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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 40  |  Issue : 2  |  Page : 92-98

Direct immunofluorescence of the hair follicle in pemphigus: a less invasive method for diagnosis


1 Departments of Dermatology, Venereology and Andrology, Faculty of Medicine, Assiut University, Assiut, Egypt
2 Departments of Histology, Faculty of Medicine, Assiut University, Assiut, Egypt
3 Departments of Dermatology, Venereology and Andrology, Assiut Police Hospital, Assiut, Egypt

Date of Submission09-Sep-2019
Date of Acceptance26-Dec-2019
Date of Web Publication09-Jun-2020

Correspondence Address:
Reham Ahmed Abdel Rahim
Consultant of Dermatology and Venereology, Assiut Police Hospital, Assiut, 71511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejdv.ejdv_47_19

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  Abstract 


Background Demonstration of intercellular deposition of immunoglobulin (Ig)G on the cell surface of keratinocytes by direct immunofluorescence (DIF) of the skin is the gold standard in diagnosis of pemphigus. In the past few years, the intercellular deposition of IgG in the outer root sheath has shown to be useful.
Objective The aim was to compare the DIF of hair (plucked anagen and telogen and telogen obtained by combing) with that of skin for diagnosis of patients with pemphigus vulgaris (PV) and those with pemphigus foliaceus (PF).
Materials and methods A total of 30 patients [24 with PV and six with PF] with active disease were included. Clinical evaluation (ABASIS score), histopathological examination, and DIF of skin and hair were done. Presence of intercellular deposits of IgG and or C3 in skin and outer root sheath of the hair was considered positive.
Results DIF of skin was positive in all patients for IgG and in 25 patients for C3 and also DIF in anagen hair was positive in all patients, whereas in telogen hair, it was positive in 23 patients (17 with PV for both IgG and C3, and six with PF for IgG, and four of them for C3). All patients showed a positive relation between clinical, histopathological, DIF skin, and DIF of anagen hair in both PV and PF. Regarding telogen hair, the same findings was found in all six (100%) patients with PF, but in PV, only 17 (70.83%) patients showed these positive relations, whereas the other seven (29.2%) had positive DIF of both skin and anagen hair only. The sensitivity of hair DIF was 100% for anagen hair and 76.67% for telogen hair in patients with pemphigus.
Conclusion Anagen and telogen hair DIF is a simple, noninvasive, and cost-effective procedure and can be used as an additional procedure for diagnosis in patients with PV and PF.

Keywords: anagen and telogen hair, hair immunofluorescence, pemphigus


How to cite this article:
Mohamed HZ, Abdel Hafez HZ, Mohamed NA, Abdel Rahim RA. Direct immunofluorescence of the hair follicle in pemphigus: a less invasive method for diagnosis. Egypt J Dermatol Venerol 2020;40:92-8

How to cite this URL:
Mohamed HZ, Abdel Hafez HZ, Mohamed NA, Abdel Rahim RA. Direct immunofluorescence of the hair follicle in pemphigus: a less invasive method for diagnosis. Egypt J Dermatol Venerol [serial online] 2020 [cited 2020 Jul 6];40:92-8. Available from: http://www.ejdv.eg.net/text.asp?2020/40/2/92/286289




  Introduction Top


Pemphigus is a chronic autoimmune bullous disorder characterized by autoantibodies directed against desmoglein (Dsg) 3 and/or 1. Direct immunofluorescence (DIF) of the skin or mucosa is considered as the gold standard in the diagnosis of pemphigus [1]. DIF of the perilesional skin demonstrates intercellular deposition of immunoglobulin G (IgG) with or without complement 3 (C3) in the epidermis. This requires expensive and sophisticated equipment and specific expertise; in addition, it is not always possible to take biopsy such as in mucosa and flexural areas and in children. Moreover, this facility is not available in every setting [2].

DIF of the skin or mucosa is an invasive and expensive procedure, and the patient may not be willing to it. Pemphigus-specific immunofluorescence pattern has been demonstrated in the outer root sheath (ORS) of hair follicles, which is structurally analogous to epidermal keratinocytes, with a sensitivity ranging from 85 to 100% [3].

A scalp lesion is not a prerequisite for performing DIF on hair as the test may be positive even in the absence of scalp involvement [4]. Hence, DIF of hair may be an ideal substrate for diagnosis and assessment of immunological remission, because it is a simple, non-invasive, and cost-effective procedure [3]. DIF of plucked hair bulbs is considered a reliable diagnostic method in patients with pemphigus [5].


  Materials and methods Top


After obtaining approval from the Institutional Ethics Committee and consent from the patients, a cross-section study was conducted in the Department of Dermatology in Assiut University Hospital over a period of 2 years. A total of 30 patients with pemphigus who fulfilled the following criteria were included in the study: first, patients showing the clinical and histological findings, as well as positive DIF of the skin biopsy, diagnostic for pemphigus; second, patients of all age groups; and third, patients of either sex. The exclusion criteria were as follows: first, patients who refused to be included in the study protocol; second, patients with paraneoplastic pemphigus; third, and patients with any other dermatological disease.

Age, sex, type of lesions, site, mucous membrane affection, duration, associated symptoms, previous treatment, and other system affection were evaluated.

Two skin biopsies (one from the bullous lesion and the other one from adjacent area) were taken from each patient after local anesthesia using a 5 mm punch biopsy.

Lesional biopsy specimens were collected in 10% formalin solution and used for histopathological examination after standard processing. The sections were stained with hematoxylin and eosin and examined under the light microscope. Perilesional skin samples were used for performing DIF using a 5 mm punch biopsy from perilesional skin. Samples were received in fresh state without any fixative to avoid dehydration. The tissues were wrapped in saline-moistened gauze, were placed in a labeled plastic container, and were snap-frozen in liquid nitrogen as soon as possible (−196°C). The container was cooled rapidly in liquid nitrogen until the edges were frozen until the consistency of the block is appropriate for cryosectioning. For sectioning, the metal chunk bearing the frozen tissue specimen was placed in the cryostat. The number of sections to be cut was dependent on the number of fluorescent antisera used (in our study we used tow) and additional sections to be used as controls.

Sections of 4–6 µm thickness were cut in a cryostat and taken off the cryostat by gently touching with a glass slide. As many as five to six sections can be applied on a standard microscopic slide. Tissue sections were separated from each other by straight lines, by wax pencil, to prevent intermixing of fluorescent antisera. The slides were then allowed to air dry for 15 min.

Incubation with fluorescent isothiocyanate-conjugates

After rinsing in a PBS, slides were overlaid in a moist chamber with FLTC-conjugate for 20–30 min at 37°C.

The following antisera were used: anti-IgG and C3. The unreacted antiserum was washed off in phosphate buffer solution for 30 min. The slides were allowed to drain, and the excess buffer was wiped from the bottom of the slide and around the sections with dry cotton gauze. Then the slides were cover-slipped using a drop of buffered glycerin (90% glycerin in-PBS) and examined under the fluorescence microscope (fluorescent microscope Olympus BX51 in Histology Department, Faculty of Medicine, Assiut University). Regarding hair samples, ∼30 hair strands was obtained in the same way as for the trichogram. Telogen hairs were selected by combing the hair and collecting the loose strands of hair from the comb. The hair type was further confirmed by microscopy. The hairs with their ORS were immediately frozen in liquid nitrogen at −196°C, in Eppendorf lock tubes (Eppendorf, Hamburg, Germany) without medium or optimal cutting temperature (OCT). The samples were transported to the department of histology and subjected to DIF staining with fluorescent isothiocyanate (FITC)-labeled rabbit anti-IgG (Thermofisher Scientific, Loughborough, Leicestershire, UK) and anti-C3 antibodies (SH-1923-R2). The samples were washed in PBS three times; each wash took 10 min. Following which, they were incubated with FITC-labeled anti-IgG and anti-C3 for 1 h at 37°C and once again washed in PBS three times for 10 min each, and then air dried and mounted on slide with PBS-glycerol solution for examination. Then were imaged by Olympus camera attached to Olympus epifluorescence microscope (Olympus, Tokyo, Japan). Evaluation of biopsy sections was made by positive and negative marks only.


  Results Top


Among patients with pemphigus, pemphigus vulgaris (PV) was the most frequent disease [24/30 (80%)], followed by pemphigus foliaceus (PF) [6/30 (20.0%)]. The age of patients with pemphigus ranged from 21 to 75 years, with a mean±SD age of 47.73±16.56 years. Among patients having PV, female patients with pemphigus were more frequent, whereas in patients with PF, both females and males are equally affected ([Table 1]).
Table 1 Age and sex distribution in patients with pemphigus

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Histopathological examination

All patients were clinically diagnosed as having PV (n=24), confirmed histopathologically. Their biopsies showed suprabasal bullae, acantholytic cells, and the dermal papillae project as villi into suprabasal cavities. Dermal papillae were lined by a single layer of basal keratinocytes.

All patients were clinically diagnosed as having PF (n=6), confirmed histopathologically. Their biopsies showed intraepidermal subcorneal bulllae with the presence of inflammatory cells.

Direct immunofluorescence of skin and hair

DIF of patients with PV was positive in all patients, showing intraepidermal deposition of IgG in all patients (100%) and C3 was observed in 87.5% ([Table 2], [Figure 1]a and b).
Table 2 Skin, anagen hair and telogen hair direct immunofluorescence with immunoglobulin G and C3 in patients with pemphigus groups

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Figure 1 (a) Direct immunofluorescence of pemphigus vulgaris of perilesional skin showing intercellular deposits of immunoglobulin G in the epidermis (E) and eccrine glands (C) (×200); (b) direct immunofluorescence of pemphigus foliaceus of perilesional skin showing intercellular deposits of C3 in the epidermis (arrow) (×200).

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DIF in patients with PF was positive in all patients (100%), showing intraepidermal deposition of IgG. Regarding skin C3, 66.67% of cases in PF group were positive mainly in the upper part of the epidermis and eccrine glands ([Table 2], [Figure 1]c and d).

In patients with PV, DIF of anagen hair regarding IgG and C3 was positive in 24 (100%) patients. In PF group, 100% of cases in study group has positive anagen IgG and anagen C3 ([Table 2], [Figure 2]a and b).
Figure 2 (a) Direct immunofluorescence of anagen scalp hair in pemphigus vulgaris showing intercellular deposition of immunoglobulin G in outer root sheath (×400); (b) direct immunofluorescence of anagen scalp hair in Pemphigus foliaceus showing deposition of C3 in the outer root sheath (×400).

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Regarding telogen hair, DIF in PV group was positive for both IgG and C3 ([Table 2], [Figure 3]a and b). Overall, 100% of cases in P.F group has positive telogen IgG & telogen C3 ([Table 2], [Figure 2]c and d).
Figure 3 Direct immunofluorescence of telogen scalp hairs showing Intercellular deposition of immunoglobulin G in the outer root sheath in pemphigus vulgaris (a) (×200) and in pemphigus foliaceus (b) (×400) and showing intercellular deposition of C3 in the outer root sheath in pemphigus vulgaris (c) (×200) and in pemphigus foliaceus (d) (×400).

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  Discussion Top


Pemphigus is a rare group of blistering autoimmune diseases affecting the skin and mucous membranes, where autoantibodies are directed against the cell surface of keratinocytes mainly Dsg1 and Dsg3. DIF test is a very sensitive test and is the gold standard in the diagnosis of pemphigus [6].

Desmogleins are the main target antigens in pemphigus. They are calcium-dependent transmembrane adhesion proteins belonging to the cadherin family [1].

Dsg1 is expressed in the suprabasal cells of the epidermis and inner root sheath as well as the innermost layers of ORT of the hair follicle. Dsg3 is present in the basal and suprabasal cells of the epidermis and throughout the ORS of the hair follicle except in the areas of epidermal-like keratinization such as in the infundibulum, where it is confined to the basal layer of ORS [7].

Dsg3 is also responsible for anchoring the telogen hair in the follicle [8]. In the past few years, DIF of plucked hair demonstrating intercellular deposition of IgG in ORS has shown to be useful [1]. Pemphigus-specific immunofluorescence pattern has been demonstrated in ORS of hair follicles, which is structurally analogous to epidermal keratinocytes, with a sensitivity ranging from 85 to 100% [3].

Regarding skin DIF with IgG in PV & PF was positive in all patients in most of studies, while with C3 encountered marked variation in their incidence among different studies, whereas C3 encountered marked variation in their incidence among different studies. In the present study, DIF staining of the skin for IgG and C3 in patients with pemphigus resulted in intraepidermal deposition of IgG in 30 (100%) of cases and of C3 in 25 (83.3%) of cases. These results agree with Robati and colleagues, who reported DIF study from perilesional skin in patients with pemphigus and showed mainly IgG and C3 deposits. There were 19 of 32 patients of pemphigus who had intraepidermal deposition of both intercellular IgG and C3, and 13 had only IgG deposition. Both PF and PV had 100% positive skin IgG, and regarding skin C3, 66.67% of patients were positive in P.F group versus 87.5% in P.V group [6]. This agrees with Ghanadan and colleagues, who reported the DIF assay estimated 90–100% of skin specimens taken from patients with PV were positive for antibody [9]. Other studies done by Kabir and colleagues and Khannan and Baht found that IgG was positive in all patients with PF (100%) in the intercellular substance (ICS) in the full thickness of epidermis [10],[11].

In present study, we reexamined the possibility of using hair DIF instead of conventional DIF in relation to sensitivity and utility as a less invasive technique for diagnosis of pemphigus disease. Sano and colleagues described the sensitivity of DIF on ORS in pemphigus as 85–100%. The sensitivity of mucosal or skin DIF in pemphigus patients with active disease is 90–100% [12],[13].

Difficulties in processing the hair samples have been cited as the reason for negative DIF findings in earlier reports [13]. When we processed the hair samples, we used whole rather than sectioned hairs, and also underlined the importance of taking the adequate hair samples that include the hair follicle ORS structures and immediately froze them in liquid nitrogen to avoid the degradation of the samples, and for examination, they were mounted on slides, and then processed directly for DIF without prior sectioning of the hair follicles, with the hairs remaining on the slide for examination. We found this technique an easier and more rapid method. By using these precautions, we eliminated the possibility of examining inadequate hair samples that would determine false-negative results of hair DIF.

In the present study, immunological deposit characteristics of pemphigus (intercellular deposition of IgG and C3 in ORS) were found in the ORS of anagen hair in 100% and of telogen hair in 70.8% of patients with PV and in 100% of patients with PF for both anagen and telogen hairs. The sensitivity of anagen hair DIF for IgG and C3 in PV and PF was 100% as that for cutaneous DIF.

For the sensitivity of telogen hair DIF with IgG, it was 70.83% and with C3, it was 80.95% in PV, and was 100% with both IgG and C3 in PF.

This agrees with Alexandru and colleagues who used a similar technique by directly mounting hair samples on slides then processed them for DIF without freezing, and the sensitivity of anagen hair DIF in patients with PV was 100% (12/12 patients with PV) similar to that of cutaneous DIF [14].

Moreover, DIF was positive on Tzanck smear in 23 (76.7%) patients. Intercellular deposition of IgG was seen in the ORS of anagen hair in 27 out of 30 (90%) patients [4].

Badran and colleagues reported that in pemphigus patients, all examined skin slides were positively stained for DIF on using FITC-labeled anti-human IgG, showing lace-like pattern of intercellular deposits in the epidermis, whereas 28 of the 30 hair slides (93.3%) were positively stained for DIF on using FITC-labeled antihuman IgG, showing pemphigus-specific pattern with a sensitivity of 93% [15].


  Conclusion Top


In our study, the sensitivity of anagen hair DIF in pemphigus patients was 100%, similar to that of the cutaneous DIF (100%), and also near to that found in telogen hair DIF (76.67%).

Regarding patients with PV, the sensitivity of anagen hair DIF was 100%, as that of the cutaneous DIF (100%) and also near to that found in telogen hair DIF (70.83%).

Regarding patients with PF, the sensitivity of anagen and telogen hair DIF was 100% for both, as their cutaneous DIF sensitivity was 100%.

Telogen hairs are readily obtained by combing the hair or by gentle pulling rather than plucking the hair (which can be painful).

There is a possibility that our simple, economical, and noninvasive technique for taking hair samples may be performed in remote health centers and transported without a medium to specialized laboratories for further analysis.

DIF of hair is a simple, specific, and noninvasive test and provides the opportunity to avoid repeated skin biopsies in patients with pemphigus.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Rai R, Harikumar MV. Comparison of direct immunofluorescence of plucked hair and skin for evaluation of immunological remission in pemphigus. Indian Dermatol Online J 2017; 8:319–322.  Back to cited text no. 1
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2.
Sayedatun NM, Kabir N, Barua AR, Kumar PH. Role of direct immunofluorescence on outer root sheath of hair follicle in monitoring disease activity of pemphigus vulgaris. Pathol Nepal 2017; 7:1162–7.  Back to cited text no. 2
    
3.
Kumaresan M, Rai R, Sandhya V. Immunofluorescence of the outer root sheath in anagen and telogen hair: an aid to diagnosis in pemphigus. Int J Trichol 2009; 1:138–9.  Back to cited text no. 3
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4.
Tahir K, Batool S, Shahid M, Aman S. Role of direct immunofluorescence on Tzanck smear and plucked hair in the diagnosis of pemphigus vulgaris. JPAD 2018; 28:290–295.  Back to cited text no. 4
    
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Sar-Pomian M, Rudnicka L, Olszewska M. The significance of scalp involvement in pemphigus. Biomed Res Int 2018; (12):1–8.  Back to cited text no. 5
    
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Robati RM, Ayatollahi A, Toossi P, Younespour S. Serum angiotensin converting enzyme in pemphigus vulgaris. Indian J Dermatol 2014; 59:348–51.  Back to cited text no. 6
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Wu H, Brandling-Bennett HA, Harrist TJ. Noninfectious vesiculobullous and vesicopustular diseases. In: Elder DE, Elenitsas R, Johnson BL, Murphy GF, Xu X, editors. Lever’s Histopathology of the Skin. 10th ed. Philadelphia, PA: Wolters Kluwer; 2009. 245–54  Back to cited text no. 7
    
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Rao R, Dasari K, Shenoi S, Balachandran C. Demonstration of pemphigus-specific immunofluorescence pattern by direct immunofluorescence of plucked hair. Int J Dermatol 2009; 48:1187–9.  Back to cited text no. 8
    
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Ghanadan A, Saghazadeh A, Daneshpazhooh M, Rezaei N. Direct immunofluorescence for immunobullous and other skin diseases. Expert Rev Clin Immunol 2015; Early online, 1–8.  Back to cited text no. 9
    
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Kabir AN, Das RK, Kamall M. Direct immunofluorescence test of skin biopsy samples − results of 204 cases. Dinajpur Med Coll J 2009; 2:8–12.  Back to cited text no. 10
    
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Khannan CK, Bhat R. A retrospective study of clinical, histopathological and direct immunofluorescence spectrum of immunobullous disorders. Int J Sci Res Pub 2015; 5:2250.  Back to cited text no. 11
    
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Sano SM, Quarracino MC, Aguas SC, González EJ, Harada L, Krupitzki H et al. Sensitivity of direct immunofluorescence in oral diseases.Study of 125 cases. Med Oral Patol Oral Cir Bucal 2008; 13:287–91.  Back to cited text no. 12
    
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Kumaresan M, Rai R, Sandhya V. Immunofluorescence of the outer root sheath: an aid to diagnosis in pemphigus. Clin Exp Dermatol 2011; 36:298–301.  Back to cited text no. 13
    
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Alexandru A, Zurac S, Salavastru CM. Direct immunofluorescence on hair follicles − present and future perspectives. Am J Dermatopath 2013; 35:472–476.  Back to cited text no. 14
    
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Badran F, Moniem EA, Abdo L, Genedy R, Ismail S. Assessment of the role of direct immunofluorescence of the outer root sheath in the diagnosis of pemphigus patients. J Egypt Women Dermatol Soc 2016; 13:83–91.  Back to cited text no. 15
    


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