Simultaneous automated screening and confirmatory testing for vasculitis-specific ANCA.

Mandy Sowa,Dimitrios P Bogdanos,Ursula Anderer,Pier Luigi Meroni,Peter Schierack,Dirk Roggenbuck,Maria Orietta Borghi,Ilka Knütter,Karsten Conrad,Rico Hiemann,Kai Grossmann,Dirk Reinhold,Elena Csernok,Nadja Röber,Massimo Pietropaolo

doi:10.1371/journal.pone.0107743

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.

Highlights

Autoimmune vascular disorders comprising granulomatosis with polyangiitis (GPA, formerly Wegener’s granulomatosis), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA, formerly Churg-Strauss syndrome) are characterized by microvascular inflammation, tissue necrosis, and the appearance of anti-neutrophil cytoplasmic antibody (ANCA) [1,2,3,4,5,6]
ANCA indirect immunofluorescence (IIF) patterns as well as proteinase 3 (PR3)- and MPO-ANCA can be observed in other inflammatory conditions and several ANCA-specific targets apart from MPO and PR3 have been reported which lowers the specificity of ANCA testing by IIF [11,12]
A cytoplasmic ANCA (C-ANCA) pattern confirmed by PR3-ANCA enzyme-linked immunosorbent assays (ELISAs) positivity is indicative for GPA [1,3], whereas a perinuclear ANCA (P-ANCA) pattern confirmed by a positive MPO-ANCA ELISA finding supports the diagnosis of MPA and EGPA [11]

Summary

Introduction

Autoimmune vascular disorders comprising granulomatosis with polyangiitis (GPA, formerly Wegener’s granulomatosis), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA, formerly Churg-Strauss syndrome) are characterized by microvascular inflammation, tissue necrosis, and the appearance of anti-neutrophil cytoplasmic antibody (ANCA) [1,2,3,4,5,6]. The term ANCA-associated vasculitis has been coined for this distinct disease group characterized by loss of tolerance to neutrophilic targets. The C- and P-ANCA in human patients with ANCA-associated vasculitis are mainly directed against proteinase 3 (PR3) and myeloperoxidase (MPO), respectively, and seem to be associated with disease activity [9,10]. A C-ANCA pattern confirmed by PR3-ANCA ELISA positivity is indicative for GPA [1,3], whereas a P-ANCA pattern confirmed by a positive MPO-ANCA ELISA finding supports the diagnosis of MPA and EGPA [11].

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Conclusion