Cicatricial (scarring) alopecias are an uncommon group of permanent patchy hair loss conditions clinically characterised by the loss of visible follicular ostia, associated with variable degrees of visible inflammation. Histologically, the main feature is inflammatory destruction of the hair follicle epithelium with replacement by scar-like fibrous tissue. They are broadly divided into primary or secondary cicatricial alopecias (CA) depending on the target of the pathological process. In Primary CA (PCA), inflammation specifically targets the hair follicle epithelium, whereas in secondary CA the hair follicle is damaged as part of a wider destructive process within the skin (e.g., radiation, thermal burns, etc.)—see Nanda et al. [3]. Permanent alopecia may also occur in traditionally non-scarring alopecia disorders, where follicular drop out occurs in long-standing cases. Here the fibrous tract seen may be subtle and easily overlooked but histologically is reminiscent of follicular scarring seen in PCA. This “biphasic” presentation can be seen in longstanding cases of androgenetic alopecia, alopecia areata and traction alopecia [4].

Due to overlapping and changing clinical signs, securing a diagnosis on clinical features alone can often be challenging in PCA. Thus, scalp biopsy is regarded as the key investigation of any suspected cases of scarring alopecia [5].

To optimise the diagnostic yield, scalp biopsies should be taken from an affected area where the hair follicles are clinically inflamed and reduced in number, but still present (NB. usually toward the edge of the patch). Trichoscopy can help to identify subtle signs of inflammation and guide biopsy site selection [6]. Samples should be taken using a 4 mm punch biopsy orientated parallel to the angle of the hair shaft growth (to prevent cross-cutting the HFs) and deep enough to include the entire HF within the sample (i.e., into subcutis) [7]. It is important to recognise that the punch biopsy device used can influence adequacy of specimen size as the internal diameter of some punch instruments can render a smaller specimen than anticipated [8]. Ideally two biopsies should be obtained for horizontal and vertical sections and sent in separate cassettes [9]. Although some advocate a triple biopsy technique to reduce the risk of inconclusive findings and a further procedure, this will result in greater scarring and (in the authors opinion) is usually not required with careful biopsy site selection [10].

If only a single biopsy is obtained, either vertical or horizontal sectioning can be adopted. The advantage of horizontal sectioning is that this technique results in visualization of all the follicles sampled, allowing for an appreciation of the diagnostic features even if only focally present within the specimen. In contrast, vertical sections offer a more complete morphological overview of the follicle. Specifically, inflammatory changes at the dermato-epidermal junction (DEJ) can be identified precisely; an attribute not found inhorizontal sections. However, in vertical sectioning, sampling errors occur more frequently as only a limited number of hair follicles within each sample are usually visualised; this limitation can be at least partially mitigated against by taking further levels if necessary. Interestingly, a study of 76 cases comparing both approaches [11] favoured vertical sectioning for diagnosing scarring alopecias when only one method was available.

One option when tissue is limited is to take three vertical sections from a single punch and then re-embed for horizontal sectioning, on the understanding this results in a smaller sample. As the experience of assessing horizontal sections varies, discussion with the local pathologist about the preferred option may be helpful.

Many techniques exist in the laboratory for processing alopecia specimens. In our experience, it is preferable that bisecting of horizontal biopsies is not performed at the time of cut up, but rather carried out during embedding by experienced skilled staff. Horizontal samples are bisected and both halves are embedded, cut surface down, with red ink used to mark the cut surfaces (Figure 1).

It is important that the epidermis and the subcutaneous fat is included in both the horizontal and vertical sections and this requires education and training of laboratory staff.

Vertical sections may show between 2–3 follicular units in a 4mm punch biopsy and, as hair grows at an angle, tangential follicular cuts are frequently produced. However, a good biopsy technique (see above), along with appropriate sample orientation at the embedding stage, can reduce this risk. Although vertical sections do not allow for the interpretation of follicular density or hair counts (to assess hair cycle phases and miniaturisation), key attributes essential in making the diagnosis of a non-scarring pathology [1], these hair cycle parameters are less important in PCA assessment as the hair density seen is entirely dependent on the biopsy site selected. Six levels are usually adequate for vertical sections and twelve levels are typically required for horizontal sections to study all areas of the skin from the epidermis through to subcutis.

Special stains are available as a panel and can be used in line with experience, with H&E, EVG and PAS in standard use. Elastin staining (e.g., Verhoeff-Van Gieson (VVG)) on vertical specimens allows for a distinction between normal dermis and scarred areas in later stage scarring, with scarred areas lacking elastic fibres [12]. Further, distinct patterns of elastin loss are reported in different PCA entities and may help differentiate the underlying cause in later stage disease [13]. Thickened basement membrane zone, apoptotic cells and fungal elements are highlighted by Periodic Acid Schiff (PAS) staining [14,15]. Toluidine blue can help to visualise premature desquamation of the inner root sheath (IRS), helpful for the diagnosis of central centrifugal cicatricial alopecia (CCCA)—see below [16]. Moreover, toludine blue and colloidal iron are used to highlight dermal mucin, a useful marker for distinguishing cutaneous lupus erythematosus from lichen planopilaris. Direct immunofluorescence studies (DIF) is of particular value where cutaneous lupus erythematosus and autoimmune blistering conditions are considered in the differential diagnosis. It is important that careful attention is paid to site selection with DIF sampling taking place at the periphery of an active site of inflammation [2].