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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 11


Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera

Alessandra Dellavance / Danielle C. Baldo / Bing Zheng
  • Department of Oral Biology, University of Florida, Gainesville, FL, USA
  • Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
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/ Rodrigo A. Mora / Marvin J. Fritzler
  • Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
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/ Falk Hiepe
  • Department of Rheumatology and Clinical Immunology, Charité – Universitätmedizin Berlin, Corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
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/ Johan Rönnelid / Minoru Satoh
  • Department of Clinical Nursing, University of Occupational and Environmental Health, Kitakyushu, Japan
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/ Ignacio Garcia-De La Torre
  • Department of Immunology and Rheumatology, Hospital General de Occidente and University of Guadalajara, Guadalajara, Mexico
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/ Mark H. Wener
  • Division of Rheumatology and Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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/ Edward K.L. Chan / Luis E.C. AndradeORCID iD: https://orcid.org/0000-0001-8742-9931
  • Corresponding author
  • Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu 740 3° andar, 04023-062 São Paulo, Brazil
  • Immunology Division, Fleury Medicine and Health Laboratory, São Paulo, Brazil
  • orcid.org/0000-0001-8742-9931
  • Email
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Published Online: 2019-04-22 | DOI: https://doi.org/10.1515/cclm-2019-0087



International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies.


Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer ≥1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization.


A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 μL-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate anti-DFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation.


This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.

Keywords: antinuclear antibodies; autoantibodies; autoimmunity testing; DFS70; HEp-2 cells; reference standards


  • 1.

    Chan EK, Damoiseaux J, Carballo OG, Conrad K, Cruvinel WM, Francescantonio PL, et al. Report of the first international consensus on standardized nomenclature of antinuclear antibody HEp-2 cell patterns 2014–2015. Frontiers Immunol 2015;6:1–13.Web of ScienceGoogle Scholar

  • 2.

    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;10:1783–8.Web of ScienceGoogle Scholar

  • 3.

    Herold M, Klotz W, Andrade LE, Conrad K, Cruvinel WM, Damoiseaux J, et al. International Consensus on Antinuclear Antibody Patterns: defining negative results and reporting unidentified patterns. Clin Chem Lab Med 2018;10:1799–802.Web of ScienceGoogle Scholar

  • 4.

    Chan EK, Damoiseaux J, de Melo Cruvinel W, Carballo OG, Conrad K, Francescantonio PL, et al. Report on the second International Consensus on dense fine speckled Pattern (ICAP) workshop in Dresden 2015. Lupus 2016;25:797–804.CrossrefGoogle Scholar

  • 5.

    Damoiseaux J, Andrade LE, Carballo OG, Conrad K, Francescantonio PL, Fritzler MJ, et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis 2019; pii: annrheumdis-2018-214436. doi: 10.1136/annrheumdis-2018-214436.Web of ScienceGoogle Scholar

  • 6.

    Cozzani E, Drosera M, Gasparini G, Parodi A. Serology of lupus erythematosus: correlation between immunopathological features and clinical aspects. Autoimmune Dis 2014;2014:321359.PubMedGoogle Scholar

  • 7.

    Dellavance A, Gallindo C, Soares MG, Silva NP, Mortara RA, Andrade LE. Redefining the Scl-70 indirect immunofluorescence pattern: autoantibodies to DNA topoisomerase I yield a specific immunofluorescence pattern. Rheumatology (Oxford) 2009;48:632–8.CrossrefPubMedGoogle Scholar

  • 8.

    Douvas AS, Achten M, Tan EM. Identification of a nuclear protein (Scl-70) as a unique target of human antinuclear antibodies in scleroderma. J Biol Chem 1979;254:10514–22.PubMedGoogle Scholar

  • 9.

    Shero JH, Bordwell B, Rothfield NF, Earnshaw WC. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science 1986;231:737–40.PubMedCrossrefGoogle Scholar

  • 10.

    Dellavance A, Viana VS, Leon EP, Bonfa ES, Andrade LE, Leser PG. The clinical spectrum of antinuclear antibodies associated with the nuclear dense fine speckled immunofluorescence pattern. J Rheumatol 2005;32:2144–9.PubMedGoogle Scholar

  • 11.

    Mariz HA, Sato EI, Barbosa SH, Rodrigues SH, Dellavance A, Andrade LE. ANA HEp-2 pattern is a critical parameter for discriminating ANA-positive healthy individuals and patients with autoimmune rheumatic diseases. Arthritis Rheum 2011;63:191–200.CrossrefPubMedGoogle Scholar

  • 12.

    Ochs RL, Stein TW Jr, Peebles CL, Gittes RF, Tan EM. Autoantibodies in interstitial cystitis. J Urol 1994;3:587–92.Google Scholar

  • 13.

    Ochs RL, Muro Y, Si Y, Ge H, Chan EK, Tan EM. Autoantibodies to DFS 70 kd/transcription coactivator p75 in atopic dermatitis and other conditions. J Allergy Clin Immunol 2000;105:1211–20.PubMedCrossrefGoogle Scholar

  • 14.

    Ochs RL, Mahler M, Basu A, Rios-Colon L, Sanchez TW, Andrade LE, et al. The significance of autoantibodies to DFS70/LEDGFp75 in health and disease: integrating basic science with clinical understanding. Clin Exp Med 2016;16:27393.Web of ScienceGoogle Scholar

  • 15.

    Yamada K, Senju S, Shinohara T, Nakatsura T, Murata Y, Ishihara M, et al. Humoral immune response directed against LEDGF in patients with VKH. Immunol Lett 2001;78:161–8.PubMedCrossrefGoogle Scholar

  • 16.

    Ayaki M, Ohoguro N, Azuma N, Majima Y, Yata K, Ibaraki N, et al. Detection of cytotoxic anti-LEDGF autoantibodies in atopic dermatitis. Autoimmunity 2002;35:319–27.PubMedCrossrefGoogle Scholar

  • 17.

    Okamoto M, Ogawa Y, Watanabe A, Sugiura K, Shimomura Y, Aoki N, et al. Autoantibodies to DFS70/LEDGF are increased in alopecia areata patients. J Autoimmun 2004;23:257–66.PubMedCrossrefGoogle Scholar

  • 18.

    Watanabe K, Muro Y, Sugiura K, Tomita Y. IgE and IgG (4) autoantibodies against DFS70/LEDGF in atopic dermatitis. Autoimmunity 2011;44:511–9.Web of ScienceCrossrefPubMedGoogle Scholar

  • 19.

    Watanabe A, Kodera M, Sugiura K, Usuda T, Tan EM, Takasaki Y, et al. Anti-DFS70 antibodies in 597 healthy hospital workers. Arthritis Rheum 2004;50:892–900.PubMedCrossrefGoogle Scholar

  • 20.

    Mahler M, Parker T, Peebles CL, Andrade LE, Swart A, Carbone Y, et al. Anti-DFS70/LEDGF antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases. J Rheumatol 2012;39:2104–10.CrossrefWeb of SciencePubMedGoogle Scholar

  • 21.

    Mahler M, Hanly JG, Fritzler MJ. Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases. Autoimmun Rev 2011;11:642–45.Web of ScienceGoogle Scholar

  • 22.

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

  • 23.

    Damoiseaux JG, Tervaert JW. From ANA to ENA: how to proceed? Autoimmun Rev 2006;5:10–7.CrossrefPubMedGoogle Scholar

  • 24.

    Fritzler MJ, Rattner JB, Luft LM, Edworthy SM, Casiano CA, Peebles C, et al. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F. Autoimmun Rev 2011;4:194–200.Web of ScienceGoogle Scholar

  • 25.

    Bentow C, Fritzler MJ, Mummert E, Mahler M. Recognition of the dense fine speckled (DFS) pattern remains challenging: results from an international internet-based survey. Autoimmun Highlights 2016;7:8.CrossrefWeb of ScienceGoogle Scholar

  • 26.

    Sheldon J, Dellavance A. Strategies for building reference standards for autoantibodies. Front Immunol 2015;6:194.Web of SciencePubMedGoogle Scholar

  • 27.

    Chan EK, Fritzler MJ, Wiik A, Andrade LE, Reeves WH, Tincani A, et al. Autoantibody Standardization Committee in 2006. Autoimmun Rev 2007;6:577–80.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 28.

    Jacobs JF, Bossuyt X. Standardization and harmonization of autoimmune diagnostics. Clin Chem Lab Med 2018;56:1563–7.Web of ScienceCrossrefPubMedGoogle Scholar

  • 29.

    Bizzaro N, Pesente F, Cucchiaro F, Infantino M, Tampoia M, Villalta D, et al. Anti-DFS70 antibodies detected by immunoblot methods: a reliable tool to confirm the dense fine speckles ANA pattern. J Immunol Methods 2016;436:50–3.Web of ScienceCrossrefPubMedGoogle Scholar

  • 30.

    Bonroy C, Schouwers S, Berth M, Stubbe M, Piette Y, Hoffman I, et al. The importance of detecting anti-DFS70 in routine clinical practice: comparison of different care settings. Clin Chem Lab Med 2018;56:1090–9.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 31.

    Shovman O, Gilburd B, Chayat C, Amital H, Langevitz P, WatadA, et al. Prevalence of anti-DFS70 antibodies in patients with and without systemic autoimmune rheumatic diseases. Clin Exp Rheumatol 2017;36:121–6.PubMedGoogle Scholar

  • 32.

    Shoenfeld Y, Cervera R, Haass M, Kallenberg C, KhamashtaM, Meroni P, et al. EASI – the European Autoimmunity Standardisation Initiative: a new initiative that can contribute to agreed diagnostic models of diagnosing autoimmune disorders throughout Europe. Ann N Y Acad Sci 2007;1109:138–44.CrossrefWeb of ScienceGoogle Scholar

  • 33.

    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 antinuclear antibodies. Ann Rheum Dis 2014;73:17–23.PubMedCrossrefGoogle Scholar

  • 34.

    Meroni PL, Biggioggero M, Pierangeli SS, Sheldon J, Zegers I, Borghi MO. Standardization of autoantibody testing: a paradigm for serology in rheumatic diseases. Nat Rev Rheumatol 2014;10:35–43.CrossrefPubMedWeb of ScienceGoogle Scholar

  • 35.

    Seeling M, Bruckner C, Nimmerjahn F. Differential antibody glycosylation in autoimmunity: sweet biomarker or modulator of disease activity? Nat Rev Rheumatol 2017;13:621–30.PubMedWeb of ScienceCrossrefGoogle Scholar

  • 36.

    Merlini G, Blirup-Jensen S, Johnson AM, Sheldon J, Zegers I. IFCC Committee on Plasma Proteins (C-PP). Standardizing plasma protein measurements worldwide: a challenging enterprise. Clin Chem Lab Med 2010;48:1567–75.PubMedGoogle Scholar

  • 37.

    Infantino M, Shovman O, Pérez D, Grossi V, Manfredi M, Benucci M, et al. A better definition of the anti-DFS70 antibody screening by IIF methods. J Immunol Methods 2018;461:110–6.Web of SciencePubMedCrossrefGoogle Scholar

  • 38.

    Pena SD, Bastos-Rodrigues L, Pimenta JR, Bydlowski SP. DNA tests probe the genomic ancestry of Brazilians. Braz J Med Biol Res 2009;42:870–6.PubMedCrossrefGoogle Scholar

  • 39.

    Parra FC, Amado RC, Lambertucci JR, Rocha J, Antunes CM, Pena SD. Color and genomic ancestry in Brazilians. Proc Natl Acad Sci USA 2003;100:177–82.CrossrefGoogle Scholar

About the article

Corresponding author: Luis E.C. Andrade, MD, PhD, Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu 740 3° andar, 04023-062 São Paulo, Brazil; and Immunology Division, Fleury Medicine and Health Laboratory, São Paulo, Brazil

Received: 2019-01-25

Accepted: 2019-03-14

Published Online: 2019-04-22

Published in Print: 2019-10-25

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

Research funding: Fleury Medicine and Health Laboratory provided the material support for the selection and testing of all samples (NP-084). Luis Eduardo C. Andrade receives a research grant (#232711/2014-3) from the Brazilian Research Agency CNPq.

Employment or leadership: None declared.

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. MJF is a consultant or received honoraria form Inova Diagnostics, Euroimmun GmbH and Alexion Canada.

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 57, Issue 11, Pages 1754–1763, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2019-0087.

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