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.