The CytoBead assay – a novel approach of multiparametric autoantibody analysis in the diagnostics of systemic autoimmune diseases

Abstract

If there is a suspicion of a systemic autoimmune disease, a two-step assessment of autoantibodies (AAb) is recommended for the serological diagnosis thereof. First, AAb will be determined using sensitive, cell-based indirect immunofluorescence. Then, a positive result must be confirmed with a more specific test due to the possibility of false-positive results. This gradual approach is necessary because there is currently no assay technique that fulfills the requirements for a one-stage procedure for sensitivity and specificity. For effective AAb analysis, simultaneous determination of several AAb with multiparametric confirmatory assays significantly shortens serological diagnosis, compared with conventional monoparametric testing. Yet, currently available multiparametric AAb detection techniques do not offer the combination of screening and confirmatory testing. Thus, a new approach based on digital fluorescence was developed by applying a novel CytoBead technology that is presented here. The aim was to combine the recommended stepwise approach consisting of sensitive screening and confirmation of specific diagnosis in a reaction environment and thereafter the possibility of adaptation to the serological diagnosis of several autoimmune diseases. Using standard microscopic glass slides and the combination of native cellular or tissue substrates with autoantigen-loaded fluorescent microparticles (beads) in a reaction environment, along with the possibility of manual and automatic evaluation by IIF and the quantitative measurement of fluorescent signals, the disadvantages of currently existing test systems could be overcome. This novel concept is applicable for the determination of various multiparametric AAb, e.g., the determination of antinuclear antibodies and the corresponding AAb in molecular cytoplasmic and nuclear autoantigenic structures. Further, this becomes the basis for the simultaneous multiparametric AAb determination for the serology of celiac disease or ANCA-associated vasculitides.

Next-Generation Autoantibody Testing by Combination of Screening and Confirmation—the CytoBead® Technology

“…This situation changed dramatically by the development of digital fluorescence and its implementation in IIF testing. The breathtaking new options of pattern recognition combined with progress in automated fluorescence microscopy paved the way for the evolvement of an entirely new generation of automated interpretation systems [ 206 ]. Different commercially available IIF platforms for autoAb testing were designed and applied for ANA and ANCA reading in particular.…”

“…For instance, a positive anti-dsDNA finding in ELISA in combination with ANA negativity cannot be regarded as relevant regarding diagnosis of SLE. However, the possibility of false-negative findings using the two-tier strategy especially for ANA reading in terms of sera positive for autoAbs to SS antigen A (SS-A/Ro) is still eminent at hand and represents an essential drawback of such approach [ 206 ]. Only the combination of both stages in one multiplex test would overcome these shortcomings and provide an ideal solution for autoAb testing addressing key clinical and laboratory needs.…”

“…Consequently, automated multiplex IIF combining screening and confirmatory ANCA testing in one test may replace the time-consuming current two-stage ANCA testing strategy by a one-step multiplexing CytoBead® analysis [ 206 ]. In context of the emergency diagnostics required for rapidly progressive glomerulonephritis, the novel multiplex ANCA analysis by CytoBead® appears to be an attractive approach to meet the clinical need for comprehensive ANCA testing in the fastest way possible.…”

Close Spectroscopic Look at Dye-Stained Polymer Microbeads

“…Moreover, they can be read out with the existing instrumentation like flow cytometers initially developed for the analysis of cells, 7 or alternatively, with a microscope using microtiter plates. 5,8 The use of fluorescence methods for the readout of beadbased assays requires dye-encoded carrier beads, with the spectral or intensity codes signaling the surface chemistry or target-specific ligands at the bead surface. 9−12 Also, novel encoding strategies, utilizing the parameter fluorescence lifetime, are emerging 13−15 that encompass additional parameters to be controlled.…”

“…Among the many advantages of the bead-based assays are their high throughput and the ease of automation. Moreover, they can be read out with the existing instrumentation like flow cytometers initially developed for the analysis of cells, or alternatively, with a microscope using microtiter plates. , …”