New Platform Technology for Comprehensive Serological Diagnostics of Autoimmune Diseases

Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology

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

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

Comparison of classical with automated anti-neutrophil cytoplasmic antibody (ANCA) endpoint-titer analysis.

<p>ANCA endpoint titers were determined by serial dilution of the 592 samples included in the study by classical indirect immunofluorescence (IIF) and compared to those detected by automated CytoBead IIF on the digital IIF interpretation system Aklides using a 1 to 20 serum dilution only.</p><p>Comparison of classical with automated anti-neutrophil cytoplasmic antibody (ANCA) endpoint-titer analysis.</p

Receiver-operating characteristic curve analysis of anti-neutrophil cytoplasmic antibodies (ANCAs) to proteinase 3 (PR3) and myeloperoxidase (MPO) by CytoBead ANCA.

<p>465 sera from 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 20 with Crohn's disease, 20 with autoimmune hepatitis and 125 blood donors were included. PR3- and MPO-ANCA were determined simultaneously by microbead immunoassay employing 90 patients with granulomatosis with polyangiitis and 28 patients with microscopic polyangiitis as positive criterion, respectively.</p

Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) and myeloperoxidase (MPO) levels by classical and automated multiplex microbead assay analysis in one reaction environment.

<p>PR3- and MPO-ANCA were determined by enzyme-linked immunosorbent assay (ELISA) (A, C, respectively) and multiplex CytoBead microbead assay (B, D, respectively) in 118 patients with ANCA-associated vasculitis, 300 with autoimmune and gastrointestinal diseases, 49 with infectious disorders, and 125 blood donors (BD). (Data are displayed in Box-and-Whisker plots with <i>far out</i> values, defined as values that are smaller than the lower quartile minus 3 times the interquartile range, or larger than the upper quartile plus 3 times the interquartile range, displayed as red circles.). AIH, autoimmune hepatitis; CD, Crohn's disease; GPA, granulomatosis with polyangiitis; ID, infectious diseases; MPA, microscopic polyangiitis; PSC, primary sclerosing cholangitis; RA, rheumatoid arthritis; UC, ulcerative colitis.</p

CytoBead ANCA assay principle.

<p>Microscopic glass slides with ethanol-fixed human neutrophils (ethN; middle compartment of the well) and proteinase 3 (PR3) as well as myeloperoxidase (MPO) coated microbeads (right compartment of the well) are used for detection of anti-neutrophil cytoplasmic antibodies (ANCAs) by ethN-based indirect immunofluorescence and simultaneous analysis of PR3- and MPO-ANCA by microbead immunoassay. PR3-ANCA positive sera show cytoplasmic fluorescence patterns on ethN and a green fluorescence halo on the surface of PR3-coated microbeads (9 µm). In contrast, MPO-ANCA positive sera show perinuclear fluorescence patterns on ethN and a green fluorescence halo on the surface of MPO-coated microbeads (15 µm).</p

Receiver-operating characteristic curve analysis of anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) and myeloperoxidase (MPO) levels by classical and automated multiplex microbead assay testing.

<p>PR3- and MPO-ANCA were determined by enzyme-linked immunosorbent assay (ELISA) and multiplex CytoBead microbead assay (MIA) in 118 patients with ANCA-associated vasculitis as disease criterion and in 300 patients with autoimmune and gastrointestinal diseases, 49 with infectious disorders, and 125 blood donors (BD) as control criterion. MPO-ANCA detected by MIA demonstrated a significantly higher AUC compared with those determined by ELISA. AUC, area under the curve; CI, confidence interval.</p