Role of direct immunofluorescence on outer root sheath of hair follicle in monitoring disease activity of pemphigus vulgaris

Direct immunofluorescence; Hair follicle; Outer root sheath; Pemphigus vulgaris Background: Since the outer root sheath of hair follicle is structurally analogous to epidermal keratinocytes, direct immunofluorescence of hair may be an alternate method for monitoring disease activity of pemphigus vulgaris. Our objective was to find the effectiveness of direct immunofluorescence of outer root sheath as an alternate method in monitoring patients with pemphigus vulgaris.


INTRODUCTION
The bullous diseases have a history as old as that of medicine.In the early 1950's, Lever was able to differentiate most of these by using histological criteria.Pemphigus vulgaris (PV) is a very serious skin disease that causes blisters.Pemphigus vulgaris affects 0.1-0.5 patients per 1,00,000 population per year 1 and 0.58-0.80 per 1,00,000 person each year in the UK. 2 Incidence of PV is increasing and the reasons for the changes in incidence are not clearly understood but have implications for identifying causative factors. 2 Most cases develop in people aged over 50 years.Men and women are equally affected.It is more common mucosal areas, or in children. 10

MATERIALS AND METHODS
This study was carried out at the Department of pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka and in the Dermatology Department of BSMMU, Dhaka during the period of September'2012 to August' 2014.Approval of the study protocol by the Institutional Review Board of Bangabandhu Sheikh Mujib Medical University, Dhaka was obtained.
A total number of 16 clinically, histologically and immunologically diagnosed cases of pemphigus vulgaris on clinical remission patients were included in this study.The patients were selected from recorded data of DIF on pemphigus vulgaris of Pathology Department of BSMMU and they were followed up in the out-patient Department of Dermatology of BSMMU.The patient was considered to be remission when there was no mucosal or cutaneous lesion, including blisters, erosions, crusts, or vegetative in certain groups of people -those from the Mediterranean, Indian subcontinent or of Jewish origin.
Large flaccid bullae develop on the oral mucosa, face, scalp, central chest, and inter-triginous zones in older individuals.Oral lesions are the first manifestation in 10% to 15% of patients and almost invariably develop during the course of the disease. 3mphigus is a group of autoimmune blistering disease of skin and mucous membranes which are characterized histologically by intraepidermal blisters due to acantholysis. 4Acantholysis was first demonstrated as the characteristic feature of the bullae of pemphigus in 1943. 5The autoantibodies in pemphigus are directed against desmosomal proteins both desmoglein (Dsg) 1 and desmoglein (Dsg) 3 resulting in acantholysis, loss of cohesion between keratinocytes and the appearance of bullae.Complement fixation may potentiate the acantholysis.The outer root sheath (ORS) of the hair follicle is structurally analogous to epidermal keratinocytes. 6Pemphigus antigens are distributed throughout the ORS and in the dermal bulb matrix cells. 6Pemphigus-specific immunofluorescence pattern seen in the skin has been demonstrated in the ORS of a plucked hair follicle. 7It characteristically shows the deposition of immunoglobulin G (IgG) with or without complement (C3) in the intercellular region of the epidermis.As the acantholytic process extends into the wall of the hair follicle, immune reactants should also be demonstrable in the follicular wall.Dsg 3 is responsible for anchoring the hair to the follicle.The increase in volume of the target antigen (Dsg 3 and Dsg 1) in the follicular epithelium could be a factor in determining scalp involvement in pemphigus.The gold standard for the demonstration of tissue-bound auto-antibodies is direct immunofluorescence (DIF) of perilesional skin. 8DIF pattern of ORS in cases of PV resembles the DIF pattern of the peri-lesional skin. 9The sensitivity of hair DIF in PV patients was 100%, similar to that of the cutaneous DIF. 10 Negative direct immunofluorescence (DIF) has been repeatedly shown to be a better predictor of immunologic remission compared with indirect immunoflurescence. 11taining a sample for DIF from oral or skin biopsy is a relatively invasive and unpleasant procedure to the patient.The need for repeating the test until positive results are obtained further complicates the problem; many patients are reluctant to accept it.Hence finding a less-invasive way for collecting a suitable substrate would be of much help.Recently, Schaerer and Trueb et al 7 showed that ORS of plucked hair could be used as substrate for DIF in the diagnosis of pemphigus.The value of this method was proven in further studies on patients with untreated or relapsed pemphigus. 12Using an alternative substrate for DIF instead of skin or mucosa would be beneficial because it eliminates the need for multiple biopsies in pemphigus patients especially in circumstances of difficult biopsies like   Direct immunofluorescence on hair follicle in pemphigus vulgaris lesions for at least 3 months while the patient was receiving maintenance therapy with oral prednisolone.
Fulfilling the criteria the patient was selected for collection of specimen.Skin biopsy and plucked scalp hair samples were obtained for direct immunofluorescence stain.Hairs were plucked from the scalp using a rubber tipped artery forceps after applying spray anaesthetic.About five scalp hairs were selected and transported in normal saline and processed for DIF without sectioning.First, the hair samples were taken on a Petri dish and then washed in phosphate buffered saline (PBS) for three times, each wash taking ten minutes.Then the hair samples were placed on a ring glass slide and they were incubated with Fluorescein isothiocyanate conjugated (FITC) rabbit anti-sera against human IgG and complement 3 (Dako, Glostrusp, Denmark) for at least 30 minutes.At the end of the procedure they were washed again in PBS, with three cycles of fifteen minutes each and that time, wash was done over ring glass slide.At that time extra care was taken so that the hair would not float away from the slide.
To avoid this, ring slides were placed on a clean plastic box which was kept on slightly slanting position on the table.After washing, the slides were mounted with buffered glycerin mountant and cover slipped and examined under fluorescence microscope (Olympus BX51).
Full thickness of skin with small subcutaneous tissue as required, by elliptical excisional biopsy specimen was also taken after application of 2% lidocaine local anesthetic.Specimen was taken in normal saline and after quick freezing; 4-5 micrometer thick sections were cut in the cryostat (Minotome, International Equipment Company, Damon, USA) at -20 0 C and were taken into glass slides.These were air-dried and kept at -20 0 C until staining was performed.Staining procedure of skin was same as that of conventional DIF procedure.During this procedure DIF of skin also performed in an equal number of controls.Positive controls were taken from known positive cases and negative controls were taken from other dermatoses.
Result of DIF of skin and ORS of plucked hair was recorded by the same observer and the intensity was arbitrarily graded on a scale from (+) to (+ + +), or negative (-).Statistical analysis was carried out by using the Statistical Package for Social Sciences (SPSS) version 16. negative (fig.6).One case showed false negative result as ORS of hair follicle was negative (fig.7) but positive on skin DIF (fig.8).Two patients were negative on both hair and skin DIF test.On DIF of skin for C3, 3(18.25%)patients had weakly positive and 13(81.25%)had negative result.DIF of hair for C3 of the study patients showed negative result in all (100%) cases.
Table 1 shows DIF of skin (IgG) and hair (IgG) of the study patients, it was observed that three fourth (75.0%) patients had weakly positive, one (6.25%)had moderately positive and three (18.75%)had negative on DIF of both skin (IgG) and hair(IgG).
Conventional DIF is known as the best method for assessing immunologic remission in PV and was used as the gold standard in statistical analysis with which hair DIF was compared.Out of 16 patients with pemphigus in clinical remission 02 showed true negative result on DIF e.g.DIF was negative both in skin and hair.Twelve patients were true positive e.g.DIF was positive in both skin and hair.One of them was false positive e.g.DIF was positive in hair but negative in skin.One of them was false negative as DIF

RESULTS
A total of 16 patients clinically, histologically and immunologically diagnosed cases of pemphigus vulgaris on clinical remission were included in this study and scalp hair and perilesional skin biopsy were taken and DIF was done to compare both.A slight preponderance of male patient was observed in the study with male female ratio of 1.7:1.The age distribution of patients varied between 23 to 72 years with mean age of 38.94(SD=12.28)years.The main age group involved was fourth decade.Seven cases (43.75) belonged to this group.
On direct immunofluorescence test of hair (IgG), among the 16 patients 13 showed intercellular IgG deposition in outer root sheath of hair follicle DIF (fig.1).Three cases showed no such IgG deposition in ORS of hair follicle (fig.2).On DIF test of skin (IgG), among the 16 patients 13 showed lace like deposition of IgG in squamous intercellular space of epidermis (fig.3).Similarly 3 cases showed no such IgG deposits (fig.4).Twelve patients were positive in both hair and skin DIF.There was one case with false positive as ORS of hair follicle DIF was positive (fig.5) but skin DIF was was negative in hair but positive in skin.Table 2 shows the distribution of frequency of hair and conventional direct immunofluorescence in patient with pemphigus in clinical remission.
Direct immunofluorescence of hair evaluation for patients with pemphigus vulgaris in clinical remission is described in table 2. There were 12 true positive cases, 1 false positive case, 1 false negative case and 2 true negative cases in identification by DIF of skin.The results of the interpreter analysis are Kappa = 0.590 with p<0.05 (0.018) with moderate agreement and statistically significant.
Sensitivity of hair DIF (i.e. the percentage of patients with positive conventional DIF whose hair DIF was positive), specificity of hair DIF (i.e. the percentage of patients with negative conventional DIF whose hair DIF was negative), positive predictive value of hair DIF (ie, the percentage of patients with positive hair DIF whose conventional DIF was also positive), and negative predictive value of hair DIF (i.e. the percentage of patients with negative hair DIF whose conventional DIF was also negative) were calculated (Table 3).Positive predictive value enables us to tell how likely it is that the patient is not in immunologic remission if his/ her hair DIF is positive.Negative predictive value enable us to tell how likely it is that the patient is in immunologic remission if his/her hair DIF is negative.

DISCUSSION
The goal of therapy in pemphigus is to achieve clinical and immunological remission.Clinical remission is achieved in most patients with immunosuppressive agents.The most difficult management decision is how to maintain remission with the least number of medications.Although many pemphigus authorities rely on clinical status to discontinue treatment, assessing immunologic remission by DIF has its own advocates.Actually, many studies have shown that the rate of relapse in patients in clinical remission after discontinuing therapy was higher when DIF was positive. 13,14o the treatment should be continued in the patients with positive DIF results.On the other hand, repeated negative DIF during remission is a possible marker for apparent cure of the disease, and treatment can be stopped in such group of patients. 11Patients in remission may refuse to undergo repeated skin biopsies.The present study was undertaken with the goal to find out an alternate reliable source of substrate for DIF test in monitoring of pemphigus vulgaris.
In this study the main age group involved was fourth decade.
Seven cases (43.75) belong to this group.The age range was 23 to 72 years with mean age of 38.94 (SD = 12.28) years.Male female ratio was 1.7:1.These finding differ from Raoet al 15 and Daneshpazhooh et al 16 where mean age was around 45 and female was more than male.This difference may be due to small sample size.
In this study on direct immunofluorescence test of hair (IgG), among the 16 patients 13 (81.25%)were positive and 3(18.75%) were negative.On DIF test of skin (IgG), among the 16 patients 13 (81.25%)were positive and 3 (18.75%)were negative.Twelve patients were positive in both hair and skin DIF considered as true positive.There was one false positive case whose hair DIF was positive but skin DIF was negative.One patient was false negative who was negative on hair DIF but positive on skin DIF.Two patients were true negative i.e. negative on both hair and skin DIF test.Schaerer and Trueb 7 first demonstrated the practicability of using DIF on plucked hair, and they were able to demonstrate the presence of the pemphigus specific DIF pattern in the ORS of all of their 15 (100%) pemphigus patients.Rao et al 15  least medication.Long-term remission of all therapy has been reported to occur in 10 -75% of the patients. 17he differentiation between drug induced clinical control of the disease in the presence of autoantibodies, from immunological inactivity can help us to detect the subgroup of patients who maintain clinical remission after discontinuation of treatment.However immunological inactivity of the disease may also be drug induced and relapse may occur after discontinuation of therapy even in these patients.Furthermore, immunological remission of disease after discontinuation of drugs does not necessarily indicate a cure.According to a previous report, many patients with negative DIF findings (73.3%) remained in clinical remission after discontinuation of treatment.All patients with positive DIF findings during remission had a relapse within 3 months after discontinuation of treatment. 13us, DIF findings may be indicative of either further regression or eminent exacerbation.Therapy should be continued in patients with positive DIF results to avoid relapse. 14On the other hand, treatment can be stopped in those groups of patients showing repeated negative DIF during remission which indicates apparent cure of the disease.David et al 11 studied 24 PV patients and treatment was discontinued in seven patients with repeated negative results of DIF.One out of seven patients (14%) relapsed in a 14 months follow-up period.
Patients need repeated DIF testing during diagnosis and in the follow up after clinical remission to be sure of immunological remission.DIF of skin is still the gold standard for the diagnosis of pemphigus vulgaris.But patients in remission stage may don't agree to do repeat skin biopsies as it is an invasive procedure.DIF of hair may be an ideal choice of substrate as it is a simple and non-invasive test with an equal degree of sensitivity and specificity.

CONCLUSION
This study proved the value of plucked hair as an appropriate substrate for DIF for the disease monitoring of PV.In addition to its relatively high sensitivity DIF of hair is a simple, specific and noninvasive test, and provides the opportunity to avoid repeated skin biopsies in patients with pemphigus vulgaris.

ACKNOWLEDGMENTS
Our sincere gratitude all the patients whose sample has helped conduct us this study.We would also like to thank all the faculties of department of pathology and dermatology and technical staffs for their support.My deepest regards and sincerest gratitude to respected teacher and guide Professor Ashim Ranjan Barua, department of pathology, BSMMU, Dhaka, for all his able guidance and whole hearted co-operation in making this study a success.

Figure 2 :Figure 4 :Figure 1 :Figure 3 :
Figure 2: Hair stained by DIF showing no deposition of intercellular IgG in outer root sheath of hair follicle (x 200)

Table 3 : Sensitivity, specificity, accuracy, positive and negative predictive values of DIF of hair evaluation for patients with pemphigus vulgaris in clinical remission
DOI : 10.3126/jpn.v7i2.17994 16und 85% DIF positivity in the ORS of plucked anagen hair.Daneshpazhooh et al16reported 91% DIF positivity in ORS of plucked hair from 110 patients with PV.In our study, the DIF pattern in the ORS of scalp hair was 81.25% which is almost similar with other studies.
Pemphigus vulgaris can be controlled with treatment in most instances but difficult to maintain control with the DOI : 10.3126/jpn.v7i2.17994Nessa M et al.