Direct immunofluorescence pattern and histopathological staging in alopecia areata.

AIM: The present study was designed to categorize alopecia areata (AA) into various stages based on histopathology and further study the direct immunofluorescence (DIF) pattern.
MATERIALS AND METHODS: The current study is noninterventional, prospective study on 25 consecutive patients suspected of AA based on clinical assessment. Histomorphologic features and immunoreactivity for IgG, IgM, IgA, and C3, was studied on biopsy material.
RESULTS: Age of the patients ranged from 6 years to 48 years with a mean age of 28.56 ± 21.8 years. Majority of patients, 9 (36%) were in the age group of 21-30 years. Of 25 patients, 13 (52%) were males and 12 (48%) were females. Male: female ratio was 1.1:1. On histopathology majority of cases were in subacute stage 9 (36%), followed by chronic 7 (28%), acute 5 (20%) and recovery stages 4 (16%). Three (12%) of 4 cases showed characteristic swarm of bees appearance. Two (8%) of the cases showed presence of giant cells. Increased numbers of catagen hair were seen in 12 (48%) cases. Of 25 cases, 9 (36%) cases showed positive DIF with granular deposits. The most common immunoreactant was IgG in 7 (28%) cases, followed by IgA in 4 (16%), C3 in 6 (24%) and IgM in 3 (12%) cases. Of 9 cases showing positive staining, 3 (12%) were in acute stage and 2 (8%) each in subacute, chronic and recovery stages.
CONCLUSION: The observations further reiterate that immune mechanisms play a role in the pathogenesis of AA.

INTRODUCTION

Alopecia areata (AA) is a common disorder that often produces sudden patchy hair loss. It is thought to be an organ specific autoimmune disorder. It commonly occurs in association with other autoimmune diseases.[1] AA affects all age groups with equal sex distribution. Frequency of AA ranges from 0.7% to 3.8% of patients attending dermatology clinics.[2] The lifetime risk of acquiring AA is approximately 1.7%.[1] Family history of the disease is found in 10-20% of patients.[34]

The diagnosis is usually made on clinical grounds. In some cases, the diagnosis is elusive, and biopsies are necessary. In other cases, biopsies are useful from prognostic point of view to determine whether there are enough follicles left for future regrowth.[3] Direct immunofluorescence (DIF) studies have reported deposits of C3, IgG, IgM in varying combinations along the basement membrane zone (BMZ) of hair follicle (HF) in AA.[5678] The purpose of this study is to evaluate the involvement of immune mechanisms in AA.

MATERIALS AND METHODS

The present study was conducted in the Department of Pathology, in collaboration with the Department of Dermatology and Venereology. The study included 25 patients suspected of AA on clinical assessment. The study population was screened for connective tissue disorders and autoimmune diseases. Patients with connective tissue disorders and autoimmune diseases were excluded from the study. An informed consent was obtained from all the patients included in the study. Twenty-five skin biopsies from nonalopecia subjects without any autoimmune disease acted as control for DIF. Four mm punch biopsy was obtained from the margin of alopecic area. The biopsy was received in normal saline or Michele's medium. For immunofluorescence the biopsy was embedded in Cryomatrix medium (Shandon), frozen in cryostat (Model: Crytotome, Make: Shandon, UK). Sections of 4-5 mm were cut and layered onto poly-L-lysine coated slides. The slides were stored at −20°C until being stained.[9] Fluorescein isothionate labeled monospecific immunoglobulins to human IgG, IgM, IgA and C3 were applied. The remaining specimen was put in 10% buffered formalin for histopathological processing by paraffin embedding method.

Diagnosis of acute AA was made when mild to moderate peribulbar lymphocytic infiltrate was present with or without hair follicular pigment incontinence and dysmorphic hair. Subacute stage was reported when there were increased numbers of catagen hair along with some inflammation. Chronic AA was diagnosed when miniaturised HFs and/or fibrous stelae were present with a variable inflammation. Stage of recovery was diagnosed when there was minimal inflammation with lack of other features. Furthermore, in the recovery stage, the terminal to vellus ratio was normal, and the percentage of anagen hair increased.

RESULTS

Age of the patients ranged from 6 years to 48 years with a mean age of 28.56 ± 21.8 years. Majority of patients, 9 (36%) were in the age group of 21-30 years. Of 25 patients, 13 (52%) were males and 12 (48%) were females. Male to female ratio was 1.1:1. All the 25 (100%) cases presented with a complaint of loss of hair over the scalp and combined scalp and other body sites such as eyebrows in 2 (8%) cases. Nail abnormalities were present in 3 (12%) cases, 2 females and 1 male. The abnormalities included brittleness in 2 (8%) cases and longitudinal ridges in 1 (4%) case. History of atopy was present in 2 (8%) cases. Family history of diabetes mellitus, hair loss and hypothyroidism was present in 8 (32%), 3 (12%) and 1 (4%) case respectively in first-degree relatives.

Based on the histopathologic findings AA was categorized into 4 stages; acute, subacute, chronic and recovery stage. Majority of cases were in subacute stage accounting for 9 (36%), followed by chronic stage 7 (28%), acute stage 5 (20%) and recovery stage 4 (16%). Lymphocytic infiltrate was present in all the 25 (100%) cases and was graded as minimal, mild and moderate. Lymphocytic infiltrate was minimal in 11 (44%), mild in 10 (40%) and moderate in 4 (16%) cases. Three (12%) of 4 cases showing moderate lymphocytic infiltrate, showed characteristic swarm of bees appearance [Figure 1]. Two (8%) of the cases showing moderate lymphocytic infiltrate also showed the presence of giant cells. Pigment incontinence of hair bulbs was seen in 11 (44%) cases. Three (12%), 5 (20%), 2 (8%) and 1 (4%) cases were in acute, subacute, chronic and recovery stages respectively. Increased numbers of catagen hair were seen in 12 (48%) cases [Figure 2]. They were seen in subacute and chronic stages in 8 (32%) and 4 (16%) cases respectively. Dysmorphic hair were seen in 7 (28%) cases. Four (8%), 1 (4%) and 2 (8%) cases were in acute, subacute and chronic stages respectively. Eosinophils were seen occasionally in 1 (4%) case, of acute stage. Miniaturized hair were seen in 2 (8%) cases. Both were in the chronic stage. Follicular stelae were seen in 5 (20%) cases. Four (16%) and 1 (4%) case were in chronic and subacute stages respectively.

Figure 1

Perifollicular lymphocytic infiltrate resembling a swarm of bees in acute stage of alopecia areata (H and E, ×100)

Figure 2

Photomicrograph of subacute alopecia areata showing increased number of catagen hair (H and E, ×100)

Of 25 cases 9 (36%) cases showed positive DIF results [Table 1]. All controls were negative. Of 9 positive cases 4 (16%) were males, and 5 (20%) were females. In all positive cases, deposits were granular. The most common immunoreactant was IgG in 7 (28%) cases [Figure 3]. The deposits were located at intercellular spaces of the epidermis (EC) in 4 (16%), BMZ in 3 (12%) and HF in 3 (12%) cases. IgM was least common accounting for 3 (12%) cases. In these cases, the deposits were located at both intercellular spaces of epidermis and HF [Figure 4].

Table 1

Morphologic categories of AA with their DIF staining pattern

Figure 3

Direct immunofluorescence showing focal granular positivity of IgG at intercellular spaces in epidermis and hair follicle (Direct Immunofluorescence, ×200)

Figure 4

Intercellular spaces of epidermis and hair follicle showing focal granular positivity of IgM in alopecia areata (Direct Immunofluorescence, ×100)

IgA was positive in 4 (16%) cases. The deposits were located at intercellular spaces of the epidermis in 3 (12%) and BMZ in 1 (4%) case. C3 was positive in 6 (24%) cases. Deposits were located at intercellular spaces of the epidermis in 4 (16%) and BMZ in 2 (8%) cases. Of 9 cases showing positive staining, 3 (12%) were in acute stage and 2 (8%) cases each in subacute, chronic and recovery stages.

DISCUSSION

Alopecia areata is a chronically relapsing inflammatory disorder, like most other autoimmune diseases which suggests a cyclic recurrence of disease-promoting events.[10] Exact etiology of AA is not known but evidence exists to support genetic, immune and environmental factors. The development of hair loss involves aberrant modulation of the hair growth cycle.[2] Collapse of the anagen hair bulb immune privilege may play a crucial part in the pathogenesis of AA.[11]

The earliest evidence that autoimmunity may play a role in the pathogenesis of AA stems from observed association between classical autoimmune disorders and AA, in particular vitiligo and thyroid diseases. The efficacy of immunomodulatory agents in the treatment further implicates immune mechanisms in the disease development. Associations between specific human leukocyte antigen genes and AA lend further circumstantial evidence that autoimmune mechanisms are involved in the pathogenesis.[121314] While the studies strongly support the hypothesis that AA is an autoimmune disease, direct evidence is currently lacking.[12]

The histopathologic picture varies are depending on disease duration. A peribulbar lymphocytic infiltrate “swarm of bees” characterizes the acute phase of AA. In subacute cases, large numbers of catagen and telogen hairs will be present. HF miniaturization with minimal or no inflammation is seen in chronic cases accompanied by a decrease in follicle density.[212131415] Histopathological features observed in the present study are consistent with the other studies.[23151617] Histopathologic features suggest an immune mechanism in the pathogenesis as all the clinically diagnosed cases of AA showed a variable degree of peribulbar lymphocytic infiltrate. Two cases showed presence of giant cells as observed in other studies.[314] The characteristic presence of lymphocytes around lesional HFs has served as evidence that abnormal immune responses may be involved in AA. The infiltrate consists predominantly of activated T lymphocytes along with macrophages and Langerhans cells. A peribulbar lymphocytic infiltrate is the expected histologic feature, but it is absent in many scalp biopsy specimens. Other diagnostic criteria are hence needed.[16] In the present study, presence of occasional eosinophils was seen in only 1 (4%) case. In a study by Elston et al.[18] eosinophils were present in 38 of the 71 cases and were present in 13 of 27 cases that lacked peribulbar infiltrate.

A role for humoral immunity in AA has also been investigated, producing disparate results. Heterogeneous auto-antibodies to HFs have been reported, but they are not specific for any one antigen and target multiple structures in anagen HFs.[5] Immunofluorescence is a valuable auxiliary diagnostic tool, detects in situ and circulating immune deposits that may be involved in the pathogenesis of skin diseases.[19]

The present study was performed to assess if positive DIF was a consistent finding in patients of AA in view of the limited literature on DIF studies.[68] Variable results on immunofluorescence ranging from no deposition of immunoglobulins to deposition in 11 of 12 cases (92%) have been reported by other researchers.[678] In the current study, of 25 cases 9 (36%) showed positive results. The most common immunoreactant was IgG in 7 (28%) cases. However, in a study by Bystryn et al.[6] C3 was the commonest immunoreactant. Among the positive cases in our study, 7 (28%) were positive for more than one immunoreactant. IgA was positive in 4 (16%) cases in our study. However, isolated IgA positivity was not seen in any of the cases. Within biopsies of clinically normal areas of the scalp in AA patients, moderate linear junctional staining for IgA was found in a patient in another study.[8] The IgA immunopositivity was seen in the BMZ and epidermis without deposits in the HFs in our study. Such an event is difficult to explain. Inconsistent deposition (IgM and C3) along the basement membrane and intercellular areas of the epidermis without immunodeposits in the HF has been reported in a previous study.[6] Immunoglobulin deposition has been shown to be an infrequent finding in patients without the disease.

To conclude, abnormal deposits of immunoglobulins and C3 are often present at intercellular spaces of the epidermis, HFs and BMZ of patients with AA. It must be emphasized that there is an abnormal accumulation of immunoglobulins and C3 deposits in AA and the staining reaction is more common than is observed on DIF of normal skin.[20] The observations of the present study are consistent with the concept that immune mechanisms play a role in the pathogenesis of AA.