Method of quantitating proteins and genes in cells using a combination of immunohistochemistry and in situ hybridization

A computer program on computer readable medium comprising instructions to cause a computer to:

• measure a plurality of colors in a biological sample on a slide, wherein at least a first color is associated with an IHC stained polypeptide and a second color is associated with a CISH stained polynucleotide;
• determine an amount of IHC stained polypeptide in the sample and an amount of CISH stained polynucleotide by comparing the first color to a first standard and the second color to a second standard;
• and
• outputting an indication of the amounts of polypeptide and polynucleotide.

A machine vision system for automated analysis of a biological sample on a slide comprising:

• a computer comprising:
• a system processor;
• a computer program on computer readable medium, the computer program comprising an image algorithm comprising instructions to cause the computer to:
• measure a color channel value in a plurality of pixels from a plurality of control samples comprising a single color of interest;
• define a vector for each of the plurality of control samples, wherein each vector comprises an average of each color channel value present in the control;
• define a matrix comprising each of the averages for each of the color channels;
• define a conversion matrix comprising the inverse of the matrix based upon the control measurements;
• measure color channel values in an image of an experimental sample comprising a plurality of colors of interest, each of the pixels comprising a plurality of color channels;
• determine an amount of an IHC stained polypeptide in the sample and an amount of a CISH stained polynucleotide by calculating the amount of a color in the experimental sample by converting the channel values in the experimental sample using the conversion matrix;
• and
• outputting an indication of the amounts of polypeptide and polynucleotide.
• a monitor in operable communication with the computer;
• and
• an input device in communication with the computer;
• an optical system in operable communication with the computer, comprising:
• a movable stage;
• an automated loading and unloading member for loading and unloading of a slide;
• an identification member;
• an optical sensing array in optical communication with the stage configured to acquire an image at a location on a slide and in electrical communication with the processor;
• a storage member for storing the location of a candidate object or area of interest;
• and
• a storage device for storing each image.