Neutrophil fixation protocols suitable for substrates to detect anti-neutrophil cytoplasmic antibodies by indirect immunofluorescence.

Anti-neutrophil cytoplasmic antibodies (ANCAs) are autoantibodies that recognize neutrophil cytoplasmic antigens. The major ANCA antigens are myeloperoxidase and proteinase 3. Necrotizing small vessel vasculitis accompanied by ANCA production is called ANCA-associated vasculitis (AAV). In addition to AAV, ANCA is sometimes produced in patients with connective tissue diseases, such as systemic lupus erythematosus, and inflammatory bowel diseases. Indirect immunofluorescence (IIF) and enzyme immunoassay (EIA) have been used to detect ANCAs. Recently, the accuracy of EIA has improved and it has become the gold standard for ANCA detection. However, IIF does not lose its role in ANCA detection because EIA cannot detect ANCAs that recognize antigens other than those coated on the plate. For IIF, neutrophil substrates prepared with two different fixations, namely, ethanol fixation and formalin fixation, are used. There is a recommended protocol for ethanol fixation but not for formalin fixation. This study prepared neutrophil substrates according to the recommended protocol for ethanol fixation and protocols in the literature and original protocols for formalin fixation and then examined ANCA specificity and how storage period would influence the number of cells, antigen distribution, and antigenicity of the substrates. As a result, the number of cells and antigen distribution did not change after storage for up to 2 months regardless of fixation protocols, whereas a time-dependent decline in ANCA antigenicity and a fixation protocol-dependent difference in ANCA specificity were observed. How neutrophils are fixed on the glass slide needs to be checked upon evaluation of ANCAs by IIF.