ACQUISITION
Selecting the biopsy site is the most difficult and important part of the process.
The most fruitful site will vary depending on the disease, and often the clinician is uncertain of the diagnosis. Sampling the center of a lesion of alopecia areata or trichotillomania would be appropriate. Sampling the bald center of a lesion of scarring alopecia is seldom useful, and the ‘active’ peripheral margin would be a more suitable target. Compounding the difficulty of site selection is sampling error. Clinicians cannot see below the surface of the skin, and even the most experienced physician may choose an unrewarding spot. If a recently involved area of scalp showing early clinical changes is selected, the diagnostic yield will be higher. Highly inflamed sites (pustules or papules) are often very advanced lesions and are frequently non-diagnostic. Multiple separate specimens chosen from several sites in or around the lesion will increase the diagnostic yield.
However, clinicians may not have the luxury of obtaining multiple specimens.
In some cases the patient’s ‘normal’ scalp can serve as a basis for comparison with the abnormal scalp. For example, a specimen from the mid-occiput can help establish a diagnosis of common balding in a patient with hair loss on the crown.
Once the site is selected, it should be anesthetized with lidocaine with epinephrine (adrenaline). A generous amount of anesthetic (1–3 ml) should be injected into the deep dermis and superficial fat, and allowed to act for 15–30 minutes before the biopsy is performed. This will minimize bleeding.
The blade of the punch biopsy tool should extend through the dermis down into the fat, so that intact bulbs of deeply rooted terminal hairs can be removed.
A 4-mm biopsy wound can be easily closed with 3–0 suture because the needle can traverse the wound in a single pass. A suture color that contrasts with the patient’s hair will assist in suture removal 1 week after the biopsy is performed.
HANDLING
Once obtained, the scalp biopsy specimen should be allowed to fix in formalin for at least 24 hours before sectioning. Biopsy specimens obtained for direct immunofluorescence testing should of course be placed in the appropriate transport solution.
PROCESSING
The required tools include a sharp blade, a blade holder, a pair of fine-toothed forceps, marking ink, a cotton-tipped applicator and the standard plastic specimen cassette. Sponges should be used inside the cassette to prevent the thin slices of tissue from escaping. The sharpest blade for the job is a disposable, flexible shaving blade (‘blue blade’). These can easily be snapped in half for economy and safety (Figure 2.1). The author prefers to use red marking ink, but
SPECIMEN ACQUISITION, HANDLING AND PROCESSING 33
any color will do. A specially colored cassette will alert the histology technician that embedding must be performed in a particular way.
There are several ways to slice the tissue into transverse sections. The technique used by Headington and Whiting involves a single transverse slice about 1 mm below the epidermal surface. Both cut sides of the specimen are embedded down in the cassette. As the microtome cuts deeper into the tissue block, the sections become progressively more superficial in one half of the specimen and deeper in the other. Simply sectioning deeper into the block allows one to obtain sections as superficial or as deep as required.
An alternative method is that of Frishberg and Sperling. One treats the biopsy specimen like a cylindrical loaf of bread, and cuts it into three or four slices (Figures 2.2 and 2.3). The ‘slices’ are about 1 mm thick. The deep surface of each slice is inked (Figure 2.4) and the inked sides are placed down in the cassette. Once the ink has been removed by the microtome, a single section is taken. In this way, the specimen is sampled at several different depths (Figure 2.5). Because only a single section is needed to view multiple levels on a Figure 2.1 Tools for performing transverse sections. The flexible shaving blade has been snapped in half
Figure 2.2 The specimen is placed on its side like a cylindrical loaf of bread, and is gently stabilized with forceps. A flexible, disposable shaving blade is ideal for taking the slices 34 AN ATLAS OF HAIR PATHOLOGY
single slide, the remainder of the tissue in the block is available for ‘recuts’ and special stains.
Yet another but more tedious method involves embedding the epidermal and/or fat end down in the cassette, and taking multiple horizontal sections through the entire specimen. Dozens of sections can be required to section through the entire block, and the block is exhausted in the process. However, every possible level of every follicle in the specimen can be carefully studied, and this technique is useful for research purposes.
BIBLIOGRAPHY
Frishberg DP,Sperling LC,GuthrieVM. Transverse scalp sections: a proposed method for laboratory processing. J Am Acad Dermatol 1996; 35:220–2
Headington J. Transverse microscopic anatomy of the human scalp. Arch Dermatol 1984;
120:449–56
Figure 2.3 The specimen is cut into three or four slices, which are placed with the cut surfaces up, so that they are ready for inking
Figure 2.4 The inked slices are placed in a cassette, packed between sponges. They will be embedded in paraffin, ink side down
SPECIMEN ACQUISITION, HANDLING AND PROCESSING 35
Whiting DA. Diagnostic and predictive value of horizontal sections of scalp biopsy specimens in male pattern androgenetic alopecia. J Am Acad Dermatol 1993; 28:
755–63
Whiting DA, Howsden FL Color Atlas of Differential Diagnosis of Hair Loss. Cedar Grove, NJ: Canfield Publishing, 1996
Figure 2.5 The final product is a single slide containing disks of tissue from multiple levels (superficial to deep)
36 AN ATLAS OF HAIR PATHOLOGY