title: Characterization for anti-cytoplasmic antibodies specificity by morphological and molecular techniques.
abstract: OBJECTIVE
  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.
authors.affiliation: Servizio Analisi Chimico Cliniche Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Alpini
authors.affiliation: Laboratorio Analisi A.O. Ospedale Civile di, Legnano, Milan Italy.
authors.forename: Milvia
authors.initials: M
authors.lastname: Lotzniker
authors.affiliation: Servizio Analisi Chimico Cliniche Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy.
authors.forename: Serenella
authors.initials: S
authors.lastname: Valaperta
authors.affiliation: Dipartimento di Biologia e Biotecnologie "L.Spallanzani", Laboratorio di Biologia Cellulare e Neurobiologia, Università di Pavia, Pavia, Italy ; Istituto di Genetica Molecolare del CNR, Pavia, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Bottone
authors.affiliation: Dipartimento di Biologia e Biotecnologie "L.Spallanzani", Laboratorio di Biologia Cellulare e Neurobiologia, Università di Pavia, Pavia, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Malatesta
authors.affiliation: Laboratorio Analisi IRCCS Istituto Clinico Humanitas, Rozzano, Milan, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Montanelli
authors.affiliation: Servizio Analisi Chimico Cliniche Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy.
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Merlini
keywords: Anti-Golgi apparatus antibodies; Anti-endoplasmatic reticulum antibodies; Anti-lysosome/endosome antibodies