Evaluation strategy of anti-mitochondrial antibodies M2-negative: the role of multiplex rodent tissues and related clinical implications.

Indirect immunofluorescence on HEp-2 cell line (HEp-2-IIF) remains "gold standard" method for the detection of antinuclear antibodies (ANA). ANA is an operative definition, showing the possibility of autoantibodies (Aab) to bind nuclear, and cytoplasmic antigens. One of the major examples is represented by anti-mitochondrial antibodies (AMAs), which target proteins of the inner and outer mitochondrial membranes, located into the cytoplasm. The standard IIF on rat kidney/stomach/liver tissue sections, with the combined use of other commercial assays, may all be used in ordinary lab life to validate the AC-21 pattern on Hep-2 cells. The routine lab experience teaches that commercial kits cannot always be detected and define specific AMAs. In these cases the literature proposes the use of other homemade assays to detect AMAs as immunoprecipitation (IP) and Western blot (IP-WB). However, using IP or IP-WB is difficult to apply in a routine laboratory, because of numerous cases to process and the related troubles. Where find confirmation of the AC-21 pattern if line-immunoblot and other routine methods (ELISA, CLIA/FEIA assays) fail? We review AC-21 AMA-like sera from our patients (year 2022) and propose a revised diagnostic algorithm based on the combined use of IIF on Hep-2 cells, line immunoblot and IIF on rodent tissue as a third line method. We demonstrated that, particularly in cases where the second level test was unsuccessful, the application of IFI on rodent tissues became indispensable to verify the existence of AMAs.