*3.3.2.1. Digitization of the sections*

is a natural dye obtained from lichens which are found to stain elastic fibers dark brown

CD31 is a single chain type 1 transmembrane protein with a molecular mass of approximately 135 kDa, belonging to the immunoglobulin superfamily. CD34 can also be applied to a subgroup, but this marker also stains cells other than endothelial. CD31 is expressed on endothelial cells of epithelial origin (all continuous endothelia, including those of arteries, arterioles, venules, veins, and capillaries, but it is not completely expressed on discontinuous endothelium in, for example, splenic red pulp). In addition, CD31 is expressed diffusely on the surfaces of megakaryocytes, platelets, myeloid cells, natural killer cells, and some subsets of T cells, as well as on B‐cell precursors. Cells labeled by the antibody predominantly display

Finally, IHC is used to detect diverse subpopulations of lymphocytes in tumoral tissues. For example, CD45 is a transmembrane glycoprotein expressed on most nucleated cells of hematopoetic origin, *i.e.*, all human leucocytes; CD20 reacts with an epitope located on the surface of B cells and appears early during B‐cell maturation; CD3 is a pan‐T cell marker for identification of T cells. It is well‐suited for labeling reactive T cells in tissue with lymphoid infiltrates, and for classification of T‐cell neoplasms; CD7 is expressed by the majority of peripheral blood T cells, NK cells, and all thymocytes. It is one of the earliest surface antigens on T and NK‐cell lineages; CD4 is a transmembrane glycoprotein, expressed on normal thymocytes, T‐helper cells, majority of mature peripheral T cells, and a subset of suppressor or cytotoxic T cells; CD8 is a 68 kDa transmembrane glycoprotein expressed as a heterodimer by a majority of thymocytes, and by class I major histocompatibility complex restricted, mature, suppressor/cytotoxic T cells; CD68 labels human monocytes and macrophages, but not myeloid cells; CD163 has been shown to mark cells of monocyte/macrophage lineage; CD11b is expressed on the surface of many leukocytes including monocytes, neutrophils, natural killer cells, granulocytes and macrophages, as well as on 8% of spleen cells and 44% of bone marrow cells; and CD11c is expressed prominently on the plasma membranes of

membrane staining with weaker cytoplasmic staining (**Figure 6**).

268 Composition and Function of the Extracellular Matrix in the Human Body

monocytes, tissue macrophages, NK cells, and most dendritic cells (**Figure 7**).

The samples contained in TMAs or in whole slides can subjectively be analyzed by a pathol‐ ogist to assess the amount of each ECM element as non‐informative: artefact, scant material, lost cylinder; negative: no expression or <5% of stained area is detected; positive 1+: mild staining, 5–10% of the area; positive 2+: moderate staining, 10–50% of the area.; and positive

The growing size and number of medical diagnostic images requires the use of computer‐ automated segmentation algorithms for the delineation of ECM structures of interest, which

**3.3. Evaluation of the samples**

3+: strong staining, >50% of the area.

*3.3.2. Automated quantification*

*3.3.1. Subjective assessment*

(**Figure 5**).

The digitization of the samples is required to perform morphometric analysis. The method available in most laboratories is the capture of several images per sample with a photomicro‐ scope. Sequential photos can be done at 20x or 40x magnifications with a photomicroscope and then carefully merged with Adobe Photoshop to reconstruct a single whole cylinder image (**Figure 2**). Following our experience, this method needs approximately two weeks to digitize 30 samples (2 cylinders per sample) for a single staining at 20x magnification, or approximately 6 to 7 weeks at 40x magnification.

**Figure 2.** Process of hematoxylin & eosin and CD31 immunostaining TMA image capture. **A)** At 20x magnification, 6 individual images of the cylinder must be captured to reconstruct the whole cylinder image. **B)** The whole 1mm cylin‐ der image at 40x magnification must be reconstructed from 20 individual images.

The use of a slide scanner is the only method that permits the digitization of a high number of cases for a reasonable time when developing a routine image biobank [82, 83] or a research project. Additionally, the use of slide scanners provides the possibility to standardize the image quality using preserved light conditions.

In order to enhance the standardization of the image capture conditions and therefore, the quality of the measurements and to save time, whole slide scanning is advised, as described next. Our group used the ScanScope XT scanner, Aperio technologies, but increasing alterna‐ tives such as Panoramic Midi from3D Histech and the Ventana iScann Coreo Au from Roche, among others can also be considered [82, 84]. ScanScope XT scanner is a brightfield scanner that digitizes whole sections at 20x or 40x magnification providing high resolution images in approximately 15 to 30 minutes per slide, depending on the magnification and the size of the tissue. 40x magnification was used, originating images with a resolution of 0.25 μm/pixel. Given to the enormous amount of pixels scanned, the images were compressed in JPEG2000 to 100–200 megabytes for the average size of a TMA, and saved to a proprietary TIFF format (SVS).

The sections were placed in a mobile plate one by one. An option for TMAs digitization provided by the scanner driver was used. This option recognizes the tissue cylinders and places several points per sample where the objective adjusts the focus to obtain clear and focused images. The mobile plate with the section moves in consecutive stripes, until it sweeps the whole section while the objective scans. Individual scanned stripes are originated and stitched together automatically to reconstruct the whole image, which can be visualized up to 40x magnifications with the Image Scope viewer software (Aperio technologies). The process is briefly shown in **Figure 3**.

**Figure 3.** Digitization process. **A)** ScanScope XT, Aperio technologies. **B)** Mobile plate with one section on it. The plate is introduced in the scanner and placed under the objective to start the process. **C)** Preview of an area of the section. Blue arrows show the pre‐set points where the scanner shall readjust the focus. Green points have already been prop‐ erly focused and yellow points still have to be focused. The red arrow shows different marks corresponding to the different horizontal stripes which are going to be scanned individually and then stitched together. **D)** 4 single consecu‐ tive stripes are shown corresponding to the section in E. **E)** All the stripes are stitched to form a single image of the whole section. The image is opened in the free viewer ImageScope, Aperio technologies.

### *3.3.2.2. Design of automated image analysis algorithms*

In order to enhance the standardization of the image capture conditions and therefore, the quality of the measurements and to save time, whole slide scanning is advised, as described next. Our group used the ScanScope XT scanner, Aperio technologies, but increasing alterna‐ tives such as Panoramic Midi from3D Histech and the Ventana iScann Coreo Au from Roche, among others can also be considered [82, 84]. ScanScope XT scanner is a brightfield scanner that digitizes whole sections at 20x or 40x magnification providing high resolution images in approximately 15 to 30 minutes per slide, depending on the magnification and the size of the tissue. 40x magnification was used, originating images with a resolution of 0.25 μm/pixel. Given to the enormous amount of pixels scanned, the images were compressed in JPEG2000 to 100–200 megabytes for the average size of a TMA, and saved to a proprietary TIFF format

270 Composition and Function of the Extracellular Matrix in the Human Body

The sections were placed in a mobile plate one by one. An option for TMAs digitization provided by the scanner driver was used. This option recognizes the tissue cylinders and places several points per sample where the objective adjusts the focus to obtain clear and focused images. The mobile plate with the section moves in consecutive stripes, until it sweeps the whole section while the objective scans. Individual scanned stripes are originated and stitched together automatically to reconstruct the whole image, which can be visualized up to 40x magnifications with the Image Scope viewer software (Aperio technologies). The process is

**Figure 3.** Digitization process. **A)** ScanScope XT, Aperio technologies. **B)** Mobile plate with one section on it. The plate is introduced in the scanner and placed under the objective to start the process. **C)** Preview of an area of the section. Blue arrows show the pre‐set points where the scanner shall readjust the focus. Green points have already been prop‐ erly focused and yellow points still have to be focused. The red arrow shows different marks corresponding to the different horizontal stripes which are going to be scanned individually and then stitched together. **D)** 4 single consecu‐ tive stripes are shown corresponding to the section in E. **E)** All the stripes are stitched to form a single image of the

whole section. The image is opened in the free viewer ImageScope, Aperio technologies.

(SVS).

briefly shown in **Figure 3**.

Depending on the staining to be measured and the availability of morphometric systems, different methods for image analysis can be used, all following a common workflow (**Figure 4**).

**Figure 4.** Flowchart showing the multi‐resolution image analysis system for TMA with two cylinders per sample. Im‐ ages belonging to different samples stained with different markers have been quantified by image analysis following a common process including segmentation (differential recognition of the staining) with specific input parameters for each marker and method and extraction of some given parameters. Adapted from Tadeo *et al.* [85]*.*
