title: Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens.
abstract: OBJECTIVE
  Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low.
  
  
  METHODS
  We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue.
  
  
  RESULTS
  Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin:spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis.
  
  
  CONCLUSIONS
  This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.
authors.affiliation: Center for Gene Therapy, Nationwide Children's Hospital, Columbus, OH, USA.
authors.forename: L E
authors.initials: LE
authors.lastname: Taylor
authors.forename: Y J
authors.initials: YJ
authors.lastname: Kaminoh
authors.forename: C K
authors.initials: CK
authors.lastname: Rodesch
authors.forename: K M
authors.initials: KM
authors.lastname: Flanigan
mesh_terms: D000328:Adult; D016189:Dystrophin / Q000032:analysis* / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism*; D013049:Spectrin / Q000032:analysis / Q000378:metabolism
chemical_list: D016189:Dystrophin; D013049:Spectrin