Evaluation and Correlation of Clinical, Histopathological and Direct Immunofluorescence Findings in Vesicobullous Disorders of Skin: A Cross Sectional Study with Review of Literature

Background: To assess and correlate the clinical, histopathological and DIF features and compare the sensitivity of DIF with that of histopathology in autoimmune bullous disorders of skin. Methods: A cross-sectional descriptive hospital based study was conducted on 45 patients who had active vesicobullous lesions. After a detailed cutaneous examination, two punch biopsies were taken, one from lesional skin for histopathological study and another from perilesional skin for DIF. Biopsies from 31 patients were deemed fit to be included in the study. Result: Based on clinical, histopathological and DIF findings the most common final diagnosis was Pemphigus Vulgaris (PV), 18/31 cases. On histopathology, characteristic histopathological features were seen in 15/18 cases of PV, 6/11 cases of Pemphigus Foliaceous (PF), 3/4 cases of Bullous Pemphigoid (BP) and a single case of Dermatitis Herpitiformis (DH) while 4/31 cases showed non specific findings (NS). DIF was positive in 30/31 cases (96.77%) except in a single case of DH. Good clinico–histo-immunological correlation was seen in 21/31 cases (67.7%). In 25/31 cases (80.06%) good histo-immunological correlation (p < 0.05; significant) was observed while 6/31 cases (19.3%) showed discordance between histological and DIF findings. The senstivity of the histopathology in the pemphigus group (PV + PF + Paraneoplastic Pemphigus), BP and DH was 88%, 75% and 100% respectively while on DIF it was 100% for the pemphigus group and BP. Single case of DH was negative on DIF. Conclusion: As compared to histopathology, DIF has better sensitivity and it is an indispensible tool especially in vesicobullous skin lesions that are difficult to diagnose on the basis of clinical and histopathological features. Original Article *Corresponding author: Dr. Geetika Sharma, House number 362, Sector 8, Panchkula, Haryana, India, Pincode -134109 Phone : +919560835225, +919013952791 Email: [email protected] A-142 Direct Immunofluorescence in Skin Annals of Pathology and Laboratory Medicine, Vol. 03, No. 03, July September 2016 Introduction Autoimmune bullous disorders are a heterogeneous group of diseases in which components of the epidermis and basement membrane zone (BMZ) are the focus of attack. [1] Their accurate diagnosis requires detailed clinical examination, histopathological evaluation followed by direct immunofluorescence study (DIF). [2] DIF plays an essential role in the diagnosis, classification and treatment of various immunobullous disorders as it shows distinct immunofluorescence patterns. [3] Patients in clinical remission with positive DIF findings show early relapse of disease which also emphasizes the role of DIF in prognosticating and monitoring of disease activity. [1,4,5] Thus, DIF is considered gold standard for the diagnosis of autoimmune bullous disorders, specially in patients with clinical and/or histopathological dilemma. [4,6,7,8] However, in developing countries like India, DIF is done only in the few centers due to its high cost, requirement of technical skill and difficulty in maintaining the facility. Therefore, this study was undertaken to assess and correlate the clinical, histopathological and DIF features of various vesicobullous diseases of the skin and compare the sensitivity of DIF with that of histopathology. Materials and Methods A cross-sectional descriptive hospital based study was conducted on 45 patients attending the Departments of Dermatology and Pathology, Lady Hardinge Medical College and associated Hospitals, New Delhi over a period of 2 years who were clinically diagnosed with active vesicobullous diseases, irrespective of age and sex. Patients with no active lesions and on systemic steroids/ immunosuppressive therapy for the last three months were excluded from the study. In all the patients, two punch biopsies from skin were taken. Biopsy for histopathological examination was taken from the lesional skin, put in 10% neutral formalin solution and stained with haematoxyline and eosin. On histopathological examination the lesions were categorized based on:  Site of blister separation plane (Subcorneal/granular, Intraepidermal/spinous, Suprabasal, Subepidermal)  Nature of inflammatory infiltrate (Neutrophil, Eosinophil, Lymphocytes & Mast cells) within the bulla cavity  Presence/Absence of acantholytic cells within the bulla cavity Another biopsy for DIF was taken from the perilesional skin (i.e. 3-4 mm punch biopsy from clinically normal appearing skin within 2 cm from the lesion except from hands, feet, neck, groin and mucous membrane), put in Michel’s medium and kept at -70 °C to -20°C until cut. Before cutting, the biopsy for DIF was washed thrice in phosphate–buffered saline (PBS) at pH 7.2 for 15 minutes each and was embedded in OCT in cryostat and 4-6 micron sections were obtained on poly–L-Lysine slides at -20°C to -25°C (minimum of six sections per biopsy). For DIF staining, sections were brought at room temperature after rinsing thrice with PBS for 10 min each and five frozen sections of each biopsy were overlaid with 40-50 μl each of optimally diluted fluorescein isothiocyanate (FITC) conjugated monoclonal anti human antisera (IgG, IgA, IgM, C3 and fibrinogen, supplied by Diagnostic biosystems) and sixth section with PBS (Control) for at least 1 hour. After washing the sections again thrice in PBS they were mounted with Buffered glycerin mountant and finally examined under NIKON fluorescence microscope fitted with an ultraviolet lamp source, under ideal citation and barrier filter combination.  On DIF examination the following parameters were evaluated:  Site of deposition of immunoreactants (Epidermis/ BMZ/Dermis)  Pattern of Immunofluorescence (Linear/Granular/ both) Intensity of fluorescence (graded as“+++”: Strongly positive, “++”: Moderately positive, “+”: Weakly positive and “-”: Negative) Result Among the 45 vesicobullous skin biopsies received, 14 biopsies were excluded from the study. Amongst these 14 biopsies, 8 biopsies without epidermis were inadequate for opinion, 4 biopsy samples were dried up & in 2 biopsies, patients were later found to be on steroids. Finally in the 31 cases studied, the most common age group was 4th – 5thdecade (29.03%) with M:F ratio of 0.82:1. Single definitive clinical diagnosis given in 24/31 cases (77.4%) were: PV(13/31), PF(6/31), BP(3/31), PNP(1/31) & DH(1/31) which were consistent with the final diagnosis after histopathology and DIF findings. In 7/31 (22.5%) cases, differential clinical diagnosis were considered . Definitive histopathological diagnoses were made in 27/31 cases (87.1%) while, 4/31 (12.9%) cases showed non-specific findings on histopathology. DIF positivity was seen in 30/31 cases (96.77%). Based on clinical, histopathological and DIF findings the most common final diagnosis was PV 18/31cases (58.06%). (Table 1) In 18 clinically diagnosed cases of PV, females were affected twice more commonly than males. The most common