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Table 2 The compatibility of ImmunoRatio with variable staining and image acquisition settings

From: ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67

Immunostaining/image acquisition feature Compatibility with ImmunoRatio Comments
Primary antibody dilution
(defined for MIB-1 Ki-67)
  
   too dilute (1:400) + too low labeling index (Figure 6a)
   optimal (1:100) + + + best match with visual counting (Figure 6b)
   too strong (1:25) + cytoplasmic background causing overly high labeling indexes (Figure 6c)
Hematoxylin counterstaining   
   weak - insufficient nuclear segmentation (Figure 6d)
   optimal + + + best match with visual counting (Figure 6e)
   strong + false segmentation of cytoplasmic structures (Figure 6f)
Microscope objective magnification
(using 1× phototube)
  
   10× + non-carcinomatous cells often included*
   20× + + + for accurate result, an average of three images per sample is recommended
   40× + + for accurate result, averaging several images per sample is recommended
Image brightness   
   underexposed (too dim) + mean gray intensity of the blankfield image <200
   in optimal range + + + as guided by the Camera Adjustment Wizard of ImmunoRatio
   overexposed (too bright) - mean gray intensity of the blankfield image >250
Image contrast   
   too low - foreground mean gray intensity over 85% of the background mean gray intensity
   in optimal range + + + as guided by the Camera Adjustment Wizard of ImmunoRatio
   too high + foreground mean gray intensity under 50% of the background mean gray intensity
Image compression   
   uncompressed (lossless) + + slow network transmission (slower overall analysis time)
   JPEG, quality factor 50 to 100 (lossy) + + + optimal for ImmunoRatio
   JPEG, quality factor <50 (lossy) - visible image artifacts
  1. + + + = optimally compatible, + + = compatible, + = compatible with possible chance of analytical errors, - = not compatible with ImmunoRatio.
  2. * Region of interest function can be used to exclude unwanted tissue areas.