Comparison of Direct Immunofluorescence of Plucked Hair and Skin for Evaluation of Immunological Remission in Pemphigus : Indian Dermatology Online Journal

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Comparison of Direct Immunofluorescence of Plucked Hair and Skin for Evaluation of Immunological Remission in Pemphigus

Rai, Reena; Harikumar, Manu V.

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Indian Dermatology Online Journal 8(5):p 319-322, Sep–Oct 2017. | DOI: 10.4103/idoj.IDOJ_280_16
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Pemphigus is a chronic autoimmune bullous disorder characterized by autoantibodies directed against desmoglein (Dsg) 3 and/or 1.[1] Direct immunofluorescence (DIF) of the skin or mucosa is considered as the gold standard in the diagnosis of pemphigus. Systemic steroids alone or in combination with other immunosuppressive agents are the main stay of treatment, and long-term administration of the same is associated with significant adverse effects, morbidity, and mortality.

Therefore, a system is required to monitor disease activity to lower the dosage of the drugs and eventually withdraw treatment. Various methods for assessment of immunological remission include DIF, indirect immunofluorescence, and anti-desmoglein enzyme-linked immunosorbent assay (ELISA) titres.[234567]

DIF of the skin or mucosa is an invasive and expensive procedure and the patient may not be willing for the same. Recently, 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 85–100%.[891011]

Hence, DIF of hair may be an ideal substrate for assessment of immunological remission because it is a simple, non-invasive, and cost effective procedure. We conducted this study to compare the DIF of plucked hair and skin for the evaluation of immunological remission in pemphigus vulgaris patients in clinical remission.

Materials and Methods

After obtaining approval from the institutional ethics committee and consent from the patients, a prospective study was conducted in the Department of Dermatology in the PSG institute of medical sciences and research (PSG IMS&R) over a period of 1 year. A total of 30 patients with pemphigus vulgaris who showed intercellular deposits of IgG antibodies against Dsg3 and/orDsg1 and/or Complement 3 (C3) in the DIF of skin and hair during the active stage of disease fulfilling the following inclusion criteria were included in the study: (a) patients in clinical remission, defined as those who had no new or non-healing skin or mucosal lesions for the past 6 months or more and (b) with or without daily prednisolone dosage equal to or less than 10 mg with or without adjuvant immunosuppressive therapy such as azathioprine 50mg or cyclophosphamide 50 mg daily.

Patients with new or non-healing skin or mucosal lesions in the preceding 6 months and patients with other bullous disorders were excluded from the study.

The presence of lace-like pattern in DIF in the ORS of the hair follicle and in the epidermis was considered as positive [Figure 1].

Figure 1:
Fish net-like pattern of immunofluorescence in the outer root sheath of hair on DIF

Skin and hair DIF was performed in all the patients. The skin sample was obtained from the upper trunk. Trichogram was done and anagen hair was collected. The samples were transported in the Michels medium to the Department of Pathology in PSG IMS&R and subjected to DIF staining with fluorescent isothiocyanate (FITC) labeled rabbit anti IgG and C3 antibodies. The samples were washed in PBS three times, each wash taking 10 minutes. Following which they were incubated with FITC labeled anti-IgG and anti-C3 for 1 hour at 37°C and once again washed in PBS three times for 10 minutes each, and then air dried and mounted on slide with PBS-glycerol solution for examination. The PBS contained propidium bromide as a counterstain.

Patients demographic parameters, phenotype of pemphigus vulgaris, history of scalp involvement, duration of disease, duration of remission, and past and current treatment were recorded.


Patient characteristics and hair and skin DIF findings are shown in Tables 1 and 2, respectively. The age of patients in our study ranged from 17–69 years, and majority (33.3%) of the patients were in the age group of 41–50 years. All the 30 patients had a history of scalp involvement. Of the 30 patients, DIF of skin was positive in 10 patients and hair was positive 14 patients. The findings of hair and skin DIF correlated with each other in 22 patients. In 6 patients, DIF of hair was positive and skin was negative. Whereas in 2 patients, DIF of the skin was positive and hair was negative, as seen in Table 2.

Table 1:
Patient characteristics
Table 2:
Hair and skin DIF skin findings in pemphigus patients in clinical remission

The skin DIF was considered as the gold standard, and the sensitivity and specificity of hair DIF was calculated. The sensitivity (true positive/true positive + false negative) of hair DIF was 80% and specificity (true negative/true negative + false positive) was 70%. There was a statistically significant association between skin and hair DIF positivity with a P value of 0.010 [Table 2]. Positive predictive value of hair DIF (true positive/true positive + false positive), which refers to the percentage of patients with a positive test who actually have the disease was 57.14%, and negative predictive value (true negative/true negative + false negative), which refers to the percentage of patients with a negative test who do not have the disease was 87.5%.

A total of 18 patients were undergoing treatment in our study. In these patients, the hair and skin DIF was negative in 10 and 13 patients, respectively, which was higher than that in patients not undergoing treatment. However, the findings were not statistically significant. There was no significant association between hair DIF and duration of disease, remission, and treatment.


Desmogleins are the main target antigens in pemphigus. These desmogleins are calcium dependent transmembrane adhesion proteins belonging to the cadherin family. In the epidermis and hair, the distribution of desmogleins correlates with the degree of differentiation. Desmoglein 1 is expressed in the suprabasal cells of the epidermis and inner root sheath as well as the innermost layers of the ORS of the hair follicle.[12] Desmoglein 3 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 its confined to the basal layer of ORS.[12] Desmoglein 3 is also responsible for anchoring the telogen hair in the follicle.[13]

Wilson et al. in 1991 demonstrated that the human hair follicle is rich in the target antigens of pemphigus.[14] In 2003, Schaerer and Trueb first reported positive DIF findings in the ORS and the matrix of hair follicle in 100% of pemphigus patients.[8] Subsequently, Rao et al. reported 80% positivity and Daneshpazhooh et al. reported 91% positivity of DIF in the anagen hair.[910] Kumaresan et al. and Tanasilovic et al. also demonstrated positive DIF findings in 100% of their patients with active pemphigus in ORS of both anagen and telogen hair.[1115]

David et al. in 1989, based on their study suggested that repeated negative DIF in pemphigus patients on clinical remission could be a sign of immunological remission.[16] Similar findings were also reported by Balighi et al. and Ratnam et al.[617]

Ratnam et al. also noted that the patients with positive DIF findings during clinical remission had a significantly higher relapse rate after the discontinuation of treatment.[17]

Various studies have shown that the rate of relapse was approximately 44–100% in patients with positive DIF findings during remission and 13–27% in patients with negative DIF.[1618]

Rao et al. in 2012 conducted a study to assess the role of hair DIF in monitoring the disease activity in pemphigus; they suggested that, in patients in clinical remission, DIF of hair could be an ideal substrate for assessment of immunological remission.[19]

Only limited studies are available till date to analyze the role of hair DIF for the assessment of immunological remission. Apart from the study published by Rao et al., one study by Daneshpazhooh et al. in 2013 demonstrated the usefulness of hair DIF in the assessment of immunological remission in pemphigus patients.[20]

In their study, the sensitivity and specificity of hair DIF was 79% and 48%, respectively. Similarly, our study had a sensitivity of 80%, however, the specificity was 70% which is comparatively higher.

Even though skin or mucosal DIF is considered as the ideal substrate for the assessment of immunological remission, 6 patients in our study had positive hair DIF even though skin DIF was negative, as seen in Table 1. Thus indicating that these patients were not yet in immunological remission. Had the clinician relied only on the DIF of skin, this finding could have been missed. The sensitivity of hair DIF in our study was not high enough to suggest it as an alternative to skin DIF for the assessment of immunological remission. However, hair DIF, being a non-invasive procedure can be used initially for patients in clinical remission to assess immunological status and if it is positive then the DIF of skin could be avoided.


Hence, DIF of hair is a simple, non-invasive, and cost effective procedure, and can be used as an additional procedure for assessment of immunological remission in pemphigus vulgaris. However, further studies are needed to validate the same.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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Hair immunofluorescence; immunological remission; pemphigus

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