title: An investigation of the added value of an ACPA multiplex assay in an early rheumatoid arthritis setting.
abstract: BACKGROUND
  Recently, arrays have become available that allow the simultaneous analysis of several anti-citrullinated protein antibody (ACPA) reactivities using distinct citrullinated peptides. Such assays are designed for exploratory studies. The interpretation of positive antibody reactivities can best be made if the diagnostic and prognostic value of a multiplex array in an early arthritis setting is known and if the multiplex-positive patients who are negative according to three commonly used commercial ACPA assays are characterized.
  
  
  METHODS
  Using Thermo Scientific's ImmunoCap ISAC (Immuno Solid-phase Allergen Chip) system, a multiplexed array that determines reactivities to 11 citrullinated peptides, we analysed serum/plasma of 195 healthy controls and 1282 early arthritis patients from two independent cohorts: the Leiden Early Arthritis Clinic (n = 1013) and the IMPROVED (n = 269) cohort. Findings were compared with results primarily of the anti-citrullinated cyclic peptide 2 (anti-CCP-2) assay but also with anti- CCP-3 and anti-mutated citrullinated vimentin (anti-MCV) assays. The associations between ACPA reactivities and patient characteristics, risk factors (shared epitope, smoking) and disease outcomes (progression of undifferentiated arthritis to rheumatoid arthritis (RA) and severity of joint destruction) were assessed.
  
  
  RESULTS
  Thirty-one percent of anti-CCP-2-negative RA patients displayed reactivity toward citrullinated peptides in the multiplex assay. These patients had a positive signal toward a more restricted peptide repertoire than anti-CCP-2-positive RA patients (median of 1 versus 5). Within anti-CCP-2-negative patients, ACPA reactivity as detected by multiplex array was not significantly associated with known risk factors or clinical or prognostic parameters. The frequency of sera from anti-CCP-2-negative RA patients who were positive for the multiplexed peptides was comparable to the frequency in non-RA arthritic patients (27 %).
  
  
  CONCLUSIONS
  Additive citrulline peptide reactivities detected by the current multiplex system did not reach significant power to be RA-specific. The presence of residual citrulline reactivities detected by this multiplex system in arthritis patients who are negative in commercial ACPA assays needs to be interpreted with caution.
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. j.van_heemst@lumc.nl.
authors.forename: Jurgen
authors.initials: J
authors.lastname: van Heemst
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. l.a.trouw@lumc.nl.
authors.forename: Leendert A
authors.initials: LA
authors.lastname: Trouw
authors.affiliation: Laboratory of Epidermis differentiation and Rheumatoid Autoimmunity, Centre National de la Recherché Scientifique, Avenue de Grande-Bretagne 330, TSA 40031, Toulouse, 31059, Cedex 9, France. nogueira.l@chu-toulouse.fr.
authors.forename: Leonor
authors.initials: L
authors.lastname: Nogueira
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. h.w.van_steenbergen@lumc.nl.
authors.forename: Hanna W
authors.initials: HW
authors.lastname: van Steenbergen
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. a.h.m.van_der_helm@lumc.nl.
authors.forename: Annette H M
authors.initials: AH
authors.lastname: van der Helm-van Mil
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. c.f.allaart@lumc.nl.
authors.forename: Cornelia F
authors.initials: CF
authors.lastname: Allaart
authors.affiliation: Laboratory of Epidermis differentiation and Rheumatoid Autoimmunity, Centre National de la Recherché Scientifique, Avenue de Grande-Bretagne 330, TSA 40031, Toulouse, 31059, Cedex 9, France. guy.serre@udear.cnrs.fr.
authors.forename: Guy
authors.initials: G
authors.lastname: Serre
authors.affiliation: Department of Medical Biochemistry and Biophysics, Medical Inflammation Research Unit, Karolinska Institutet, Stockholm, SE-171 77, Sweden. rikard.holmdahl@ki.se.
authors.forename: Rikard
authors.initials: R
authors.lastname: Holmdahl
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. t.w.j.huizinga@lumc.nl.
authors.forename: Tom W J
authors.initials: TW
authors.lastname: Huizinga
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. r.e.m.toes@lumc.nl.
authors.forename: René E M
authors.initials: RE
authors.lastname: Toes
authors.affiliation: Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, PO Box 9600, Leiden, 2300 RC, The Netherlands. d.van_der_woude@lumc.nl.
authors.forename: Diane
authors.initials: D
authors.lastname: van der Woude
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D002956:Citrulline / Q000276:immunology*; D015331:Cohort Studies; D042241:Early Diagnosis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D010456:Peptides, Cyclic / Q000276:immunology*; D011379:Prognosis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014746:Vimentin / Q000276:immunology
chemical_list: D001323:Autoantibodies; D010456:Peptides, Cyclic; D014746:Vimentin; C487763:cyclic citrullinated peptide; D002956:Citrulline