In vivo Reflectance Confocal Microscopy
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This tutorial is provided to describe the unifying concept of confocal microscopy with complete definitions of the parameters and example images. Afer completion, please proceed to the training area, where a set with 45 images is available for the refinement and final elaboration of the list. All tutorial set images cover an area of 470x350 μm.
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RCM features description

Superficial (granulosum/spinosum) layers

General aspect

The superficial layer is evaluated identifying three possible patterns:

1. Honeycombed pattern

Typically is formed by 10 to 20 μm polygonal cells with dark nuclei and bright and thin cytoplasm (Fig. 1-3).

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Figure 1. Honeycombed pattern

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Figure 2. Honeycombed pattern

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Figure 3. Honeycombed pattern

It is called Atypical, when irregularity in size of the cells and thickness of the contour is present (Fig. 4,6).

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Figure 4. Atypical honeycombed pattern, also showing a dendritic pagetoid cell and bright spots

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Figure 6. Atypical honeycombed pattern, with broadened and irregular keratinocyte contours

Broadened honeycombed corresponds to a honeycombed pattern with bright enlarged and broadened intercellular spaces.

2. Cobblestone pattern

Typically consisting of small polygonal cells with refractive cytoplasm separated by a less refractive border (Fig. 7-9).

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Figure 7. Typical cobblestone pattern

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Figure 8. Typical cobblestone pattern

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Figure 9. Typical cobblestone pattern (few nucleated cells are visible, but they are not «multiple»)

It is termed Atypical when irregularity in size and/or refractivity of the cells is present (Fig. 10,11).

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Figure 10. Atypical cobblestone pattern: irregular and uneven cell refractivity is present

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Figure 11. Atypical cobblestone pattern with small multiple nucleated cells

Moreover, the presence of small multiple nucleated cells is also reported (Fig. 12).

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Figure 12. Atypical cobblestone pattern with small multiple nucleated cells, irregular in shape and size, with the contemporary presence of larger cells (pagetoid melanocytosis)

3. Disarranged pattern

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. 13-15).

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Figure 13. Disarranged pattern: not honeycombed nor cobblestone structure is recognizable

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Figure 14. Disarranged pattern: some bright spots and a pagetoid cells are also present

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Figure 15. Disarranged pattern, with some pagetoid cells, dendritic in shape and pleomorphic

Grainy image

Grainy image indicates superficial layers predominantly constituted by bright refractive particles not associated or pertaining to any cell type, dispersed throughout the image.

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Figure 16. Grainy image

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. 17-23).

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Figure 17. Pagetoid cells: Roundish, Large, num. 1-3

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Figure 18. Pagetoid cells: Roundish, Large, Pleomorphic, num. >6

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Figure 19. Pagetoid cells: Roundish and Dendritic, Large, Pleomorphic, num. 1-3

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Figure 20. Pagetoid cells: Dendritic, Large, not Pleomorphic, num. >6

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Figure 21. Pagetoid cells: Roundish and Dendritic, Small and Large, Pleomorphic, num. >6

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Figure 22. Pagetoid cells: Roundish and Dendritic, Small and Large, Pleomorphic, num. >6

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Figure 23. Pagetoid cells: Roundish, Small, not Pleomorphic, num. 3-5

Cell shape is evaluated as «roundish» and/or «dendritic».

Cell size is considered «small» for cells with the maximum diameter lower than 50 μm, and «large» if greater.

Pleomorphism is the variability of pagetoid cell morphology in the same image (3 or more pagetoid cells are needed). If less than 3 cells are present it will not be scored since not evaluable. If a combination of round and dendritic pagetoid cells are seen this should be considered pleomorphic.

Cell density is classified as «low» for less than 1-3 cells per image, «medium» for 4-6 cells or «high» for more than 6 cells.

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 All images are propriety of G. Pellacani and protected by copyright. Reproduction is not permitted without permission.