Tissue analysis

Quantitative histology and tissue analysis

The automatic quantification of tissues serves to reproduce the interpretation of pathologists, by means of algorithms. The tissue sections are arranged on microscope slides and digitized with a slide scanner. Automatic quantification is a complex task because:

  • Images are huge: slide scanner generates virtual slides combining thousands of microscope fields (tiles) that correspond to the entire tissue sample
  • Slide to  slide variability: the inclusion steps, staining steps and acquisitions steps can lead to important differences between identical slides. Thus it is important to process carefully images in order to reduces slide to slide differences. Internal normalization can help to standardize colors and intensities
  • Several segmentation steps are required to achieve good quantification: sample segmentation (to remove the background of the sample), tissue structure segmentation (like epithelium area, necrotic area, lumens, tissue lesions), cells segmentation (to obtain individual intensity, shape, number of spots)
  • Result visualization is not trivial: the visual inspection of slide quantification is very important and the user has to verify the results at different resolutions. He has to be able to navigate with ergonomic tools to zoom in, zoom out and pane the original image and superimpose resulting segmentation
  • In many situation, the analysis has to be guided by the expert or the anatomopathologist. They define region of interest, include or exclude areas according to their understanding. So it is important to combine automatic quantification with expert supervision

The tissue slides and Tissue Micro Array (TMA)

A tissue slide can contain : 

histology
A tissue section
tissue microarray
An assembly of hundreds or thousands of samples called TMA (Tissue Microarray)

Histological stainings

Common stainings are :

histological stainings

Histochemistry: staining based on chemical reactions between reagents and components of the tissue under study (e.g.: hematoxylin-eosin staining, Periodic Acid-Shiff (PAS), Masson’s Trichrome de Masson)

histochemistry

Immunohistochemistry (IHC): Immunological recognition of any given antigenic target (e.g. protein). Antibodies are coupled to an enzyme that convert a chromogenic substrate in a colored precipitate. Detection can be direct (enzyme-coupled primary antibody to antigen) or indirect (enzyme-coupled secondary antibody recognizes non-coupled primary antibody).

immunohistochemistry

Immunofluorescence (IF): Immunological recognition of any given antigenic target (e.g. protein). Antibodies are coupled to a fluorochrome. Detection can be direct (fluorochrome-coupled primary antibody to antigen) or indirect (fluorochrome-coupled secondary antibody recognizes non-coupled primary antibody)

immunofluorescence

In situ hybridisation in situ (ISH): A probe recognizes a particular DNA or RNA sequence, which allows the localization or quantification of this sequence at the cellular level. The cDNA probe is labeled with an isotope or a fluorochrome (FISH)

Quantitative histological analyses

The quantitative information that is extracted from a tissue can be of different types:

  • Description of tissue morphology and organization
  • Description of cellular organization within a tissue
  • Localization and expression of biomarkers in a tissue of in cells

 

The gain of automated tissue analysis

Automated tissue analysis facilitates:

  • Analysis of a unlimited number of histological sections
  • Overall quantification of a slide
  • Quantification of each tissue structure
  • Precise quantification of each cell
  • The selection of unusual structures of interest
  • Easy navigation through the mass of images

 

3D Histology

To obtain 3D information in tissue analysis, sequential slides are cut and acquired to perform:

  • 3D reconstruction of the object, organ, tissue structure (bio-materials, tumor, vessels)
  • Measure 3D volumes
  • 3D visualization (see more …)

3D histology is achieved using image registration tool boxes like ITK or Elastix.

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