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Utility of Direct Immunofluorescence Studies in Subclassification of Autoimmune Sub-Epidermal Bullous Diseases: a 2-Year Study in a Tertiary Care Hospital

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Utility of Direct Immunofluorescence Studies in Subclassification of Autoimmune Sub-Epidermal Bullous Diseases: a 2-Year Study in a Tertiary Care Hospital Original Article doi: 10.5146/tjpath.2015.01345 Utility of Direct Immunofluorescence Studies in Subclassification of Autoimmune Sub-Epidermal Bullous Diseases: A 2-Year Study in a Tertiary Care Hospital Supriya JAIN1, Vijaya BasavaRaj2, Manjunath GubbaNNA VimaLA2 Department of Pathology 1Bharati Vidyapeeth Deemed University Medical College and Hospital, SANGLI, INDIA and 2JSS Medical College (JSS University), MYSORE, INDIA ABSTRACT Objective: Sub-epidermal bullous disorders belong to immunobullous diseases which develop as a result of autoantibody action against epidermal basement membrane proteins. Clinically, they are tense bullae and do not rupture easily. They are classified into various forms based on histopathology and direct immunofluorescence patterns. This study was undertaken to assess the incidence of various sub-epidermal bullous disorders and the utility of direct immunofluorescence in accurately classifying them, and to study the intensity and pattern of immunofluorescence in various sub-epidermal bullous disorders Material and Method: A 2-year study of 38 cases of sub-epidermal bullous disorders sent for direct immunofluorescence studies formed the study group. The specimens were processed as per standard protocols. The clinical details were obtained from case files and requisition sent for histopathological and direct immunofluorescence studies. Results: Thirty-eight patients were diagnosed to have sub-epidermal bullous disorders over the period of 2 years. Twenty five cases of Bullous Pemphigoid, 5 cases of Dermatitis Herpetiformis, 3 cases of Linear IgA Bullous disorder, 2 cases of Bullous Systemic Lupus Erythematoses and 1 case each of Epidermolysis Bullosa Acquisita, Cicatricial Pemphigoid and Pemphigus Gestationis was diagnosed. Positive direct immunofluorescence was seen in 91.3% of the cases. Conclusion: Histopathology alone cannot classify sub-epidermal bullous disorders and direct immunofluorescence studies are mandatory in all of them. Bullous Pemphigoid needs to be distinguished from Epidermolysis Bullosa Acquisita which requires Salt split direct immunofluorescence studies. Dermatitis Herpetiformis, Bullous Systemic Lupus Erythematosus and Linear IgA Bullous disorder show more or less similar histological picture with neutrophilic microabscess. Direct immunofluorescence studies help in the majority of cases but further testing such as immunoblotting, immunoelectron microscopy or indirect immunofluorescence becomes essential in cases with overlapping features. Key Words: Direct immunofluorescence, Bullous skin diseases, Bullous Pemphigoid, Dermatitis Herpetiformis INTRODUCTION of immunoglobulins and/or complement at the basement membrane zone, with the exception of DH, where the Sub-epidermal bullous disorders (SEBDs) are characterized deposits are seen in the dermal papillae. by autoantibodies specifically against the components of the epidermal basement membrane zone (1). SEBDs MATERIALS and METHODS embrace the following entities: Bullous Pemphigoid (BP), The study was conducted during a 2-year period. Two 5 Cicatricial Pemphigoid (CP), Epidermolysis Bullosa mm punch skin biopsies, one in 10% formalin and the Acquisita (EBA), Dermatitis Herpetiformis (DH), Linear other in normal saline, were sent from patients with a IgA Bullous Dermatoses (LABD) and Bullous Systemic suspected clinical diagnosis of vesiculobullous disease Lupus Erythematosus (SLE). to the department of pathology for both histopathology SEBDs are characterized clinically by the presence of and DIF studies, respectively. Biopsy specimens sent in cutaneous and/or mucosal blisters on a hemorrhagic or formalin were processed and serial haematoxylin and eosin non-hemorrhagic base, associated with intense pruritus. sections were analyzed. The biopsy for DIF studies was sent Histology shows all SEBD’s characterized by sub-epidermal in normal saline and was processed as follows. Specimens split with inflammatory infiltrate in the bullous cavity. were embedded in a cryomatrix embedding medium Direct immunofluorescence (DIF) studies show deposition and snap-frozen at -400C until sectioned. Cryostat tissue (Turk Patoloji Derg 2016, 32:91-98) Correspondence: Supriya JAIN Department of Pathology, Bharati Vidyapeeth Deemed University Medical Received : 06.09.2015 Accepted : 30.11.2015 College and Hospital, SANGLI, INDIA E-mail: [email protected] Phone: +91 998 699 99 68 91 Turkish Journal of Pathology JAIN S et al: DIF Studies in Subepidermal Bullous Diseases sections of 4 microns were air-dried and washed thrice with RESULTS phosphate-buffered saline (PBS), pH 7.4, for 10 minutes Thirty-eight patients were diagnosed as SEBD over the before being overlaid for 30 minutes with fluorescein period of 2 years. Twenty five cases were of BP (65.7%), isothiocyanate-conjugated rabbit anti-human IgG, IgA, 5 cases were of DH (13.1%), 3 cases were of LABD (7.8%), 2 IgM and C3. Sections were then incubated in a humidified cases were of Bullous SLE (5.2%), and there was 1 case each chamber at room temperature, washed thrice with PBS at of EBA, CP and Pemphigus Gestationis (PG) (2.6%). pH 7.4 for 10 minutes and mounted with glycerine before viewing with a fluorescent microscope (Olympus BX-40). BP was the most common lesion among SEBDs followed by DH, LABD and Bullous SLE. The clinical features A total of 75 cases of immunobullous diseases were and histopathological findings are shown in Table I. evaluated of which 38 cases were from the SEBD group Histopathologically all the cases of BP except for three and were included in the study. The remaining 44 cases cases showed a sub-epidermal bulla with fibrin and belonged to the pemphigus group and were excluded from inflammatory infiltrate in the bullous cavity. Three the study. Clinical data provided along with the biopsy cases showed an intraepidermal bulla with regenerative specimen were documented separately. DIF patterns were epithelium on the floor. Eosinophils were the most common interpreted according to standard criteria. inflammatory infiltrate seen followed by neutrophils. Table I: The clinical and histopathological findings of sub-epidermal bullous diseases Histopathology Mean Disease entity Clinical findings Site predilection Adjacent Contents of age Level of split epidermis bulla Multiple fluid filled vesicles Lower limbs>upper Eosinophilic Bullous pemphigoid 60 which rupture Sub-epidermal Eosinophils limbs> whole body spongiosis with crusting and erosions Neutrophils, Neutrophilic Multiple papulo- eosinophils, Dermatitis Chest>scapular collections 37 pruritic lesions Sub-epidermal lymphocytes herpetiformis region>back in papillary with severe itching and dermis karyorrhexis. Neutrophilic Multiple fluid collections Bullous SLE 28 filled lesions with Chest, upper back Sub-epidermal in papillary urticarial plaques dermis and karyorrhexis. Fluid filled lesions, Neutrophilic Neutrophils, tense vesicles collections Linear IgA disease 11 All over the body Sub-epidermal eosinophils to hemorrhagic in papillary and fibrin. bullae. dermis. Multiple urticarial Abdomen, back and Pemphigoid gestationis 24 plaques and Sub-epidermal forearm. papules Eosinophils, Epidermolysis bullosa Multiple tense 67 Chest, oral cavity Sub-epidermal lymphocytes acquisita bullae and fibrin Cicatricial pemphigoid Oral cavity Fibrosis 92 Vol. 32, No. 2, 2016; Page 91-98 JAIN S et al: DIF Studies in Subepidermal Bullous Diseases Turkish Journal of Pathology A B C D Figure 1: Bullous pemphigoid: A) Scanner view showing sub-epidermal bulla with inflammatory infiltrate in bullous cavity (H&E; x40), B) Low power view showing sub-epidermal bulla with eosinophils and exudates in bullous cavity (H&E; x100), C,D) Linear deposition of C3 and IgG along the dermo-epidermal junction (IF; x200). The adjacent epidermis showed eosinophilic spongiosis in papillary dermis. DIF studies showed deposition of IgA in 8 cases. (Table I, Figure 1A-B). DIF studies showed linear granular and fibrillary pattern in the dermal papillae. deposition of C3 along the dermo-epidermal junction in LABD showed sub-epidermal bulla rich in neutrophils all the cases (100%) and deposition of IgG in 91.8% of the and eosinophils (Figure 2A). DIF studies showed a linear cases (Figure 1C-D). The fluorescence was appreciable at homogenous deposition of IgA along the basement lower magnification of the 10x objective in 66.6% of cases membrane zone. One case showed deposition of IgM along and at 40x in the remaining 33.3% of the cases representing with IgA (Figure 2B,C). a lower intensity of fluorescence. Bullous SLE showed epidermis displaying focal basal cell PG showed identical histopathological features as BP with vacuolar degeneration and a small sub-epidermal cleft was only C3 deposition along the dermoepidermal junction. CP seen with intense collection of neutrophils in the papillary on DIF studies showed deposition of IgG and IgM along and mid dermis with karyorrhexis (Figure 3A). DIF studies the dermo-epidermal junction. The histopathology showed showed granular and linear deposits of IgG, IgA, IgM and fibrosis of the subepithelial region. C3 along the dermoepidermal junction and around hair DH showed sub-epidermal clefting with presence of fibrin follicle epithelium (Figure 3B-D). and neutrophils. There was denudation of the epidermis EBA showed deposition of IgG and C3 along DEJ in with collection of neutrophils and fibrin in the underlying continous and linear pattern (Figure 4) Vol. 32, No. 2, 2016; Page 91-98 93 Turkish Journal of Pathology JAIN S et al: DIF
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