Please cite this article in press as: Russo I, et al. The use of biochip immunofluorescence microscopy for the diagnosis of Pemphigus vulgaris. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2013.12.012 ARTICLE IN PRESS G Model ACTHIS-50815; No. of Pages 4 Acta Histochemica xxx (2014) xxx–xxx Contents lists available at ScienceDirect Acta Histochemica jou rn al homepage: www.elsevier.de/acthis The use of biochip immunofluorescence microscopy for the diagnosis of Pemphigus vulgaris Irene Russo, Andrea Saponeri, Andrea Peserico, Mauro Alaibac ∗ Dermatology Unit, Department of Medicine, University of Padua, Via Battisti 206, 35121 Padua, Italy a r t i c l e i n f o Article history: Received 21 October 2013 Received in revised form 21 December 2013 Accepted 23 December 2013 Available online xxx Keywords: Pemphigus vulgaris Immunofluorescence Desmoglein 3 BIOCHIP technology a b s t r a c t Pemphigus vulgaris is an autoimmune intraepithelial blistering skin disease characterized by the presence of circulating autoantibodies directed against surfaces of keratinocytes. Diagnosis is generally based on clinical features, histology, direct and indirect immunofluorescence and ELISA. This study describes a new BIOCHIP mosaic-based indirect immunofluorescence technique based on recombinant antigenic substrates and transfected cells. We investigated the diagnostic use of BIOCHIP for the serological diag- nosis of Pemphigus vulgaris. Autoantibodies against desmoglein 3 were detected in 97.62% of patients (41/42) with P. vulgaris. There were no positive results in the negative control group. Our study revealed that BIOCHIP has high sensitivity and specificity comparable to that of the ELISA assays. Therefore the BIOCHIP technique seems to be an appropriate method for the diagnosis of P. vulgaris as it has been shown to be a simple, standardized and readily available novel tool, which could facilitate the diagnosis of this autoimmune bullous disease. We suggest that it could be used as an initial screening test to identify patients with P. vulgaris before using the ELISA approach. © 2013 Elsevier GmbH. All rights reserved. Introduction Pemphigus is a rare autoimmune intraepithelial blistering skin disease characterized by the presence of circulating autoantibodies directed against surfaces of keratinocytes, resulting in loss of the normal epithelial cell-to-cell adhesion, through a process called acantholysis (Sitaru and Zillikens, 2005). There are two main sub- types of Pemphigus: P. vulgaris (PV) and P. foliaceus (PF), which have distinct clinical, histological and immunopathological pro- files (Joly and Litrowski, 2011). In PV, blisters develop just above the basal-cell layer and are associated with autoantibodies against desmoglein 3 (DSG3) and desmoglein 1 (DSG1), whereas in PF the blisters are just below the Stratum corneum and are associ- ated with autoantibodies against desmoglein 1 (DSG1) (Bystryn and Rudolph, 2005). PV, which if left untreated can be fatal, is the most common form in both Europe and USA (Bystryn and Rudolph, 2005). The diagnosis of PV is generally based on clini- cal features, histology and immunological tests, notably direct and indirect immunofluorescence and enzyme linked immunosorbent assay (ELISA) (Schmidt and Zillikens, 2010). The typical clinical find- ings of PV are chronic painful oral erosions, multiple flaccid blisters arising from healthy skin and a positive Nikolsky sign (Bystryn and Rudolph, 2005). Histologically, there are intraepithelial ∗ Corresponding author. E-mail address: mauro.alaibac@unipd.it (M. Alaibac). blisters associated with acantholytic cells. Direct immunofluores- cence (DIF) and classic indirect immunofluorescence (IIF) using monkey esophagus or salt-split normal human skin sections have been for a long time the main tests utilized for diagnosis (Schmidt and Zillikens, 2010). DIF demonstrates in vivo a net-like pattern deposit of IgG and C3 on the plasma membrane of keratinocytes of both lesional and normal-appearing perilesional skin (Pohla- Gubo et al., 2011), whereas circulating intercellular antibodies are detected using IIF in 80–90% of patients with PV (Jiao and Bystryn, 1997). Human recombinant DSG1 and DSG3 proteins have been applied in the development of sensitive and specific ELISA for the detection of circulating autoantibodies (Amagai et al., 1999a,b). Here, we describe a new BIOCHIP mosaic-based indirect immunofluorescence technique for the determination of anti-DSG3 autoantibodies based on recombinant antigenic substrates and transfected cells. In this pilot study we investigate the sensitivity and specificity of this innovative immunoassay in order to evaluate its diagnostic use for the laboratory diagnosis of this autoimmune skin condition. Materials and methods The study comprised 42 Caucasian patients with PV, 22 males, age ranging from 20 to 89, and 20 females age ranging from 22 to 80 (Table 1). Serum samples from 10 normal healthy individuals, 9 patients with Bullous pemphigoid and 1 patient with Epidermoly- sis bullosa acquisita were used for negative controls (Table 2). The 0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.12.012