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

• US 20060041385A1
• Filed: 08/18/2004
• Published: 02/23/2006
• Est. Priority Date: 08/18/2004
• Status: Abandoned Application

• Current Assignee: Carl Zeiss Microimaging Ais, Inc. (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microimaging Ais, Inc. (Carl-Zeiss-Stiftung)
• Inventors: Bauer, Kenneth D., Webster, Scott, Gottlieb, Keith
• Application Number:
  US10/922,121
• Publication Number:
  US 20060041385A1
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 21/27 using photo-electric detection G01N21/31 takes precedence; circuits for computing concentration logarithmic circuits G06G7/24; photometric circuits in general G01J
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/90 Determination of colour characteristics
• CPC Class Codes: G06V 20/695 Preprocessing, e.g. image segmentation

Abstract

The disclosure provides a system for and method of accurately determining the status of a protein target and a nucleic acid target in a biological sample (e.g., a tissue or cell sample). Provided are methods of quantitating a protein target and a nucleic acid target in a tissue or cell sample using image analysis to correlate the optical density of a chromogenic stain to the concentration of a target molecule in a biological sample. The use CISH in combination with IHC to accurately determine the status of two independent target molecules is provided. A computer-aided image analysis system is used to process and analyze optical images of a chromagen-stained tissue or cell sample and to determine the optical density of the stained tissue or cell sample.

Claims

1. A method comprising:

measuring 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;

determining 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.

2. The method of claim 1, wherein the measuring is by an automated microscope system.

3. The method of claim 1, wherein the first color and the second color are the same color.

4. The method of claim 1, wherein the first color and the second color are different colors.

5. The method of claim 1, wherein the determining comprises

measuring a color channel value in a plurality of pixels from a plurality of standard samples, wherein each standard comprises a single color of interest;

defining a vector for each of the plurality of control samples, wherein each vector comprises an average of each color channel value present in the standard;

defining a matrix comprising each of the averages for each of the color channels;

defining a conversion matrix comprising the inverse of the matrix based upon the control measurements;

measuring color channel values in an image of the biological sample comprising the IHC stained polypeptide and a CISH stained polynucleotide, each of the pixels comprising a plurality of color channels; and

calculating the amount a polypeptide or polynucleotide by converting the channel values in the experimental sample using the conversion matrix.

6. The method of claim 1, wherein the plurality of colors are measured simultaneously.

7. The method of claim 1, wherein the plurality of colors are measured sequentially.

8. A computer implemented method of claim 1.

9. 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.

10. The computer readable program of claim 9, wherein the plurality of colors comprise red, green, and blue.

11. The computer readable program of claim 9, further comprising instructions to display an image of the biological sample.

12. The computer readable program of claim 9, further comprising instructions to cause the computer to:

measure a color channel value in a plurality of pixels from a plurality of standard samples, wherein each standard comprises 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 standard;

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 the biological sample comprising the IHC stained polypeptide and a CISH stained polynucleotide, each of the pixels comprising a plurality of color channels; and

calculate the amount a polypeptide or polynucleotide by converting the channel values in the experimental sample using the conversion matrix.

13. The computer readable program of claim 9, further comprising rendering a digital display of the experimental sample.

14. 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.