Automated direct immunofluorescence analyses of skin biopsies.

BACKGROUND: Diagnosis of autoantibody- and immune complex-induced skin diseases is primarily based on direct immunofluorescence (DIF) microscopy. DIF staining is usually performed manually and, therefore, is labor intensive. The quality of immunohistochemical results considerably depends on the experience of the person conducting the tests. The novel EUROTide(™) technique in combination with the biochip-based system EUROPath represents a new technology for automation of DIF staining. METHODS: Frozen sections of previously characterized skin biopsies from bullous pemphigoid and pemphigus vulgaris patients were incubated with fluorescein-labelled anti-human IgG and complement C3c following the standard manual procedure and, for comparison, applying EUROTide/EUROPath in an automated version. RESULTS: Both, the manual and the automated procedure, detected IgG and C3c deposits in all samples. However, DIF stainings performed with EUROTide/EUROPath displayed more intense specific IF signals and distinctly less background staining. The detecting antibody could be used at a ×4 higher dilution. CONCLUSION: EUROTide/EUROPath applied in an automated system improves diagnostic accuracy and saves reagents. Larger studies in other routine laboratories may further explore the value of the EUROTide/EUROPath technology and may include comparison with other automated stainers.

EUROPLUS ANCA BIOCHIP mosaic: PR3 and MPO antigen microdots improve the laboratory diagnostics of ANCA-associated vasculitis.

INTRODUCTION: The consensus on anti-neutrophil cytoplasmic antibody (ANCA) testing requires screening with indirect immunofluorescence (IIF) and confirmation in MPO- and PR3-ANCA specific assays. The EUROPLUS system combines in one incubation the conventional cell substrates with microdots of single antigens, i.e., MPO and PR3. We evaluated the diagnostic applicability of this new system for ANCA-associated vasculitis (AAV). METHODS: To assess the diagnostic performance of the EUROPLUS Granulocyte Mosaic, sera from 249 AAV patients, 85 disease controls and 27 healthy controls were analysed. Results were compared with a reference multi-testing algorithm based on IIF with ethanol-fixed granulocytes, direct and capture ELISAs for both MPO- and PR3-ANCA. RESULTS: Based on the reference multi-testing algorithm 123 AAV patients were defined as having PR3-ANCA and 68 AAV patients as having MPO-ANCA (diagnostic sensitivity: 76.7%). For the EUROPLUS MPO and PR3 microdots the diagnostic sensitivity was 77.1% in the same AAV cohort. The concordance between both methods for PR3- and MPO-ANCA was 96.8% and 99.2%, respectively. In the control cohort the diagnostic specificity was 99.1% for the multi-testing algorithm and 98.2% for the EUROPLUS microdots. CONCLUSIONS: The combination of conventional cell substrates and single MPO and PR3 antigen microdots greatly facilitates the identification of ANCA reactivity clinically relevant for AAV. Since our results obtained after a single incubation in the EUROPLUS system are highly concordant with the reference multi-testing algorithm (based on IIF, direct and capture ELISAs) the EUROPLUS system is advocated as an efficient test system.