title: Quantification of vascular density using a semiautomated technique for immunostained specimens.
abstract: OBJECTIVE
  To develop a semiautomated, quantitative techniquefor the assessment of vascular density in immunohistochemically stained tissue sections using diaminobenzidine tetrahydrochloride (DAB) and hematoxylin as chromagens.
  
  
  METHODS
  A semiautomated thresholding technique was developed to quantitate vascular density in tissue sections stained with anti-CD31 (1 degrees antibody). The immunohistochemically stained specimens were digitally imaged using a 24-bit color camera. The blue component of the RGB image was segmented using a variable high-pass filter. After thresholding, the segmented areas (CD31 positive) were quantified and vascular density determined. The validity of the method was verified by calculating the precision of the technique using the coefficient of repeatability and by quantifying its agreement with manual analysis according to the Bland-Altman approach.
  
  
  RESULTS
  Vascular endothelial cells were specifically selected using anti-CD31 as the primary antibody and the appropriate horseradish peroxidase-conjugated secondary antibody. Utilizing the semiautomated thresholding technique, the separation of DAB-stained tissuefrom non-DAB-stained tissue was achieved. The method developed possesses a low coefficient of repeatability (0.49%), agrees well with manual assessment (mean difference = -0.29 +/- 0.92%), is highly automated and is user friendly.
  
  
  CONCLUSIONS
  A novel semiautomated technique for the quantification of vascular density was developed. This technique provides a method for reproducible measurement of immunostaining procedures (immunohistochemistry, immunocytochemistry and in situ hybridization) utilizing immunoperoxidase techniques with DAB as a chromagen.
authors.affiliation: Department of Plastic Surgery, University of Texas M. D. Anderson Cancer Center, and University of Texas Center for Biomedical Engineering, Houston 77030, USA.
authors.forename: Timothy W
authors.initials: TW
authors.lastname: King
authors.forename: Eric M
authors.initials: EM
authors.lastname: Brey
authors.forename: Adel A
authors.initials: AA
authors.lastname: Youssef
authors.forename: Carol
authors.initials: C
authors.lastname: Johnston
authors.forename: Charles W
authors.initials: CW
authors.lastname: Patrick
mesh_terms: D015100:3,3'-Diaminobenzidine; D000818:Animals; D001808:Blood Vessels / Q000166:cytology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007124:Immunoenzyme Techniques; D020007:Implants, Experimental; D017403:In Situ Hybridization; D018919:Neovascularization, Physiologic; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D015203:Reproducibility of Results; D013194:Staining and Labeling
chemical_list: D019408:Platelet Endothelial Cell Adhesion Molecule-1; D015100:3,3'-Diaminobenzidine