Special Techniques and Considerations

Immunofluorescence: immunofluorescent examinations are required for the diagnosis of chronic blistering diseases and are useful in connective tissue diseases. The site of biopsy is important for immunofluorescence, particularly in the blistering disorders. In dermatitis herpetiformis a biopsy for immunofluorescence should be taken from clinically normal skin away from the area of blistering. In the other blistering disorders, perilesional skin is submitted. The skin should have an intact epidermal/dermal junction. In most of the connective tissue disorders lesional skin is submitted for immunofluorescence except for the lupus band test, where normal non-sun-exposed skin is used. Most patients having skin submitted for immunofluorescence should

Figure 37.5. Quadrant blocks of a skin ellipse.

a and c b lesion margins

Figure 37.6. Wedge resection of skin.

also have a serum sample sent to the laboratory for examination for autoantibodies related to the disease process. Skin biopsy specimens for immunofluorescence are either snap frozen in liquid nitrogen or sent in the correct transport medium to the laboratory. Most laboratories doing immunofluorescence will supply the clinical area with the appropriate transport medium. Formalin fixation is inappropriate for immunofluorescence and will render the specimen unsuitable for examination. Specimens for immunofluorescence are either punch or elliptical biopsies.

Mohs' micrographic surgery: for the surgical removal of the tumour under microscopic control. The aim of the technique is to remove all the tumour with the minimum of surrounding normal tissue. This is a time-consuming and slow procedure for the patient, dermatological surgeon and laboratory staff but it is useful in a small number of cases. Mohs' micrographic surgery is used primarily for the treatment of basal cell carcinoma, but may be used for squamous cell carcinoma, some sarcomas of the skin especially dermatofibrosarcoma protuberans and, rarely, some types of desmoplastic melanoma and other malignant skin appendage tumours. It is especially useful for tumours occurring on the face around the eyelids, nose and mouth where a good cosmetic result is required. The technique involves examination of frozen sections of surgical margins with the patient and the surgeon awaiting the results. If limits are involved a further excision of this area is carried out and examined by frozen section. This is repeated until the margins are clear. The defect is then repaired by the surgeon on the same day. In some units the tissue is fixed, processed to paraffin, and margin sections examined the next day. If the margins are involved further tissue is removed, processed to paraffin and sections examined. Only when the margins are clear is repair carried out. This is a slower procedure over a period of days in which the patient has a defect which has to wait for confirmation of clearance before repair can take place. This technique is useful for rarer types of tumour where there may be an infiltrate of single spindle cells such as a desmoplastic malignant melanoma, or where immunocytochemistry is required to identify tumour cells.

Mohs' laboratory procedure: it is essential that there is clear communication between the surgeon and the pathologist examining the specimen by Mohs' micrographic surgery. The specimen should be laid out flat on a dish or board and the margins indicated either by sutures or pins of different colours. It is useful if the surgeon also draws a diagram of the lesion and its location on the patient with appropriate landmarks. The pathologist ensures that the complete surgical margins are examined. It is often necessary to divide the specimen into smaller blocks to be examined microscopically. They may need to be marked so that the area involved by tumour can be clearly pinpointed. On an ellipse skin margins can be marked in relation to the clockface or to compass points. The surgical margins are marked with different-coloured inks, to aid locating the correct area with tumour involvement (Figure 37.7).

Surgical margins: on excision biopsies the pathologist should comment on the adequacy of excision and, in line with protocols, measure the tumour distance from the margins. In punch biopsies the specimen is either embedded intact or bisected and embedded. The pathologist can comment on two lateral and deep margins. The edge of the punch biopsy can be inked to indicate microscopically the true excision margins, which are also often associated with red blood cells.

Similarly, in an elliptical biopsy the margin status is documented by the pathologist. Quadrant blocks result in four lateral margins and a deep margin being examined. Bread-loaf slicing through the ellipse results in all the margins being seen microscopically but this is only suitable for relatively small ellipses. The margins can be inked to assist microscopic identification, although usually red blood cells are present. To examine all the surgical margins in a large skin biopsy the best approach is a modified Mohs' technique. The pathologist sections the margins and these are marked with different-coloured inks to aid identification.

Sutures and markers: the surgeon will often mark margins with sutures to help orientate the specimen and an accompanying diagram is also useful. Techniques of margin sampling in large excisions may need to be modified in the light of attached sutures or clinical request form information.

N Ink colour 1

E Ink colour 2

Ink colour 1

| Ink colour 2

Vf j

Ink colour 1

Figure 37.7. Sections for Moh's micrographic surgery.

Grafts: tumours may recur under and around an area of skin grafting. These specimens are dealt with in the usual manner for a skin ellipse.

Re-excisions: The most common cause for re-excision is when a malignant tumour is incompletely excised, or in the case of a malignant melanoma, despite complete primary excision, the margins are not wide enough to follow standard guidelines. Re-excision biopsies are sampled as for primary excisions. Tumour, if present, is usually at the edge of the previous biopsy scar. Again, margins of excision are commented on.

Diagrams: diagrams are also useful in orientating specimens.

Transmission Electron Microscopy (TEM): As in other branches of pathology, the role of diagnostic TEM is declining. Immunocytochemistry has reduced the need for it in diagnosing undifferentiated tumours and viral infections, although it is still useful for inborn errors of metabolism. All such samples of skin should be placed in a glutaraldehyde fixative. Other indications for TEM are in the diagnosis and subclassification of: (1) congenital anomalies, e.g., icthyosis, (2) blistering diseases as in the epidermolysis bullosa group (it may be necessary to obtain a fresh blister by rubbing up with an eraser) and (3) acquired blistering disorders (in conjunction with immunohistochemistry).

Scanning Electron Microscopy: Scanning electron microscopy is useful in the diagnosis of hair shaft anomalies. The hair sample should be sent unfixed to the laboratory. Microprobe Analysis: A microprobe attached to the scanning electron microscope can be useful to detect small amounts of elements present in the skin that may be causing increased abnormal pigmentation.

Skin scrapings: scrapings from the skin surface can be examined for fungal particles or scabies mites. This may be a wet preparation by putting the scrapings in potassium hydroxide or fixing the tissue and processing it. This is a useful way to make a diagnosis without a full surgical biopsy.

Fixation: the usual fixation for skin biopsies for histology is 10% formalin. Mast cells are easier to demonstrate if the skin biopsy has been fixed in alcohol. Where a mast cell lesion is suspected the biopsy should be divided and halves placed in formalin and alcohol. However, if mast cells are present in large numbers they can still be seen in formalin-fixed tissue. Similarly, the ureate crystals in gout dissolve in formalin. It is still possible to diagnose gout on formalin-fixed tissue but it is easier to demonstrate the crystals if the tissue has been placed in alcohol fixative.

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