Histopathology is the examination of tissue under the microscope.

Tissue removed at surgery is usually sent for histopathological examination. In addition very small samples of tissue can be obtained by needle or core biopsy. The microscopic features of individual cells are used to determine the nature of the suspected melanoma.

(Pathologists who specialise in the assessment of surgical material are called histopathologists.)

Below is a schematic example of how the pathologist examines the excised surgical specimen:

Skin containing the melanoma, the re-excised specimen, lymph nodes and other surgical specimens are processed in similar ways. The preparation of tissue for examination by microscopy is technically demanding and time consuming.  It usually takes 5-7 days for the final report to be issued. Additional special techniques including immunohistochemistry (use of antibodies that help identify specific tissues or tumours) may be required.

The pathologist receives the surgically removed tissue and divides it into a series of smaller blocks. These are then processed with fixatives and embedded in paraffin wax.

The paraffin blocks are then placed in a device (microtome) that cuts extremely thin slices (approximately 1/100th of a millimetre thick). These tissue sections are placed on glass slides and stained with chemicals such as haematoxylin and eosin to identify various cell structures.

Microscopy

The pathologist examines tissue on the glass slide under the microscope at magnifications ranging from 10 to 400 times. Pigment producing cells in the skin are called melanocytes.  Abnormal melanocytes are melanoma cells.  The distribution of melanoma cells within the skin layers is assessed to determine the thickness of the melanoma.

This melanoma is shown as the pathologists sees it through the microscope. The measuring scale (graticule) is visible. The tumour is microstaged to have a Breslow thickness of 1.6mm, Clark Level 3.

Immunohistology

It can also be helpful to use special stains called immunohistochemistry to identify specific types of cells and tissue elements. These techniques may help the pathologist better identify details such as the thickness of the primary melanoma, adequacy of excision or spread to lymph nodes. Monoclonal antibody stains such as S100, HMB 45 and Melan A preferentially react with melanoma cells.

Lymph nodes are examined to identify any evidence of melanoma cells which may have spread from the primary tumour. Special techniques (immunohistochemistry) can identify very small clusters of tumour cells (micrometastases).

Micro-deposit of melanoma is difficult to identify in standard tissue preparation.

Micro-deposit of melanoma stained red with immunohistochemistry (antibody that identifies melanoma)