Direct immunofluorescence study in autoimmune bullous disorders of skin

Background and objectives: This study was taken up to know the utility of application of Direct Immunofluorescence on skin biopsies with particular reference to Autoimmune Bullous disorders. The objectives of the present study was to detect specific patterns of DIF in different Autoimmune bullous disorders, to supplement clinical and histopathological features, and hence for confirmatory diagnosis of these disorders. Methods: DIF using fluorescent labelled antibodies IgG, IgM, IgA and C3 - was carried out in 60 clinically suspected cases of Autoimmune bullous disorders referred to Department of Pathology, KIMS Hospital and Research Center, Bangalore, over a period of 18 months from January 2011 to July 2012. Results: Out of 60 cases analysed, 39 cases were given a final diagnosis as Autoimmune bullous disorder based on clinical, histopathological and DIF findings. DIF positivity was observed in 34 cases. DIF findings correlated with clinical and histopathological findings in 37 cases (94.8%). Interpretation and Conclusion: In this study we found out that DIF serves as a simple, highly sensitive, cost-effective and hence gold standard test for Autoimmune bullous disorders. The technique is essential to supplement clinical findings and histopathology in the diagnosis of these Immunobullous disorders.


Introduction
Immunofluorescence is a histochemical laboratory staining technique used for demonstrating the presence of antibodies bound to antigens in tissue or circulating body fluids 1 .
Coons et al 2 first described the technique of fluorescent antibody studies in 1942, while Burnham et al 3 were the first to report a fluorescent band in the dermoepidermal junction in Lupus Erythematosus. The method used by Coons is known as the 'direct technique', where the antibody was conjugated directly with the fluorochrome.
The technique is essential to supplement clinical findings and histopathology in the diagnosis of Immunobullous disorders. It permits early diagnosis, treatment and subsequent monitoring of disease activity in patients with these lifethreatening diseases 1 . The direct method is the most widely used by most dermatologists for diagnosis of bullous disorders 3 .
It is important to pursue classification as accurately as possible so that useful clinical trends may become apparent, eg. differences in response to therapy 4 .
Considering this, we apply the technique of Direct Immunofluorescence to supplement clinical and histopathological findings in the investigation of Immunobullous disorders.

Method of collection of Data:
A detailed history was taken and clinical examination done of patients and those who were clinically diagnosed to have immmuno bullous disorder were selected and biopsy done from the fresh vesicle for histopathology and from the adjacent skin for DIF.
Biopsy specimen was snap frozen immediately. In case of delay between biopsy and snap freezing, it was refrigerated. Four frozen sections of 4microns taken on a cryostat, placed on a glass slide and fixed in cold acetone. Slides were then overlaid in a moist chamber and incubated for 2hours with fluorescein conjugated antibodies with following specificities: Anti IgG, Anti IgM, Anti IgA and Anti C3. Slides were then washed gently in phosphate buffer saline, mounted in buffer glycerin mixture and examined under fluorescence microscope.
The Immunofluorscence pattern was studied and reported, considering the following parameters: (1) Nature of immune deposits -IgG, IgA, IgM, C3 (2) Site of immune deposits -Dermoepidermal junction / Intercellular spaces in epidermis The immunoreactants include antibodies, Complement components and fibrinogen. Immunoreactants are deposited in 2 main patterns: 1. In the epidermal intercellular space 2. Along the basement membrane zone

Statistics
The findings were tabulated and basic statistics of calculating percentages was used from Master chart windows excel 2000.

Results
A prospective study with 60 cases of clinically suspected Autoimmune Bullous diseases was undertaken over a period of 18 months. .
Out of 60 cases analyzed, 39 cases (65%) were diagnosed as AIBDs based on clinical, histopathological and DIF findings.
There was nearly equal incidence of AIBDs in males and females. 20 males (51.2%) and 19 females (48.8%) were diagnosed as AIBDs and gender distribution in individual AIBDs. Generalised skin lesions were observed in 13 cases (33.3%). The lesions were localized to head and neck, trunk and/or extremities in 26 cases (66.7%). Mucosal involvement was noted in 14 cases, all of which were Pemphigus vulgaris. One of these patients had nasal involvement, while the rest had oral mucosa involved.
Bulla was noted on histopathological examination in 31 out of 39 cases. Papillary micro abscess was seen in 4 cases, which includes 3 cases of DH and 1 case of LAD. (Table 2) Lichenoid infiltrate was seen in 4 cases, including 2 cases each Bullous LE and Bullous EMF. One of the cases showed focal acantholysis with spongiosis and dyskeratosis, which was diagnosed as TAD. One case showed dilapidated brick wall due to acantholysis with subepidermal clefting, typical of HH disease.Out of 31 cases showing bulla on histopathology, most common site of bulla was suprabasal, observed in 15 cases, corresponding to 15 cases of PV, followed by subepidermal bulla in 11 cases and subcorneal bulla in 5 cases.

Discussion
The present study is a prospective study over a period of 18 months. Total of 60 clinically suspected cases of Autoimmune Bullous disorders, were included in the study.
Autoimmune Bullous disorders are a heterogenous group of diseases characterized by antibodies to structural components of the skin and mucous membranes 5 .

Age incidence:
Most Autoimmune bullous disorders occur during the ages of 40 to 60. Our study found a peak in the age group (41-50) years. 72% of the cases are between 31 -60 years in accordance with the Lieferman et al study 6 .
Peak age incidence in Pemphigus vulgaris is between 4 th to 6 th decade as per the study by Vodegel 7 . In our study also peak incidence was between 31 -50 years.

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Bullous pemphigoid is found in older age group, mainly in the elderly, between 50-80 years as reported by Korman N J et al 8 . In our study also most cases were between 41-70 years.

Gender distribution:
Autoimmune disorders did not show sex predilection in the study by Korman N J 8 and Scott J E 9 . In our study also, overall sex distribution in AIBDs was equal, though 3:1 male predominance was noted in Bullous pemphigoid, in Dermatitis herpetiformis 2:12 in accordance with Korman N J study who noted 2:1 male predominance in BP and 1:1 ratio in DH.

Clinical presentation:
Pemphigus vulgaris presents with flaccid blisters, most rupture leaving behind denuded areas 10. A study by Rados J reported 100% cases of PV presenting with mucosal involvement 5 . In our study all except one, who was on remission had mucosal lesions (93%). One of these patients had nasal involvement alone which is a rare observation according to Weedon 11 , while the rest had oral lesions. Pemphigus foliaceous cases showed similar lesions without oral lesions which correlates with Rados J study 5 .  All cases of Bullous pemphigoid showed tense blisters, and all 3 cases of Dermatitis herpetiformis had vesicles with crusted lesions and none had any symptoms of gluten-sensitive enteropathy. This is in accordance with the reports by Nousari H C 12 and Hall R P 13 studies that though all cases have histologic evidence of GSE, only 10%cases of DH aresymptomatic.

Histopathology:
Out of 39 cases, bulla was observed in 31 cases. Subcorneal bulla was noted in 5 cases, which include all 4cases of PF and 1 case of SCPD. Suprabasal bulla was seen in 15 cases, corresponding to all 15 cases of PV. Subepidermal bulla was seen in 11 cases which comprises of 8 cases of BP and one case each DH, EBA and Bullous LE. Only micropapillary abscess without bulla was found in 2 cases of DH and 1 case of LAD, which is a more important diagnostic finding as reported by Rados J study 5 .

DIF positivity:
Of 60 cases for which DIF was performed, 34 were positive, all diagnosed as Autoimmune bullous disorders. 26 cases were DIF negative, out of which 5 were still diagnosed as AIBDs. These include 1case each DIFnegative AIBDs -SCPD, TAD and HH; 2DIF false negative cases -PV and BP. This is in accordance with the studies by Jablonska S 14 and Beutner et al 15

Conclusion
• Autoimmune Bullous disorders are life-threatening diseases demanding early accurate diagnosis with clinical, histopathological and DIF findings.
• Most affect middle age adults with no sex predilection.
• While pemphigus vulgaris was found in middle aged, Bullous pemphigoid was found in older age group, in the elderly.
• DIF positivity in Autoimmune Bullous disorders was found in 87% cases.
• DIF positivity was 93.3% for Pemphigus vulgaris and 100% for Dermatitis herpetiformis, which is in accordance with other studies.
• DIF positivity for Bullous pemphigoid was 87.5%, slightly lower in comparison to other studies, probably due to fewer cases encountered in our study and false negativity.
• inical and histopathological features correlated with DIF findings in 37 of 39 cases -94.8% correlation. The present study reaffirms that apart from new sophisticated tests (immunoblotting, immunoprecipitation, immunoelectron microscopy), the diagnosis of Autoimmune bullous disorders still relies on DIF findings in most laboratories. It is cost effective. Thus DIF has been the gold standard.
It is important to integrate clinical findings, histopathology and DIF in reaching an accurate diagnosis of Autoimmune bullous disorders.