Methods and systems for assessing histological stains
The present disclosure includes methods of assessing a histologically stained specimen based on a determined color signature of a region of interest of the specimen. Such assessments may be performed for a variety of purposes including but not limited to assessing the quality of the histological stain, as part of identifying one or more biologically relevant features of the image, as part of differentiating one feature of the image from other features of the image, identifying an anomalous area of the stained specimen, classifying cells of the specimen, etc. Also provided are systems configured for performing the disclosed methods and computer readable medium storing instructions for performing steps of the disclosed methods.
1. A method of assessing a histologically stained specimen, the method comprising:
a) obtaining a digital color image of the specimen;
b) defining on the image a region of interest (ROI) based on a biological feature of the specimen;
c) separating the digital color image into individual color channels;
d) determining a color signature for the ROI, wherein the color signature comprises:
a mean intensity for a first color channel, a second color channel, and a third color channel;
a first color coefficient calculated by determining the ratio of the mean intensity value for the first color channel to the mean intensity value for the second color channel;
a second color coefficient calculated by determining the ratio of the mean intensity value for the third color channel to the mean intensity for the first or the second color channel; and
a third color coefficient calculated by determining the ratio of the first color coefficient to the second color coefficient; and
e) comparing the determined color signature to a reference color signature that is specific to the biological feature and the histological stain to assess the histologically stained specimen.
2. The method of claim 1 , wherein the separating is performed after defining the ROI.
3. The method of claim 1 , wherein the ROI comprises a cell or a portion thereof.
4. The method of claim 1 , wherein the ROI comprises a plurality of cells.
5. The method of claim 1 , wherein the ROI is the cytoplasm of a cell or a portion thereof.
6. The method of claim 1 , wherein the ROI is the nucleus of a cell or a portion thereof.
7. The method of claim 1 , wherein the reference color signature is derived from a reference histologically stained specimen.
8. The method of claim 1 , wherein the reference color signature comprises a threshold value.
9. The method of claim 1 , wherein the assessment comprises establishing whether the determined color signature is within a predetermined range and, when within the range, the specimen is further processed.
10. The method of claim 1 , wherein the assessment comprises establishing whether the determined color signature is outside of a predetermined range and, when outside the range, the specimen is not further processed.
11. The method of claim 1 , wherein the assessment is an evaluation of histological stain quality.
12. The method of claim 1 , wherein the assessment is used in identifying the ROI as a feature of the image.
13. The method of claim 1 , wherein the assessment is used in differentiating the ROI from other features of the image.
14. The method of claim 1 , wherein the method further comprises quantifying additional ROIs of the image having a substantially similar color signature.
15. The method of claim 1 , wherein the histological specimen is stained with a Romanowsky stain.
16. The method of claim 1 , wherein the first color coefficient is calculated by determining the ratio of the mean intensity value for a red color channel to the mean intensity value for a green color channel.
17. The method of claim 1 , wherein the second color coefficient is calculated by determining the ratio of the mean intensity value for a blue color channel to the mean intensity value for a red color channel.
18. The method of claim 1 , wherein the first color coefficient is calculated by determining the ratio of the mean intensity value for a red color channel to the mean intensity value for a green color channel and the second color coefficient is calculated by determining the ratio of the mean intensity value for a blue color channel to the mean intensity value for a red color channel.
19. The method of claim 18 , wherein the third color coefficient is calculated by determining the ratio of the first color coefficient to the second color coefficient.
20. The method of claim 1 , wherein the histological specimen is derived from a biological sample selected from the group consisting of: a blood sample, a bodily fluid sample and a tissue sample.
21. The method of claim 1 , wherein the biological feature is a cell, wherein the cell is selected from the group consisting of: an erythrocyte, a mast cell, a megakaryocyte, a basophil, a neutrophil, an eosinophil, a macrophage, a NK cell, a T cell, a B cell and a blood cell progenitor.
22. The method of claim 1 , wherein the method further comprises calibrating an illumination source used in obtaining the digital color image, wherein calibrating comprises quantification of one or more color parameters over an ROI defined on a blank slide.