Research paper Evaluation of a multiplex flow cytometric immunoassay to detect PR3- and MPO-ANCA in active and treated vasculitis, and in inflammatory bowel disease (IBD) ☆ Michelle Trevisin a,b , Wendy Pollock b,c , Wayne Dimech d , Judy Savige b, ⁎ a Pathology Department, Austin Health, Heidelberg, VIC 3084, Australia b The University of Melbourne, Department of Medicine, Northern Health, Epping VIC 3076, Australia c Gribbles Pathology, Clayton, VIC 3168, Australia d National Serology Reference Laboratory, Fitzroy, VIC 3065, Australia article info abstract Article history: Received 14 November 2007 Received in revised form 21 March 2008 Accepted 28 March 2008 Available online 25 April 2008 This study compared the performance of a flow cytometric immunoassay for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO), with indirect immunofluorescence (IIF) and ELISAs from 12 different manufacturers. Sera were from patients with active (n =55) or treated (n = 68) small vessel vasculitis, or inflammatory bowel disease (IBD, n = 22). The immunoassay specificity was 88% compared with 96% for IIF and 94% (median, range 91– 96%) for both ELISAs. Its sensitivity in treated disease was 82% compared with 84% for IIF and 69% (median, range 57–82%) for the ELISAs. The immunoassay's specificity was 88% which was the same as the median for both ELISAs (range 84–95%). The PR3- and MPO-ANCA immunoassay was almost as sensitive as IIF, and more sensitive than, but just as specific as, most ELISAs, in detecting ANCA in active and treated vasculitis. A major advantage of this assay is its ability to be further modified to simultaneously screen for a panel of autoantibodies relevant to vasculitis. © 2008 Elsevier B.V. All rights reserved. Keywords: Antineutrophil cytoplasmic antibodies Proteinase 3 Myeloperoxidase Flow cytometric immunoassay 1. Introduction The ‘International Consensus Statement on Testing and Reporting Antineutrophil Cytoplasmic Antibodies (ANCA)’ requires sera from patients with suspected small vessel vasculitis to be screened by indirect immunofluorescence (IIF) on ethanol-fixed neutrophils, and all IIF-positive sera to be confirmed in antigen-specific ELISAs (Savige et al., 1999). The IIF assay is highly sensitive for ANCA but also labour- intensive, expensive, subjective and lacking in specificity (Savige et al., 1998). The direct and capture ELISAs are less sensitive and more specific than IIF for small vessel vasculitis (Csernok et al., 2002; Csernok et al., 2004), but their performance is varied in treated disease where antibody levels are low (Trevisin et al., 2008). The usefulness of both IIF and the ELISAs is further limited by their inability to demonstrate antibodies directed against minor ANCA anti- gens and other vasculitis-associated antibodies, including antiglomerular basement membrane (GBM) antibodies. The early detection of both ANCA and antiGBM antibodies is critical in patients with rapidly progressive glomerulone- phritis where renal function recovers with timely therapeu- tic intervention. The multiplex flow cytometric immunoassay for vasculitis (FIDIS, Biomedical Diagnostics, France) uses a novel technique Journal of Immunological Methods 336 (2008) 104–112 ☆ This work was presented in part at the 13th International Vasculitis and ANCA Workshop in Cancun Mexico, April 2007. The evaluation of the 12 PR3- and MPO-ANCA ELISAs has been described separately. ⁎ Corresponding author. The University of Melbourne, Department of Medicine (Austin/Northern), The Northern Hospital, Epping VIC 3076, Australia. Tel.: +613 8405 8823; fax: +613 8405 8724. E-mail address: jasavige@unimelb.edu.au (J. Savige). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.03.012 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim