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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 56, Issue 10


Reference standards for the detection of anti-mitochondrial and anti-rods/rings autoantibodies

S. John Calise / Bing Zheng / Tomoko Hasegawa
  • Department of Clinical Nursing, University of Occupational and Environmental Health, Kitakyushu, Japan
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/ Minoru Satoh
  • Department of Clinical Nursing, University of Occupational and Environmental Health, Kitakyushu, Japan
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/ Natasa Isailovic
  • Laboratory of Autoimmunity and Metabolism, Division of Rheumatology and Clinical Immunology, Humanitas Research Hospital, Rozzano, Milan, Italy
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/ Angela Ceribelli
  • Laboratory of Autoimmunity and Metabolism, Division of Rheumatology and Clinical Immunology, Humanitas Research Hospital, Rozzano, Milan, Italy
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/ Luis E.C. Andrade
  • Division of Rheumatology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
  • Immunology Division, Fleury Medicine and Health Laboratories, São Paulo, Brazil
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/ Katherine Boylan / Ilaria Cavazzana
  • Department of Clinical and Experimental Science, University of Brescia and ASST-Spedali Civili di Brescia, Brescia, Italy
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/ Marvin J. Fritzler / Ignacio Garcia de la Torre
  • Department of Immunology and Rheumatology, Hospital General de Occidente and University of Guadalajara, Guadalajara, Mexico
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/ Falk Hiepe
  • Charité – Universitätsmedizin Berlin and Deutsches Rheumaforschungszentrum Berlin, Berlin, Germany
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/ Kathryn Kohl / Carlo Selmi
  • Laboratory of Autoimmunity and Metabolism, Division of Rheumatology and Clinical Immunology, Humanitas Research Hospital, Rozzano, Milan, Italy
  • BIOMETRA Department, University of Milan, Milan, Italy
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/ Yehuda Shoenfeld / Angela Tincani
  • Department of Clinical and Experimental Science, University of Brescia and ASST-Spedali Civili di Brescia, Brescia, Italy
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/ Edward K.L. Chan /
Published Online: 2018-02-26 | DOI: https://doi.org/10.1515/cclm-2017-1152



Anti-mitochondrial antibodies (AMA) are found in >90% of primary biliary cholangitis patients. Anti-rods/rings antibodies (anti-RR) are most commonly associated with interferon-α and ribavirin treatment in hepatitis C patients. Clinical laboratories routinely screen for AMA and anti-RR using indirect immunofluorescence on HEp-2 cells (HEp-2-IFA). Therefore, we sought to establish reference materials for use in AMA and anti-RR testing.


AMA-positive and anti-RR-positive human plasma samples (AMA-REF and RR-REF), identified as potential reference materials based on preliminary data, were further validated by multiple laboratories using HEp-2-IFA, immunoprecipitation (IP), western blotting, IP-western, line immunoassay (LIA), addressable laser bead immunoassay (ALBIA) and enzyme-linked immunosorbent assay (ELISA).


AMA-REF showed a strong positive cytoplasmic reticular/AMA staining pattern by HEp-2-IFA to ≥1:1280 dilution and positive signal on rodent kidney/stomach/liver tissue. AMA-REF reacted with E2/E3, E3BP, E1α and E1β subunits of the pyruvate dehydrogenase complex by IP and western blotting and was positive for AMA antigens by LIA, ALBIA and ELISA. RR-REF showed a strong positive rods and rings staining pattern by HEp-2-IFA to ≥1:1280 dilution. RR-REF reacted with inosine monophosphate dehydrogenase by IP, IP-western and ALBIA. RR-REF also produced a nuclear homogenous staining pattern by HEp-2-IFA, immunoprecipitated proteins associated with anti-U1RNP antibody and reacted weakly with histones, nucleosomes, Sm and nRNP/Sm by LIA.


AMA-REF and RR-REF are useful reference materials for academic or commercial clinical laboratories to calibrate and establish internal reference standards for immunodiagnostic assays. AMA-REF and RR-REF are now available for free distribution to qualified laboratories through Plasma Services Group.

Keywords: anti-mitochondrial antibody; anti-rods/rings antibody; autoantibody; autoimmunity; hepatitis C; primary biliary cholangitis


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About the article

Corresponding author: Professor Edward K.L. Chan, Department of Oral Biology, University of Florida, 1395 Center Drive, Gainesville, FL 32610-0424, USA, Phone: (+1) 352-273-8849; Fax: (+1) 352-273-8829

aThe IUIS Autoantibody Standardization Committee: www.AutoAb.org; echan@ufl.edu

Received: 2017-12-10

Accepted: 2018-01-24

Published Online: 2018-02-26

Published in Print: 2018-09-25

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

Research funding: SJC is supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1315138 and was previously supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under Award No. 2T90DE021990-06 while this work was performed. MS is supported by JSPS KAKENHI (Grants-in-Aid for Scientific Research) grant number 15K08790. MJF is a coinvestigator on the UCAN-CANDU project funded by the Canadian Institutes of Health Research. IGDLT receives support from the Mexican National Research System (SNI) from Conacyt (National Council of Science and Technology). CS is supported by the Italian Ministry of Foreign Affairs (PGR00807) and receives research funding from AESKU.Diagnostics GmbH and Menarini Diagnostics (Florence, Italy). The IUIS Autoantibody Standardization Committee has received unrestricted grants from Bio-Rad, Inova Diagnostics and Euroimmun. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health.

Employment or leadership: None declared.

Honorarium: LECA has received speaking honoraria from Inova Diagnostics and Werfen International (Barcelona, Spain). MJF has received speaking honoraria from Inova Diagnostics, Werfen International, Amgen Canada (Mississauga, ON, Canada) and Alexion Pharmaceuticals (New Haven, CT, USA). CS has received speaking honoraria from AESKU.Diagnostics GmbH, Menarini Diagnostics and Grifols (Barcelona, Spain). AC and EKLC have also received speaking honoraria from Grifols.

Competing interests: MJF was and/or continues to be a consultant to Inova Diagnostics, Werfen International, Alexion Pharmaceuticals and Bio-Rad. CS is a consultant for AESKU.Diagnostics GmbH and Grifols. The funding organizations 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 10, Pages 1789–1798, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2017-1152.

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