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ORIGINAL ARTICLE
Year : 2018  |  Volume : 10  |  Issue : 1  |  Page : 11-16  

Evaluation of alopecia: A new processing technique combining vertical and transverse sections from a single scalp biopsy specimen


Department of Pathology, PSG Institute of Medical Sciences and Research, Coimbatore, Tamil Nadu, India

Date of Web Publication17-Jan-2018

Correspondence Address:
Dr. Chaitra Venkataswamy
Department of Pathology, PSG Institute of Medical Sciences and Research, Coimbatore - 641 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijt.ijt_66_17

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   Abstract 

Background: Histopathological study of alopecia generally requires both vertical and transverse sections (VS and TS) and this may demand the need for multiple scalp biopsy samples. Here, we suggest a new processing technique which provides both VS and TS from a single biopsy, thus making the interpretation easier. Materials and Methods: All formalin-fixed scalp biopsy specimens were processed routinely. Nine VS were taken and mounted on 3 glass slides. The block was then melted and the tissue reembedded with the subcutaneous tissue facing downward and the epidermal aspect toward the technician to obtain TSs. Serial 4 μ sections were cut until the tissue was exhausted and every 25th section was mounted on glass slides and stained with H and E. Results: 100 scalp biopsy specimens with the history of alopecia have been processed using this technique. The technique provides proper visualization of both VS and TS from a single scalp biopsy without compromising the quality. Further, evaluation of the remaining sections with application of special stains can also be possible with this technique which will maximize the information and contribute to the accurate diagnosis of various types of alopecia. Conclusions: This technique can be used to obtain diagnostically satisfactory VS and TS from a single scalp biopsy specimen by modifying the embedding procedure, thereby avoiding the need for a second biopsy. All types of alopecia can be diagnosed by this effective method.

Keywords: Embedding technique, histopathology, nonscarring alopecia, scalp biopsy, scarring alopecia


How to cite this article:
Gurusamy U, Venkataswamy C, Sivaraman A. Evaluation of alopecia: A new processing technique combining vertical and transverse sections from a single scalp biopsy specimen. Int J Trichol 2018;10:11-6

How to cite this URL:
Gurusamy U, Venkataswamy C, Sivaraman A. Evaluation of alopecia: A new processing technique combining vertical and transverse sections from a single scalp biopsy specimen. Int J Trichol [serial online] 2018 [cited 2018 Sep 24];10:11-6. Available from: http://www.ijtrichology.com/text.asp?2018/10/1/11/223383


   Introduction Top


Conventionally, histopathologists have relied entirely on vertical sections (VSs) of most tissues, except nerve, skeletal muscle and blood vessel, for diagnostic purposes. Although VS are satisfactory for the study of dermatologic diseases, their utility in the evaluation of alopecia is questionable. The hair follicle lies obliquely in relation to the epidermis. As a result of this, the follicle is often missed, cut tangentially, or incomplete. In 1984, Headington described and illustrated the morphology of hair follicles in transverse sections (TSs) of the scalp. He indicated that the morphometric analysis of TSs would provide information that could be used to study hair follicles in health, disease, and assess response to therapy.[1] Subsequent workers have shown that horizontal scalp sections are the preferred method for evaluating all forms of hair loss.[2],[3]

The histologic diagnosis of alopecia can be challenging. To maximize the diagnostic yield, multiple biopsies may be necessary. Elston have also demonstrated that combined vertical and horizontal sections increase the diagnostic yield.[2]

When only a single biopsy specimen is submitted, the pathologist is faced with a choice. Either VS or TS may be performed. A consensus from a working group of alopecia experts stated that combining VS and TS are optimal, but suggested that when a single biopsy is submitted it should always be sectioned transversely.[4] VSs may be superior for some forms of alopecia (especially scarring alopecia) and TSs may be superior for other forms (especially nonscarring alopecia).

The purpose of this study is to assess the histological changes in the various types of alopecia and to evaluate the impact of combining vertical and horizontal sections in a single 4 mm scalp biopsy specimen by a new tissue processing technique.


   Materials and Methods Top


Scalp biopsies were received in the Histopathology Department from 100 patients with alopecia, from July 2006 to July 2016. Except for 2 biopsies that were 3 mm in diameter, the rest were 4 mm punch biopsies, taken from sites where the disease was active, yet not too advanced. This was usually at the periphery of the lesion. They were sent in 10% formalin and processed in an automatic tissue processor before paraffin embedding. Nine VS were taken and mounted on 3 glass slides [Figure 1] and stained with H and E, Verhoeff van Gieson (VVG) or Alcian blue/periodic acid-Schiff (AB/PAS) as required. The block was then melted and the tissue reembedded with the subcutaneous tissue facing downward and the epidermal aspect toward the technician to obtain horizontal sections [Figure 2]. On a microtome (LEICA RM22255) with a disposable blade until the tissue was exhausted and every 25th section was mounted on glass slides and each slide had 4 levels, for example, 25,50,75, and100 and stained with H and E. Unmounted sections were saved in the refrigerator between filter paper sheets until reporting was completed [Figure 3]. The steps are depicted in line diagram [Figure 4].
Figure 1: (a) Paraffin block for vertical sections (b) 4 slides, 3 vertical sections on each. Thickness of section = 4 μ

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Figure 2: (a) Melting the paraffin block (b and c) Tissue reembedded with the subcutaneous tissue facing downward and the epidermal aspect toward the technician

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Figure 3: (a) Serial 4 μ sections were cut until the tissue was exhausted. (b and c) Unmounted sections were saved in the refrigerator between filter paper sheets until reporting was completed

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Figure 4: Line diagram representing the embedding and sectioning steps for vertical sections and transverse sections, scalp biopsy

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   Results Top


One hundred cases were included in the study.The distribution of these cases is depicted in pie diagram [Figure 5]. The group of nonscarring alopecia consisted of alopecia areata [Figure 6] 22 cases, androgenetic alopecia 11 cases, trichotillomania 8 cases, and 2 cases each of tinea capitis and psoriatic alopecia.
Figure 5: Pie diagram depicting histological types of alopecia

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Figure 6: Alopecia areata – (a) peribulbar inflammation, vertical sections (H and E, ×10). (b) – nanogen hair follicle, transverse sections (H and E, ×40)

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Among the 44 cases of primary scarring alopecia, there were 19 cases of lichen planopilaris (LPP) [Figure 7] and 8 cases of discoid lupus erythematosus (DLE) [Figure 8]. 2 cases showed overlap features of LPP and DLE. 6 cases of end-stage scarring alopecia, 3 cases of folliculitis decalvans, 2 cases each of acne keloidalis, keratosis follicularis spinulosa decalvans, and fibrosing alopecia in a pattern distribution were diagnosed.
Figure 7: Lichen planopilaris – (a) Subepidermal wedge-shaped scar, vertical sections, (Verhoeff van Gieson, ×10). (b) Lichenoid inflammation around hair follicle, transverse sections (H and E, ×40)

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Figure 8: Discoid lupus erythematosus – (a) Basement membrane thickening, vertical sections (Alcian blue/periodic acid-Schiff, ×10) (b) Perieccrine inflammation, transverse sections (H&E, ×40)

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There were 11 cases of secondary scarring alopecia and consisted of 3 cases of scleroderma, 2 cases of lichen sclerosis et atrophicus and 1 case of atypical necrobiosis lipoidica. The unusual causes of secondary scarring alopecia included 2 cases each of nevus sebaceus, Vitamin D associated alopecia and 1 case of pemphigus vulgaris.

The diagnosis of nonscarring alopecia (n = 45) could be made exclusively on TS and VS in 44 (98%) cases and 33 cases (73.3%), respectively. When combining the TS and VS, the diagnosis was possible in all the cases (100%). The availability of more number of hair follicles, better visualization of the follicular variation, miniaturization and ratio of anagen and telogen hair follicles were possible only in the TS.

Of the 55 patients with scarring alopecia (both primary and secondary), 39 (70.9%) and 30 (54.5%) patients were diagnosed solely by TS and VS, respectively. The study of both the sections was necessary to arrive at a diagnosis in 53 out of 55 patients. The remaining two patients showed overlap features of LPP and DLE which underscores the importance of immunofluorescence study. TSs were superior in cases of lupus erythematosus, LPP with focal follicular involvement, fibrosing alopecia in a pattern distribution, Vitamin D associated alopecia, and keratosis follicularis spinulosa decalvans. Stelae and blank spots were more prominent in TSs. Interface changes and lupus panniculitis were better demonstrated in VSs. The histological types of primary scarring, secondary scarring including unusual causes and non scarring alopecia are listed in [Table 1],[Table 2] and [Table 3] respectively.
Table 1: Histological types of primary scarring alopecia

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Table 2: Histological types of secondary scarring and unusual causes of alopecia

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Table 3: Histological types of nonscarring alopecia

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   Discussion Top


Histopathologic examination is routinely done for the diagnosis of many dermatologic diseases. Although hair loss is distressing and not infrequent, scalp biopsies form a minor component of specimens received in the histopathology laboratory. For decades, the microscopic examination of hairs that have been pulled, plucked, or spontaneously shed have been used for the evaluation of hair loss. This is useful in diseases of the hair shaft and to an extent in nonscarring alopecia, but of limited value in most cases of alopecia. Attempts to study the histological changes in alopecia, by conventional VS, were disappointing as no useful information was forthcoming and no clinical correlates could be established. Both dermatologists and pathologists groped for a diagnostic approach to the problem of alopecia.

In 1984, Headington established the morphology of hair follicles in TSs of the scalp. Subsequent workers have shown that horizontal scalp sections are the preferred method for evaluating all forms of hair loss.[5],[6],[7],[8]

The main advantage of VS is that the dermoepidermal junction, papillary dermis, and subcutis are better demonstrated. Interface dermatitis, lupus panniculitis, miniaturized hairs, and trichomalacia are more obvious.

The advantages of TSs, in addition to the higher yield of follicles are the rapid, easy, and accurate assessment of follicular density, follicle and shaft diameters, anagen, telogen, terminal, and vellus hairs. Since each sections have their own advantages, many authors [2],[3] have clearly demonstrated that the increased diagnostic utility of combining the vertical and horizontal sections.

Clinicians usually perform two biopsies; one for vertical and the other one for transverse sectioning. Due to difficulty in convincing the patient for two biopsies and to limit the morbidity, the clinicians started doing a single biopsy. In that situation, the drawbacks are the need to use a different protocol for specimen preparation and familiarity with the transverse microanatomy of the scalp.

There are several ways to slice the single scalp biopsy specimen for TSs.[9],[10],[11] However, the novel HoVert technique is an effective method in diagnosing alopecia by combining the advantages of both VS and TS in a single biopsy sample.[12]

When the study commenced, we were unfamiliar with the transverse microanatomy of the scalp in health and disease. Since we are accustomed to VS, we thought it would be better to have a few VS, in all biopsies for orientation. This was found to be particularly useful in the detection of interface dermatitis in primary scarring alopecia and for demonstrating the subepidermal wedge-shaped scars of LPP.[13] Since we had 2 more unstained VS, special stains such as AB-PAS (mucin) and VVG (elastic) can also be done when required, apart from the H and E section.

The diagnosis of scarring alopecia [14],[15] was easily made on VS. However, the TS may provide an insight into the mechanism of hair loss. LPP was the most common variant of scarring alopecia. It was possible to distinguish it from DLE by the absence of inflammation in the deep dermis, diffuse deposition of dermal mucin, and extensive dermal scarring. However, in 2 cases, there was an overlap of microscopic features and this underscores the need for immunofluorescence studies in cases of scarring alopecia.

Unusual forms of secondary scarring alopecia-scleroderma, lichen sclerosus et atrophicus, and necrobiosis lipoidica were also studied.

The diagnosis of nonscarring alopecia requires TSs since they are related to abnormalities in the growth cycling of hair follicles. The features such as variation in size of the hair follicles with miniaturization and presence of nanogen follicles in case of alopecia areata were easily studied only on TSs. Alopecia areata was the most common cause of hair loss and was diagnosed on the presence of peribulbar inflammation and/or nanogen hair follicles.

The present technique allows us to analyze the epidermis and entire thickness of the dermis with subcutis on VSs. However, which is not possible in the previously described methods, where the biopsy is transected and used for vertical and TSs separately.

The main drawback of this method includes a need for a trained technical staff to obtain proper TS without having oblique sections.


   Conclusion Top


This new tissue processing technique is useful in the diagnosis of all types of alopecia (both scarring and nonscarring) as all the aspects of the hair follicles including the size (terminal, vellus, and indeterminate), phase of the hair cycle (anagen, catagen, and telogen), bulbs, follicular units (evenly spaced/blank spots etc.), presence of inflammation (at the level of bulb, suprabulbar, isthmus, and infundibulum) are clearly demonstrated. In addition to that, this technique may also be useful in studying the epidermis, dermoepidermal junction, and subcutis. This technique also provides unmounted sections at all levels of the hair follicles which are saved in the refrigerator between filter paper sheets until reporting was completed.

Although it is cumbersome, a more complete and thorough examination is possible by combining vertical and transverse sectioning of the same punch biopsy specimen by this effective technique.

To summarize, this is a histological study of scalp biopsies, sectioned vertically and horizontally, in 100 cases of alopecia. The series is fairly representative of the various clinicopathological types of alopecia. Alopecia areata was the most common cause of alopecia, both types, put together. LPP was the most common type of scarring alopecia. TSs were particularly useful in the histology of nonscarring alopecia. Since most patients are reluctant/refuse to permit a second punch biopsy, a single 4 mm punch can be used to obtain diagnostically satisfactory vertical and horizontal sections by modifying the embedding procedure, thereby avoiding the need for the second biopsy.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patients have given his their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Headington JT. Transverse microscopic anatomy of the human scalp. A basis for a morphometric approach to disorders of the hair follicle. Arch Dermatol 1984;120:449-56.  Back to cited text no. 1
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2.
Elston DM. Vertical vs. Transverse sections: Both are valuable in the evaluation of alopecia. Am J Dermatopathol 2005;27:353-6.  Back to cited text no. 2
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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.  Back to cited text no. 3
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4.
Olsen EA, Bergfeld WF, Cotsarelis G, Price VH, Shapiro J, Sinclair R, et al. Summary of North American Hair Research Society (NAHRS)-sponsored workshop on cicatricial alopecia, Duke university medical center, February 10 and 11, 2001. J Am Acad Dermatol 2003;48:103-10.  Back to cited text no. 4
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Özcan D, Özen Ö, Seçkin D. Vertical vs. Transverse sections of scalp biopsy specimens: A pilot study on the comparison of the diagnostic value of two techniques in alopecia. Clin Exp Dermatol 2011;36:855-63.  Back to cited text no. 6
    
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Singh K, Sharma S, Singh UR, Bhattacharya SN. A comparison of vertical and transverse sections in the histological diagnosis of alopecia areata scalp biopsy specimens. Int J Trichology 2016;8:111-5.  Back to cited text no. 7
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McKee PH, Calonje E, Granter SR. Pathology of the Skin: With Clinical Correlations. 4th ed. UK: Elsevier Health Publisher; 2011.  Back to cited text no. 9
    
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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;38:401-6.  Back to cited text no. 12
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Elston DM, McCollough ML, Warschaw KE, Bergfeld WF. Elastic tissue in scars and alopecia. J Cutan Pathol 2000;27:147-52.  Back to cited text no. 13
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Templeton SF, Solomon AR. Scarring alopecia: A classification based on microscopic criteria. J Cutan Pathol 1994;21:97-109.  Back to cited text no. 14
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

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