Diagnosis and disease severity assessment of epidermolysis bullosa acquisita by ELISA for anti‐type VII collagen autoantibodies: an Italian multicentre study

Background Epidermolysis bullosa acquisita (EBA) is a rare autoimmune mucocutaneous bullous disease caused by autoantibodies against type VII collagen, a component of anchoring fibrils that stabilizes dermoepidermal adherence. Type VII collagen is composed of a collagenous domain linked by the noncollagenous (NC)1 and NC2 domains. Objectives To assess the repeatability, sensitivity and specificity of a recently developed enzyme‐linked immunosorbent assay (ELISA) for detection of anti‐type VII collagen autoantibodies, and to ascertain whether they may be a marker of disease activity in EBA. Methods Using this ELISA, which was able to recognize autoantibodies against the NC1 and NC2 epitopes of type VII collagen, we tested 14 EBA sera, 30 healthy control sera and 113 disease control sera. Results In the EBA sera group, 12 out of the 14 samples were positive in ELISA, with autoantibody titres varying from 7·2 to 127·9 U mL^?1 (cutoff value < 6), the sensitivity of the method being 86%. Among the controls, only two bullous pemphigoid sera tested positive, the specificity being 98·6%. A good correlation was found between EBA disease severity, expressed as autoimmune bullous skin disorder intensity score, and the serum levels of anti‐collagen VII autoantibodies, measured by ELISA (n = 14; r = 0·965; P = 0·0001). The intra‐ and interassay coefficients of variation of the ELISA method ranged from 6·3% to 18·3%. Conclusions This NC1 + NC2 ELISA can be a practical assay for the diagnosis of EBA. The correlation between autoantibody titres and disease severity suggests its usefulness as a marker of disease activity in EBA However, this should be confirmed by studies on larger series of patients.     

Case of epidermolysis bullosa acquisita with concomitant anti‐laminin‐332 antibodies

Subepidermal autoimmune blistering disease including bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid, anti‐laminin‐γ1 pemphigoid, linear immunoglobulin A bullous disease and epidermolysis bullosa acquisita (EBA), are all characterized by direct immunofluorescence microscopy or immunoglobulin deposition on the basement membrane zone. Among them, EBA is a rare acquired subepidermal autoimmune blistering disease of the skin and mucous membranes reactive with type VII collagen, a major component of the epidermal basement membrane zone. Anti‐laminin‐332‐type mucous membrane pemphigoid has pathogenic autoantibodies against laminin‐332, which is a basement membrane heterotrimeric protein composed of α3, β3 and γ2 laminin chains. We describe a 73‐year‐old Japanese man presenting with multiple, annular, tense blisters on the lower legs and oral lesions. Despite the severe clinical manifestations, the disease was successfully controlled by combination therapy of oral prednisolone and mizoribine. This case was confirmed to have autoantibodies to both type VII collagen and laminin‐332 α3 chain by indirect immunofluorescence of 1 mol NaCl‐split normal human skin, various immunoblot analyses and enzyme‐linked immunosorbent assays. This case was a rare case of EBA with concomitant anti‐laminin‐332 antibodies.     

Anti‐type VII collagen autoantibodies,detected by enzyme‐linked immunosorbent assay,fluctuate in parallel with clinical severity in patients with epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease caused by autoantibodies against type VII collagen. An enzyme‐linked immunosorbent assay (ELISA) is currently available to detect autoantibodies in EBA. There have been reports suggesting generically that ELISA indices reflect EBA disease severity; however, there is, as yet, no conclusion as to whether ELISA indices fluctuate with disease activity over time in each EBA patient. This study aimed to investigate whether ELISA titers fluctuate with EBA disease activity and to validate the clinical significance of checking ELISA values in EBA by monitoring type VII collagen ELISA titers and disease severity, evaluated in terms of numbers of blisters and erosions as a clinical score, over time in three Japanese patients with EBA. All three cases in this study, which were treated successfully, showed titers of anti‐type VII collagen autoantibodies detected by ELISA that fluctuated in parallel with disease activity. Especially in case 1, we could determine that the expanding erosions were not due to flare‐ups of EBA because the ELISA indices stayed low, although new lesions continued to appear. In fact, control of infection and nutrition helped the lesions to become epithelialized. In conclusion, we found that repeated ELISA measurements are useful in monitoring disease activity and making decisions in EBA treatment plans.     

Place of human amniotic membrane immunoblotting in the diagnosis of autoimmune bullous dermatoses

Background Fine analysis of antiskin autoantibodies can contribute to the differential diagnosis of autoimmune bullous dermatoses. Objectives To develop a high‐performance immunoblotting method using human amniotic membrane as the antigen source, and to compare it with current laboratory methods. Methods Sera from 113 patients were tested by immunoblotting (IB), rat and monkey oesophagus and salt‐split skin indirect immunofluorescence (IIF), and enzyme‐linked immunosorbent assay (ELISA) quantification of anti‐BP180‐NC16a and anti‐BP230, or antidesmoglein (Dsg) 1 and 3 antibodies. There were 56 cases of bullous pemphigoid (BP), 22 cases of mucous membrane pemphigoid (MMP), eight cases of epidermolysis bullosa acquisita (EBA), two cases of bullous systemic lupus erythematosus (BSLE), 17 cases of pemphigus vulgaris (PV), and four cases each of pemphigus foliaceus (PF) and paraneoplastic pemphigus (PNP). Results In BP, the three methods had similar sensitivity (84–89%) for both anti‐BP180‐NC16a and anti‐BP230 antibody detection. In MMP, autoantibodies (mainly directed against BP180 or laminin 332 subunits) were detected in 77% of patients by IB, compared with only 9% by IIF on rat and monkey oesophagus and 36% on salt‐split skin, and 14% by anti‐BP180‐NC16a and anti‐BP230 ELISA. In patients with pemphigus, ELISA had 92% sensitivity for anti‐Dsg1 and 3, but IB and rat bladder IIF were necessary to confirm PNP by revealing specific and rare patterns (antidesmoplakin I/II, antienvoplakin and antiperiplakin antibodies). IB also revealed anticollagen VII antibodies in 60% of patients with EBA and BSLE, and antibodies to BP180, BP230 and Dsg3 in a few patients who were negative using the other two techniques. Conclusion Amniotic membrane immunoblotting is an interesting diagnostic tool for bullous diseases, as the entire panel of autoantibodies can be detected with a single extract. This method improves the identification of complex and heterogeneous autoimmune processes in conjunction with IIF and ELISA, and is particularly useful for MMP characterization.     

Association between effective dose of prednisolone, alone or in conjunction with other immunosuppressants, and titre of anti-bullous pemphigoid 180 antibody: a retrospective study of 42 cases

Background. Corticosteroids, especially prednisolone or prednisone, are the most commonly used drugs for the treatment of bullous pemphigoid (BP). However, the appropriate initial effective prednisolone dose has not been established. Recently, a highly sensitive and specific ELISA for detection of autoantibodies to the non‐collagenous extracellular domain (NC16A) of the 180 kDa transmembrane hemidesmosome component [bullous pemphigoid (BP)180] was developed, and the titre of anti‐BP180 antibody was found to be closely related to disease activity. Aim. To investigate the relationship between anti‐BP180 antibody titre and effective prednisolone dose alone or in conjunction with other immunosuppressants. Methods. Anti‐BP180 antibody titres were measured by ELISA for the NC16A domain of BP180 in the sera of patients with BP (n = 42) at the start of treatment. The effective prednisolone dose was calculated from the patients’ records. Results. Higher anti‐BP180 antibody titres correlated with a higher effective prednisolone dose. In particular, patients with antibody titres > 200 required a significantly higher effective prednisolone dose than did those with antibody titres ≤ 200. Conclusions. A higher effective prednisolone dose may be necessary for patients who have both a high titre of anti‐BP180 antibody and severe clinical disease.     

Development of a simple enzyme‐linked immunosorbent assay for the detection of autoantibodies in anti‐p200 pemphigoid

Background Anti‐p200 pemphigoid is a subepidermal blistering skin disease characterized by autoantibodies against a 200‐kDa protein (p200) of the dermal–epidermal junction. The laminin γ1 chain has recently been identified as target antigen in this disease and the C‐terminus was described as an immunodominant region of laminin γ1. Diagnosis of anti‐p200 pemphigoid requires detection of serum IgG at the dermal side of 1 mol L^?1 salt‐split skin by indirect immunofluorescence microscopy and labelling of a 200‐kDa protein by Western blotting of dermal extract. However, preparation of dermal extract is not widely available, limiting the possibility of diagnosing this disease to a few laboratories. Objectives To develop a simple, sensitive and specific diagnostic tool for anti‐p200 pemphigoid. Methods Sera from patients with anti‐p200 pemphigoid (n = 35), bullous pemphigoid (BP, n = 101), epidermolysis bullosa acquisita (EBA, n = 10), antilaminin 332 mucous membrane pemphigoid (MMP, n = 14), pemphigus vulgaris (PV, n = 51) and healthy volunteers (HV, n = 131) were tested by a novel enzyme‐linked immunosorbent assay (ELISA) that employed a recombinant monomeric C‐terminal fragment of human laminin γ1 (hLAMC1‐cterm) expressed in Escherichia coli. Results Serum reactivity with hLAMC1‐cterm was detected in sera from 24 of 35 (69%) patients with anti‐p200 pemphigoid, two of 101 (2%) with BP, 0 of 10 with EBA, two of 14 (14%) with anti‐laminin 332 MMP, 0 of 51 with PV, and 0 of 131 HV. Conclusions This novel ELISA will facilitate the diagnosis of anti‐p200 pemphigoid.     

Epidermolysis bullosa acquisita with combined features of bullous pemphigoid and cicatricial pemphigoid.

Epidermolysis bullosa acquisita (EBA) is an acquired subepidermal blistering disease associated with autoantibodies against type VII collagen. The classical or mechanobullous form of EBA is characterized by skin fragility, trauma-induced blisters and erosions with mild mucous membrane involvement and healing with scars. Furthermore, bullous-pemphigoid-like and cicatricial pemphigoid-like features have been described. We report a patient who developed a bullous skin disease with upper airway obstruction requiring tracheotomy. The diagnosis of EBA was established by immunoblot, showing a band at 290 kD (collagen VII), and NaCl-split skin immunofluorescence (IgG deposition at the floor of the split). This case presented with clinical features of both bullous pemphigoid and cicatricial pemphigoid which to our knowledge is the first report of such a combination in EBA. The patient also presented tracheal involvement that has never been described either.     

Inflammatory variant of epidermolysis bullosa acquisita with IgG autoantibodies against type VII collagen and laminin alpha3

BACKGROUND: The inflammatory variant of epidermolysis bullosa acquisita (EBA) may clinically closely resemble bullous or cicatricial pemphigoid. Patients with inflammatory EBA have IgG autoantibodies against type VII collagen. Patients with anti-epiligrin cicatricial pemphigoid have IgG autoantibodies against laminin 5. OBSERVATION: We describe a patient with inflammatory EBA exhibiting nonscarring oral and vaginal involvement. Indirect immunofluorescence using skin substrate lacking an epidermal basement membrane molecule, direct immunoelectron microscopy, immunoblot, and immunoprecipitation studies revealed the simultaneous presence of circulating IgG autoantibodies against type VII collagen and laminin alpha3. A final diagnosis of EBA was based on the sublamina densa level of blister formation. CONCLUSION: This case illustrates the clinical and immunological overlap between EBA and anti-epiligrin cicatricial pemphigoid, a unique finding that may have developed as a consequence of epitope spreading.     

Childhood epidermolysis bullosa acquisita with autoantibodies against the noncollagenous 1 and 2 domains of type VII collagen: case report and review of the literature

Epidermolysis bullosa acquisita (EBA) is an acquired subepidermal bullous disease characterized by IgG autoantibodies to type VII collagen, a major component of anchoring fibrils. Most patients with EBA are adult and develop autoantibodies to the noncollagenous (NC) 1 domain of type VII collagen. We describe a 4-year-old Japanese boy presenting pruritic vesicles and tense blisters over his whole body. Immunofluorescence studies revealed linear IgG/C3 deposits along the dermal-epidermal junction of the patient's skin, and circulating IgG autoantibodies mapping to the dermal side of 1 M NaCl-split skin. By immunoblotting analysis using dermal extracts as a substrate, the patient's IgG antibodies labelled a 290-kDa protein corresponding to type VII collagen. Immunoblotting studies using recombinant proteins demonstrated that the patient's circulating autoantibodies recognized not only the NC1 but also the NC2 domain of type VII procollagen. Review of the previously reported cases and the present case suggested that patients with EBA with autoantibodies to regions other than the NC1 domain are all children younger than 10 years of age with clinical features of an inflammatory phenotype.     

Development of NC1 and NC2 domains of type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients

Background

Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain.

Objective

The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA.

Methods

We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera.

Results

When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity.

Conclusion

We conclude that the NC1 + NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients.     

Correlation of clinical severity and ELISA indices for the NC16A domain of BP180 measured using BP180 ELISA kit in bullous pemphigoid

BACKGROUND: Titres of circulating autoantibodies detected by indirect immunofluorescence (IIF) have been used for the diagnosis and evaluation of disease activity in bullous pemphigoid (BP). In BP, the major pathogenic epitope is known to be the non-collagenous extracellular domain (NC16A) of the 180-kDa transmembrane hemidesmosomal protein (BPAG2). Recently, an enzyme-linked immunosorbent assay (ELISA) kit using the NC16A domain recombinant protein (BP180 ELISA kit) has become commercially available to measure the quantities of pathogenic autoantibodies circulating in BP patients. OBJECTIVE: To investigate the correlation of clinical severity and ELISA indices in BP. METHODS: Fourteen patients with a typical form of BP and one refractory BP patient who died despite extensive treatment were included in this study. Antibody titres in sera from these patients were measured using BP180 ELISA kit and an analysis of ELISA indices with disease activity was performed. RESULTS: ELISA indices were significantly reduced after successful therapy, although IIF titres did not always show apparent correlations. In the patient with refractory BP, ELISA indices also showed a good correlation with disease course. ELISA indices measured using the BP180 ELISA kit were well correlated with the disease activity. CONCLUSION: This commercially available kit more closely followed disease activities than the IIF titres. The BP ELISA system may be a useful tool to evaluate the disease activity and to assess the effectiveness of the treatment of BP.     

Clearance rates of circulating and tissue‐bound autoantibodies to type VII collagen in experimental epidermolysis bullosa acquisita

Background Epidermolysis bullosa acquisita (EBA) is a severe autoimmune skin disease characterized by autoantibodies to type VII collagen, the major component of anchoring fibrils. In this and other autoimmune bullous dermatoses, specific autoantibody detection systems are not only of diagnostic use but also allow monitoring of circulating and skin‐bound autoantibodies during the course of the disease. However, little is known about their natural clearance rates in these different compartments. Objectives To study clearance rates of circulating and tissue‐bound autoantibodies to type VII collagen in experimental EBA. Methods Using offspring from mice with experimentally induced EBA, we examined retention times of diaplacentally transmitted autoantibodies to type VII collagen in serum of neonatal mice by enzyme‐linked immunosorbent assay and of immunoreactant deposits in skin by direct immunofluorescence microscopy. Additionally, the pathogenic potential of transmitted autoantibodies was evaluated in descendant mice. Results Immediately after birth, comparable levels of pathogenic antibody concentrations were observed in maternal and neonatal mice. The clearance time of skin‐bound autoantibodies was twice as long as that of circulating autoantibodies (8 and 4 weeks, respectively). Maternofetal transfer of pathogenic autoantibodies produced specific immunopathological (IgG, IgG1, IgG2a/b and complement C3 deposits) but not histological or clinical alterations in skin of offspring mice. Conclusions Although still to be confirmed in humans, our findings add to the knowledge on turnover rates of circulating and skin‐bound autoantibodies in autoimmune bullous dermatoses, which in turn may facilitate a more specific monitoring of these antibodies during the disease course, reduce the need for repeated skin biopsies, and may also be helpful in guiding treatment decisions.     

Role of BP180NC16a-enzyme-linked immunosorbent assay (ELISA) in the diagnosis of bullous pemphigoid in China

Background Autoantibodies of bullous pemphigoid (BP) patients react with two components of the hemidesmosome of stratified epithelia: the BP antigen 230 (BP230) and the BP antigen 180 (BP180). Recently, strong evidence has been provided that autoantibodies to BP180 play a key role in subepidermal blister formation in BP patients, and NC16A contains an important antigen determinant of BP. Objective To study the role of BP180NC16a enzyme‐linked immunosorbent assay (BP180NC16a‐ELISA) in the diagnosis of BP in China. Methods Sera from BP patients (n = 42) and control subjects (normal controls, n = 24; pemphigus patients, n = 18) were measured by BP180NC16a‐ELISA. All BP sera were obtained at presentation from patients who had not received previous systemic treatment. The values of immunoglobulin G (IgG) antibody levels measured by ELISA were compared with those measured by indirect immunofluorescence (IIF) (gold standard for the diagnosis of BP) on salt‐split skin. Results Using BP180NC16a‐ELISA, 41 of the 42 BP sera were positive, whereas only one of the serum samples from 24 normal controls was positive and all the pemphigus sera showed a negative result. Thus, the sensitivity and specificity of BP180NC16a‐ELISA were both 97.62%. There was no correlation between the mean ELISA values and IIF titers. The ELISA and IIF results were further compared and analyzed using a 2 × 2 contingency table, which showed that they were not significantly different. Conclusions It is suggested that BP180NC16a‐ELISA is a useful tool for the diagnosis of BP.     

Fluorescence overlay antigen mapping using laser scanning confocal microscopy differentiates linear IgA bullous dermatosis from epidermolysis bullosa acquisita mediated by IgA

Background Linear IgA bullous dermatosis (LABD) and epidermolysis bullosa acquisita (EBA) mediated by IgA antibodies belong to the group of autoimmune subepidermal bullous diseases mediated by IgA autoantibodies. Early and correct diagnosis is crucial because the management and prognosis of the diseases are different. Objectives To determine whether fluorescence overlay antigen mapping using laser scanning confocal microscopy (FOAM‐LSCM) is helpful in the differentiation between these diseases. Methods FOAM‐LSCM and immunoblot studies were performed in 19 patients with disseminated tense blisters who presented with in vivo bound and circulating IgA antibasement membrane zone (BMZ) antibodies on immunofluorescence. Results Using FOAM‐LSCM, in vivo bound IgA above type IV collagen, which is characteristic for LABD, was seen in 14 of the 19 cases, whereas five of the 19 cases had IgA deposits below type IV collagen, typical for EBA. Immunoblot studies showed that IgA antibodies in 11 of the 14 patients with deposits above type IV collagen reacted with different epitopes on BP180, mainly with LAD‐1, which is a target antigen in LABD. Among the five patients with deposits below type IV collagen, one showed IgA antibodies to the 200‐kDa laminin γ‐1 and one had antibodies to the 290‐kDa type VII collagen, EBA antigen. Additionally, enzyme‐linked immunosorbent assay with recombinant type VII collagen was positive in three of the five cases who presented with IgA deposits below type IV collagen on FOAM‐LSCM. Conclusions The results using FOAM‐LSCM were consistent with those obtained on immunoblotting. FOAM‐LSCM is useful in routine diagnostics in cases with undetectable circulating anti‐BMZ antibodies, and can differentiate LABD from IgA‐EBA, the former with in vivo bound IgA above type IV collagen and the latter with IgA deposits below type IV collagen.     

Epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a rare, acquired, chronic subepidermal bullous disease of the skin and mucosa characterized by autoantibodies to type VII collagen (C7) structures, a major component of anchoring fibrils, which attach the epidermis to the dermis. EBA patients have tissue-bound and circulating antitype C7 autoantibodies that attack type C7 and result in a reduction or perturbation of normally functioning anchoring fibrils. Patients with EBA have skin fragility, blisters, erosions, scars, milia, and nail loss, all features reminiscent of genetic dystrophic epidermolysis bullosa. These immunoglobulin G antitype C7 antibodies are pathogenic, because when they are injected into mice, the mice develop an EBA-like blistering disease. In addition to the classical mechanobullous presentation, EBA also has several other distinct clinical syndromes similar to bullous pemphigoid, Brunsting-Perry pemphigoid, or cicatricial pemphigoid. Although treatment for EBA is often unsatisfactory, some therapeutic success has been achieved with colchicine, dapsone, plasmapheresis, photopheresis, infliximab, and intravenous immunoglobulin.     

Identification of the target antigen in chronic bullous disease of childhood and linear IgA disease of adults

Disease-associated autoantibodies to basement membrane proteins have been used to characterize structural components of the epidermal basement membrane such as bullous pemphigoid (BP) antigen and epidermolysis bullosa acquisita (EBA) antigen (type VII collagen). The autoimmune bullous diseases characterized by IgA autoantibodies to the basement membrane zone (BMZ), i.e. linear IgA disease of adults (LAD) and chronic bullous disease of childhood (CBDC) may have circulating antibodies. Previous studies of tissue distribution and ultrastructural binding have suggested that the LAD and CBDC antigens are similar, if not identical, and differ from the target antigens of the other bullous diseases. We present the molecular characterization of the LAD/CBDC antigens by Western blotting of a large series of antisera. Seven of 33 sera (21%) were positive on immunoblotting and bound to the same antigen which has a molecular weight (MW) of 285 kDa. Using both defined polyclonal antisera to BP and LH 7.2 monoclonal antibody to type VII collagen (carboxy terminal) we have shown that the LAD and CBDC antisera both bind to an identical molecular weight protein which clearly differs from both the BP and EBA (type VII collagen) antigens. Although detectable in dermal tissue extracts like EBA, the MW of 285 kDa is heavier than type VII collagen (250 kDa, in our system, using non-collagenous standards). This study confirms the identity of LAD and CBDC antigens to be the same and to differ from previously described basement membrane proteins.     

Epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune subepidermal bullous disease with clinical features similar to the genetic form of dystrophic epidermolysis bullosa. EBA is characterized by the presence of autoantibodies against type VII collagen which is a major component of the anchoring fibrils at the dermal‐epidermal junction. EBA can be divided into two main clinical types; mechanobullous and inflammatory EBA. Mechanobullous EBA, referred to as classic EBA, presents with skin fragility, blisters and dystrophic changes on trauma‐prone areas. Inflammatory EBA resembles other autoimmune subepidermal bullous diseases. Compelling evidence from mouse models supports a pathogenic role of autoantibodies against type VII collagen in EBA. Treatment of EBA is often unsatisfactory. The most widely used systemic treatment is corticosteroids. Colchicine and dapsone have been reported to be good treatment modalities when combined with corticosteroids. Some intractable cases of EBA have successfully been treated with intravenous immunoglobulin or rituximab.     

Acquired bullous diseases of childhood: re-evaluation of diagnosis by indirect immunofluorescence examination on 1 M NaCl split skin and immunoblotting

Summary Acquired autoimmune bullous diseases of childhood are rare, and can be difficult to distinguish clinically. We have studied 12 children, with an initial diagnosis of bullous pemphigoid (BP) in eight patients, cicatricial pemphigoid (CP) in one, chronic bullous disease of childhood (CBDC) in one, and epidermolysis bullosa acquisita (EBA) in two.
All patients had positive indirect immunofluorescence (HF) of the BMZ with IgG. Using 1 M NaCl split skin, six patients showed epidermal binding of IgG, with additional IgA in three cases, and in five patients IgG antibodies bound a dermal protein. Immunoblotting studies revealed an antibody to type VII collagen (EBA antigen) in three patients who had a dermal pattern on IIF. Six sera reacted with an epidermal protein of 180 and/or 220 kDa, characteristic of BP and CP. One of the three IgA-positive sera detected 220-and 180-kDa epidermal proteins using anti-IgA antibody. Following these studies the diagnosis was changed in three of the children. The diagnosis of CBDC was changed to either BP or EBA because of the presence of circulating IgG autoantibodies. In two children with an initial diagnosis of BP the diagnosis was changed to EBA.
We conclude that the clinical picture in bullous disorders of childhood shows considerable overlap, and is often misleading. Additional circulating IgA autoantibodies seem to be more common in BP than has been recognized previously. Indirect immunofluorescence investigation on 1 M NaCl split skin may be helpful in differentiating between BP and EBA, but does not replace immunoblotting studies. EBA is apparently more common in children than in adults. No difference was found between the children with BP and EBA with regard to the duration of disease. The long-term outlook is good, although the course may be protracted.     

U-serrated immunodeposition pattern differentiates type VII collagen targeting bullous diseases from other subepidermal bullous autoimmune diseases

BACKGROUND: Epidermolysis bullosa acquisita (EBA) can be differentiated from other subepidermal bullous diseases by sophisticated techniques such as immunoelectron microscopy, salt-split skin antigen mapping, fluorescence overlay antigen mapping, immunoblot and enzyme-linked immunosorbent assay. OBJECTIVES: To determine whether the diagnosis can also be made by routine direct immunofluorescence microscopy. METHODS: We studied frozen skin biopsies from 157 patients with various subepidermal immunobullous diseases. RESULTS: We found three distinct 'linear' fluorescence patterns at the basement membrane zone: true linear, n-serrated and u-serrated. The true linear pattern, often seen in conjunction with either the n- or the u-serrated pattern, was found in any subepidermal immunobullous disease with nongranular depositions. In bullous pemphigoid, mucous membrane pemphigoid, antiepiligrin cicatricial pemphigoid, p200 pemphigoid and linear IgA disease the n-serrated pattern was found, corresponding with depositions located in hemidesmosomes, lamina lucida or lamina densa. However, in EBA and bullous systemic lupus erythematosus the u-serrated staining pattern was seen, corresponding with the ultralocalization of type VII collagen in the sublamina densa zone. The diagnosis of EBA with IgG or IgA autoantibodies directed against type VII collagen was confirmed by immunoelectron microscopy, salt-split skin antigen mapping, fluorescence overlay antigen mapping or immunoblotting. CONCLUSIONS: Using this pattern recognition by direct immunofluorescence microscopy we discovered several cases of EBA which would otherwise have been erroneously diagnosed as a form of pemphigoid or linear IgA disease.     

Enzyme‐linked immunosorbent assay (ELISA) and indirect immunofluorescence testing in a bullous pemphigoid and pemphigoid gestationis

Enzyme‐linked immunosorbent assay (ELISA) is an excellent tool for detection of circulating antibodies against the NC16A portion of BP180 antigen. We compared the sensitivity and specificity of a commercially available BP180‐NC16a domain ELISA with that of an indirect immunofluorescence (IIF) testing in the evaluation of bullous pemphigoid (BP) and pemphigoid gestationis (PG), and analyzed the relationship between ELISA results and the presence of IgG deposition, in an epidermal or combined pattern, on direct immunofluorescence (DIF) testing of salt‐split skin. ELISA was performed on serum from 28 patients (24 BP, 4 PG) and 50 controls. IIF testing was performed on serum from 27 patients and 98 controls. For the group of 28 patients with BP or PG, ELISA had a sensitivity of 93% and specificity of 96% (P < 0.001), while sensitivity was 74% and specificity 96% (P < 0.001) for IIF testing. In these patients, ELISA has a higher sensitivity than IIF testing, but similar specificity. Evaluation of controls who had IgG deposition on the dermal side of salt‐split skin on DIF testing showed specificity for the ELISA of 100% (all four cases negative) and 80% for IIF testing (one of five positive). Positive ELISA correlated with a diagnosis of BP or PG only in patients who had IgG at the basement membrane zone (BMZ) by DIF testing. Overall, ELISA appears to have greater sensitivity and specificity for BP or PG than does IIF testing.