title: Quantitative image analysis of immunohistochemical stains using a CMYK color model.
abstract: BACKGROUND
  Computer image analysis techniques have decreased effects of observer biases, and increased the sensitivity and the throughput of immunohistochemistry (IHC) as a tissue-based procedure for the evaluation of diseases.
  
  
  METHODS
  We adapted a Cyan/Magenta/Yellow/Key (CMYK) model for automated computer image analysis to quantify IHC stains in hematoxylin counterstained histological sections.
  
  
  RESULTS
  The spectral characteristics of the chromogens AEC, DAB and NovaRed as well as the counterstain hematoxylin were first determined using CMYK, Red/Green/Blue (RGB), normalized RGB and Hue/Saturation/Lightness (HSL) color models. The contrast of chromogen intensities on a 0-255 scale (24-bit image file) as well as compared to the hematoxylin counterstain was greatest using the Yellow channel of a CMYK color model, suggesting an improved sensitivity for IHC evaluation compared to other color models. An increase in activated STAT3 levels due to growth factor stimulation, quantified using the Yellow channel image analysis was associated with an increase detected by Western blotting. Two clinical image data sets were used to compare the Yellow channel automated method with observer-dependent methods. First, a quantification of DAB-labeled carbonic anhydrase IX hypoxia marker in 414 sections obtained from 138 biopsies of cervical carcinoma showed strong association between Yellow channel and positive color selection results. Second, a linear relationship was also demonstrated between Yellow intensity and visual scoring for NovaRed-labeled epidermal growth factor receptor in 256 non-small cell lung cancer biopsies.
  
  
  CONCLUSIONS
  The Yellow channel image analysis method based on a CMYK color model is independent of observer biases for threshold and positive color selection, applicable to different chromogens, tolerant of hematoxylin, sensitive to small changes in IHC intensity and is applicable to simple automation procedures. These characteristics are advantageous for both basic as well as clinical research in an unbiased, reproducible and high throughput evaluation of IHC intensity.
authors.affiliation: Division of Applied Molecular Oncology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Canada. npham@uhnres.utoronto.ca
authors.forename: Nhu-An
authors.initials: NA
authors.lastname: Pham
authors.forename: Andrew
authors.initials: A
authors.lastname: Morrison
authors.forename: Joerg
authors.initials: J
authors.lastname: Schwock
authors.forename: Sarit
authors.initials: S
authors.lastname: Aviel-Ronen
authors.forename: Vladimir
authors.initials: V
authors.lastname: Iakovlev
authors.forename: Ming-Sound
authors.initials: MS
authors.lastname: Tsao
authors.forename: James
authors.initials: J
authors.lastname: Ho
authors.forename: David W
authors.initials: DW
authors.lastname: Hedley