Characterization for anti-cytoplasmic antibodies specificity by morphological and molecular techniques

PURPOSE: The aim of our study was the characterization of anti-cytoplasmic antibodies by home-made morphological and biochemical techniques. Indeed, indirect immunofluorescence (IIF) on HEp-2 cell line is not always exhaustive in relation to the complexity of the antigens involved. METHODS: Nine serum samples with anti-cytoplasmic antibodies (2 anti-Golgi apparatus, 3 with diffuse pattern and 4 with lysosome/endosome-like pattern) were tested with fluorescent confocal microscopy, Western blot analysis and, when necessary, with electron microscopy technique. RESULTS: Confirmation of the IIF staining pattern was performed in confocal microscopy by comparison with the respective antibody marker. The anti-endoplasmatic reticulum positivity was also confirmed by electron microscopy evaluation. Both anti-lysosome/endosome and anti-endoplasmatic reticulum positivity have been definitely identified by Western blot through clear reactivity with calreticulin and LC3B, respectively. CONCLUSIONS: These results do not aim at representing a standard routine laboratory procedure. Electron microscopy evaluation cannot be proposed as a routine approach, but confocal microscopy technique may be offered in centralized reference laboratories. Newer technologies, especially multiplex immunoassay, can also lead to an easier identification of these autoantibodies, without recurring to a home-made immunoblotting. Only with a complete characterization we will be able to define the clinical relevance of anti-cytoplasmic antibodies, which are still considered as “esoteric” and not as “diagnostic” antibodies.