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Superficial Layer

General aspect

The superficial layer is evaluated identifying different patterns:

Epidermal Pattern

1. Regular Honeycombed pattern

Typically is formed by 10 to 20 μm polygonal cells with dark nuclei and bright and thin cytoplasm (Fig. 1-3). This is the pattern of normal skin and corresponds to unaltered epidermis with small amount of melanin within keratinocytes.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 1. Honeycombed pattern

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 2. Honeycombed pattern

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 3. Honeycombed pattern

2. Regular Cobblestone pattern

It consists of small polygonal cells with refractive cytoplasm separated by a less refractive border (Fig. 4-6). Cobblestone pattern is more frequently detected in pigmented melanocytic lesions, owing to the consistent amount of melanin within the keratinocyte cytoplasm.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 4. Typical cobblestone pattern

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 5. Typical cobblestone pattern

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 6. Typical cobblestone pattern (few nucleated cells are sometimes visible)

3. Broadened Honeycombed pattern

Broadened honeycombed corresponds to a honeycombed pattern with bright enlarged and broadened intercellular spaces (Fig. 7). Although an exact histologic correlate was not found so far, this pattern is frequently observed in association with eosinophilic hyperkeratosis, frequently observable in nodular lesions, such as Nodular Melanomas and nodular non melanocytic skin cancers.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 7. Atypical honeycombed pattern, with broadened and irregular keratinocyte contours

4. Irregularly Shaped Keratinocytes

The overall epidermal pattern is constituted by Irregular shaped keratinocytes when irregularity in size of the cells and thickness of the contour is present.

An overall honeycombed architecture characterized by atypical and irregular keratinocytes is observed in melanocytic (Fig. 8) and non melanocytic lesions (Fig. 9), such as Actinic Keratoses, Bowen's Disease, and Squamous Cell Carcinomas.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 8. Irregularly shaped keratinocytes give rise to an atypical honeycombed pattern, also showing a dendritic pagetoid cell and bright spots in a melanocytic lesion (in situ melanoma).

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 9. Irregularly shaped keratinocytes in an Actinic Keratosis.

An overall cobblestone architecture showing irregular in size and/or refractivity keratinocytes may also define an «irregularly shaped keratinocytes» pattern (Fig. 10).

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 10. Irregularly shaped keratinocytes with bright cytoplasm giving rise to an atypical cobblestone pattern in a melanocytic lesion.

5. Disarranged pattern

It is characterized by disarray of the normal architecture of the superficial layers with unevenly distributed bright granular particles and cells, in the absence of honeycombed or cobblestone pattern (Fig. 11-13).

This pattern is more frequently observable in invasive Melanomas, usually associated with pagetoid cells, although other skin malignancy, such as Squamous Cell and, rarely, Basal Cell Carcinomas.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 11. Disarranged pattern: not honeycombed nor cobblestone structure is recognizable

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 12. Disarranged pattern: some bright spots and a pagetoid cells are also present

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 13. Disarranged pattern, with some pleomorphic pagetoid cells

6. Undefined pattern

Superficial layers predominantly constituted by a more or less homogeneous reflectivity, not associated or pertaining to any cell type, dispersed throughout the image, giving rise to an aspect not referable to any of the previous ones (Fig. 14).

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 14. Undefined pattern. None of the above aspects is observable

Presence of polarized elongated meshes

Some degree of keratinocytic atypia and architectural disarray within a generally honeycombed pattern, showing elongated cells with their nuclei oriented along the same principal axis, thus manifesting a polarized appearance (Fig. 15). The pattern is frequently observable in Basal Cell Carcinomas, although sometimes present in benign lesions, such as dermatofibromas.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 15. Polarized elongated cells within an honeycombed pattern showing irregularly shaped keratinocytes

Erosion-ulceration

Dark areas, usually with sharp borders and irregular contours, filled with amorphous substance and/or clotted bright small particles (Fig. 16).

Small erosions or ulcerated areas are frequently observable in non melanocytic skin tumors, such as Actinic Keratoses, Bowen's disease, Basal Cell and Squamous Cell Carcinomas, although sometimes present in melanocytic lesions too, such as ulcerated Melanomas or traumatized Nevi.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 16. Erosion-ulceration in a Basal Cell Carcinoma. It corresponds to the roundish dark area (1.5x1.5 mm) with sharply outlined contours, containing amorphous and clotted material

Presence and aspects of pagetoid cells

Pagetoid cells are considered when large nucleated cells, twice the size of keratinocytes, with a dark nucleus and bright cytoplasm, are observable within superficial layers (Fig. 17-22). Pagetoid cells, especially the «roundish» ones, represent the most accurate pattern for Melanoma diagnosis.

Melanomas are frequently characterized by numerous pagetoid cells, scattered throughout the lesions, usually roundish or pleomorphic in shape.

A small percentage of Clark nevi, as well as some Spitz/Reed nevi, shows pagetoid cells, usually few and localized in the center of the lesion.

Dendritic pagetoid cells have a lower diagnostic power, since they are sometimes observable in benign melanocytic lesions and in non melanocytic ones. They are referable to melanocytes spreading upword in a pagetoid fashion and/or to Langherans cells. Within Melanomas, Lentigo Maligna type may be characterized by the presence of dendritic pagetoid cells, frequently surrounding and infiltrating the hair follicles.

Concerning «Pagetoid Cells» some parameters will be considered:

1. Cell Type / Aspect

Aspect is evaluated, according to the predominant cell shape, as «roundish» or «dendritic». «Pleomorphism» is the variability of pagetoid cell morphology, characterized by the presence of both roundish and dendritic cells, and/or the presence of cells with bizarre shapes.

2. Numerosity

Numerosity corresponds to the density of pagetoid cells in the region where they are located. It is evaluated approximately per «square millimeter», corresponding to an area of 4 adjacent full resolution images (squares) in the mosaic. It is classified into three different levels, for the presence of less than 5 cells per square millimeter, «<5sqm», for 5 to 10 cells per square millimeter, «5-10sqm», and for more than 10 cells per square millimeter, «>10sqm», respectively.

3. Distribution

Distribution takes into account the localization of pagetoid cells onto the lesion area. It is considered as «localized» when pagetoid spread is present and concentrated in a limited portion of the lesion, as «sparse» when it is possible to observe different, but not extended, foci of pagetoid infiltration, and as «widespread» when pagetoid cells are scattered throughout the whole lesion area.

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 17. Pagetoid cells: Roundish

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 18. Pagetoid cells: numerous Roundish cells

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 19. Pagetoid cells: Dendritic cells

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 20. Pagetoid cells: Pleomorphism (presence of both Roundish and Dendritic)

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 21. Pagetoid cells: Pleomorphism (presence of both Roundish and Dendritic)

Confocal Laser Scanning Microscopy tutorial - superficial layers

Figure 22. Pagetoid cells: Pleomorphism (presence of cells with bizarre shapes)

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