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Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos
MINI-SYMPOSIUM: DERMATOPATHOLOGY
Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos
represent a reversible response of the sebaceous glands to overlying epidermal psoriasiform changes. A peribulbar lymphoid cell infiltrate ‘clue’ for alopecia areata may be part of an alopecia areata-like pattern associated with different clinicopathologic scenarios, or possibly even incidental. Clues to ‘silent’ (invisible) alopecias will also be discussed. Awareness of the increased complexity in presentation due to coexistence of multifactorial etiologies is essential while bearing in mind the time-frame factor (lives of lesions) in alopecia diagnostics. The boundaries between evolving end-stage non-scarring alopecia and scarring alopecia prove to be closer than previously thought.
Catherine M Stefanato
Abstract In recent years the increased number of scalp biopsies in alopecia has afforded the dermatopathologist improved confidence in distinguishing the histopathologic characteristics of ‘classic’ scarring and nonscarring alopecias, as well as the spectrum of their presentation. Occasionally, however, histopathologic ‘dogmas’ have been disproven. The loss of sebaceous glands considered to be a clue to scarring alopecia or the peribulbar lymphoid cell infiltrate diagnostic of alopecia areata may present in unconventional scenarios, and thus, represent pitfalls. Clues to diagnose histopathologically ‘silent’ (invisible) alopecias, the identification of multiple etiologies, and awareness of the ‘time-factor’ are herein discussed.
The St John’s diagnostic update and technical considerations Our role, as dermatopathologists, is to offer the best diagnostic options for the biopsies provided, in the light of clinicopathological correlation.1 At times, however, scalp biopsies of alopecia lack adequate clinical history, and clinical photographs are not provided. In this instance, the use of our St John’s combined scarring and non-scarring alopecia protocols has proved to be a ‘safety net’ enabling diagnosis at the microscope.1,2 In the absence of clinical history, helpful factors to reach a meaningful diagnosis include (a) the number of scalp biopsies provided, (the more biopsies provided the more likely an accurate diagnosis can be reached), (b) knowledge of scalp biopsy anatomical sites (certain alopecias have a predilection for distinct anatomical sites3), (c) combined use of horizontal and vertical sections in order to approach the study from all perspectives, in a ‘kaleidoscopic’ way, and (d) ancillary studies. A structured microscopic evaluation of multiple serial levels with careful histopathologic examination of the four key levels of the hair follicle, (hair bulb, suprabulbar, isthmus, and infundibulum), is a ‘conditio sine qua non’ paving the pathway to the diagnosis. In our St John’s scarring alopecia protocol, two 4 mm punch biopsies are taken at the peripheral ‘active’ edge of the scarring alopecia. Of the two biopsies, one specimen is sectioned transversely in the laboratory while the second one is sectioned vertically by the dermatologist in the clinic, with 1/2 of the skin biopsy placed in Michel’s medium for direct immunofluorescence study, and the other half sent to the dermatopathology laboratory for vertical sections examination.1,2 Two 4 mm punch biopsies are also taken in non-scarring alopecia. In this case, one biopsy is taken from the center of the involved area of alopecia, usually the vertex, the other biopsy at the occiput, a nonandrogenic area, acting as a normal control and helpful for comparison purposes. Since Headington’s seminal paper in 1984, transverse sectioning of scalp biopsies has become the preferred method in the context of non-scarring alopecia, owing to the quantitative and morphometric data that can be generated.4 There are many examples of how to optimize the diagnostic yield from a single biopsy, such as the HoVert5 and the Tyler6 techniques. Our 15year experience with the St John’s protocol based on combined transverse and vertical sections of two 4 mm punch biopsies has proven to be diagnostically effective, and further validates what Elston et al.7 had already reported, as a biopsy
Keywords alopecia areata-like; clues; follicular mycosis fungoides; multifactorial alopecia; non-scarring alopecia; pitfalls; psoriasis; scarring alopecia
Introduction Although the type of alopecia may be diagnosed with reasonable certainty clinically and dermoscopically, a scalp biopsy can, nonetheless, be paramount in the assessment of a patient with hair loss. When possible, the multi-team clinical-pathological approach, including accurate biopsy site selection, communication between the dermatologist, the dermatopathology laboratory and the dermatopathologist trained in hair histopathology, are ideal factors. However, this is not always the case, and the dermatopathologist may be left to interpret alopecia slides from biopsies provided with minimal, if any, clinical information. Histopathologically, diagnostic ‘tools’ for the dermatopathologist in the evaluation of alopecia comprise the localization level of the inflammatory process, (peri-bulbar/suprabulbar/isthmic/ infundibular), the degree of the cellular component (mild/moderate/severe), and the quality and predominance of the inflammatory cell infiltrate (lymphocytes, plasma cells, neutrophils, eosinophils, mast cells). Moreover, assessment of the presence/ absence of sebaceous glands and of perifollicular fibrosis and observation of anomalies of the hair cycle are all part of the routine evaluation. Pitfalls, such as the absence of sebaceous glands in a scalp biopsy considered a ‘clue’ for scarring alopecia, may instead
Catherine M. Stefanato MD, FRCPath Consultant in Dermatopathology, Department of Dermatopathology, St John’s Institute of Dermatology, St Thomas’ Hospital, Guy’s and St Thomas’ NHS Trust, London, UK. Conflicts of interest: none declared.
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Please cite this article as: Stefanato CM, Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.12.003
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when the scalp site chosen to biopsy is not truly representative of the disease or b) biopsies taken from late, ‘burnt out’ areas, since, in late scarring, immune-deposition is lost, and the immunodeposits are no longer detected. The DIF yield is also influenced by laboratory technical considerations. Monoclonal antibody detection sensitivities used may vary between different laboratories,19 but also the choice of the transport medium for the scalp biopsy (Michel’s medium vs saline or honey),20 along with the time to reach the laboratory, are all factors that play a role. To optimize diagnostic yield, it is thus critical to obtain the DIF skin sample from a clinically ‘active’ area of scalp inflammation at the periphery of the scarring process, truly representative of the disease. In this instance, the DIF yield will likely be positive.
combination is superior to either transverse or vertical biopsy examination alone.8 The choice of which combination of biopsies and sectioning to use may vary, based on the presence or absence of the clinical data provided. Our flexible, ‘kaleidoscopic’ approach not only incorporates the “best of two worlds” but also allows us to obtain fresh tissue for direct immunofluorescence studies.2,9 The use of vertical sections has shown to be helpful in adding morphologic information to the horizontal sections, as well as providing morphological insights, particularly when the horizontal sections are not contributory. Although vertical sections show a limited number of tangentially sectioned hair follicles, they outline the dermal eepidermal junction and demonstrate full-thickness dermis and subcutaneous tissue, allowing for precise localization of an inflammatory infiltrate.2
The role of special stains The role of direct immunofluorescence
Special stains further supply valuable information.21 The histopathologic findings in alopecia may be focal, such as the presence, by ‘serendipity’ of fungal hyphae in a single hair follicle in a scarring alopecia (Figure 5) or in non-inflammatory tinea,21 or at times be misleading, when tinea mimics dissecting cellulitis.22,23 Thus, the use of periodic acid-Schiff (PAS) is essential.23 The use of PAS will also help to identify subtle basement membrane zone thickening in the evaluation of lupus. Elastic stains are invaluable in assessing subtle perifollicular fibrosis and highlighting fibrous tracts,24e26 as well as mucin stains for distinguishing the mucinous perifollicular early fibroplasia in scarring LPP27 or highlighting the interstitial dermal mucin diagnostic of lupus.21,28,29
The implementation of direct immunofluorescence (DIF) in the diagnosis of primary scarring alopecia has been recently challenged,10 with reservation suggested for its use.11 In our experience, however, DIF has shown to be an important aspect in the diagnostic process, not only aiding in distinguishing lupus (Figure 1) from lichen planopilaris (LPP) (Figure 2),12 but also in the identification of other causes of scalp alopecia such as those seen in the setting of autoimmune blistering disease,13 including pemphigus14e17 (Figure 3) and bullous pemphigoid (Figure 4), and its localized Brunsting-Perry variant.18 Possible explanations as to why in many cases the scalp biopsy DIF yield is often negative may be ascribed to: a) biopsy sampling bias,
Figure 1 Scalp, Lupus, Active stage, Isthmus. Scanning magnification showing predominately anagen hair follicles, with a moderately dense perifollicular lymphoid cell infiltrate and focal tufting (40) (a). Higher magnification highlighting perifollicular lymphocytes with nuclear dust and with lymphocytic exocytosis in follicular epithelium (400) (b). Perineural inflammation, a feature commonly seen in lupus scalp biopsies (400) (c). Direct immunofluorescence showing a linear granular deposition of IgM at the basement membrane zone. (200). This case was also positive for IgG, IgA and C3 (lupus band test positive) (d).
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Please cite this article as: Stefanato CM, Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.12.003
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Figure 2 Scalp, Lichen planopilaris, Isthmus. Scanning magnification showing different stages of the disease within the same horizontal section. Anagen hair follicles with an active moderately dense perifollicular lymphoid cell infiltrate (upper left) which is gradually reduced to be replaced by perifollicular fibrosis, consistent with a more advanced stage of the disease (lower right) (40) (a). High power detail of an anagen hair follicle with perifollicular lymphoid cell infiltrate, lymphocytic exocytosis and perifollicular fibrosis. (400) (b). Direct immunofluorescence staining with fibrinogen showing follicular involvement of the hair follicle (400) (c).
Figure 3 Scalp, Pemphigus vulgaris. Scanning magnification showing suprabasal acantholysis with complete detachment of the epidermal roof (40) (a). Higher magnification detail showing suprabasal acantholysis of a hair follicle (200) (b) Direct immunofluorescence showing follicular net-like intercellular deposition of IgG. This case also showed similar intercellular deposition of C3 (400) (c).
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Please cite this article as: Stefanato CM, Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.12.003
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Figure 4 Scalp, Bullous pemphigoid. Scanning magnification showing epidermal hyperkeratosis and acanthosis, with patchy papillary dermal and perifollicular lymphoid cell infiltrate and epidermal-dermal separation involving the hair follicle (40) (a). Higher power detail of the follicular split (400) (b). Direct immunofluorescence documents linear deposition of IgG at the epidermal basement membrane zone which is extending into the hair follicle. This case was also positive for similar linear deposition of C3 (200) (c).
seborrheic dermatitis or eczema of the scalp. Miniaturization of sebaceous glands has been recently documented in pemphigus vulgaris and pemphigus foliaceus of the scalp,32 and atrophy of the sebaceous lobules reported as a histologic clue for TNFinhibitor associated psoriatic alopecia.33 Loss of sebaceous glands is commonly regarded as one of the key diagnostic morphologic features of scarring alopecia,1 occurring secondary to perifollicular fibrosis and perifollicular
Clues and pitfalls Sebaceous gland atrophy: a triple pitfall Sebaceous gland atrophy of the scalp was originally reported by Headington et al. in 1989 in association with psoriasis.30 This phenomenon is reversible, and felt to be secondary to abnormal sebaceous gland function,31 likely in response to the overlying epidermal changes. It may be seen also as secondary to any psoriasiform-spongiotic epidermal reaction pattern, such as
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Figure 5 Scalp, incidental tinea on lichen planopilaris. Scanning magnification showing a reduced number of hair follicles, with two hair follicles exhibiting perifollicular fibrosis with perifollicular lymphoid cell infiltrate (40) (a). Medium power highlighting focal follicular infundibular acanthosis and hyperkeratosis of a hair follicle involved by lichen planopilaris at the edge of the biopsy (100) (b). Higher magnification documents the presence of fungal hyphae within the hyperkeratotic follicular infundibulum (400) (c). The fungal hyphae are highlighted with periodic acid-Schiff (PAS) stain (400) (d).
the vicinity of nevi or in follicles involved by malignant melanoma (Figure 7). In recent years, there have been an increasing number of reports describing alopecia areata-like histopathology in contexts other than ‘classic’ alopecia areata. These include reactions to medications such as carbocysteine,38 immune checkpoint inhibitors,39 anti-tumor necrosis factor alpha drugs,40 (Figures 8 e10), taxanes in patients undergoing chemotherapy for breast carcinoma41 (Figure 11), and heavy metal thallium poisoning.42 Alopecia areata-like peribulbar lymphoid cell infiltrate is observed in the non-cicatricial variant of systemic lupus erythematosus.43 An increased telogen/catagen shift, also mimicking the ‘shift’ out of anagen of acute/subacute alopecia areata, may be seen in the affected areas. Distinguishing histopathologic clues in support of lupus include a peribulbar lymphoid cell infiltrate which is denser than commonly seen in alopecia areata, a vacuolar-interface change, and perieccrine and perivascular lymphoid cell infiltrate containing plasma cells.43 Syphilis, the ‘great mimicker’ also comes into play, with a peribulbar lymphoid cell infiltrate, miniaturization of the hair follicles, and a shift out of anagen of almost all hair follicles in the catagen/telogen phase.44 These are all features seen in classic alopecia areata. However, clues to the diagnosis of syphilitic alopecia are the presence of peribulbar plasma cells (which are absent in alopecia areata), as well as, -in opposition to alopecia areata where the infiltrate remains confined to the hair bulbs-, extension of the infiltrate to the upper follicular levels, reaching the infundibulum. Moreover, when these findings are viewed in
lymphoid cell infiltrate, as seen in the setting of immunemediated alopecias (lichen planopilaris and lupus). However, sebaceous gland regression leading to complete atrophy and apparent loss may represent a potential pitfall. If the sebaceous gland atrophy is complete, the process mimics a scarring alopecia. Regressed sebaceous glands, morphologically reverted into basaloid epithelial aggregates, resemble telogen hair follicles. If only few sebaceous glands are atrophic, a misdiagnosis of telogen effluvium may be rendered, but if all sebaceous glands are regressed to basaloid epithelial aggregates, the histopathologic picture may resemble subacute alopecia areata. 34,35 Immunoperoxidase stains for androgen receptors and Adipophilin will help distinguish atrophic sebaceous glands from telogen hair follicles by highlighting the residual sebocytes within the basaloid epithelial aggregates.34 (Figure 6). Peribulbar lymphoid cell infiltrate: not only alopecia areata Hair follicle bulbs are sites of immune-privilege, the collapse of which, together with exposure of the major histocompatibility complex (MHC) class I, initiates an autoimmune response leading to a perifollicular and intrafollicular T-cell lymphoid infiltrate affecting hair bulbs of anagen-stage hair follicles.36,37 This morphologically corresponds to the peribulbar lymphoid cell infiltrate (also known as a ‘swarm of bees’), which is the histopathologic feature we commonly look for to diagnose alopecia areata.1 However, peribulbar lymphoid cell infiltrate may be encountered also incidentally, in unrelated conditions such as in
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Figure 6 Scalp, psoriasis: Vertical section showing hyperkeratosis and a psoriasiform epidermis. The underlying dermis exhibits hair follicles in anagen phase and atrophic sebaceous glands mimicking telogen hair follicles (X40) (a). Horizontal section, isthmus: diffuse atrophy of the sebaceous glands regressed to basaloid epithelial aggregates mimicking telogen hair follicles (X40) (b). A follicular unit with atrophic sebaceous glands where residual sebocytes can still be seen (X200) (c). Another follicular unit with more advanced sebaceous gland atrophy (X100) (d). Detail of an atrophic sebaceous gland with residual sebocytes (e). Immunoperoxidase stain for androgen receptor highlights the residual sebocytes in the atrophic sebaceous glands. (400) (feg).
diagnosis and therapeutic intervention, given the less favorable survival rate when compared with non folliculotropic mycosis fungoides.45,51
a clinical context, syphylitic alopecia is far less common than alopecia areata.44 Folliculotropic cutaneous T-cell lymphoma (mycosis fungoides): alopecia areata-like folliculotropic mycosis fungoides is an uncommon variant of cutaneous T-cell lymphoma with distinct clinical and pathologic findings (Figures 12e13). Clinically, alopecia is a typical finding, occurring on the face (eyebrows) and scalp in 65% of patients.45 Patchy alopecia areatalike changes with peribulbar lymphoid cell infiltrate histopathology has been reported in up to 34%.46e48 This may involve the genital area,49 as well as other body sites,47 and often represents an early presenting sign of mycosis fungoides.49 Histopathologically, folliculotropism occurs predominantly at the infundibulum and isthmus, with epidermotropism presenting in fewer than half of patients.45 Syringotropism may also be present. Various histopathologic patterns of follicular involvement at all follicular levels have been described,45 including the recently reported spiky hyperkeratotic variant.50 Awareness of these patterns is a diagnostic clue to follicular mycosis fungoides, as often they may coexist in the same biopsy, or occur in different biopsies of the same patient (Figure 14). The immunohistochemical profile is similar to that of classic mycosis fungoides, with a CD4 predominance over CD8; although rare, alopecia areata-like mycosis fungoides can be CD8 predominant.47 Recognition of the alopecia areata-like pattern in follicular mycosis fungoides is paramount, as it may result in earlier
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‘Silent’ (invisible) non-scarring alopecias with scant inflammatory cell infiltrate A ‘cumbersome’ scenario for the dermatopathologist is the interpretation of the histopathology in biopsies of non-scarring alopecia with a minimal inflammatory cell infiltrate and a limited clinical history of hair loss. The differential diagnoses commonly offered include telogen effluvium (TE), alopecia areata (AA), and female pattern hair loss (FPHL). In this setting, transverse sections are the preferred method of evaluation, as all the hair follicles can be quantified and studied morphologically at all the key follicular levels.2 Awareness of the telogen hair follicle counts and of the presence/absence of miniaturization will help in distinguishing them. It is helpful to be reminded that the histopathology of an episode of acute TE will characteristically resemble normal scalp, likely reflecting morphologically the recovery phase of the disease.52,53 Chronic telogen effluvium (CTE), on the other hand, occurs in later years of age, has a fluctuating course, and eventually burns itself out; CTE, in its active stages, may also show slightly increased numbers of telogen hair follicles, typically in the absence of miniaturization. But, if miniaturization is identified, then a CTE uncovering a background of FPHL should be considered.
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Figure 7 Congenital nevus. Scanning magnification of this intradermal nevus shows incidental neighboring hair follicles which exhibit peribulbar lymphoid cell infiltrate and pigment casts, simulating alopecia areata (40) (a). Details of the hair follicles in anagen and telogen phase with alopecia areata-like peribulbar lymphoid cell infiltrate (100, 400, 400) (bed). Follicular malignant melanoma. The hair follicle involved by malignant melanocytes (left) shows a dense peribulbar lymphoid cell infiltrate simulating alopecia areata. The adjacent anagen hair follicle (right) is unaffected. (200, 400) (eef).
In diffuse FPHL the number of telogen hair follicles is expected to be higher than in TE. Moreover, FPHL is associated with increased vellus hair follicles and miniaturization.54 Diffuse alopecia areata may be misdiagnosed as telogen effluvium, clinically and histopathologically.55,56 Hair bulbs examination, with identification of the peribulbar lymphoid cell infiltrate (‘swarm of bees’), is diagnostic; however, in long-standing chronic lesions of alopecia areata, peribulbar and intrabulbar lymphoid cell infiltrate is minimal to absent,57,58 and the diagnosis of a silent (invisible) alopecia areata is missed. Awareness of the morphological time-frame changes hair follicles undergo in the acute and chronic stages, including miniaturization, high percentage in
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catagen/telogen hair follicles, (‘shift out of anagen’), trichomalacia and pigment casts will point to the diagnosis.58 The identification of peribulbar mast cells has been regarded as an additional clue in alopecia areata59 (Figure 15) with the suggestion that mast cells ‘cross-talk’ with the pathogenic CD8 (þ) T-cells involved in hair follicle immune privilege collapse.37,59 More recently, degranulating mast cells were also reported in telogen effluvium, and felt to be a response to stress-induced hair loss.60 A helpful tool to distinguish diffuse AA from diffuse FPHL is the use of the immunostain CD3.61 This will highlight the rare intra-bulbar lymphoid cells (Figure 16) which may be also identified within empty follicular fibrous tracts.61
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Figure 8 Infliximab alopecia. Scalp, Isthmus. Scanning magnification showing follicular units with hair follicles in anagen phase and atrophic sebaceous glands (40) (a). Detail of a follicular unit sebaceous with gland atrophy and lymphoplasmacellular infiltrate (200) (b). Peribulbar lymphoplasmacellular infiltrate simulating alopecia areata (400) (c). Eccrine glands with lymphoplasmacellular cell infiltrate (400) (d).
Figure 9 Infliximab alopecia, Scalp (same case as in Figure 8). Scanning magnification showing epidermal acanthosis with dense lymphoid cell infiltrate and underlying dermal sebaceous gland atrophy (40) (a). There is a band-like papillary dermal lymphoid cell infiltrate with prominent epidermal lymphocytic exocytosis, mimicking cutaneous T-cell lymphoma (200) (b). No cytologic atypia was present, and both immunostains CD4 and CD8 highlight reactive lymphocytes in the epidermis and papillary dermis (200, 200) (ced).
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Figure 10 Infliximab alopecia, Scalp, Isthmus. Scanning magnification of another case showing hair follicles in anagen with atrophic sebaceous glands (40) (a). Higher power details of the different stages of sebaceous gland atrophy (200 and 400) (bec). Immunoperoxidase antibody for Adipophilin highlights the atrophic sebaceous glands (200) (d).
Thoughts ‘out of the box’: multifactorial alopecia
same biopsy.3 Multiple serial levels throughout the biopsy and examination at all the key follicular levels are important. The study of the subcutaneous tissue to evaluate the hair bulbs is critical to rule out AA and the examination of the isthmus to rule
When the dermatopathologist is asked to answer a diagnostic question, the complexity is often compounded by more than one type of alopecia coexisting in the same patient, and even in the
Figure 11 Taxane alopecia. Scalp, Isthmus. Alopecia areata-like morphology with a ‘shift out of anagen’ characterized by increased miniaturized telogen hair follicles (40, 200) (aeb). Dysmorphic telogen hair follicles, stigmata of taxane alopecia, are identified both in horizontal sections (400) (c) as well as in vertical sections (400) (d).
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Figure 12 Folliculotropic cutaneous T-cell lymphoma, Scalp. ‘Shift out of anagen’ with all hair follicles in telogen phase (40) (a). Higher magnification highlights a telogen hair follicle with dense peribulbar lymphoid cell infiltrate in an alopecia areata-like pattern (400) (b). Immunoperoxidase stains confirm the infiltrate is CD4 predominant (400) (c), with only a few CD8-positive reactive lymphocytes (400) (d).
Figure 13 Folliculotropic cutaneous T-cell lymphoma, arm, sub-isthmus. Scanning magnification showing alopecia areata-like peribulbar lymphoid cell infiltrate (40, 400) (aeb). Detail of an eccrine gland with atypical epithelium and with eccrinotropism of atypical lymphocytes (400) (c). Isthmus: Follicular units showing follicular epithelium acanthosis, perifollicular lymphoid cell infiltrate, folliculotropism (40) and follicular mucinosis (inset 400) (d).
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Figure 14 Follicular cutaneous T-cell lymphoma variants. Follicular infundibular acanthosis with lymphocytic folliculotropism (100, 400) (aeb). Follicular cystic variant simulating epidermal inclusion cysts (40) (c), but with atypical lymphocytes (400) (d).
out an immune-mediated scarring alopecia such as lichen planopilaris or discoid lupus erythematosus, which may coexist, although rarely,62,63 but then DIF studies will help in the differentiation.63
Knowledge of specific types of alopecia having a predilection for different scalp zones will also aid in the diagnosis.3 For example, the occipital and parietal scalp zones are often sites of traction alopecia, and the crown and the vertex are sites of
Figure 15 Alopecia areata. Scalp, Isthmus. Follicular units with diffuse miniaturization and increased telogen hair follicles, characteristic of alopecia areata (20) (a). Detail on the telogen hair follicles (200) (b). Control biopsy of uninvolved scalp with anagen hair follicles (40) (c). Higher power showing a fibrous tract with numerous mast cells (d).
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Figure 16 Alopecia areata incognita. Scalp, Isthmus. Diffuse miniaturization mimicking female pattern hair loss (40) (a). Subisthmus: only three follicular bulbs are present (40) (b). Higher power shows focal peribulbar lymphoid cell infiltrate (400) (c). Immunoperoxidase stain CD3 highlights intrabulbar lymphocytes diagnostic of alopecia areata (400) (d).
study,67 a significant reduction of follicular counts occurred in 10.6% of male patients with androgenic alopecia at age 50. In females with FPHL, follicular counts were reduced in 5.7% at age 70 years and 2.0% in diffuse FPHL at age 80. This data supports that reduced hair follicle density in the elderly is androgendriven67 and not due to old age.
predilection for central centrifugal cicatricial alopecia, whereas FPHL occurs on frontal, temporal, crown and vertex zones but not in the occiput. Conversely, both AA and TE may occur in any site.3 Thus, thinking ‘out of the box’ and accuracy in identifying ‘kaleidoscopically’ the subtle clues to other concomitant diagnoses, whilst still answering the main diagnostic query, is helpful.3
Conclusion Lessons from the past: ‘lives of lesions’ in alopecia
Although this review addresses only a few aspects of hair loss disorders, alopecia histopathology remains a complex topic. The increased number of publications in recent years has contributed new entities to the field, as well as new approaches that revisit old entities. However, much still needs to be explored and discovered to provide new aspects and diagnostic clues for the dermatopathologist. A
Clinically, non-scarring and scarring alopecias have different modalities of presentation. They may be cyclic (AA), evolve slowly (FPHL), and, in time, burn themselves out (LPP/FPHL). Understanding the ‘lives of lesions’ and knowledge of the morphologic features associated with their chronology in its different stages is a principle taught to us by A. Bernard Ackerman in 1984.64 This principle applies also to alopecia. It can enable a diagnosis, in spite of a scanty clinical history, but only provided that no biopsy sampling bias has occurred. We commonly classify alopecias into scarring (cicatricial) with permanent hair loss and non-scarring, with non-permanent hair loss. However, this classification65,66 would imply that nonscarring alopecias are non-permanent. Although there are instances in which hair follicles ‘synchronize’ (such as in the ‘shift out of anagen’ observed in subacute alopecia areata, the pathogenic pathway leading to hair follicle loss is quite distinct in scarring alopecias. On the other hand, evolving, long-standing end-stage non-scarring alopecias do also undergo hair follicle loss, with fibrosis of the follicular stelae, resulting in scarring and permanent alopecia (biphasic alopecias).65,66 With age, the number of follicles also decreases and characterizes what is known as senescent alopecia.67 In a large
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Practice points C
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The gold standard in alopecia diagnostics is based on accurate clinical-pathological correlation In the absence of clinical history or photographs, a multifaceted structured diagnostic approach to alopecia is possible Direct immunofluorescence and special stains are valuable supporting diagnostic tools Sebaceous gland atrophy in response to overlying psoriasiform epidermal hyperplasia may mimic scarring alopecia Atrophic sebaceous basaloid epithelial aggregates may mimic telogen hair follicles with the consequent risk of a misdiagnosis of telogen effluvium and alopecia areata
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Please cite this article as: Stefanato CM, Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.12.003
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Immunoperoxidase stains for androgen receptor and Adipophilin can help distinguish atrophic sebaceous glands from telogen hair follicles Alopecia areata-like histopathology may be seen in a variety of clinico-pathologic scenarios unrelated to and distinctive from classic alopecia areata Careful examination of telogen hair follicle counts and identification of the presence/absence of miniaturization will help in distinguishing diffuse TE versus FPHL versus AA When evaluating diffuse telogen effluvium versus diffuse female pattern hair loss, the chronic phase of a non-inflammatory ‘silent’ (invisible) alopecia areata should be considered in the differential diagnosis In multifactorial alopecia, a single scalp biopsy may harbor more than one concomitant diagnosis Different alopecias have a predilection for different scalp zones Non-scarring alopecia, when end-stage, is characterized by permanent hair follicle loss and becomes scarring (biphasic alopecia)
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20 REFERENCES 1 Stefanato CM. Histopathology of alopecia: a clinicopathological approach to diagnosis. Histopathology 2010; 56: 24e38. 2 Tailor A, Asher RG, Craig PJ, Groves RW, Fenton DA, Stefanato CM. The current state of play in the histopathologic assessment of alopecia: two for one or one for two? J Cutan Pathol 2013; 40: 298e304. 3 Wohltmann WE, Sperling L. Histopathologic diagnosis of multifactorial alopecia. J Cutan Pathol 2016; 43: 483e91. 4 Headington JT. Transverse microscopic anatomy of the human scalp. A basis for a morphometric approach to disorders of the hair follicle. Arch Dermatol 1984; 120: 449e56. 5 Nguyen JV, Hudacek K, Whitten JA, Rubin AI, Seykora JT. The hovert technique: a novel method for the sectioning of alopecia biopsies. J Cutan Pathol 2011; 38: 401e6. 6 Elston D. The ’Tyler technique’ for alopecia biopsies. J Cutan Pathol 2012; 39: 306. 7 Elston DM. Vertical vs. Transverse sections: both are valuable in the evaluation of alopecia. Am J Dermatopathol 2005; 27: 353e6. 8 Elston DM, Ferringer T, Dalton S, Fillman E, Tyler W. A comparison of vertical versus transverse sections in the evaluation of alopecia biopsy specimens. J Am Acad Dermatol 2005; 53: 267e72. 9 Trachsler S, Trueb RM. Value of direct immunofluorescence for differential diagnosis of cicatricial alopecia. Dermatology 2005; 211: 98e102. 10 Jaulim SZA, Abdelkarim M, Abdullah A, Maheshwari MB. Diagnosis of primary cicatricial alopecia: is direct immunofluorescence necessary? Br J Dermatol 2017; 177: 92. 11 Donati A, Gupta AK, Jacob C, Cavelier-Balloy B, Reygagne P. The use of direct immunofluorescence in frontal fibrosing alopecia. Skin Appendage Disord 2017; 3: 125e8. 12 Agarwal R, Singh M, Dahiya R, et al. Direct immunofluorescence findings in cicatricial alopecia: a retrospective study of 155 cases. Indian J Pathol Microbiol 2019; 62: 103e6. 13 Xie D, Bilgic-Temel A, Abu Alrub N, Murrell DF. Alopecia in autoimmune blistering diseases: a systematic review of
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pathogenesis and clinical features of disease. Skin Appendage Disord 2019; 5: 263e75. Hadayer N, Ramot Y, Maly A, Zlotogorski A. Pemphigus vulgaris with loss of hair on the scalp. Int J Trichol 2013; 5: 157e8. Sar-Pomian M, Rudnicka L, Olszewska M. The significance of scalp involvement in pemphigus: a literature review. BioMed Res Int 2018; 2018: 6154397. Gaitanis G, Patmanidis K, Skandalis K, Alexis I, Zioga A, Bassukas ID. Scaring alopecia in pemphigus vulgaris: a rare or underdiagnosed presentation? Eur J Dermatol 2013; 23: 253e5. Veraitch O, Ohyama M, Yamagami J, Amagai M. Alopecia as a rare but distinct manifestation of pemphigus vulgaris. J Eur Acad Dermatol Venereol 2013; 27: 86e91. Reis Gavazzoni Dias MF, Aparecida Guedes Vilar E, de Oliveira Bento C, et al. Brunsting-Perry type pemphigoid causing secondary cicatricial alopecia in 2 patients. Skin Appendage Disord 2018; 4: 308e11. Perez JL, Garcia A, Niubo J, Salva J, Podzamczer D, Martin R. Comparison of techniques and evaluation of three commercial monoclonal antibodies for laboratory diagnosis of varicella-zoster virus in mucocutaneous specimens. J Clin Microbiol 1994; 32: 1610e3. Kumudhini S, Pai S, Rao C, Rao R. A comparative study of Michel’s medium versus honey as a transport medium for skin specimens prior to direct immunofluorescence microscopy and antigen mapping. J Cutan Pathol 2019; 46: 729e35. LaSenna C, Miteva M. Special stains and immunohistochemical stains in hair pathology. Am J Dermatopathol 2016; 38: 327e37. Sperling LC. Inflammatory tinea capitis (kerion) mimicking dissecting cellulitis. Occurrence in two adolescents. Int J Dermatol 1991; 30: 190e2. Miletta NR, Schwartz C, Sperling L. Tinea capitis mimicking dissecting cellulitis of the scalp: a histopathologic pitfall when evaluating alopecia in the post-pubertal patient. J Cutan Pathol 2014; 41: 2e4. Elston DM, McCollough ML, Warschaw KE, Bergfeld WF. Elastic tissue in scars and alopecia. J Cutan Pathol 2000; 27: 147e52. Elston CA, Kazlouskaya V, Elston DM. Elastic staining versus fluorescent and polarized microscopy in the diagnosis of alopecia. J Am Acad Dermatol 2013; 69: 288e93. Horenstein MG, Jacob JS. Follicular streamers (stelae) in scarring and non-scarring alopecia. J Cutan Pathol 2008; 35: 1115e20. Tandon YK, Somani N, Cevasco NC, Bergfeld WF. A histologic review of 27 patients with lichen planopilaris. J Am Acad Dermatol 2008; 59: 91e8. Goldberg LJ, Sekhri V, Bhawan J. Dermal mucin in alopecia areata-tell tale sign or incidental finding? J Cutan Pathol 2008; 35: 757e60. Nambudiri VE, Vleugels RA, Laga AC, Goldberg LJ. Clinicopathologic lessons in distinguishing cicatricial alopecia: 7 cases of lichen planopilaris misdiagnosed as discoid lupus. J Am Acad Dermatol 2014; 71: 135e8. Headington JT, Gupta AK, Goldfarb MT, et al. A morphometric and histologic study of the scalp in psoriasis. Paradoxical sebaceous gland atrophy and decreased hair shaft diameters without alopecia. Arch Dermatol 1989; 125: 639e42. Rittie L, Tejasvi T, Harms PW, et al. Sebaceous gland atrophy in psoriasis: an explanation for psoriatic alopecia? J Investig Dermatol 2016; 136: 1792e800.
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32 Sar-Pomian M, Czuwara J, Rudnicka L, Olszewska M. Miniaturization of sebaceous glands: a novel histopathological finding in pemphigus vulgaris and pemphigus foliaceus of the scalp. J Cutan Pathol 2017; 44: 835e42. 33 Afanasiev OK, Zhang CZ, Ruhoy SM. Tnf-inhibitor associated psoriatic alopecia: diagnostic utility of sebaceous lobule atrophy. J Cutan Pathol 2017; 44: 563e9. 34 Sperling LC, Cowper SE, Knopp EA. Psoriatic alopecia. In: Sperling LC, Cowper SE, Knopp EA, eds. An atlas of hair pathology with clinical correlations 2nd. Edition. London, UK: Informa Heathcare, 2012; 96e101. 35 Clayton NlF, Fenton DA, Stefanato CM. Sebaceous gland atrophy: a potential pitfall in the histopathologic evaluation of nonscarring alopecia associated with psoriasiform dermatitis of the scalp. J Investig Dermatol 2013; 133. 1405-1405. 36 Ito T, Ito N, Saatoff M, et al. Maintenance of hair follicle immune privilege is linked to prevention of nk cell attack. J Investig Dermatol 2008; 128: 1196e206. 37 McElwee KJ, Gilhar A, Tobin DJ, et al. What causes alopecia areata? Exp Dermatol 2013; 22: 609e26. 38 Mashima E, Sawada Y, Inoue A, et al. Alopecia areata-like hair loss accompanying toxic epidermal necrolysis. Acta Derm Venereol 2018; 98: 906e7. 39 Sibaud V, Boulinguez S, Pages C, et al. [dermatologic toxicities of immune checkpoint inhibitors]. Ann Dermatol Venereol 2018; 145: 313e30. 40 Doyle LA, Sperling LC, Baksh S, et al. Psoriatic alopecia/alopecia areata-like reactions secondary to anti-tumor necrosis factoralpha therapy: a novel cause of noncicatricial alopecia. Am J Dermatopathol 2011; 33: 161e6. 41 Fonia A, Cota C, Setterfield JF, Goldberg LJ, Fenton DA, Stefanato CM. Permanent alopecia in patients with breast cancer after taxane chemotherapy and adjuvant hormonal therapy: clinicopathologic findings in a cohort of 10 patients. J Am Acad Dermatol 2017; 76: 948e57. 42 Bandino JP, Elston DM. Acute-onset alopecia. Cutis 2019; 103: 24e6. 43 Sperling LC, Cowper SE, Knopp EA. Noncicatricial alopecia from systemic lupus erythematosus. In: Sperling LC, Cowper SE, Knopp EA, eds. An atlas of hair pathology with clinical correlations 2nd. Edition. London, UK: Informa Heathcare, 2012; 111e4. 44 Sperling LC, Cowper SE, Knopp EA. Syphilitic alopecia. In: Sperling LC, Cowper SE, Knopp EA, eds. An atlas of hair pathology with clinical correlations 2nd. Edition. London, UK: Informa Heathcare, 2012; 108e10. 45 Gerami P, Rosen S, Kuzel T, Boone SL, Guitart J. Folliculotropic mycosis fungoides: an aggressive variant of cutaneous T-cell lymphoma. Arch Dermatol 2008; 144: 738e46. 46 Pileri A, Agostinelli C, Bruni F, et al. Alopecia areata-like mycosis fungoides: lions for lambs. G Ital Dermatol Venereol 2018; 153: 293e5. 47 Amin SM, Tan T, Guitart J, Colavincenzo M, Gerami P, Yazdan P. CD8þ mycosis fungoides clinically masquerading as alopecia areata. J Cutan Pathol 2016; 43: 1179e82. 48 Bi MY, Curry JL, Christiano AM, et al. The spectrum of hair loss in patients with mycosis fungoides and sezary syndrome. J Am Acad Dermatol 2011; 64: 53e63.
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49 Iorizzo M, El Shabrawi Caelen L, Vincenzi C, Misciali C, Tosti A. Folliculotropic mycosis fungoides masquerading as alopecia areata. J Am Acad Dermatol 2010; 63: 50e2. 50 Tomasini C, Kempf W, Novelli M, et al. Spiky follicular mycosis fungoides: a clinicopathologic study of 8 cases. J Cutan Pathol 2015; 42: 164e72. 51 Benton EC, Crichton S, Talpur R, et al. A cutaneous lymphoma international prognostic index (clipi) for mycosis fungoides and sezary syndrome. Eur J Cancer 2013; 49: 2859e68. 52 Eudy G, Solomon AR. The histopathology of noncicatricial alopecia. Semin Cutan Med Surg 2006; 25: 35e40. 53 Whiting DA. Chronic telogen effluvium: increased scalp hair shedding in middle-aged women. J Am Acad Dermatol 1996; 35: 899e906. 54 Whiting DA. Chronic telogen effluvium. Dermatol Clin 1996; 14: 723e31. 55 Watanabe-Okada E, Inazumi T, Matsukawa H, Ohyama M. Histopathological insights into hair loss in Cronkhite-Canada syndrome: diffuse anagen-telogen conversion precedes clinical hair loss progression. Australas J Dermatol 2014; 55: 145e8. 56 Ong S, Rodriguez-Garcia C, Grabczynska S, et al. Alopecia areata incognita in Cronkhite-Canada syndrome. Br J Dermatol 2017; 177: 531e4. 57 Dy LC, Whiting DA. Histopathology of alopecia areata, acute and chronic: why is it important to the clinician? Dermatol Ther 2011; 24: 369e74. 58 Peckham SJ, Sloan SB, Elston DM. Histologic features of alopecia areata other than peribulbar lymphocytic infiltrates. J Am Acad Dermatol 2011; 65: 615e20. 59 Bertolini M, Zilio F, Rossi A, et al. Abnormal interactions between perifollicular mast cells and CD8þ T-cells may contribute to the pathogenesis of alopecia areata. PLoS One 2014; 9: e94260. 60 Grace SA, Sutton AM, Abraham N, Armbrecht ES, Vidal CI. Presence of mast cells and mast cell degranulation in scalp biopsies of telogen effluvium. Int J Trichol 2017; 9: 25e9. 61 Kolivras A, Thompson C. Distinguishing diffuse alopecia areata (aa) from pattern hair loss (phl) using CD3(þ) T cells. J Am Acad Dermatol 2016; 74: 937e44. 62 Nascimento LLD, Enokihara M, Vasconcellos MRA. Coexistence of chronic cutaneous lupus erythematosus and frontal fibrosing alopecia. Anais Brasil Dermatol 2018; 93: 274e6. 63 Khan S, Fenton DA, Stefanato CM. Frontal fibrosing alopecia and lupus overlap in a man: guilt by association? Int J Trichol 2013; 5: 217e9. 64 Ackerman BA. Preface. In: Ackerman BA, Ragaz A, eds. The lives of lesions: chronology in Dermatopathology. NY, USA: Masson Publishing New York, 1984. vi-viii. 65 Sellheyer K, Bergfeld WF. Histopathologic evaluation of alopecias. Am J Dermatopathol 2006; 28: 236e59. 66 Solomon AR. The transversely sectioned scalp biopsy specimen: the technique and an algorithm for its use in the diagnosis of alopecia. Adv Dermatol 1994; 9: 127e57. discussion 158. 67 Whiting DA. How real is senescent alopecia? A histopathologic approach. Clin Dermatol 2011; 29: 49e53.
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Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos
represent a reversible response of the sebaceous glands to overlying epidermal psoriasiform changes. A peribulbar lymphoid cell infiltrate ‘clue’ for alopecia areata may be part of an alopecia areata-like pattern associated with different clinicopathologic scenarios, or possibly even incidental. Clues to ‘silent’ (invisible) alopecias will also be discussed. Awareness of the increased complexity in presentation due to coexistence of multifactorial etiologies is essential while bearing in mind the time-frame factor (lives of lesions) in alopecia diagnostics. The boundaries between evolving end-stage non-scarring alopecia and scarring alopecia prove to be closer than previously thought.
Catherine M Stefanato
Abstract In recent years the increased number of scalp biopsies in alopecia has afforded the dermatopathologist improved confidence in distinguishing the histopathologic characteristics of ‘classic’ scarring and nonscarring alopecias, as well as the spectrum of their presentation. Occasionally, however, histopathologic ‘dogmas’ have been disproven. The loss of sebaceous glands considered to be a clue to scarring alopecia or the peribulbar lymphoid cell infiltrate diagnostic of alopecia areata may present in unconventional scenarios, and thus, represent pitfalls. Clues to diagnose histopathologically ‘silent’ (invisible) alopecias, the identification of multiple etiologies, and awareness of the ‘time-factor’ are herein discussed.
The St John’s diagnostic update and technical considerations Our role, as dermatopathologists, is to offer the best diagnostic options for the biopsies provided, in the light of clinicopathological correlation.1 At times, however, scalp biopsies of alopecia lack adequate clinical history, and clinical photographs are not provided. In this instance, the use of our St John’s combined scarring and non-scarring alopecia protocols has proved to be a ‘safety net’ enabling diagnosis at the microscope.1,2 In the absence of clinical history, helpful factors to reach a meaningful diagnosis include (a) the number of scalp biopsies provided, (the more biopsies provided the more likely an accurate diagnosis can be reached), (b) knowledge of scalp biopsy anatomical sites (certain alopecias have a predilection for distinct anatomical sites3), (c) combined use of horizontal and vertical sections in order to approach the study from all perspectives, in a ‘kaleidoscopic’ way, and (d) ancillary studies. A structured microscopic evaluation of multiple serial levels with careful histopathologic examination of the four key levels of the hair follicle, (hair bulb, suprabulbar, isthmus, and infundibulum), is a ‘conditio sine qua non’ paving the pathway to the diagnosis. In our St John’s scarring alopecia protocol, two 4 mm punch biopsies are taken at the peripheral ‘active’ edge of the scarring alopecia. Of the two biopsies, one specimen is sectioned transversely in the laboratory while the second one is sectioned vertically by the dermatologist in the clinic, with 1/2 of the skin biopsy placed in Michel’s medium for direct immunofluorescence study, and the other half sent to the dermatopathology laboratory for vertical sections examination.1,2 Two 4 mm punch biopsies are also taken in non-scarring alopecia. In this case, one biopsy is taken from the center of the involved area of alopecia, usually the vertex, the other biopsy at the occiput, a nonandrogenic area, acting as a normal control and helpful for comparison purposes. Since Headington’s seminal paper in 1984, transverse sectioning of scalp biopsies has become the preferred method in the context of non-scarring alopecia, owing to the quantitative and morphometric data that can be generated.4 There are many examples of how to optimize the diagnostic yield from a single biopsy, such as the HoVert5 and the Tyler6 techniques. Our 15year experience with the St John’s protocol based on combined transverse and vertical sections of two 4 mm punch biopsies has proven to be diagnostically effective, and further validates what Elston et al.7 had already reported, as a biopsy
Keywords alopecia areata-like; clues; follicular mycosis fungoides; multifactorial alopecia; non-scarring alopecia; pitfalls; psoriasis; scarring alopecia
Introduction Although the type of alopecia may be diagnosed with reasonable certainty clinically and dermoscopically, a scalp biopsy can, nonetheless, be paramount in the assessment of a patient with hair loss. When possible, the multi-team clinical-pathological approach, including accurate biopsy site selection, communication between the dermatologist, the dermatopathology laboratory and the dermatopathologist trained in hair histopathology, are ideal factors. However, this is not always the case, and the dermatopathologist may be left to interpret alopecia slides from biopsies provided with minimal, if any, clinical information. Histopathologically, diagnostic ‘tools’ for the dermatopathologist in the evaluation of alopecia comprise the localization level of the inflammatory process, (peri-bulbar/suprabulbar/isthmic/ infundibular), the degree of the cellular component (mild/moderate/severe), and the quality and predominance of the inflammatory cell infiltrate (lymphocytes, plasma cells, neutrophils, eosinophils, mast cells). Moreover, assessment of the presence/ absence of sebaceous glands and of perifollicular fibrosis and observation of anomalies of the hair cycle are all part of the routine evaluation. Pitfalls, such as the absence of sebaceous glands in a scalp biopsy considered a ‘clue’ for scarring alopecia, may instead
Catherine M. Stefanato MD, FRCPath Consultant in Dermatopathology, Department of Dermatopathology, St John’s Institute of Dermatology, St Thomas’ Hospital, Guy’s and St Thomas’ NHS Trust, London, UK. Conflicts of interest: none declared.
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when the scalp site chosen to biopsy is not truly representative of the disease or b) biopsies taken from late, ‘burnt out’ areas, since, in late scarring, immune-deposition is lost, and the immunodeposits are no longer detected. The DIF yield is also influenced by laboratory technical considerations. Monoclonal antibody detection sensitivities used may vary between different laboratories,19 but also the choice of the transport medium for the scalp biopsy (Michel’s medium vs saline or honey),20 along with the time to reach the laboratory, are all factors that play a role. To optimize diagnostic yield, it is thus critical to obtain the DIF skin sample from a clinically ‘active’ area of scalp inflammation at the periphery of the scarring process, truly representative of the disease. In this instance, the DIF yield will likely be positive.
combination is superior to either transverse or vertical biopsy examination alone.8 The choice of which combination of biopsies and sectioning to use may vary, based on the presence or absence of the clinical data provided. Our flexible, ‘kaleidoscopic’ approach not only incorporates the “best of two worlds” but also allows us to obtain fresh tissue for direct immunofluorescence studies.2,9 The use of vertical sections has shown to be helpful in adding morphologic information to the horizontal sections, as well as providing morphological insights, particularly when the horizontal sections are not contributory. Although vertical sections show a limited number of tangentially sectioned hair follicles, they outline the dermal eepidermal junction and demonstrate full-thickness dermis and subcutaneous tissue, allowing for precise localization of an inflammatory infiltrate.2
The role of special stains The role of direct immunofluorescence
Special stains further supply valuable information.21 The histopathologic findings in alopecia may be focal, such as the presence, by ‘serendipity’ of fungal hyphae in a single hair follicle in a scarring alopecia (Figure 5) or in non-inflammatory tinea,21 or at times be misleading, when tinea mimics dissecting cellulitis.22,23 Thus, the use of periodic acid-Schiff (PAS) is essential.23 The use of PAS will also help to identify subtle basement membrane zone thickening in the evaluation of lupus. Elastic stains are invaluable in assessing subtle perifollicular fibrosis and highlighting fibrous tracts,24e26 as well as mucin stains for distinguishing the mucinous perifollicular early fibroplasia in scarring LPP27 or highlighting the interstitial dermal mucin diagnostic of lupus.21,28,29
The implementation of direct immunofluorescence (DIF) in the diagnosis of primary scarring alopecia has been recently challenged,10 with reservation suggested for its use.11 In our experience, however, DIF has shown to be an important aspect in the diagnostic process, not only aiding in distinguishing lupus (Figure 1) from lichen planopilaris (LPP) (Figure 2),12 but also in the identification of other causes of scalp alopecia such as those seen in the setting of autoimmune blistering disease,13 including pemphigus14e17 (Figure 3) and bullous pemphigoid (Figure 4), and its localized Brunsting-Perry variant.18 Possible explanations as to why in many cases the scalp biopsy DIF yield is often negative may be ascribed to: a) biopsy sampling bias,
Figure 1 Scalp, Lupus, Active stage, Isthmus. Scanning magnification showing predominately anagen hair follicles, with a moderately dense perifollicular lymphoid cell infiltrate and focal tufting (40) (a). Higher magnification highlighting perifollicular lymphocytes with nuclear dust and with lymphocytic exocytosis in follicular epithelium (400) (b). Perineural inflammation, a feature commonly seen in lupus scalp biopsies (400) (c). Direct immunofluorescence showing a linear granular deposition of IgM at the basement membrane zone. (200). This case was also positive for IgG, IgA and C3 (lupus band test positive) (d).
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Figure 2 Scalp, Lichen planopilaris, Isthmus. Scanning magnification showing different stages of the disease within the same horizontal section. Anagen hair follicles with an active moderately dense perifollicular lymphoid cell infiltrate (upper left) which is gradually reduced to be replaced by perifollicular fibrosis, consistent with a more advanced stage of the disease (lower right) (40) (a). High power detail of an anagen hair follicle with perifollicular lymphoid cell infiltrate, lymphocytic exocytosis and perifollicular fibrosis. (400) (b). Direct immunofluorescence staining with fibrinogen showing follicular involvement of the hair follicle (400) (c).
Figure 3 Scalp, Pemphigus vulgaris. Scanning magnification showing suprabasal acantholysis with complete detachment of the epidermal roof (40) (a). Higher magnification detail showing suprabasal acantholysis of a hair follicle (200) (b) Direct immunofluorescence showing follicular net-like intercellular deposition of IgG. This case also showed similar intercellular deposition of C3 (400) (c).
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Figure 4 Scalp, Bullous pemphigoid. Scanning magnification showing epidermal hyperkeratosis and acanthosis, with patchy papillary dermal and perifollicular lymphoid cell infiltrate and epidermal-dermal separation involving the hair follicle (40) (a). Higher power detail of the follicular split (400) (b). Direct immunofluorescence documents linear deposition of IgG at the epidermal basement membrane zone which is extending into the hair follicle. This case was also positive for similar linear deposition of C3 (200) (c).
seborrheic dermatitis or eczema of the scalp. Miniaturization of sebaceous glands has been recently documented in pemphigus vulgaris and pemphigus foliaceus of the scalp,32 and atrophy of the sebaceous lobules reported as a histologic clue for TNFinhibitor associated psoriatic alopecia.33 Loss of sebaceous glands is commonly regarded as one of the key diagnostic morphologic features of scarring alopecia,1 occurring secondary to perifollicular fibrosis and perifollicular
Clues and pitfalls Sebaceous gland atrophy: a triple pitfall Sebaceous gland atrophy of the scalp was originally reported by Headington et al. in 1989 in association with psoriasis.30 This phenomenon is reversible, and felt to be secondary to abnormal sebaceous gland function,31 likely in response to the overlying epidermal changes. It may be seen also as secondary to any psoriasiform-spongiotic epidermal reaction pattern, such as
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Figure 5 Scalp, incidental tinea on lichen planopilaris. Scanning magnification showing a reduced number of hair follicles, with two hair follicles exhibiting perifollicular fibrosis with perifollicular lymphoid cell infiltrate (40) (a). Medium power highlighting focal follicular infundibular acanthosis and hyperkeratosis of a hair follicle involved by lichen planopilaris at the edge of the biopsy (100) (b). Higher magnification documents the presence of fungal hyphae within the hyperkeratotic follicular infundibulum (400) (c). The fungal hyphae are highlighted with periodic acid-Schiff (PAS) stain (400) (d).
the vicinity of nevi or in follicles involved by malignant melanoma (Figure 7). In recent years, there have been an increasing number of reports describing alopecia areata-like histopathology in contexts other than ‘classic’ alopecia areata. These include reactions to medications such as carbocysteine,38 immune checkpoint inhibitors,39 anti-tumor necrosis factor alpha drugs,40 (Figures 8 e10), taxanes in patients undergoing chemotherapy for breast carcinoma41 (Figure 11), and heavy metal thallium poisoning.42 Alopecia areata-like peribulbar lymphoid cell infiltrate is observed in the non-cicatricial variant of systemic lupus erythematosus.43 An increased telogen/catagen shift, also mimicking the ‘shift’ out of anagen of acute/subacute alopecia areata, may be seen in the affected areas. Distinguishing histopathologic clues in support of lupus include a peribulbar lymphoid cell infiltrate which is denser than commonly seen in alopecia areata, a vacuolar-interface change, and perieccrine and perivascular lymphoid cell infiltrate containing plasma cells.43 Syphilis, the ‘great mimicker’ also comes into play, with a peribulbar lymphoid cell infiltrate, miniaturization of the hair follicles, and a shift out of anagen of almost all hair follicles in the catagen/telogen phase.44 These are all features seen in classic alopecia areata. However, clues to the diagnosis of syphilitic alopecia are the presence of peribulbar plasma cells (which are absent in alopecia areata), as well as, -in opposition to alopecia areata where the infiltrate remains confined to the hair bulbs-, extension of the infiltrate to the upper follicular levels, reaching the infundibulum. Moreover, when these findings are viewed in
lymphoid cell infiltrate, as seen in the setting of immunemediated alopecias (lichen planopilaris and lupus). However, sebaceous gland regression leading to complete atrophy and apparent loss may represent a potential pitfall. If the sebaceous gland atrophy is complete, the process mimics a scarring alopecia. Regressed sebaceous glands, morphologically reverted into basaloid epithelial aggregates, resemble telogen hair follicles. If only few sebaceous glands are atrophic, a misdiagnosis of telogen effluvium may be rendered, but if all sebaceous glands are regressed to basaloid epithelial aggregates, the histopathologic picture may resemble subacute alopecia areata. 34,35 Immunoperoxidase stains for androgen receptors and Adipophilin will help distinguish atrophic sebaceous glands from telogen hair follicles by highlighting the residual sebocytes within the basaloid epithelial aggregates.34 (Figure 6). Peribulbar lymphoid cell infiltrate: not only alopecia areata Hair follicle bulbs are sites of immune-privilege, the collapse of which, together with exposure of the major histocompatibility complex (MHC) class I, initiates an autoimmune response leading to a perifollicular and intrafollicular T-cell lymphoid infiltrate affecting hair bulbs of anagen-stage hair follicles.36,37 This morphologically corresponds to the peribulbar lymphoid cell infiltrate (also known as a ‘swarm of bees’), which is the histopathologic feature we commonly look for to diagnose alopecia areata.1 However, peribulbar lymphoid cell infiltrate may be encountered also incidentally, in unrelated conditions such as in
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Figure 6 Scalp, psoriasis: Vertical section showing hyperkeratosis and a psoriasiform epidermis. The underlying dermis exhibits hair follicles in anagen phase and atrophic sebaceous glands mimicking telogen hair follicles (X40) (a). Horizontal section, isthmus: diffuse atrophy of the sebaceous glands regressed to basaloid epithelial aggregates mimicking telogen hair follicles (X40) (b). A follicular unit with atrophic sebaceous glands where residual sebocytes can still be seen (X200) (c). Another follicular unit with more advanced sebaceous gland atrophy (X100) (d). Detail of an atrophic sebaceous gland with residual sebocytes (e). Immunoperoxidase stain for androgen receptor highlights the residual sebocytes in the atrophic sebaceous glands. (400) (feg).
diagnosis and therapeutic intervention, given the less favorable survival rate when compared with non folliculotropic mycosis fungoides.45,51
a clinical context, syphylitic alopecia is far less common than alopecia areata.44 Folliculotropic cutaneous T-cell lymphoma (mycosis fungoides): alopecia areata-like folliculotropic mycosis fungoides is an uncommon variant of cutaneous T-cell lymphoma with distinct clinical and pathologic findings (Figures 12e13). Clinically, alopecia is a typical finding, occurring on the face (eyebrows) and scalp in 65% of patients.45 Patchy alopecia areatalike changes with peribulbar lymphoid cell infiltrate histopathology has been reported in up to 34%.46e48 This may involve the genital area,49 as well as other body sites,47 and often represents an early presenting sign of mycosis fungoides.49 Histopathologically, folliculotropism occurs predominantly at the infundibulum and isthmus, with epidermotropism presenting in fewer than half of patients.45 Syringotropism may also be present. Various histopathologic patterns of follicular involvement at all follicular levels have been described,45 including the recently reported spiky hyperkeratotic variant.50 Awareness of these patterns is a diagnostic clue to follicular mycosis fungoides, as often they may coexist in the same biopsy, or occur in different biopsies of the same patient (Figure 14). The immunohistochemical profile is similar to that of classic mycosis fungoides, with a CD4 predominance over CD8; although rare, alopecia areata-like mycosis fungoides can be CD8 predominant.47 Recognition of the alopecia areata-like pattern in follicular mycosis fungoides is paramount, as it may result in earlier
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‘Silent’ (invisible) non-scarring alopecias with scant inflammatory cell infiltrate A ‘cumbersome’ scenario for the dermatopathologist is the interpretation of the histopathology in biopsies of non-scarring alopecia with a minimal inflammatory cell infiltrate and a limited clinical history of hair loss. The differential diagnoses commonly offered include telogen effluvium (TE), alopecia areata (AA), and female pattern hair loss (FPHL). In this setting, transverse sections are the preferred method of evaluation, as all the hair follicles can be quantified and studied morphologically at all the key follicular levels.2 Awareness of the telogen hair follicle counts and of the presence/absence of miniaturization will help in distinguishing them. It is helpful to be reminded that the histopathology of an episode of acute TE will characteristically resemble normal scalp, likely reflecting morphologically the recovery phase of the disease.52,53 Chronic telogen effluvium (CTE), on the other hand, occurs in later years of age, has a fluctuating course, and eventually burns itself out; CTE, in its active stages, may also show slightly increased numbers of telogen hair follicles, typically in the absence of miniaturization. But, if miniaturization is identified, then a CTE uncovering a background of FPHL should be considered.
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Figure 7 Congenital nevus. Scanning magnification of this intradermal nevus shows incidental neighboring hair follicles which exhibit peribulbar lymphoid cell infiltrate and pigment casts, simulating alopecia areata (40) (a). Details of the hair follicles in anagen and telogen phase with alopecia areata-like peribulbar lymphoid cell infiltrate (100, 400, 400) (bed). Follicular malignant melanoma. The hair follicle involved by malignant melanocytes (left) shows a dense peribulbar lymphoid cell infiltrate simulating alopecia areata. The adjacent anagen hair follicle (right) is unaffected. (200, 400) (eef).
In diffuse FPHL the number of telogen hair follicles is expected to be higher than in TE. Moreover, FPHL is associated with increased vellus hair follicles and miniaturization.54 Diffuse alopecia areata may be misdiagnosed as telogen effluvium, clinically and histopathologically.55,56 Hair bulbs examination, with identification of the peribulbar lymphoid cell infiltrate (‘swarm of bees’), is diagnostic; however, in long-standing chronic lesions of alopecia areata, peribulbar and intrabulbar lymphoid cell infiltrate is minimal to absent,57,58 and the diagnosis of a silent (invisible) alopecia areata is missed. Awareness of the morphological time-frame changes hair follicles undergo in the acute and chronic stages, including miniaturization, high percentage in
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catagen/telogen hair follicles, (‘shift out of anagen’), trichomalacia and pigment casts will point to the diagnosis.58 The identification of peribulbar mast cells has been regarded as an additional clue in alopecia areata59 (Figure 15) with the suggestion that mast cells ‘cross-talk’ with the pathogenic CD8 (þ) T-cells involved in hair follicle immune privilege collapse.37,59 More recently, degranulating mast cells were also reported in telogen effluvium, and felt to be a response to stress-induced hair loss.60 A helpful tool to distinguish diffuse AA from diffuse FPHL is the use of the immunostain CD3.61 This will highlight the rare intra-bulbar lymphoid cells (Figure 16) which may be also identified within empty follicular fibrous tracts.61
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Figure 8 Infliximab alopecia. Scalp, Isthmus. Scanning magnification showing follicular units with hair follicles in anagen phase and atrophic sebaceous glands (40) (a). Detail of a follicular unit sebaceous with gland atrophy and lymphoplasmacellular infiltrate (200) (b). Peribulbar lymphoplasmacellular infiltrate simulating alopecia areata (400) (c). Eccrine glands with lymphoplasmacellular cell infiltrate (400) (d).
Figure 9 Infliximab alopecia, Scalp (same case as in Figure 8). Scanning magnification showing epidermal acanthosis with dense lymphoid cell infiltrate and underlying dermal sebaceous gland atrophy (40) (a). There is a band-like papillary dermal lymphoid cell infiltrate with prominent epidermal lymphocytic exocytosis, mimicking cutaneous T-cell lymphoma (200) (b). No cytologic atypia was present, and both immunostains CD4 and CD8 highlight reactive lymphocytes in the epidermis and papillary dermis (200, 200) (ced).
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Figure 10 Infliximab alopecia, Scalp, Isthmus. Scanning magnification of another case showing hair follicles in anagen with atrophic sebaceous glands (40) (a). Higher power details of the different stages of sebaceous gland atrophy (200 and 400) (bec). Immunoperoxidase antibody for Adipophilin highlights the atrophic sebaceous glands (200) (d).
Thoughts ‘out of the box’: multifactorial alopecia
same biopsy.3 Multiple serial levels throughout the biopsy and examination at all the key follicular levels are important. The study of the subcutaneous tissue to evaluate the hair bulbs is critical to rule out AA and the examination of the isthmus to rule
When the dermatopathologist is asked to answer a diagnostic question, the complexity is often compounded by more than one type of alopecia coexisting in the same patient, and even in the
Figure 11 Taxane alopecia. Scalp, Isthmus. Alopecia areata-like morphology with a ‘shift out of anagen’ characterized by increased miniaturized telogen hair follicles (40, 200) (aeb). Dysmorphic telogen hair follicles, stigmata of taxane alopecia, are identified both in horizontal sections (400) (c) as well as in vertical sections (400) (d).
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Figure 12 Folliculotropic cutaneous T-cell lymphoma, Scalp. ‘Shift out of anagen’ with all hair follicles in telogen phase (40) (a). Higher magnification highlights a telogen hair follicle with dense peribulbar lymphoid cell infiltrate in an alopecia areata-like pattern (400) (b). Immunoperoxidase stains confirm the infiltrate is CD4 predominant (400) (c), with only a few CD8-positive reactive lymphocytes (400) (d).
Figure 13 Folliculotropic cutaneous T-cell lymphoma, arm, sub-isthmus. Scanning magnification showing alopecia areata-like peribulbar lymphoid cell infiltrate (40, 400) (aeb). Detail of an eccrine gland with atypical epithelium and with eccrinotropism of atypical lymphocytes (400) (c). Isthmus: Follicular units showing follicular epithelium acanthosis, perifollicular lymphoid cell infiltrate, folliculotropism (40) and follicular mucinosis (inset 400) (d).
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Figure 14 Follicular cutaneous T-cell lymphoma variants. Follicular infundibular acanthosis with lymphocytic folliculotropism (100, 400) (aeb). Follicular cystic variant simulating epidermal inclusion cysts (40) (c), but with atypical lymphocytes (400) (d).
out an immune-mediated scarring alopecia such as lichen planopilaris or discoid lupus erythematosus, which may coexist, although rarely,62,63 but then DIF studies will help in the differentiation.63
Knowledge of specific types of alopecia having a predilection for different scalp zones will also aid in the diagnosis.3 For example, the occipital and parietal scalp zones are often sites of traction alopecia, and the crown and the vertex are sites of
Figure 15 Alopecia areata. Scalp, Isthmus. Follicular units with diffuse miniaturization and increased telogen hair follicles, characteristic of alopecia areata (20) (a). Detail on the telogen hair follicles (200) (b). Control biopsy of uninvolved scalp with anagen hair follicles (40) (c). Higher power showing a fibrous tract with numerous mast cells (d).
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Figure 16 Alopecia areata incognita. Scalp, Isthmus. Diffuse miniaturization mimicking female pattern hair loss (40) (a). Subisthmus: only three follicular bulbs are present (40) (b). Higher power shows focal peribulbar lymphoid cell infiltrate (400) (c). Immunoperoxidase stain CD3 highlights intrabulbar lymphocytes diagnostic of alopecia areata (400) (d).
study,67 a significant reduction of follicular counts occurred in 10.6% of male patients with androgenic alopecia at age 50. In females with FPHL, follicular counts were reduced in 5.7% at age 70 years and 2.0% in diffuse FPHL at age 80. This data supports that reduced hair follicle density in the elderly is androgendriven67 and not due to old age.
predilection for central centrifugal cicatricial alopecia, whereas FPHL occurs on frontal, temporal, crown and vertex zones but not in the occiput. Conversely, both AA and TE may occur in any site.3 Thus, thinking ‘out of the box’ and accuracy in identifying ‘kaleidoscopically’ the subtle clues to other concomitant diagnoses, whilst still answering the main diagnostic query, is helpful.3
Conclusion Lessons from the past: ‘lives of lesions’ in alopecia
Although this review addresses only a few aspects of hair loss disorders, alopecia histopathology remains a complex topic. The increased number of publications in recent years has contributed new entities to the field, as well as new approaches that revisit old entities. However, much still needs to be explored and discovered to provide new aspects and diagnostic clues for the dermatopathologist. A
Clinically, non-scarring and scarring alopecias have different modalities of presentation. They may be cyclic (AA), evolve slowly (FPHL), and, in time, burn themselves out (LPP/FPHL). Understanding the ‘lives of lesions’ and knowledge of the morphologic features associated with their chronology in its different stages is a principle taught to us by A. Bernard Ackerman in 1984.64 This principle applies also to alopecia. It can enable a diagnosis, in spite of a scanty clinical history, but only provided that no biopsy sampling bias has occurred. We commonly classify alopecias into scarring (cicatricial) with permanent hair loss and non-scarring, with non-permanent hair loss. However, this classification65,66 would imply that nonscarring alopecias are non-permanent. Although there are instances in which hair follicles ‘synchronize’ (such as in the ‘shift out of anagen’ observed in subacute alopecia areata, the pathogenic pathway leading to hair follicle loss is quite distinct in scarring alopecias. On the other hand, evolving, long-standing end-stage non-scarring alopecias do also undergo hair follicle loss, with fibrosis of the follicular stelae, resulting in scarring and permanent alopecia (biphasic alopecias).65,66 With age, the number of follicles also decreases and characterizes what is known as senescent alopecia.67 In a large
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The gold standard in alopecia diagnostics is based on accurate clinical-pathological correlation In the absence of clinical history or photographs, a multifaceted structured diagnostic approach to alopecia is possible Direct immunofluorescence and special stains are valuable supporting diagnostic tools Sebaceous gland atrophy in response to overlying psoriasiform epidermal hyperplasia may mimic scarring alopecia Atrophic sebaceous basaloid epithelial aggregates may mimic telogen hair follicles with the consequent risk of a misdiagnosis of telogen effluvium and alopecia areata
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Immunoperoxidase stains for androgen receptor and Adipophilin can help distinguish atrophic sebaceous glands from telogen hair follicles Alopecia areata-like histopathology may be seen in a variety of clinico-pathologic scenarios unrelated to and distinctive from classic alopecia areata Careful examination of telogen hair follicle counts and identification of the presence/absence of miniaturization will help in distinguishing diffuse TE versus FPHL versus AA When evaluating diffuse telogen effluvium versus diffuse female pattern hair loss, the chronic phase of a non-inflammatory ‘silent’ (invisible) alopecia areata should be considered in the differential diagnosis In multifactorial alopecia, a single scalp biopsy may harbor more than one concomitant diagnosis Different alopecias have a predilection for different scalp zones Non-scarring alopecia, when end-stage, is characterized by permanent hair follicle loss and becomes scarring (biphasic alopecia)
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Please cite this article as: Stefanato CM, Histopathologic diagnosis of alopecia: clues and pitfalls in the follicular microcosmos, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.12.003