Immunohistochemistry versus immunofluoresence in the diagnosis of autoimmune blistering diseases

Abstract

Introduction: In patients with autoimmune skin blistering diseases (ABDs), the diagnostic gold standard has classically been direct and indirect immunofluorescence (DIF and IIF), despite inherent technical problems of autofluorescence. Aim: We sought to overcome autofluorescence issues and compare the reliability of immunofluorescence versus immunohistochemistry (IHC) staining in the diagnoses of these diseases. Methods: We tested via IHC for anti-human IgG, IgM, IgA, IgD, IgE, Kappa light chains, Lambda light chains, Complement/C3c, Complement/ C1q, Complement/C3d, albumin and fibrinogen in 30 patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF), and 30 control biopsies from the endemic area. We also tested archival biopsies from patients with ABDs whose diagnoses were made clinically, histopathologically and by DIF/IIF studies from 2 independent dermatopathology laboratories in the USA. Specifically, we tested 34 patients with bullous pemphigoid (BP), 18 with pemphigus vulgaris (PV), 8 with pemphigus foliaceus (PF), 14 with dermatitis herpetiformis (DH) and 30 control skin samples from plastic esthetic surgery reduction surgeries. Results: The diagnostic correlation between IHC and DIF-IIF was almost 98% in most cases. IHC revealed evidence of autofluorescence around dermal blood vessels, dermal eccrine glands and neurovascular packages feeding skin appendices in ABDs; this autofluorescence may represent a non-specific immune response. Strong patterns of positivity were seen also in endothelial-mesenchymal cell junction-like structures, as well as between dermal fibrohistiocytic cells. In PV, we noted strong reactivity to neurovascular packages supplying sebaceous glands, as well as apocrine glands with edematous changes. Conclusions:We suggest that IHC is as reliable as DIF or IIF for the diagnosis of ABDs; our findings further suggest that what has previously been considered DIF/IIF autofluorescence background may be of relevance in ABDs. Our discovery of reactivity against edematous dermal apocrine glands may be related to the fact that PV has a vegetant form, with lesions present in anatomic areas where these glands exist.

Autoantibodies to full body vascular cell junctions colocalize with MYZAP, ARVCF, desmoplakins I and II and p0071 in endemic pemphigus in Colombia, South America

“…Our data correlates with a recent study demonstrating cardiovascular alterations in patients with inflammatory skin diseases (including pemphigus and pemphigoid) versus controls; 2002–2012 US national inpatient hospitalizations ( n = 72 108, 077 adults) were reviewed and correlated with cardiovascular and cerebrovascular associated diseases . In our current and previous studies, we present evidence for vascular alterations in most autoimmune skin diseases …”

“…In El Bagre‐EPF and in other autoimmune blistering diseases including PF, bullous pemphigoid (BP), pemphigus vulgaris (PV), epidermolysis bullosa acquisita, linear IgA disease, and dermatitis herpetiformis, we have noted patient vessels overexpressing HLA‐DPDQDR antigen, ribosomal protein s6‐ps240, cyclo‐oxygenase 2, and C5‐b9/MAC. These patient biopsies also revealed vascular rouleaux phenomenon and other vascular inflammatory markers, including CD3, CD20, CD1a, HAM56, CD68, and S100, and autoantibodies to the vessels . We confirmed our data by IHC, which prevents the “autofluorescence” possible when using DIF/IIF.…”

“…We confirmed our data by IHC, which prevents the “autofluorescence” possible when using DIF/IIF. In most skin biopsies on all autoimmune skin blistering diseases, dilated vessels and papillary dermal edema are noted under the blisters, with additional mild dermal perivascular inflammation …”

“…It is possible that the autoantibodies stimulate endothelial cells to express adhesion molecules and various chemokines; this may lead to a recruitment of monocytes, leukocyte recruitment with the secretion of cytokines and other inflammatory markers. Deposition of fibrin, fibrinogen and activated platelets on damaged endothelium may expresses tissue and von Willebrand factor, creating a prothrombotic milieu and leading to clinical vascular disorders . The polyclonality of the immune response may partially result from pollution by metals and metalloids in the endemic area, as well as patient exposure to tropical and parasitic diseases …”

Membrane attack complex (C5b‐9 complex or Mac), is strongly present in lesional skin from patients with endemic pemphigus foliaceus in El Bagre, Colombia

“…Of interest is the correlation we found between positivity of other autoimmune blistering and skin diseases such lupus, BP, DH, and PV and the positivity of the staining for complement C5‐b9 that perfectly matched what we had reported on IHC staining with other immunoglobulins and complement . Indeed, our findings suggest that in these other autoimmune skin diseases, the immune response may also be more complex than the classically described and currently accepted mechanisms.…”