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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


IMPACT FACTOR 2018: 3.638

CiteScore 2018: 2.44

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Volume 57, Issue 11

Issues

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
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Published Online: 2019-04-22 | DOI: https://doi.org/10.1515/cclm-2019-0087

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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

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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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