Quantitative assessment of tissue sections by Reverse Colour Coding

Stains and dyes are frequently used to emphasise tissue structures for viewing under microscopy but few simple objective quantification methods exist. We describe the derivation of a mathematical formula enabling calculation of the relative contribution of three different coloured components to an image, which can be applied to rapid batch assessment of tissue sections for quantification of area proportions of differently stained elements. In order to validate this method, termed Reverse Colour Coding (RCC), we compared RCC estimations with known area proportions in artificially created images to calculate absolute accuracy, and compared RCC with panel visual estimation (VE) for the assessment of actual NCAM-stained muscle slides. Our results indicate that RCC has an absolute accuracy of 98-98.5% and superior inter-observer agreement and both inter- and intra-observer variability compared with VE. Results also suggest that cognitive bias occurring with VE may be eliminated by use of RCC. We submit RCC as a more accurate and less labour-intensive method of quantifying area proportions of stained tissues on microscopic images.