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| ORIGINAL ARTICLE |
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| Year : 2014 | Volume
: 6
| Issue : 1 | Page : 13-18 |
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'Ho-ver'ing Over alopecia areata: Histopathological study of 50 Cases
Manjot Marwah1, Nitin Nadkarni2, Sharmila Patil2
1 Department of Dermatology, National Hair Clinic, Hamirpur, Himachal Pradesh, India
2 Department of Dermatology, Dr. D.Y. Patil Medical College and Research Centre, Mumbai, Maharashtra, India
| Date of Web Publication | 15-Jul-2014 |
Correspondence Address:
Manjot Marwah
H. No. 29, Hiranagar, Hamirpur - 177 001, Himachal Pradesh
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0974-7753.136749
 Abstract | | |
Background: Different studies have been done on this topic, most of the older studies on the vertical section confirm that peribulbar infiltrate is an important finding, however the newer studies on the horizontal section, mention decreased anagen to telogen ratio, decreased terminal to vellus hair ratio and a decreased follicular count. These studies on horizontal (transverse sections) have shown that transverse sectioning is superior and more diagnostic in studying alopecias. However, these studies are based on multiple cut sections, because in a single cut section, we may miss a few hair bulbs above or below, depending on where the section is taken. Hence this makes it a time consuming, technically challenging and expensive procedure. Objectives: 1. To compare the histopathological features of single vertical section versus a single horizontal section cut in the reticular dermis. 2. To determine if a single horizontal section is sufficient to report a biopsy of alopecia areata compared to multiple sections. Materials and Methods: To compare the findings, in 50 patients with localized alopecia areata, 25 sectioned horizontally and 25 vertically, single section. Only three of the common findings, peribulbar infiltrate, intrabulbar infiltrate and perifollicular microscarring was considered and reported. Results: Once the anagen to telogen ratio is excluded from the comparison, diagnosis made on the basis of a single horizontal section is considered sufficient to give us an aid toward the prognosis. Keywords: Alopecia areata, histopathology, horizontal sections
How to cite this article:
Marwah M, Nadkarni N, Patil S. 'Ho-ver'ing Over alopecia areata: Histopathological study of 50 Cases
. Int J Trichol 2014;6:13-8 |
| Introduction | |  |
Alopecia areata (AA) is a T-cell mediated autoimmune disease resulting in partial or total, self-resolving and nonscarring hair loss [1] [Figure 1]a]. In the histologic assessment of scalp biopsy specimens from AA, a peribulbar lymphocytic inflammation is usually considered to be an essential finding in establishing the diagnosis [Figure 1]b]. [2],[3],[4],[5],[6] However, patients with AA visit physicians at different times during and in between episodes, when the peribulbar inflammation may be absent. Hence, clinical research in AA has become difficult due to these variability as well as unpredictable nature of spontaneous hair regrowth and lack of a uniform response to various therapies. [7] It is rarely biopsied because it is easy to make a clinical diagnosis and hence, many pathologists are unfamiliar with its interpretations. [8] | Figure 1: (a) Localized alopecia areata with depigmented vellus hair present in the center. Occasional exclamatory mark hair are seen, (b) Vertical section seen with peribulbar infiltrate resembling "swarm of bees" in H and E, under ×10 power
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A peribulbar infiltrate is the expected histological feature of AA, but it is absent in many scalp biopsies. Hence, other diagnostic features are needed. In addition, there are very few studies available on AA histopathological findings in India. The published literature stresses on the importance of transverse sections for a correct diagnosis. However, these are not often performed in most laboratories. A correct histopathological diagnosis is extremely vital for a dermatologist in special situations. These include atypical presentations, cases recalcitrant to treatment, patients using topical medications that alter the clinical picture or in trying to provide prognostic information to an anxious patient. [9]
This study was conducted from June 2010 to September 2012 in Department of Dermatology, Dr. D.Y. Patil Hospital, Mumbai, Maharashtra, India. 50 patients clinically diagnosed with localized AA giving consent for lesional biopsy were included in the study.
| Aims and objectives | | |
Different studies have been done on this topic, most of the older studies on the vertical section mention peribulbar infiltrate as an important finding, however the newer studies on the horizontal section, mention decreased anagen to telogen ratio, decreased terminal to vellus hair ratio and a decreased follicular count. [9] These studies on horizontal (transverse sections) have shown that transverse sectioning is superior and more diagnostic in studying alopecias. However, these studies are based on multiple cut sections, because in a single cut section, we may miss a few hair bulbs above or below, depending on where the section is taken. Hence this makes it a time consuming, technically challenging and expensive procedure. To avoid these disadvantages we have compared the histopathological features of single vertical section versus a single horizontal section cut in the reticular dermis. To compare the findings, we considered only three of the common findings, peribulbar infiltrate, intrabulbar infiltrate, and perifollicular microscarring. Once the anagen to telogen ratio is excluded from the comparison, diagnosis made on the basis of a single horizontal section is considered sufficient to give us an aid toward the prognosis. [10]
Objectives
- To compare the relevance of the horizontal section versus vertical section in a scalp biopsy of AA for diagnosis
- To evaluate if a single section of horizontal or vertical biopsy can replace multiple sections.
| Materials and Methods | | |
- After taking consent, detailed history and examination of patients with localized AA coming to our outpatient department was taken according to the proforma
- The reference patch is selected and evaluated in terms of duration, progress, hair pull test (HPT) and hair root mount
- Patients were divided into two groups randomly by the "coin flip technique" and a single lesional 4 mm punch biopsy was performed
- One group samples were sectioned horizontally in reticular dermis (25) and other vertically (25)
- The histological findings were studied and analyzed
- The following parameters were compared vis-ΰ-vis the vertical and horizontal section
- Intrabulbar infiltrate
- Peribulbar infiltrate
- Perifollicular microscarring
- In addition, follicular counts and terminal to vellus hair ratio were calculated in the horizontally sectioned samples.
Inclusion criteria
All patients clinically diagnosed with localized AA.
Exclusion criteria
- Patients not consenting to be part of study or not willing to be biopsied
- Pregnant females
- Any subject treated with topical, intralesional, or systemic agent likely to cause regrowth in AA within the past 1 month.
| Pathogenesis and its effects on hair cycle | | |
Normally, the hair follicles are continuously transformed in a cycle of organ construction and deconstruction. A pigmented hair shaft is generated during anagen. This phase of active growth consists of six stages (I through VI). Anagen is followed by catagen; an apoptotic phase that lasts several weeks, during which melanogenesis stops and the hair shaft is transformed into a "club hair." The hair follicle then enters telogen, a phase of relative quiescence that varies in duration (e.g. lasting several months on the scalp), and then returns to anagen. [11],[12] In AA, there is shortened and disordered hair cycle, in which a characteristic inflammatory-cell infiltrate attacks primarily pigment-producing hair follicles (predominantly those in stages III through VI of anagen). The mixed inflammatory-cell infiltrate contains T-cells, mast cells, natural killer cells, and dendritic cells, among which CD8+ T-cells are typically the first inflammatory-cells seen to be entering the anagen hair bulb epithelium. [13],[14],[15]
| Observations and results | | |
Results of our study were completely one-sided. A single horizontal section showed the selected microscopic parameters in higher number of patients than the single vertical section [Graph 1]. In addition, the single horizontal sections then underwent follicular counts, results as seen in Graph 2. The normal telogen to vellus hair ratio (T: V) is 7:1; however, in our study, it was reversed to 1:3.6. [9]
Retrospectively, the horizontally sectioned slides were grouped into two groups further depending on HPT. Positive was regarded as active disease and negative as inactive. There were 17 patients in the active group and eight in the inactive group. Results and correlation are seen in Graph 3.
| Discussion | | |
Reading a vertically sectioned scalp biopsy is comparatively easier to a horizontally sectioned biopsy. A horizontally sectioned biopsy requires a high degree of expertise on the part of the technician and the dermatopathologist [Figure 2]. | Figure 2: Horizontal section showing multiple vellus hair with three terminal hairs. Four hair bulbs with perifollicular and intrafollicular infiltrate are also seen ×4
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Follicular units
A follicular unit, also known as pilosebaceous unit, involves 2-4 hair follicles along with their adnexeal structures. They are clearly seen on the transverse sections as grouped hair bulbs or shafts with interweaving connective tissue [Figure 3]. Approximately, 10 such units were seen in a 4 mm punch biopsy in our study. Hence we get a follicular count of nearly 30-40 hair per biopsy. This follicular count is decreased in AA and also miniaturization of hair is seen in these follicular units. [9],[16] | Figure 3: Horizontal section showing three follicular units seen with miniaturization and decrease in number of hair follicles. Only vellus hair are seen on this section ×10
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Anagen hair
They are identified horizontally by the histopathological finding of pigment in the hair bulb. In AA, anagen hairs go into telogen prematurely. This phenomenon is seen clearly on a vertically sectioned biopsy [Figure 4]. This occurs with the formation of a fibrous stellae. This fibrous stellae are identified by the presence of loose fibrous tissue with the presence of abundant capillaries present in them [Figure 5]. [9],[16],[17],[18] | Figure 4: Vertical section showing anagen hair going into early telogen with formation of fibrous stellae, ×10
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 | Figure 5: Horizontal section with anagen hair showing pigment and formation of fibrous stellae around it. Fibrous stellae are identified by the presence of loose fibrous tissue with abundant capillaries. Also a vellus hair is seen in the upper half of the photograph, showing hair shaft diameter thinner than the outer root sheath diameter ×10
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Telogen hair
Above the fibrous stellae, the telogen hair bulb can be seen. These hair bulbs are generally present at a higher level than the anagen hair. Telogen hair can be identified with the confidence only by viewing the telogen hair bulb on the horizontal section. This hair bulb lacks pigment and contains a serrated inner root sheath (IRS); hence the perfect circular circumference of the hair shaft is not maintained [Figure 6] and [Figure 7]. [9],[19],[20] | Figure 6: Follicular unit showing 2 catagen hair, 1 telogen hair and 1 anagen hair (clockwise from top). Catagen hair showing the apoptosis with a thick hyalinized convoluted membrane. Telogen hair showing a central mass of trichelemmal keratin surrounded by palisading basaloid cells and Anagen hair seen with pigment and fibrous stellae formation
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 | Figure 7: (Horizontal section) Telogen germinal unit, showing a central mass of trichelemmal keratin surrounded by palisading basaloid cells. Telogen hair bulb is identified by irregular morphology, lack of pigment and absence of apoptosis ×10
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Catagen hair
Trichelemmal shrinkage and apoptosis indicated with hyalinization are the hallmarks of a catagen hair bulb [Figure 8]. [9]{Figure 8}
The hair shaft of a telogen and anagen hair shaft appears nearly the same without the IRS; hence, their identification being dependent on viewing the lower follicle sections. Thus, we require multiple cut sections to give the anagen:telogen ratio. Therefore, if we exclude this parameter from our reporting, then a single section horizontal biopsy should be sufficient to aid us in giving a prognosis. [9]
Terminal hair
They are identified when the hair shaft is greater than the outer root sheath (ORS). They are decreased in number in AA.
Vellus hair
These are increased in number in AA. They are identified by the diameter of the hair shaft which is smaller than the ORS. In a true vellus hair the hair shaft diameter will be equal to the diameter of the IRS with a relatively thin ORS, however in a miniaturized vellus hair, the ORS will be many cell layer and the IRS will be smaller than the hair shaft diameter as shown in [Figure 5]. [9],[17],[20]
Perifollicular infiltrate
"Swarm of bee appearance" is the classical term used to describe perifollicular infiltrate. This finding is easy to identify; however, they are seen in more number of follicles in horizontal sections than vertical sections. Perifollicular infiltrate was seen in 19 patients in horizontal group (76%) and in six patients in the vertical group (24%). Correlating with HPT, we found that peribulbar infiltrate was found in 77% patients with HPT positive and 60% with HPT negative [16],[18] [Figure 1]b].
Intrabulbar infiltrate
It has been known that intrabulbar infiltrate is a more specific marker of AA than peribulbar infiltrate. [21] Just like peribulbar infiltrate, it is easier to identify in more number of follicles in horizontal sections. We found that 16 patients in horizontal group (64%) and six patients in vertically sectioned group (24%) should this finding. Furthermore, it was seen in 56% patients with HPT positive and 35% patients with HPT negative.
Perifollicular microscarring
Long standing cases of AA occasionally start developing fibrosis around the hair follicles. This is known as perifollicular microscarring. This was seen in 16 patients in horizontal group (64%) and three patients in vertical (12%). This finding was easier to identify in the horizontal group. 22% patients with HPT positive and 39% with HPT negative showed this finding [Figure 7]. [9],[21],[22]
Terminal: Vellus hair ratios
The normal T: V ratio in controls is known to be 7:1 and normal follicular count is known to be approximately 40. The average number of follicular units is 10; however, all these values have been studied only in Western population. There is no study to give us the normal values in Indian population. One reason for the higher follicle counts in Whiting study. [21] [Table 1] compared to the present (28 in < 6 month) could be the genetic difference in the hair follicle density as Whiting's study was done in Western population. Asians have been shown to have lesser hair density compared to Western population. [7],[8] We could not find any study in our population analyzing the normal hair follicle counts.
Follicular units were not decreased in any of the studies, this correlated with our study.
| Conclusion | | |
Firstly, a biopsy is not required for diagnosis, only prognosis in AA. Also, we could conclude with are study that peribulbar and intrabulbar infiltrate correlate with disease activity and is seen in HPT positive patients more frequently. However, perifollicular microscarring is more in patients with HPT negative and is an indicator of inactive disease that has already been established. Horizontal sections are much better than vertical sections as they are more sensitive to the above markers and single section is sufficient and more feasible [Table 2]. | Table 2: Comparison between single horizontal and single vertical section
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However, more studies are required in this field especially in the Indian population with a larger sample size. Similar study with taking both samples from the same patient and doing case control studies will help future research.[24]
| Acknowledgment | | |
Dr. Rajiv Joshi for his guidance and help, without whom this study would not have been possible.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]
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