Sowa, M., Grossmann, K., Knütter, I., Hiemann, R., Röber, N., Anderer, U., … Roggenbuck, D.
Research and Development Department, GA Generic Assays GmbH, Dahlewitz/Berlin, Germany, Faculty of Science, Brandenburg University of Technology Cottbus- Senftenberg, Senftenberg, Germany, Institute of Immunology, Technical University of Dresden, Dresden, Germany, Department of Rheumatology, University of Schleswig-Holstein Campus Lubeck and Rheumaklinik Bad Bramstedt, Bad Bramstedt, Germany, Division of Transplantation, Immunology and Mucosal Biology, King’s University College, London, United Kingdom, Department of Rheumatology, University of Milan, Milan, Italy, Institute of Molecular and Clinical Immunology, Otto-von- Guericke University Magdeburg, Magdeburg, Germany.
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 [k]=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 (k=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 (k=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.
… “400 nm and 490 nm light-emitting diodes (LED) (PrecisExcite, CoolLED, Andover, UK
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pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.
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