Laboratory Studies
The International Bullous Diseases Group (IBDG) has created a consensus for diagnostic criteria for epidermolysis bullosa acquisita (EBA). [28] To establish a diagnosis, the following tests should be performed:
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Histopathology of the entire new bullae or vesicle, if possible, or broad saucerization of the periphery [29]
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Direct immunofluorescence on normal-appearing perilesional skin
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Indirect immunofluorescence with the patient's serum on salt-split normal human skin substrate
It is important to understand that techniques of histopathology and direct immunofluorescence microscopy can only confirm a subepithelial autoimmune bullous disease, and not specifically epidermolysis bullosa acquisita. A patient with suspected epidermolysis bullosa acquisita should have a confirmation test of serration pattern analysis and fluorescent overlay antigen mapping (FOAM), although it may only be available at academic institutions. [28]
Histopathology
Histopathology documents the presence of a subepidermal blister, ie, a separation has occurred between the epidermis and the dermis. In addition, histology also reveals a mixed inflammatory cell dermal infiltrate.
Direct immunofluorescence
See the image below. Direct immunofluorescence documents the immune-mediated disease process. Usually, it detects a thick band of IgG, and to a lesser extent C3, deposited linearly at the basement membrane zone. Other immunoreactants such as IgM or IgA may be seen. A U-serrated pattern is typical of epidermolysis bullosa acquisita, whereas an n-serrated pattern is typical of pemphigoid. If the specific serration pattern cannot be discerned, a repeat perilesional skin biopsy should be pursued in order to make an accurate diagnosis.
FOAM is a technique that can be used if the serration pattern cannot be identified from the standard approach. This process uses a visual comparison of the level of antibody deposition to a known topographic marker. The two images are overlaid and then visually compared to identify a match. [30]
Direct immunofluorescence performed on perilesional skin biopsy specimen from a patient with epidermolysis bullosa acquisita detects a linear band of immunoglobulin G deposit along the dermoepidermal junction.
Indirect immunofluorescence
See the image below. Indirect immunofluorescence demonstrates the presence of IgG circulating autoantibodies in the patient's serum that target the skin basement membrane component, type VII collagen. Usually, it detects IgG circulating autoantibodies in patient's serum that bind to the dermal floor (lower part) on salt-split normal human skin substrate. This test differentiates epidermolysis bullosa acquisita from bullous pemphigoid because IgG autoantibodies from patients with bullous pemphigoid bind to the epidermal roof (upper part) of salt-split skin. The dermal floor pattern of indirect immunofluorescence on salt-split skin substrate is also found in sera of patients with bullous systemic lupus erythematosus (SLE), antiepiligrin cicatricial pemphigoid (with autoantibodies to laminin-5 and laminin-6), and anti-p105 pemphigoid (with autoantibodies to a 105-kd lower lamina lucida protein). [31]
Indirect immunofluorescence performed on salt-split normal human skin substrate using serum from a patient affected with epidermolysis bullosa acquisita detects immunoglobulin G class circulating autoantibodies that bind to the dermal (base) side of the basement membrane.
Other Tests
Other investigative studies available that further document the diagnosis of epidermolysis bullosa acquisita (EBA) include direct and indirect immunoelectron microscopy, immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and type IV collagen immunomapping.
Direct and indirect immunoelectron microscopy
Direct and indirect immunoelectron microscopy document the ultrastructural localization of in vivo‒bound IgG autoantibodies (by direct method) or the binding site of circulating IgG autoantibodies (by indirect method) at the basement membrane. This technique detects IgG autoantibodies at the lamina densa and sublamina densa areas of the skin basement membrane. In contrast, bullous pemphigoid IgG autoantibodies are localized to the hemidesmosome and upper lamina lucida. [32]
Immunoblotting
Immunoblotting documents the specific skin basement membrane antigen recognized by the patient's IgG autoantibodies. This test detects a denatured 290-kd skin basement membrane protein or globular chain of the alpha chain (145 kd) of type VII collagen.
Enzyme-linked immunosorbent assay
ELISA [33] documents the specific basement membrane antigen recognized by the patient's IgG circulating autoantibodies. ELISA is preferred over immunoblotting because the ELISA method uses nondenatured type VII collagen, usually in a recombinant form. It is a more sensitive and specific method than immunoblotting.
Immunoprecipitation
Immunoprecipitation demonstrates the specific basement membrane antigen in its native form recognized by the patient's IgG circulating autoantibodies. This method detects a 290-kd protein either from cultured keratinocytes or from fibroblasts. It is a more difficult method to use than immunoblotting.
Type IV collagen immunomapping
Collagen IV immunomapping can help distinguish epidermolysis bullosa acquisita from bullous pemphigoid and anti-epiligrin cicatricial pemphigoid (AECP). Type IV collagen is a major component of the lamina densa, and staining delineates this region of the basement membrane zone. Positive type IV collagen staining in the roof of the blister suggests autoantibodies against epitopes below the lamina densa, such as in epidermolysis bullosa acquisita. In comparison, the type IV collagen staining in bullous pemphigoid and AECP would be in the floor of the blister. Type IV collagen immunomapping has been found to be more sensitive than indirect immunofluorescence. [34]
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Direct immunofluorescence performed on perilesional skin biopsy specimen from a patient with epidermolysis bullosa acquisita detects a linear band of immunoglobulin G deposit along the dermoepidermal junction.
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Indirect immunofluorescence performed on salt-split normal human skin substrate using serum from a patient affected with epidermolysis bullosa acquisita detects immunoglobulin G class circulating autoantibodies that bind to the dermal (base) side of the basement membrane.
