title: Active centromere and chromosome identification in fixed cell lines.
abstract: BACKGROUND
  The centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells.
  
  
  RESULTS
  A panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation (cenFISH), centromere protein immunofluorescence (CENP-IF) and multicolour FISH (mFISH), and showed the usefulness of CENP-IF together with cenFISH followed by mFISH (CENP-IF-cenFISH-mFISH) with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric.
  
  
  CONCLUSIONS
  We propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay (DCA), prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.
authors.affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, VIC 3052 Australia ; Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Melbourne, VIC 3052 Australia.
authors.forename: Thian T
authors.initials: TT
authors.lastname: Beh
authors.affiliation: Victorian Cancer Cytogenetics Service, St Vincent's Hospital, Fitzroy, Melbourne, VIC 3065 Australia ; Department of Medicine, St Vincent's Hospital, University of Melbourne, Fitzroy, Melbourne, VIC 3065 Australia.
authors.forename: Ruth N
authors.initials: RN
authors.lastname: MacKinnon
authors.affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, VIC 3052 Australia ; Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Melbourne, VIC 3052 Australia.
authors.forename: Paul
authors.initials: P
authors.lastname: Kalitsis
keywords: CENP-A; CENP-C; Centromere; Dicentric; Dicentric chromosome assay (DCA); Fluorescence in situ hybridisation (FISH); Human erythroleukaemia (HEL) cell line; Immunofluorescence; Multicolour FISH (mFISH); Neocentromere