title: Simultaneous automated screening and confirmatory testing for vasculitis-specific ANCA.
abstract: Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.
authors.affiliation: Research and Development Department, GA Generic Assays GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Mandy
authors.initials: M
authors.lastname: Sowa
authors.affiliation: Research and Development Department, GA Generic Assays GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Grossmann
authors.affiliation: Research and Development Department, GA Generic Assays GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Ilka
authors.initials: I
authors.lastname: Knütter
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Rico
authors.initials: R
authors.lastname: Hiemann
authors.affiliation: Institute of Immunology, Technical University of Dresden, Dresden, Germany.
authors.forename: Nadja
authors.initials: N
authors.lastname: Röber
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Anderer
authors.affiliation: Department of Rheumatology, University of Schleswig-Holstein Campus Lübeck and Rheumaklinik Bad Bramstedt, Bad Bramstedt, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Csernok
authors.affiliation: Division of Transplantation, Immunology and Mucosal Biology, King's University College, London, United Kingdom.
authors.forename: Dimitrios P
authors.initials: DP
authors.lastname: Bogdanos
authors.affiliation: Department of Rheumatology, University of Milan, Milan, Italy.
authors.forename: Maria Orietta
authors.initials: MO
authors.lastname: Borghi
authors.affiliation: Department of Rheumatology, University of Milan, Milan, Italy.
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Meroni
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schierack
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Reinhold
authors.affiliation: Institute of Immunology, Technical University of Dresden, Dresden, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Conrad
authors.affiliation: Research and Development Department, GA Generic Assays GmbH, Dahlewitz/Berlin, Germany; Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Roggenbuck
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology*; D002648:Child; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods / Q000592:standards; D006801:Humans; D008297:Male; D008875:Middle Aged; D012372:ROC Curve; D012016:Reference Values; D015203:Reproducibility of Results; D014657:Vasculitis / Q000175:diagnosis* / Q000276:immunology*; D055815:Young Adult
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic