Application of confocal laser scanning microscopy to the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita

Abstract

Background: The differential diagnosis of bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA) presents some difficulties since both diseases have overlapping clinical and histological features, as well as immunopathological findings. Confocal laser scanning microscopy (CLSM) has been developed and has shown to be a promising tool for dermatological investigations, giving a higher degree of resolution and available co-localization analysis. Objective: The purpose of this study was to evaluate whether the new technique of CLSM could reliably identify and differentiate the binding sites of disease specific-autoantibodies (Abs) at the basement membrane zone (BMZ), with the sera from BP and EBA. Methods: An indirect immunofluorescence (IF) assay was performed to localize the binding sites of circulating Abs from 5 cases of both BP and EBA, as well as the sites of 3 BMZ markers (integral β4, laminin-5, and type IV collagen). To facilitate identification and topographic differentiation between the two groups, patients' Abs were labeled with fluorescein isothiocyanate, whereas the BMZ markers were labeled with Texas red. The tissue specimens were observed under both conventional IF microscopy and CLSM. Results: Owing to superposition of antigens and marker labels, double immuno-labeled sections under IF microscopy revealed limitations for the differentiation of patient's sera from BMZ markers even with high magnification (× 1,000). However, CLSM was able to eliminate much of the antigen overlap. In BP, the circulating autoantibody' deposits were recognized on the epidermal side of laminin-5 and type IV collagen, and codistributed with integrin β4. On the other hand, the binding of autoantibodies in EBA was on the dermal side from that of integrin β4, laminin-5 and type IV collagen. These spatial relationships are compatible with their known microstructural locations. Conclusion: Our study indicates that CLSM examination may provide more precise localization of the antigens in BP and EBA than conventional IF microscopy. CLSM would not only be an efficient tool to identify circulating anti-BMZ autoantibodies for diagnosis and differential diagnosis of blistering diseases, but also a great addition to examining tissue specimens in patients who do not have detectable circulating Abs.