Quantifying chromogen intensity in immunohistochemistry via reciprocal intensity.

Abstract

Immunohistochemistry is a routine procedure for detecting the expression of biological markers in formalin-fixed and paraffin-embedded tissues. Chromogens, which can appear as different colors (brown, blue, red) under bright field microscopy, are localized in fixed tissues to antigens of interest via an antibody-antigen detection system. The advantage of a chromogen system is that the stained tissue section is permanently fixed, and the staining quality is maintained for many years. The short-coming, however, is quantifying the intensity of such stains. Unlike immunofluorescent protocols in which the brightness of a region is directly proportional to the amount of localized antigen, chromo-gen stains appear darker in regions with more antigen. This dark staining is visible to the human eye under white light, but darker spots have lower intensity values; something that is counterintuitive and cumbersome for the purposes of quantitation. We report that this limitation can be overcome by measuring the " reciprocal intensity " of the chromogen stain. A typical red-green-blue image resulting from bright field microscopy has its maximum intensity value found in the white, non-stained area. Areas that contain any coloration, due to the chromogen or a counterstain, have an intensity of less than the maximum. By subtracting the intensity of the stained area of interest from the maximum, the staining in these areas can be represented as a quantity that is positively correlated with increas-ing darkness. This is a more intuitive means of assessing the intensity of a chromogen stain, and al-lows for more sensitivity in quantifying gradients of coloration between treatment groups. This ap-proach has the potential to stratify nuanced protein expression in previously published human speci-men data sets into cohorts with clearer clinical outcomes.