title: Computerized image analysis of Ki-67 in ductal breast carcinoma in situ.
abstract: OBJECTIVE
  To develop and determine the staining protocols and computerized image analysis methods that are the most effective combination for performing quantitative analysis of Ki-67.
  
  
  METHODS
  We compared conventional bright-field light microscopy and refractive optical enhancement methods in combination with various immunodetection and filter enhancement methods, including immunogold in combination with epipolarization refractive optics and enzymatic conversion of chromogenic substrates in combination with optical filter enhancement. Initial Ki-67 tests were performed on lymph node tissues and cultured human breast cells and then applied to 200 ductal carcinoma in situ (DCIS) samples. DCIS acini were digitally acquired, and a region of interest was manually outlined in each one with a digital stylus to include only the cellular component; then the Ki-67 staining index was quantified by segmentation analysis.
  
  
  RESULTS
  Although combining epipolarization analysis with immunohistogold staining was the most sensitive detection method, nonspecific binding was too high. The streptavidin-horseradish-peroxidase enzymatic conversion of 3,3'-diaminobenzidine (DAB) in combination with optical enhancement filters was the most effective method tested. Ki-67 stain was associated with dense fibrillar structures of the nucleoli in the less intensely staining nuclei and was most intense in paired nuclei.
  
  
  CONCLUSIONS
  The method of measuring Ki-67 expression by DAB staining combined with optical enhancement filters and quantification via computer-assisted image analysis techniques produced objective and reproducible results. As such, this method can offer (1) less intraobserver and interobserver variability, (2) a digital archival record, and (3) a baseline for digital exchange of information between studies.
authors.affiliation: Departments of Clinical Cancer Prevention and Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: D G
authors.initials: DG
authors.lastname: Menter
authors.forename: A
authors.initials: A
authors.lastname: Hoque
authors.forename: N
authors.initials: N
authors.lastname: Motiwala
authors.forename: A A
authors.initials: AA
authors.lastname: Sahin
authors.forename: N
authors.initials: N
authors.lastname: Sneige
authors.forename: R
authors.initials: R
authors.lastname: Lieberman
authors.forename: S M
authors.initials: SM
authors.lastname: Lippman
mesh_terms: D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000737:chemistry / Q000473:pathology*; D002278:Carcinoma in Situ / Q000737:chemistry / Q000473:pathology*; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry / Q000473:pathology*; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D019394:Ki-67 Antigen / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D019394:Ki-67 Antigen