Direct Immunofluorescence of IgG on Formalin-Fixed Paraffin-Embedded Tissue by Heat-Induced Antigen Retrieval as a Sensitive Method for the Diagnosis of Pemphigus

Abstract

Purpose: Direct immunofluorescence (DIF) on frozen sections (DIF-F) plays a key role in the identification and differential diagnosis of bullous dermatoses, which are a group of critical autoimmune diseases that include pemphigus, bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA). However, this technique requires specialized laboratory equipment conditions, sample acquisition and sample preservation. In this study, the application value of DIF on paraffin-embedded tissue sections (DIF-P) detecting IgG using heat-induced antigen retrieval (HIAR) in the diagnosis of bullous dermatosis was explored.

Patients and methods: Samples from 12 patients with pemphigus vulgaris (PV), 10 patients with pemphigus foliaceus (PF), 17 patients with BP, and 4 patients with EBA were retrospectively studied for DIF-P IgG detection. Formalin-fixed, paraffin-embedded tissue (FFPE) was used, and the antigen retrieval method used in the experiment was HIAR. All patients were diagnosed with the autoimmune bullous disease (AIBD) based on clinical presentation, histopathology, DIF-F, and enzyme-linked immunosorbent assay (ELISA).

Results: Intercellular staining for IgG in the epidermis was successful in paraffin-embedded tissue sections in 11 of 12 PV samples and in all 10 PF samples. IgG at the basement membrane zone (BMZ) was not detected by immunofluorescent staining in 17 BP samples and 4 EBA samples.

Conclusion: The detection of IgG by DIF-P using HIAR can be used for the diagnosis of pemphigus as an alternative method to DIF-F.

Keywords: IgG; bullous dermatoses; direct immunofluorescence; heat-induced antigen retrieval; paraffin-embedded section; pemphigus.

Figure 1

Representative examples of histopathological and immunofluorescent staining for pemphigus, bullous pemphigoid, epidermolysis bullosa acquisita and normal skin. (A) H&E staining at perilesion sites of pemphigus foliaceus. (B) DIF on paraffin tissue sections of positive IgG staining at perilesion sites of pemphigus foliaceus. (C) H&E staining at lesion sites of pemphigus foliaceus. (D) DIF on paraffin tissue sections of positive IgG staining at lesion sites of pemphigus foliaceus which is less strong than that of the perilesion site. (E) H&E staining of normal skin. (F) No IgG deposition is observed between spinous cells and along basement membrane zone by DIF on paraffin tissue sections in normal skin. (G) H&E staining at perilesion sites of bullous pemphigoid. (H) No IgG deposition is observed along basement membrane zone at perilesion sites by DIF on paraffin tissue sections of bullous pemphigoid. (I) H&E staining at lesion sites of bullous pemphigoid. (J) No IgG deposition is observed along basement membrane zone at lesion sites by DIF on paraffin tissue sections of bullous pemphigoid. (K) H&E staining at perilesion sites of epidermolysis bullosa acquisita. (L) No IgG deposition is observed along basement membrane zone at perilesion sites by DIF on paraffin tissue sections of epidermolysis bullosa acquisita. (M) H&E staining at lesion sites of epidermolysis bullosa acquisita. (N) No IgG deposition is observed along basement membrane zone at lesion sites by DIF on paraffin tissue sections of epidermolysis bullosa acquisita. DIF, direct immunofluorescence; H&E, hematoxylin and eosin.