several sites, collect plenty of material, and divide amongst several slides to make thin suspensions, which are quicker and easier to examine efficiently.
Surface adhesive tape samples
An alternative method for obtaining surface material, including Oxyuris equi eggs, surface‐living ectoparasites, hair fragments, exfoliated cellular material, and surface microorganisms for direct microscopical examination or after staining; it is less traumatic and avoids the risk of injury associated with skin scrapes. This technique is particularly useful for identification of chorioptic mange mites which are highly motile, but also allows detection of other pathogens including dermatophytes and yeasts.
A piece of clear adhesive tape (e.g. 3M Scotch Crystal, Sellotape Clear) is applied to the lesional area 3–4 times (Figure 1.5).
Tape is applied (sticky side down) to a microscope slide over a drop of liquid paraffin for direct examination or over a drop of blue dye from a rapid Romanowsky‐type stain kit.
Excessive mounting medium or stain is removed by wiping with soft paper towel prior to microscopical examination.
Figure 1.5 Surface adhesive tape sampling.
Direct smears
From fresh, exudative, crusted, excoriated, or pustular lesions, a direct impression smear can be made for cytological examination and for microorganisms.
Press a glass slide against the concave undersurface of a removed exudative crust, or against the surface of a freshly exposed lesion.
For an intact pustule, gently break the overlying skin with a 25 g needle and press a clean glass slide to the ruptured lesion, or purulent material may be collected in the needle bevel and then transferred to the glass slide.
For lesions at sites where it is difficult to apply a slide directly, material can be collected with a dry swab and then rolled onto a glass slide.
Air‐dry the slide and store in a slide box prior to heatfixing (for Gram staining) or immersion in methanol (for Romanowsky‐type staining).
Wet crust preparations
For older, crusted lesions this technique enables microscopical examination of dried exudate.
Representative sample of crust is placed on a glass slide with a few drops of normal saline.
Material is finely chopped and macerated with a scalpel blade.
Slide is left in a warm place for 20–30 min to allow rehydration of cellular material.
Any large clumps of debris are gently removed prior to thorough drying and heat fixing of the remaining suspension prior to staining with a rapid Romanowsky‐type stain kit.
Swabs
May be useful for bacteriology and fungal culture and collecting material for cytological examination.
If the sample is to be processed within 30 min of collection, a dry, sterile swab can be used for bacterial and fungal culture, and smears. Otherwise, place swabs into suitable transport media (e.g. Amies charcoal medium, or Copan ESwabs).
Samples collected from the skin surface may not be representative of the causative agent, so collect pus from an intact pustule or the underside of a freshly removed scab, or submit biopsy material for culture. Useful cultures may sometimes be obtained from a dry crust by rehydrating with sterile normal saline prior to processing.
Needle aspirates
This technique is used for sampling nodules, masses, and enlarged superficial lymph nodes.
A 20–22 g needle can be used, with or without a 5 ml syringe. The area to be aspirated should be carefully cleaned and disinfected.
The needle is inserted into the nodule (Figure 1.6), mass, or lymph node and used to probe the tissue in several places, initially without aspirating, and subsequently whilst gently aspirating.
The needle is withdrawn from the tissue and detached from the syringe, which is then filled with a small amount of air, reattached to the needle, and the sample expressed directly onto a clean slide for cytology or onto a swab for culture. A second slide is placed over the sample to spread the material. The slides are then separated gently to avoid damaging cells and air‐dried prior to staining for microscopical examination.
Biopsy samples
Skin biopsies may be collected for a variety of reasons, including histopathology, fungal or bacterial culture, viral identification with electron microscopy, and immunohistochemistry. If in doubt, consult a pathologist as to the best way to process and transport the biopsy to the laboratory.
Figure 1.6 Fine needle aspiration of a nodular lesion.
Source: Courtesy of Liz Steeves.
There are three common ways to take biopsy samples: by excision, biopsy punch (Figure 1.7), or shave biopsy (see Chapter 3). The most common is the punch biopsy technique described below.
Sedation is generally necessary, followed by local analgesia. For the distal limbs, a nerve block (low or high four‐point or abaxial sesamoid, depending on the area involved) may be performed or local infiltration below the sample site or as a ring block around the lesion. For facial and difficult to access sites, such as inguinal and perineal lesions, general anaesthesia may be required.
Areas that include primary lesions should be selected where possible and sites not marred by medication. Take multiple samples unless only one lesion type and stage is present.
Because sample orientation during histopathological processing cannot be predicted, ensure that the whole punch sample includes tissue of interest. If normal skin is to be included for comparison, this should be taken as a separate sample in an appropriately labelled pot. Where you wish to investigate the transition between lesional and healthy skin, take an elliptical excision sample with the long axis going from normal to abnormal.
