A quick and simple serum test to differentiate bullous pemphigoid, epidermolysis bullosa acquisita, and anti-epiligrin cicatricial pemphigoid
Department of Dermatology, Baylor College of Medicine, Houston, Texas. email@example.com
Subepidermal autoimmune bullous diseasessuch as bullous pemphigoid (BP), anti-epiligrin cicatricial pemphigoid (AECP), epidermolysis bullosa acquisita (EBA), and bullous lupus erythematosus (LE)are acquired blistering disorders of the skin and/or mucous membranes. These diseases possess overlapping clinical and immunopathological features. As a result, extensive diagnostic tests are often used to differentiate these conditions.
All 4 conditions can exhibit linear deposition of IgG and C3 on direct immunofluorescence microscopy (DIF) along the basement membrane zone (BMZ). Further workup needs to be performed to make the proper diagnosis. We present 2 cases in which the correct diagnosis was made after performing an indirect immunofluorescence (IIF) test on type VII collagen-deficient skin.
A 55-year-old Caucasian woman presented with extensive blistering on her skin (Figs. 1-4) and erosions in her mouth. Two 4-mm punch biopsies were performed, 1 for histopathology and the other for DIF. Histopathology revealed a subepidermal bulla with eosinophils and DIF showed linear C3 and IgG along the BMZ. A diagnosis of BP was made and the patient was started on 1 mg/kg of prednisone daily. After 8 months, when the patient's condition had not improved, a salt-split skin IIF was performed, which showed binding of C3 and IgG on the dermal side of the split. ANA was negative. An IIF on type VII collagen-deficient skin was negative. A diagnosis of EBA was made. Since then, besides prednisone, the patient has tried azathioprine 150 mg/day (her thiopurine methyltransferase was within normal range) and mycophenolate mofetil 2 g/day. The regimen of prednisone 1 mg/kg/day and azathioprine 150 mg/day has slowed the development of bullae. Several attempts to have her insurance company pay for intravenous immunoglobulin, rituximab, and infliximab have failed so far.
A 69-year-old Caucasian man presented with a few scattered vesicles on his head, shoulders, and shins. He had a few erosions on his gums and he complained of a scratchy feeling in his eyes. Two 4-mm punch biopsies were obtained, one for histopathology and the other for DIF. Histopathology revealed a subepidermal vesicle with some eosinophils and DIF showed C3 and IgG along the BMZ. A salt-split skin IIF showed C3 and IgG binding to the dermal side of the split. ANA was negative. A diagnosis of EBA was made and the patient was started on prednisone 1 mg/kg/day. After 3 months, when there was no improvement, an IIF was performed on type VII collagen-deficient skin that showed C3 and IgG along the BMZ. A diagnosis of anti-epiligrin cicatricial pemphigoid was made and the patient was referred to an internist for work-up for malignancy and to a cornea specialist.
Bullous pemphigoid is characterized by autoantibodies against BP antigen 180 and BP antigen 230. Both are components of hemidesmosomes, which function to maintain adhesion between epithelial cells and the basement membrane. Clinically, tense bullae are most commonly seen on erythematous, cutaneous, urticarial plaques; the disease typically affects older individuals.
Epidermolysis bullosa acquisita is associated with autoantibodies to type VII collagen, which is a major component of anchoring fibrils in the sublamina densa. Type VII collagen possesses 3 α chains, with each chain featuring a collagenous helical domain, and 2 noncollagenous domains termed NC1 and NC2. Antibodies are most often directed towards the NC1 domain but have also been reported to recognize all 3 domains [1, 2]. Epidermolysis bullosa acquisita is characterized clinically by spontaneous or trauma-induced blisters appearing on the skin and less commonly on mucous membranes. Lesions typically heal with scarring and milia formation. Classically, the bullae of EBA occur on non-inflamed skin, but an inflammatory variant is now thought to be more common. The inflammatory variant can mimic BP.
Anti-epiligrin cicatricial pemphigoid is an uncommon subtype of CP, primarily affecting the mucous membranes. Anti-epiligrin cicatricial pemphigoid involves autoantibodies against the α subunit of epiligrin, also known as laminin 5 or laminin 332 (α3β3γ2), a key adhesive component in the epidermal basement membrane. Compared to other types of CP, skin involvement is more frequent in AECP although it is still a minor component of the disorder .
Bullous lupus erythematosus (LE) is a manifestation of systemic lupus erythematosus (SLE). Like EBA, bullous LE is associated with autoantibodies to type VII collagen. However, since bullous LE is a manifestation of SLE, these patients will have a positive antinuclear antibody (ANA) test and should fulfill the American Rheumatism Association (ARA) criteria for SLE.
In BP, EBA, AECP, and bullous LE, DIF will reveal linear deposition of IgG and C3 along the BMZ. Generally DIF is not useful in discriminating among these various bullous disorders. Indirect immunofluorescence on 1M NaCl split skin test is classically used to differentiate BP from EBA, AECP, and bullous LE. However, salt-split skin IIF only assists in narrowing the differential diagnosis, but does not provide a definitive diagnosis. Antibody binding on the epidermal side is seen with BP, while reactivity on the dermal side of the NaCl split skin is associated with the other three disorders. Additional testing is still required for further classification. Since bullous LE can usually be differentiated from EBA and AECP by fulfilling the ARA criteria for SLE, including having positive lupus serologies, we will focus our discussion on differentiating EBA from AECP.
There are several methods of testingsuch as Western immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopythat could be used to differentiate and prevent misdiagnoses among these diseases. However, these are often expensive and time-consuming techniques. The IIF on type VII collagen-deficient skin is a simple test for the clinician, because all the clinician has to do is to send the patient's serum in a red-top tube to a laboratory that performs these tests. (The authors use Beutner Labs in Buffalo, New York.) The authors routinely receive the test results by e-mail or by fax in less than 1 week from the day the blood specimen is sent by first-class mail.
Type VII collagen-deficient skin can be obtained from recessive dystrophic EB patients. Reactivity is not seen with EBA serum on type VII collagen-deficient skin. Our 2 cases illustrate the importance of performing an IIF on type VII collagen-deficient skin. In Case 1, ultimately, the lack of antibody binding to type VII collagen-deficient skin confirmed the diagnosis of EBA, and in Case 2 the presence of antibody binding to type VII collagen-deficient skin confirmed the diagnosis of AECP and ruled out the possibility of EBA.
The importance of distinguishing among the bullous diseases stems from their unique clinical courses and outcomes. Anti-epiligrin cicatricial pemphigoid has been associated with a higher incidence of both solid cancers and mortality from treatment with systemic immunosuppressive drugs . Case reports of AECP have cited a possible association between AECP and solid cancers  as well as B-cell non-Hodgkin lymphoma and CTCL . The basis for the link between AECP and cancer is currently unknown. It has been proposed that AECP may represent a paraneoplastic process since laminin 5 can be found in skin as well as tumors. Additionally, improvement in clinical symptoms has also been observed in patients after tumor resection . Therefore, it has been recommended to screen AECP patients for age-appropriate cancer.
In 1 study of 35 patients with AECP, 10 (28.6%) developed a solid cancer . The relative risk was calculated to be 6.8 (95% CI: 3.3-12.5). The majority of these patients developed cancer within one year of diagnosis of AECP and 8 of the 10 cancer patients died from cancer-related causes within 2 years.
This association of solid cancers with AECP underscores the importance of accurately differentiating this disease from other bullous disorders. In addition, response to therapy is different among these subepidermal autoimmune blistering diseases. The IIF on type VII collagen-deficient skin is a quick and simple test to help differentiate these disorders.
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