Spectrum of ANCA-specificities in eosinophilic granulomatosis with polyangiitis. A retrospective multicentre study.

OBJECTIVES: To determine the spectrum of anti-neutrophil cytoplasmic antibody (ANCA) antigen-specificities in eosinophilic granulomatosis with polyangiitis (EGPA), an ANCA-associated vasculitis (AAV) entity. METHODS: We conducted a retrospective analysis of 73 EGPA patients from three German tertiary referral centres for vasculitis. In addition to in-house ANCA testing, pentraxin 3 (PTX3)- and olfactomedin 4 (OLM4)-ANCA were determined using a prototype cell-based assay for research (EUROIMMUN, Lübeck, Germany). Patient characteristics and clinical manifestations were evaluated and compared based on ANCA status. RESULTS: Myeloperoxidase (MPO)-ANCA positive patients (n=8; 11%) significantly more frequently displayed peripheral nervous system (PNS) and pulmonary involvement and less frequently heart involvement compared to MPO-ANCA negative patients. PTX3-ANCA positive patients (n=5; 6.8%) had a significantly higher prevalence of ear, nose and throat, pulmonary, gastrointestinal and PNS involvement, and a lower prevalence of renal and central nervous system involvement compared to PTX3-ANCA negative patients. Proteinase 3 (PR3)-ANCA and OLM4-ANCA were detected in 2 patients (2.7%) each with multiorgan involvement. One PR3-ANCA positive patient was also positive for bactericidal permeability increasing protein (BPI)-ANCA. CONCLUSIONS: In addition to MPO, the spectrum of ANCA antigen specificities includes various other target antigens such as PR3, BPI, PTX3, and OLM4, potentially segregating further EGPA subgroups. A lower prevalence of MPO-ANCA was detected in this study compared with other studies. OLM4 is reported as novel ANCA antigen-specificity in EGPA, and thus AAV.

Computer-aided classification of indirect immunofluorescence patterns on esophagus and split skin for the detection of autoimmune dermatoses.

Autoimmune bullous dermatoses (AIBD) are rare diseases that affect human skin and mucous membranes. Clinically, they are characterized by blister formation and/or erosions. Depending on the structures involved and the depth of blister formation, they are grouped into pemphigus diseases, pemphigoid diseases, and dermatitis herpetiformis. Classification of AIBD into their sub-entities is crucial to guide treatment decisions. One of the most sensitive screening methods for initial differentiation of AIBD is the indirect immunofluorescence (IIF) microscopy on tissue sections of monkey esophagus and primate salt-split skin, which are used to detect disease-specific autoantibodies. Interpretation of IIF patterns requires a detailed examination of the image by trained professionals automating this process is a challenging task with these highly complex tissue substrates, but offers the great advantage of an objective result. Here, we present computer-aided classification of esophagus and salt-split skin IIF images. We show how deep networks can be adapted to the specifics and challenges of IIF image analysis by incorporating segmentation of relevant regions into the prediction process, and demonstrate their high accuracy. Using this semi-automatic extension can reduce the workload of professionals when reading tissue sections in IIF testing. Furthermore, these results on highly complex tissue sections show that further integration of semi-automated workflows into the daily workflow of diagnostic laboratories is promising.

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

BACKGROUND: Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. METHODS: We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. RESULTS: Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. CONCLUSIONS: Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

Multiparametric serological testing in autoimmune encephalitis using computer-aided immunofluorescence microscopy (CAIFM).

Autoantibodies against neuronal cell surface antigens are tightly associated with immunotherapy-responsive autoimmune encephalitis, and a considerable number of corresponding autoantigens has been identified in recent years. Most patients initially present with overlapping symptoms, and a broad range of autoantibodies has to be considered to establish the correct diagnosis and initiate treatment as soon as possible to prevent irreversible and sometimes even life-threatening damage to the brain. Recombinant cell-based immunofluorescence allows to authentically present fragile membrane-associated surface antigens and, in combination with multiparametric analysis in the form of biochip mosaics, has turned out to be highly beneficial for parallel and prompt determination of anti-neuronal autoantibodies and comprehensive differential diagnostics. For the evaluation of recombinant cell-based IIFT, a semi-automated novel function was introduced into an established platform for computer-aided immunofluorescence microscopy. The system facilitates the microscopic analysis of the tests and supports the laboratory personnel in the rapid issuance of diagnostic findings, which is of major importance for autoimmune encephalitis patients since timely initiation of treatment may lead to their full recovery.

Automated Evaluation of Crithidia luciliae Based Indirect Immunofluorescence Tests: A Novel Application of the EUROPattern-Suite Technology.

Systemic lupus erythematosus (SLE) is a severe rheumatic autoimmune disease with various clinical manifestations. Anti-dsDNA antibodies are an important immunological hallmark of SLE and their occurrence represents a major criterion for the diagnosis. Among the commonly applied test systems for determination of anti-dsDNA antibodies, the indirect immunofluorescence test (IIFT) using the flagellated kinetoplastida Crithidia luciliae is considered to be highly disease specific at moderate sensitivity. Since IIFT, however, is claimed to be affected by subjective interpretation and a lack of standardization, there has been an increasing demand for automated pattern interpretation of immunofluorescence reactions in recent years. Corresponding platforms are already available for evaluation of anti-nuclear antibody (ANA) IIFT on HEp-2 cells, the recommended "gold standard" for ANA screening in the diagnosis of various systemic rheumatic autoimmune diseases. For one of these systems, the "EUROPattern-Suite" computer-aided immunofluorescence microscopy (CAIFM), automated interpretation of microscopic fluorescence patterns was extended to the Crithidia luciliae based anti-dsDNA IIFT.