Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

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The current practice for detection of anti-neutrophil cytoplasm antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO) has been screening by indirect immunofluorescence (IIF) followed by an antigen specific tests for PR3- and MPO-ANCA. However, ANCA diagnostics have undergone many technical developments that have affected the 1999 international consensus recommendations, and lead to a revision of the existing ANCA detection strategy. Recent European multicentre studies have compared the diagnostic performance of various ANCA detection methods and demonstrated that PR3- and MPO-ANCA immunoassays yielded the highest diagnostic accuracy. New guidelines for ANCA testing have been developed based on these data. According to the revised 2017 international consensus recommendations, testing for ANCA in small vessel vasculitis can be done by PR3- and MPO-ANCA immunoassays, without the categorical need for IIF. Thus, IIF can be discarded completely, or can be used as confirmation assays instead a screening test. Clearly, though, the new testing strategy for ANCA in vasculitis must identify the ANCA target antigen, as PR3- and MPO-ANCA serotype correlate well with disease expression. Furthermore, recent studies have shown that AAV can be classified based on ANCA serotype, since PR3- and MPO-ANCA- diseases are strongly associated with distinguishable genetic alleles, different clinical and histological features. ANCA presence and the antigen specificity also may have important value as a prognostic factor and may serve as a guide for immunosuppressive therapy. In the current review, we summarize the novelties in ANCA testing, present the 2017 revised international consensus on ANCA testing in vasculitis, evaluate the diagnostic significance of ANCA, and discuss the role of ANCA serotypes in the diagnostic work-up of patients with AAV.