar

  • 20.

    Bizzaro N, Tozzoli R, Tonutti E, Piazza A, Manoni F, Ghirardello A, et al. Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry. J Immunol Methods 1998;219:99–107.PubMedCrossrefGoogle Scholar

  • 21.

    Rigon A, Soda P, Zennaro D, Iannello G, Afeltra A. Indirect immunofluorescence in autoimmune diseases: assessment of digital images for diagnostic purpose. Cytom Part B (Clinical Cytom) 2007;72B:472–7.CrossrefGoogle Scholar

  • 22.

    Egerer K, Roggenbuck D, Hiemann R, Weyer M-G, Büttner T, Radau B, et al. Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests. Arthritis Res Ther 2010;12:R40.Web of ScienceCrossrefPubMedGoogle Scholar

  • 23.

    Perner P, Perner H, Müller B. Mining knowledge for HEp-2 cell image classification. Artif Intell Med 2002;26:161–73.PubMedCrossrefGoogle Scholar

  • 24.

    Hiemann R, Hilger N, Michel J, Nitschke J, Böhm A, Anderer U, et al. Automatic analysis of immunofluorescence patterns of HEp-2 cells. Ann N Y Acad Sci 2007;1109:358–71.PubMedCrossrefGoogle Scholar

  • 25.

    Knütter I, Hiemann R, Brumma T, Buttner T, Grossmann K, Cusini M, et al. Automated interpretation of ANCA patterns - a new approach in the serology of ANCA-associated vasculitis. Arthritis Res Ther 2012;14:R271.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 26.

    Voigt J, Krause C, Rohwäder E, Saschenbrecker S, Hahn M, Danckwardt M, et al. Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells. Clin Dev Immunol 2012;2012:651068.Web of ScienceGoogle Scholar

  • 27.

    Krause C, Ens K, Fechner K, Voigt J, Fraune J, Rohwa E. EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics. Lupus 2015;24:516–29.Web of SciencePubMedCrossrefGoogle Scholar

  • 28.

    Tonti S, Di Cataldo S, Bottino A, Ficarra E. An automated approach to the segmentation of HEp-2 cells for the indirect immunofluorescence ANA test. Comput Med Imaging Graph 2015;40:62–9.CrossrefPubMedWeb of ScienceGoogle Scholar

  • 29.

    Di Cataldo S, Tonti S, Bottino A, Ficarra E. ANAlyte: A modular image analysis tool for ANA testing with indirect immunofluorescence. Comput Methods Programs Biomed 2016;128:86–99.Web of ScienceCrossrefPubMedGoogle Scholar

  • 30.

    Fraune J, Gerlach S, Rentzsch K, Teegen B, Lederer S, Affeldt K, et al. Multiparametric serological testing in autoimmune encephalitis using computer-aided immunofluorescence microscopy (CAIFM). Autoimmun Rev 2016;15:937–42.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 31.

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

  • 32.

    Rohwäder E, Locke M, Fraune J, Fechner K. Diagnostic profile on the IFA 40: HEp-20-10-an immunofluorescence test for reliable antinuclear antibody screening. Expert Rev Mol Diagn 2015;15:451–62.CrossrefWeb of SciencePubMedGoogle Scholar

  • 33.

    Tozzoli R, Antico A, Porcelli B, Bassetti D. Automation in indirect immunofluorescence testing: A new step in the evolution of the autoimmunology laboratory. Auto Immun Highlights 2012;3:59–65.CrossrefPubMedGoogle Scholar

  • 34.

    Bizzaro N, Antico A, Platzgummer S, Tonutti E, Bassetti D, Pesente F, et al. Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems. Autoimmun Rev 2014;13:292–8.Web of ScienceCrossrefPubMedGoogle Scholar

  • 35.

    Melegari A, Bonaguri C, Russo A, Luisita B, Trenti T, Lippi G. A comparative study on the reliability of an automated system for the evaluation of cell-based indirect immunofluorescence. Autoimmun Rev 2012;11:713–6.CrossrefWeb of SciencePubMedGoogle Scholar

  • 36.

    Bonroy C, Verfaillie C, Smith V, Persijn L, De Witte E, De Keyser F, et al. Automated indirect immunofluorescence antinuclear antibody analysis is a standardized alternative for visual microscope interpretation. Clin Chem Lab Med 2013;51:1771–9.PubMedWeb of ScienceGoogle Scholar

  • 37.

    Hayashi N, Saegusa J, Uto K, Oyabu C, Saito T, Sato I, et al. Evaluation of a computer-aided microscope system and its anti-nuclear antibody test kit for indirect immunofluorescence assay. Rinsho Byori 2016;64:142–1451.PubMedGoogle Scholar

  • 38.

    Soda P, Onofri L, Iannello G. A decision support system for Crithidia Luciliae image classification. Artif Intell Med 2011;51:67–74.Web of SciencePubMedCrossrefGoogle Scholar

  • 39.

    Buzzulini F, Rigon A, Soda P, Onofri L, Infantino M, Arcarese L, et al. The classification of Crithidia luciliae immunofluorescence test (CLIFT) using a novel automated system. Arthritis Res Ther 2014;16:R71.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 40.

    Gerlach S, Affeldt K, Pototzki L, Krause C, Voigt J, Fraune J, et al. Automated evaluation of crithidia luciliae based indirect immunofluorescence tests: A novel application of the EUROPattern-suite technology. J Immunol Res 2015;2015:742402.PubMedWeb of ScienceGoogle Scholar

  • 41.

    Lakos G, Gonzalez M, Flaherty D, Bentow C, Ibarra C, Stimson D, et al. Detection of anti-dsDNA antibodies by computer-aided automated immunofluorescence analysis. J Immunol Methods 2016;433:17–22.CrossrefWeb of SciencePubMedGoogle Scholar

  • 42.

    Avery TY, van de Cruys M, Austen J, Stals F, Damoiseaux JGMC. Anti-nuclear antibodies in daily clinical practice: prevalence in primary, secondary, and tertiary care. J Immunol Res 2014;2014:401739.Web of SciencePubMedGoogle Scholar

  • 43.

    Bossuyt X, Cooreman S, De Baere H, Verschueren P, Westhovens R, Blockmans D, et al. Detection of antinuclear antibodies by automated indirect immunofluorescence analysis. Clin Chim Acta 2013;415:101–6.CrossrefWeb of SciencePubMedGoogle Scholar

  • 44.

    Damoiseaux J, Mallet K, Vaessen M, Austen J, Cohen Tervaert JW. Automatic reading of ANCA-slides: evaluation of the AKLIDES system. Clin Dev Immunol 2012;2012:762874.PubMedWeb of ScienceGoogle Scholar

  • 45.

    Buchner C, Bryant C, Eslami A, Lakos G. Anti-nuclear antibody screening using HEp-2 cells. J Vis Exp 2014;88:e51211.Web of ScienceGoogle Scholar

  • 46.

    Tozzoli R, D’Aurizio F, Villalta D, Bizzaro N. Automation, consolidation, and integration in autoimmune diagnostics. Auto Immun Highlights 2015;6:1–6.PubMedCrossrefGoogle Scholar

  • 47.

    Csernok E, Damoiseaux J, Rasmussen N, Hellmich B, van Paassen P, Vermeersch P, et al. Evaluation of automated multi-parametric indirect immunofluorescence assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). Autoimmun Rev 2016;15:736–41.Web of ScienceCrossrefPubMedGoogle Scholar

  • 48.

    Wichainun R, Kasitanon N, Wangkaew S, Hongsongkiat S, Sukitawut W. Sensitivity and specificity of ANA and anti-dsDNA in the diagnosis of systemic lupus erythematosus: A comparison using control sera obtained from healthy individuals and patients with multiple medical problems. Asian Pacific J Allergy Immunol 2013;31:292–8.Google Scholar

  • 49.

    Lutteri L, Dierge L, Pesser M, Watrin P, Cavalier E. A paperless autoimmunity laboratory: myth or reality? Ann Biol Clin 2016;74:477–89.Web of ScienceGoogle Scholar

  • 50.

    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 (ANA) analysis. Clin Chem Lab Med 2014;52:989–98.Web of SciencePubMedGoogle Scholar

About the article

Corresponding author: Jan G.M.C. Damoiseaux, PhD, Central Diagnostic Laboratory, Maastricht University Medical Centre, P. Debyelaan 25, 6229 HX Maastricht, The Netherlands, Phone: +31 43 3876655

Received: 2017-04-15

Accepted: 2017-05-08

Published Online: 2017-06-23

Published in Print: 2017-11-27

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: W.H., M.H., S.G., K.A., N.H., and K.F. are employees of EUROIMMUN medizinische Labordiagnostika AG, Lübeck, Germany.

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 56, Issue 1, Pages 86–93, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2017-0326.

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