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Preliminary evaluation of the first international reference preparation for anticitrullinated peptide antibodies
  1. Nicola Bizzaro1,
  2. Francesca Pregnolato2,
  3. Martin A M van Boekel3,
  4. Danilo Villalta4,
  5. Renato Tozzoli5,
  6. Elio Tonutti6,
  7. Antonio Antico7,
  8. Maria Orietta Borghi2,8,
  9. Allan Wiik9,
  10. Pier Luigi Meroni2,10
  1. 1Laboratory of Clinical Pathology, Civic Hospital, Tolmezzo, Italy
  2. 2Experimental Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Milan, Italy
  3. 3Afdeling Laboratorium, St. Maartenskliniek, Nijmegen, The Netherlands
  4. 4Clinical Immunology and Virology, S Maria degli Angeli Hospital, Pordenone, Italy
  5. 5Laboratory of Clinical Chemistry and Microbiology, Civic Hospital, Latisana, Italy
  6. 6Department of Immunology and Allergology, S Maria della Misericordia University Hospital, Udine, Italy
  7. 7Laboratory of Clinical Pathology, Civic Hospital, Cittadella, Italy
  8. 8Department of Internal Medicine, University of Milan, Milan, Italy
  9. 9Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark
  10. 10Division of Rheumatology, Istituto G Pini, Department of Internal Medicine, University of Milan, Milan, Italy
  1. Correspondence to Pier Luigi Meroni, Department of Rheumatology, Istituto G Pini, Piazza C Ferrari, 1 20122 Milan, Italy; pierluigi.meroni{at}


Background A lyophilised reference serum from one patient with rheumatoid arthritis (RA) diluted with serum samples from healthy subjects was evaluated as a possible first international standard for anticitrullinated peptide antibodies (ACPAs).

Methods The authors used 12 commercial ELISAs for ACPA detection in the reference serum and for testing the linearity of the assays by studying twofold serial dilutions. To test the effectiveness of the standardisation, sera from 20 RA patients with variable antibody concentrations were analysed, and the relative concentrations were calculated using both the kit's own curve and the six dilutions of the reference serum as a calibration curve. Fifty sera from normal healthy subjects were used to calculate cut-off values for the reference serum using each commercial kit.

Results The calibration curve obtained for each of the 12 methods using the reference sample dilutions as calibrator allowed harmonisation of the ACPA concentration of the 20 RA serum samples, significantly reducing the dispersion of the values. The mean coefficient of variation (CV) was reduced from 76.4% to 27.9% (p=0.018) and from 85.9% to 33.5% (p=0.028) for the medium/high and negative samples, respectively. Low positive sera CV was also reduced, but to a smaller degree, from 82.5% to 55.5% (p=0.043).

Conclusion This first evaluation of the behaviour of the ACPA reference serum demonstrated that it tested positive in all the assays and that it may be used as a reference standard for establishing calibration curves, reducing the dispersion of antibody values and better comparing results obtained from different methods/laboratories.

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  • NB and FP contributed equally to the study.

  • Competing interests MAMB was an employee of Euro-Diagnostica BV at the time the study was performed.

  • Ethics approval Ethics committee of the Istituto Auxologico Italiano.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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