Histopathology of an EBA lesion shows a subepidermal blister. In the classic mechanobullous presentation of EBA, there is fibrin in the blister cavity and an overall paucity of associated inflammatory cells within the blister cavity and dermis. Fibrosis and scaring may also be present in the underlying dermis. In the BP-like EBA presentation, the histopathology shows a more significant dermal inflammatory infiltrate of lymphocytes, macrophages, neutrophils, and eosinophils. In the IgA pemphigoid-like EBA form, there is often a predominance of neutrophils.

Krushnick et al. [3], Gibbs and Minor [4], Nieboer et al. [5], and Yaoita et al. [6] have shown that a positive direct immunofluorescence (DIF) is necessary for the diagnosis of EBA. Immunoglobulin G (IgG) and, to a lesser extent C3, deposits are present at the dermal–epidermal junction by DIF from a perilesional biopsy. However, the immunodeposits are almost identical in pattern to those seen in bullous pemphigoid. EBA may have stronger IgG deposition, while BP may have strong C3 deposition. Therefore, performing salt-split skin (SSS) DIF and SSS indirect immunofluorescence (IIF) are necessary to distinguish EBA from the pemphigoid group of disorders. To perform the SSS technique, perilesional skin showing immunodeposits during routine DIF is incubated in 1 mol/L cold NaCl for 72 h. The procedure fractures the dermal–epidermal junction through the lamina lucida of the basement membrane zone. This places the bullous pemphigoid autoantigens associated with the hemidesmosome (i.e., BPAg 1 and BPAg 2, also known as type XVII collagen) on the epidermal roof of the separation [36–45]. The EBA antigen, type VII collagen, remains with the dermal floor [47]. The immunoreactants (usually IgG and C3) are again incubated with the tissue. If the patient has EBA, the immune deposits are detected on the dermal side of the separation.

Many, but not all, EBA patients have an anti–BMZ IgG autoantibody circulating in their blood that can be detected by IIF. The EBA serum autoantibodies label frozen sections of human skin or monkey esophagus, producing a crisp linear fluorescent staining at the dermal–epidermal junction of the frozen sections after incubation with anti-IgG fluorescent-labeled antibodies. As with the routine DIF procedure, one cannot distinguish EBA from the pemphigoid group of diseases without doing salt-split IIF, which is always done on human skin substrate. Human skin is incubated in 1 mol/L NaCl, and the dermal–epidermal junction fractures cleanly through the lamina lucida zone, placing the BP antigens on the epidermal roof and the EBA antigen (type VII anchoring fibril collagen) on the dermal floor [41]. Salt-split skin substrate can be used to distinguish EBA and BP sera [42]. If the serum antibody is IgG and labels the epidermal roof, the patient does not have EBA, and BP should be considered. If, on the other hand, the antibody labels the dermal side of the separation, the patient usually has either EBA or bullous SLE. The latter can be ruled out by other serology and clinical criteria.

It was thought that only EBA and bullous SLE show dermal staining of the salt-split skin on IIF or DIF. In recent years, other very rare autoimmune diseases have been shown to have IgG deposits in the lower lamina lucida space that map to the dermal side when the skin substrate is fractured by SSS technique. These diseases include anti-laminin 5 cicatricial pemphigoid [43], a BP-like disease in which the patients have autoantibodies to a 105- kd lamina lucida glycoprotein that is unrelated to laminin-5 [44], a newly discovered disease reported by Ghohestani and colleagues [46], with IgG autoantibodies directed against the α5 chain of type IV (lamina densa) collagen in association with renal failure, and another BP-like disease called protein 200 pemphigoid in which the autoantigen is a 200-kd glycoprotein in the lower part of the lamina lucida.

Electron microscopy (EM) shows that the dermal–epidermal separation in an EBA lesion is associated with a paucity of normal anchoring fibrils and an amorphous, electron dense band beneath the lamina densa due to the IgG deposits over the anchoring fibrils [9]. Despite the sublamina densa deposits, EBA blisters frequently separate above the immune deposits within the lamina lucida [39].

Immunoelectron microscopy (IEM) localizes the EBA IgG autoantibody deposits in the BMZ to within and below the lamina densa, the location of the anchoring fibrils. Immunoelectron microscopy showing these sublamina densa IgG deposits is the gold standard for the diagnosis, as first demonstrated by Nieboer et al. [13] and Yaoita et al. [6] This localization is distinct from BP IgG deposits which are localized to the hemidesmosomes of the basal keratinocytes and the IgG autoantibody deposits in CP and are confined to the lower lamina lucida.

Antibodies in EBA sera bind to a 290-kd band in Western blots of human skin basement membrane proteins containing type VII collagen, whereas sera from other primary blistering diseases do not [13].

This band corresponds to the alpha-chain of type VII collagen. Often a second band of 145 kd will be labeled with EBA antibodies. This band is the amino-terminal globular NC1 domain of the type VII collagen alpha-chain that is rich in carbohydrate and contains the antigenic epitopes of EBA autoantibodies, bullous SLE autoantibodies, and monoclonal antibodies against type VII collagen [13, 48].

Now that purified, recombinant, human, type VII collagen is readily available, an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of EBA has been developed by Chen et al. [24, 49]. It has proven to be more sensitive for detecting anti-COL7 EBA autoantibodies in the sera of patients than either IIF or Western blotting analysis. ELISA is performed quickly and easily with a very high sensitivity in determining which circulating autoantibodies are present.

Although EBA does not have a mendelian pattern of inheritance, African-American EBA patients in the Southeastern part of the United States have a high incidence of the human leukocyte antigen (HLA)-DR2 phenotype. The calculated relative risk for EBA in HLA-DR2+ individuals is 13.1 in these patients [27]. It is thought that, although it is not the primary cause, these patients have an immune profile that makes them susceptible to the disease.

While the etiology of EBA is unknown, it appears that when IgG autoantibodies bind to the patient’s anchoring fibrils, a paucity of normal anchoring fibrils at the BMZ develops, and this is associated with poor dermal–epidermal adherence. This is exactly the same problem as hereditary dystrophic forms of EB due to a defect in the gene that encodes for type VII collagen.

Epidermolysis bullosa acquisita likely has an autoimmune etiology. Direct immunofluorescence of perilesional skin biopsies from EBA patients reveals IgG deposits at the dermal–epidermal junction [3–6]. The EBA antibodies bind to type VII collagen within anchoring fibrils [13, 14], structures that emanate perpendicularly from the BMZ and attach to the papillary dermis. This process results in decreased anchoring fibrils, but the pathway leading to this reduction is unknown. Type VII collagen has an affinity for fibronectin, a large glycoprotein in the papillary dermis and this interaction between the two may play a role in anchoring the basement membrane [22]. It is conceivable that EBA autoantibodies binding to type VII collagen interrupt the interaction between type VII collagen and fibronectin and a separation ensues.