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Itchy skin disease

Dermatopathology

Objectives

  • Understand why skin biopsies are necessary.
  • Be able to list types of skin biopsy.
  • Be able to describe how to perform a punch biopsy.
  • Understand how a skin biopsy is processed.

Key points

  • Dermatopathology is necessary to make or confirm a clinical diagnosis and determine whether margins of excision are adequate.
  • Skin specimens include excision, incision, shave, punch biopsies and curettings.
  • Punch biopsies are most often performed for diagnostic purposes using 3 or 4mm round blades; the technique is similar to using a biscuit cutter.
  • The specimen is placed in formal saline fixative for at least 24 hours.
  • A paraffin block of tissue is sliced into 3-5 micron sections using a microtome.
  • The sections are floated onto glass slides, the paraffin is removed and they are stained with H&E and sometimes other special stains.
  • Haematoxylin is alkaline and stains nuclei blue.
  • Eosin is acid and stains cell cytoplasm red.
  • Direct immunofluorescence stains immunoglobulins and is especially useful to identify immunobullous diseases.
  • Immunohistochemistry is used to label immunoreactive materials to determine cell origin of tumours and identify certain infections.

Introduction

Skin biopsies are taken for the following reasons.

After the tissue is removed it is sent to a surgical pathology (histology) laboratory. A histopathologist or a specialist dermatopathologist examines processed routine sections under a light microscope. In some cases special stains or other tests are performed. A report is issued to the referred practitioner often within 2 working days of receiving the specimen in a straightforward case.

Skin biopsy specimens

Specimens of skin removed for pathological examination may of the following types:

Skin biopsy
Specimen may contain epidermis, dermis and/or subcutaneous tissue
Excision biopsy
A skin lesion is completely cut out for diagnosis and treatment
Incisional biopsy
A segment of the lesion is removed for diagnosis only
Shave / tangential biopsy
A horizontal section of the skin is removed for diagnosis or treatment
Punch biopsy
A standard round specimen 2-6 mm in diameter for diagnosis
Curettings
Fragments of tissue are removed using a bone curette for treatment
Fine needle aspiration
A needle is inserted into the lesion and cells are aspirated for direct examination (usually to confirm suspected metastases).

Punch biopsy

This technique is similar to using a biscuit cutter. It is used in inflammatory skin diseases and skin tumours to provide a diagnostic sample for histopathology.

A disposable biopsy punch with a round 3 or 4mm diameter blade is the most suitable general-purpose instrument. The area for biopsy should be carefully selected. Choose a typical new primary lesion that is not eroded or excoriated. Avoid the lower leg if possible. Using a skin-marking pen, draw a circle a little larger than the diameter of the selected instrument.

Infiltrate local anaesthetic (usually 2% lignocaine +/- epinephrine) into the marked area. Wearing surgical gloves and using aseptic technique, hold the biopsy punch perpendicular and in contact with the skin and rotate the blade while pushing gently. When resistance reduces, the blade will have penetrated into subcutaneous tissue and it may be withdrawn, extracting a core of full thickness skin. Using fine scissors, snip off the base of the biopsy sample through the subcutaneous tissue.

Instruments for punch biopsy

Marking the sites

Infiltrating local anaesthetic

Inserting biopsy punch

Removing the specimen

Suturing the wound


Place the sample in fixative i.e. formol saline (10% neutral buffered formalin); the volume should be at least ten times that of the tissue. Make sure the pot is properly sealed. Label it with the patient’s full name and a second identifier (date of birth and/or National Health Index number), the site of the biopsy and the date. Complete the histopathology request form.

Punch biopsy wounds are usually closed with a single 5-0 monofilament nylon suture, which is removed after 7 days.

Tissue processing

The biopsy specimen remains in fixative for a minimum of 24 hours prior to processing by a histotechnologist. This requires placing the specimen in a series one or more plastic cassettes, immersing them in alcohol then xylene prior to embedding in paraffin wax..

Paraffin blocks are prepared to allow the tissue to be cut into thin microscopic sections (3-5 microns) using a microtome. The sections are applied to a glass microscope slide and the paraffin is removed.

Skin fragments in casette

Completed paraffin block

Sectioning the biopsy

Microtome

Water bath to collect section

Staining machine


The slide is stained routinely using haematoxylin and eosin (H and E). Haematoxylin is a basic dye that stains the nucleic acids of the cell nucleus in varying hues of blue. Eosin stains are acidic and dye cytoplasmic components of the cell red. In some situations, other special stains may be used.

After further processing, a resin is applied to glue a cover slip over the section. The slide is now ready for the pathologist to report the microscopic description, the diagnosis and comments.

Reporting

Pathological changes may arise in epidermis, dermis and/or subcutaneous tissue. The pattern of changes may allow a diagnosis to be made or it may be non-specific. Many skin diseases appear quite different at different stages of their development and may be altered by secondary changes.

Dermatopathology is useful for distinguishing inflammatory skin diseases when these are difficult to diagnose clinically. However, some skin diseases have very non-specific histology. Pathologic diagnosis is particularly important for bullous diseases and granulomatous dermal infiltrates. It is sometimes useful for distinguishing infections.

The pathologist is also vital in determining whether skin tumours are benign or malignant, especially in the diagnosis of melanocytic lesions.

Direct immunofluorescence

Direct immunofluorescence staining is used to detect immunoglobulins. A batch of fluorescein isothiocyanate-labelled stains including IgG, IgM, IgA, fibrin and C3 is applied to fresh tissue and examined by fluorescence microscopy.

Specific staining patterns are seen in the immunobullous diseases, lupus erythematosus and vasculitis.

Immunohistochemistry and immunocytology

Immunohistochemistry is the histochemical localization of immunoreactive substances using labelled antibodies as reagents. Often a panel of antibodies is applied. It is particularly useful for determining the cell origin of difficult tumours and for certain infections.