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Expert Review of Clinical Immunology

ISSN: 1744-666X (Print) 1744-8409 (Online) Journal homepage: https://www.tandfonline.com/loi/ierm20

Clinical features and diagnosis of epidermolysis


bullosa acquisita

Artem Vorobyev, Ralf J. Ludwig & Enno Schmidt

To cite this article: Artem Vorobyev, Ralf J. Ludwig & Enno Schmidt (2017) Clinical features and
diagnosis of epidermolysis bullosa acquisita, Expert Review of Clinical Immunology, 13:2, 157-169,
DOI: 10.1080/1744666X.2016.1221343

To link to this article: https://doi.org/10.1080/1744666X.2016.1221343

© 2016 The Author(s). Published by Informa


UK Limited, trading as Taylor & Francis
Group.

Accepted author version posted online: 31


Aug 2016.
Published online: 08 Sep 2016.

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EXPERT REVIEW OF CLINICAL IMMUNOLOGY, 2017
VOL. 13, NO. 2, 157–169
http://dx.doi.org/10.1080/1744666X.2016.1221343

REVIEW

Clinical features and diagnosis of epidermolysis bullosa acquisita


Artem Vorobyeva,b, Ralf J. Ludwig a,b
and Enno Schmidta,b
a
Department of Dermatology, University of Lübeck, Lübeck, Germany; bLübeck Institute of Experimental Dermatology (LIED), University of Lübeck,
Lübeck, Germany

ABSTRACT ARTICLE HISTORY


Introduction: Epidermolysis bullosa acquisita (EBA) is a rare autoimmune blistering disease of skin and Received 22 May 2016
mucous membranes. EBA is caused by autoantibodies against type VII collagen, which is a major Accepted 3 August 2016
component of anchoring fibrils, attaching epidermis to dermis. Binding of autoantibodies to type VII Published online
collagen leads to skin fragility and, finally, blister formation. The clinical picture of EBA is polymorphic, 8 September 2016
with several distinct phenotypes being described. Despite recent progress in understanding the KEYWORDS
pathophysiology of EBA, its diagnosis is still challenging. Autoantibody; ELISA;
Areas covered: This review provides an update on the clinical manifestations and diagnostic methods immunofluorescence;
of EBA. We searched PubMed using the terms ‘epidermolysis bullosa acquisita’ covering articles in serration pattern analysis;
English between 1 January 2005 and 31 May 2016. Relevant older publications were retrieved form type VII collagen
cited literature.
Expert commentary: While the clinical picture is highly variable, diagnosis relies on direct immuno-
fluorescence (IF) microscopy of a perilesional skin biopsy. Linear deposits of IgG, IgA and/or C3 along
the dermal-epidermal junction with an u-serrated pattern are diagnostic for EBA alike the detection of
serum autoantibodies against type VII collagen. Several test systems for the serological diagnosis of EBA
have recently become widely available. In some patients, sophisticated diagnostic approaches only
available in specialized centers are required.

1. Introduction variants have been described, the classical mechanobullous


form [10] and the inflammatory variant [11]. Within latter
In the past decades, the incidence of autoimmune diseases in
form, subtypes resembling bullous pemphigoid, mucous
developed countries is increasing resulting in a major health
membrane pemphigoid, linear immunoglobulin A (IgA) dis-
burden [1]. Autoimmune bullous diseases (AIBD) are a hetero-
ease, and Brunsting–Perry pemphigoid can be differentiated
geneous group and one of the major organ-specific autoim-
[11–14]. Diagnosis of EBA can, thus, not be based on the
mune diseases.
clinical picture alone but requires a combination of laboratory
AIBD can be separated in three different groups, namely
investigations including direct immunofluorescence (IF) micro-
pemphigoid diseases, pemphigus diseases, and dermatitis her-
scopy and serological assays. In this review, we will provide an
petiformis. Pemphigoid diseases are characterized by the pre-
update on the clinical manifestations and diagnostic options
sence of autoantibodies against structural proteins of dermal–
of EBA and will shortly summarize recent epidemiological and
epidermal junction and lead to subepidermal blistering,
pathophysiological discoveries of this disease.
whereas in pemphigus diseases, autoantibodies against des-
mosomal proteins are detected causing intraepidermal blister
formation. Dermatitis herpetiformis is always associated with 2. History
celiac disease and characterized by autoantibodies against
EBA was initially described by G.T. Elliott in 1895 in two
transglutaminase 2 and 3 [2–4].
patients clinically reminiscent of epidermolysis bullosa hered-
Epidermolysis bullosa acquisita (EBA) is a rare chronic auto-
itaria but with no affected family members [15]. In 1971, Henry
immune blistering disease of skin and mucous membranes
H. Roenigk established the first diagnostic criteria for the
characterized by circulating and tissue bound antibodies
mechanobullous form of EBA, i.e. (1) onset of the disease in
against type VII collagen [5,6]. Type VII collagen is the major
adulthood, (2) absence of familial history for bullous diseases,
component of anchoring fibrils, which provides attachment of
(3) trauma-induced or spontaneous formation of blisters
the epidermis and underlying basement membrane zone
resembling dystrophic epidermolysis bullosa, and (4) exclusion
(BMZ) to the dermis (Figure 1) [7–9]. Destruction of anchoring
of other bullous diseases. In 1984, David Woodley identified
fibrils leads to detachment of the epidermis from the dermis
the target antigen of EBA as a 290-kDa protein in an extract of
resulting in skin fragility, blister formation, erosions that fre-
human dermis [16]. This 290-kDa protein was later shown to
quently heal with scarring and milia formation. Clinical pre-
be type VII collagen [17].
sentations of EBA are heterogeneous. Two major clinical

CONTACT Enno Schmidt enno.schmidt@uksh.de Department of Dermatology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany
© 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
158 A. VOROBYEV ET AL.

cell keratin
membrane filaments plectin

hemidesmosomal
plaque BP230

α6 integrin NC16A
domain
β4 integrin
Lamina lucida
BP180
laminin 332

Lamina densa
type VII
collagen laminin γ1

Sublamina densa

dermal collagens

Figure 1. Schematic structure of the dermal-epidermal junction. Only proteins that are targeted by autoantibodies in autoimmune blistering diseases are depicted.

Traditionally, EBA has been classified as an entity outside reported in individual EBA patients [5,37,38]. Recently, an
the pemphigoid group defining pemphigoid diseases as dis- association with hematological malignancies i.e. lymphoma
orders with hemidesmosomal target antigens [18]. For derma- was observed in about 8% of EBA patients [39] in line with a
tologists not primarily involved in the care of AIBD patients as previous study with 102 AIBD patients [40].
well as other physicians, these subtle differences may hardly As triggering factors of EBA, penicillin, vancomycin, genta-
be appreciated. We therefore advocate to classify pemphigoid mycin, UV-radiation, and contact allergy to metals have been
diseases based on reactivity against structural proteins of the reported [41–44].
dermal–epidermal junction rather that against hemidesmoso-
mal proteins [3].
4. Pathophysiology
Type VII collagen is composed of three identical alpha-chains
3. Epidemiology
each consisting of a 145-kDa N-terminal non-collagenous (NC)-
EBA is one of the rarest autoimmune blistering diseases with 1 domain, a large central collagenous domain, and a smaller
an incidence of 0.2–0.5/million inhabitants/year [19–23]. In a C-terminal NC2 domain [7]. Type VII collagen molecules form
recent analysis of data from the largest German insurance antiparallel tail-to-tail dimers with the help of the NC2
company from 2014, the prevalence of EBA was calculated to domains that are subsequently removed during this process
be 2.84/million inhabitants, i.e. about 230 EBA patients in [7,45]. The NC1 domain consists of N-terminal protein with
Germany [24]. homology to cartilage matrix protein, nine fibronectin-like
The disease can affect all age groups with an average onset domains, and C-terminal portion with homology to the von
of disease at the age of 50 years [25–27]. Children and elderly Willebrand factor A domain (Figure 2). The NC1 domain inter-
can also be affected [28–30] with children making up about acts with several structural molecules of the BMZ such as type
10% of EBA patients (unpublished observation). No gender IV collagen, laminin 332, fibronectin, and type I dermal col-
predisposition is known [24,26,27,31]. lagen [46–50]. The NC1 domain was previously described as
In two studies, EBA occurred more frequently in black major antigenic portion in EBA [51–53], however, patients with
patients of African descent [25,32]. EBA is associated with antibodies against the NC2 and the collagenous domains have
the HLA-DR2 (corresponding to HLA-DRB1*15) and been described [54–59]. Perturbation of the interaction of the
DRB1*15:03, an allele found frequently in the general popula- NC1 domain with these molecules caused by binding of auto-
tion [25,32]. Korean EBA patients more frequently carried antibodies against specific subdomains of NC1 is discussed to
DRB1*13 compared to controls [33]. This finding of an associa- explain the variation of in the clinical phenotypes of EBA.
tion with the major histocompatibility complex locus is paral- However, to this moment, no clear correlation between anti-
leled in a mouse model of the disease, where susceptibility to type VII collagen autoantibody specificities and a distinct clin-
develop clinical EBA lesions after manifestations is closely ical phenotype has been described [60,61]. Another
linked to the H2s haplotype [34]. explanation of blister formation could be the direct interfer-
While in 25% and 16% of patients from USA and France, an ence with the NC2 domain and hindering of antiparallel tail-
association with inflammatory bowel disease was observed to-tail dimer formation [62].
this association was not found in Korean patients Detailed analyses of the autoantibody response in EBA
[25,27,35,36]. A variety of other diseases, e.g. rheumatoid revealed the presence of IgG in the majority of patients with
arthritis, diabetes, cryoglobulinemia, and psoriasis were predominance of the IgG1 and IgG4 subclasses [63–65]. In
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 159

Figure 2. Structure of type VII collagen. CMP: cartillage matrix protein; 1–9: fibronectin III-like repeats 1–9; vWFA2: von Willebrand factor A; NC: noncollagenous.

addition, IgA antitype VII collagen reactivity, either exclusively knuckles, toes, sacral area (Figure 3). In the course of disease,
or in combination with IgG autoantibodies, is observed in cicatricial alopecia and onychodystrophy may appear. In mild
50–60% of patients [26,27,66]. forms of the mechanobullous variant, porphyria cutanea tarda
The pathogenic relevance of antibodies against type VII and pseudoporphyria need to be excluded. Severe forms of
collagen has clearly been shown in several in vivo and in classical EBA can imitate hereditary epidermolysis bullosa [85].
vitro models [67–71] reviewed in [6]. Incubation of cryosec-
tions of normal human skin with sera of EBA patients followed
by incubation with neutrophils from healthy volunteers leads 5.2. Bullous pemphigoid-like variant
to dermal–epidermal split formation [69]. Injection of antitype Bullous pemphigoid is by far the most frequent autoimmune
VII collagen antibodies in mice results in a skin disease that blistering disease [86–88] (reviewed in [89]). Bullous pemphi-
clinically and immunopathologically mimics the human dis- goid typically presents with tense blisters, erosions on
ease [61,67,71–73]. While these models well reflect character- inflamed or otherwise unaffected skin, crusts, and urticarial-
istic features of human EBA, they only reproduce the effector like erythema [90]. Like in bullous pemphigoid, in this EBA
phase of EBA, i.e. the antitype VII collagen-mediated tissue variant, pruritus is often reported, while milia and scar forma-
destruction. To investigate the loss of tolerance to type VII tion are absent (Figure 4). Lesions mainly involve the trunk,
collagen, mice were immunized with recombinant fragments skin folds, and extremities [11,91]. This inflammatory form of
of NC1 domain, which raised murine antitype VII collagen IgG EBA is found in 25–50% of all reported EBA cases [27].
that resulted in a long-lasting autoimmune skin disease resem-
bling human EBA [68,74]. These animal models were subse-
quently used to show the pathogenic relevance of 5.3. Mucous membrane pemphigoid-like variant
complement activation at the epidermal BMZ, neutrophils,
CD18-mediated extravasation, FcγRIV, heat-shock protein 90, Mucous membrane pemphigoid is a pemphigoid disease char-
the glycosylation status of antitype VII collagen IgG, IL-1β, IL-6, acterized by the predominant or exclusive involvement of
GM-CSF, Erk 1/2, CXCL1/2, p38, Akt, RORa, HSP90, gene expres- mucous membranes [92]. All squamous cell epithelia can be
sion in myeloid cells, and the skin microbiome [75–84]. affected, including oral and nasal mucosa, larynx, esophagus,
anal and genital mucosa as well as conjunctiva [14,93–95].
While 50–65% of EBA patients have mucosal lesions
5. Clinical presentation [26,96,97] (Figure 5) in about 5–10% of patients, mucosal
involvement predominates and patients may be classified as
Five different phenotypes of EBA have been reported so far, mucous membrane pemphigoid-variant of EBA [27,85]. In
including the classical mechanobullous variant described by some EBA patients, exclusive involvement of the esophagus
Roenigk [10] and the four inflammatory forms resembling or nasal mucosa has been reported [98]. In this variant, con-
bullous pemphigoid, mucous membrane pemphigoid, linear siderable overlap occurs with mucous membrane pemphigoid
IgA-disease, and Brunsting–Perry pemphigoid, respectively itself. Many physicians will according to the consensus con-
[11–14]. About one-third of EBA patients present with the ference on mucous membrane pemphigoid classify patients
classical phenotype and about two-thirds with one of the
inflammatory variants [26,27]. Clinical subtypes, however, are
neither categorical nor static. Some patients present with (b)
(a)
mixed phenotypes and in others, the subtype changes during
the course of their disease [26]. Mucosal involvement is found
in about 60% of patients but only predominates over skin
lesions in about 5–10% of EBA, i.e. in those with the mucous
membrane pemphigoid variant [26,27].

5.1. Mechanobullous variant


This classical form of EBA demonstrates skin fragility, vesicles,
tense blisters, and erosions on non-inflamed skin, which heal
with scarring and milia formation [10]. Lesions can affect every
Figure 3. Classical variant of EBA. (a) Erythema and tense blisters on the right
region of skin and mucous membranes with predisposition to knee of a 6-year old boy. B. Erythema and erosions on the left hand of a 53-year
trauma-prone areas, such as elbows, knees, hands, feet, old female.
160 A. VOROBYEV ET AL.

with autoantibodies against type VII collagen and predomi-


(a) (b)
nant mucosal lesions as mucous membrane pemphigoid [92],
others as mucous membrane pemphigoid-like variant of EBA.
When conjunctival or laryngeal lesions are present, the
authors would recommend classification as mucous mem-
brane pemphigoid since treatment may follow protocols for
latter disease including more aggressive regimens, e.g. with
cyclophosphamide [92].

5.4. Linear IgA disease-like variant


This EBA variant presents with tense vesicles, blisters, erythema,
and urticated plaques in an annular or polycyclic pattern with
vesicles frequently localized along the edge of the lesion. This
pattern is associated with (but not limited to) linear IgA derma-
tosis and is termed ‘string-of-pearls’ or ‘crown-of-jewels’ sign.
Figure 4. Inflammatory variant of EBA. (a) Erythema, erosions, and crusts on the Scarring and milia formation are absent like in linear IgA disease.
left knee of a 76-year old patient. (b) Blisters and erosions on the right elbow of Mucosal involvement, and in 4% of cases, severe conjunctival
the same EBA patient. lesions have been described [85,99]. As in linear IgA disease,
linear deposits of IgA are visualized by direct IF microscopy of a
perilesional biopsy. It may be debated if these patients are classi-
fied as linear IgA disease with autoantibodies against type VII
(a) collagen or as linear IgA disease-like variant of EBA, also called IgA
EBA [100,101]. Interestingly, in 11% of patients with this variant, a
close relation to the initiation of a new drug has been reported
[102]. Otherwise, no difference between EBA patients with exclu-
sive or predominant IgA reactivity compared to patients with IgG
antitype VII collagen antibodies has been detected [26].

5.5. Brunsting–Perry pemphigoid-like variant


This rare clinical form is characterized by subepidermal blisters,
erosions, hemorrhagic crusts, and atrophic scars on the head and
neck without involvement of mucous membranes [103]. In most
patients with Brunsting–Perry pemphigoid, antibodies against
type VII collagen have been detected, thus classifying as
Brunsting–Perry pemphigoid-like variant of EBA
(b) [12,26,27,104–106].
In addition, a single patient with congenital EBA has been
described [107]. A mother with known EBA delivered an other-
wise healthy girl with tense blisters and erosions. In the new-
born, direct IF microscopy showed linear deposits of IgG and
C3 at the BMZ and serum antibodies against type VII collagen
were detected. Blistering ceased within 10 days under suppor-
tive care and no recurrence was noted [107]. This case clearly
demonstrates the pathogenic potential of type VII collagen-
specific autoantibodies.

6. Diagnostic methods
Due to the variety of clinical presentations, diagnosing EBA
based on the clinical picture alone is not possible. In case of
clinical suspicion of EBA, diagnosis needs to be based on
different approaches schematically shown in Figure 12.
Traditionally, the diagnostic gold standard has been direct
Figure 5. Mucosal involvement. (a) Blisters and erosions on the lower lip of a 6-
year old boy (same patient as in Figure 3(a). (b) Erosions on the tongue of a 76- immunogold electron microscopy [108]. At present, in nearly
year old male with EBA (same patient as in Figure 4(a,b) all patients, diagnosis can be made by serration pattern
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 161

analysis of direct IF microcopy or/and detection of serum pemphigoid diseases, linear binding of IgG and/or IgA, and/or
autoantibodies against type VII collagen [16,109,110]. For lat- C3 can be seen along the dermal–epidermal junction.
ter analysis, three commercial test systems are currently avail- Importantly, by close inspection of thin sections of 4–6 µm,
able [55,65]. Vodegel et al. could show that IgG/IgA linear deposits at the
BMZ show a subtle but well remarkable pattern described as
u-serrated [110]. This pattern is characterized by arches that
6.1. Histopathology are closed at the bottom giving the appearance of ‘growing
Histopathology of a lesional skin biopsy allows distinguishing grass’ or ‘upstanding arms’ (Figure 6). It is recommended to
between intraepidermal (like in pemphigus) and subepidermal perform this analysis at ×400–×600 magnification. The u-ser-
blistering characteristic for pemphigoid disorders such as EBA. rated pattern is pathognomonic for skin-bound autoantibo-
In the classical mechanobullous form, a scarce or no inflam- dies against type VII collagen as found in EBA and bullous
matory infiltrate in the dermis can be detected. In the inflam- systemic lupus erythematosus [26,124]. In all other pemphi-
matory variants, infiltration of neutrophils with variable goid diseases, a n-serrated pattern characterized by arches
numbers of eosinophils, monocytes, and lymphocytes are closed at the top giving a ‘snake-like’ appearance
found in the upper dermis. In the classical mechanobullous [110,124,125]. However, in mucosal biopsies and a number
variant, fibrosis and milia may be present which correspond to of skin samples, no u- or n-serrated pattern can be seen.
cicatricial changes seen clinically [11]. Histopathology alone Current studies are aimed at defining the best conditions for
cannot differentiate between the different pemphigoid serration pattern analysis, determine its sensitivity and estab-
disorders. lish an automated serration pattern analysis.
In samples where no serration pattern can be identified, the
perilesional biopsy can be subjected to incubation with 1 M
6.2. Immunoelectron microscopy NaCl solution which leads to cleavage within the lamina lucida
[126]. In EBA, autoantibodies and complement depositions can
Using electron microscopy ultrastructural changes in the peri- then be detected along the floor of the artificial split. This
lesional skin, such as paucity or absence of anchoring fibrils technique, however, is tricky and may result in the complete
can be demonstrated. This finding is similar to that of epider- destruction of the skin sample. Moreover, patients with anti-
molysis bullosa hereditaria and could explain skin fragility p200/laminin γ1 pemphigoid and anti-laminin 332 pemphi-
[111,112]. When performed from lesional skin, the cleavage goid also reveal autoantibodies bound at the blister floor
plane can be demonstrated. However, cleavage can appear in and cannot be differentiated from EBA by this technique
sublamina densa or lamina lucida zone [113]. Localization of [4,127].
blister in the lamina lucida has been explained by the lamina When the perilesional skin biopsy is subjected to co-incu-
lucida being the locus minoris resistentiae and thus, most bation with antibodies against e.g. BP180, laminin 332, and
vulnerable to proteolytic enzymes released during the inflam- type VII collagen, the fluorescence overlay antigen mapping
matory reaction at the BMZ [114–116]. Thus, transmission (FOAM) technique allows the detection of co-localization of
electron microscopy cannot be used in differentiating EBA autoantibodies by overlay of the different pictures [128–130].
from inherited epidermolysis bullosa and other pemphigoid In EBA, labeling of patient’s tissue-bound autoantibodies e.g.
disorders. with a green fluorescence dye and staining of the biopsy with
In contrast, direct immunogold electron microscopy allows an antihuman type VII collagen antibody labeled e.g. with a
to visualize the deposits of autoantibodies in the sublamina red fluorescence dye will show a yellowish staining along the
densa which clearly differentiates EBA from deposits in other BMZ by direct IF microscopy. This technology is only available
pemphigoid diseases located either in the lamina lucida or in few laboratories worldwide.
lamina densa [117]. Subsequently, direct immunogold electron
microscopy became the diagnostic gold standard for EBA
[85,108,118–121]. Unfortunately, this technique is available
for the diagnosis of autoimmune blistering diseases in only a 6.4. Serology
handful of laboratories. Although attempts have been made
that allow the sending of biopsies for direct immunogold
electron microscopy [122], this approach is best performed
with freshly taken biopsies. (a) (b)

6.3. Direct IF microscopy


Direct IF microscopy is performed in a perilesional biopsy, i.e.
adjacent (with a radius of 1 cm) to a macroscopic blister [123].
In a lesional biopsy, i.e. when a split formation can be seen
microscopically, direct IF microscopy can be false negative due
to the proteolytic degradation of Ig deposits at the BMZ or Figure 6. Direct immunofluorescence microscopy. (a) u-serrated deposition of
IgG along the basement membrane zone in a patient with EBA. In contrast, in all
false positive due to unspecific binding if autoantibodies at of the pemphigoid diseases, an n-serrated pattern is seen, as exemplified in a
the BMZ of the microscopic splitting [123]. In EBA, like in all patient with bullous pemphigoid (b). Magnification x 1000.
162 A. VOROBYEV ET AL.

Serology has become the major diagnostic approach in auto- (a) (b)
immune blistering diseases including EBA [131]. This develop-
ment was fostered by the establishment of several enzyme-
linked immunosorbent assay (ELISA) and indirect IF micro-
scopy-based test systems that became widely available and
are based on the use of the corresponding recombinant target
antigen [55,65,132–136]. In EBA, serological diagnosis is ham-
pered by the relatively low rate of circulating autoantibodies
which in some studies, was only found in about 60% of
Figure 8. Indirect immunofluorescence on type VII collagen-deficient skin.
patients [26,137]. Indirect immunofluorescence microscopy of a EBA patient’s serum on normal
human skin shows linear labeling of IgG at the basement membrane zone (a),
6.4.1. Indirect IF microscopy while on type VII collagen-deficient skin, no staining is seen (b; courtesy of Dr.
Hendri Pas, Department of Dermatology, University of Groningen, The
Indirect IF microscopy can be applied for the characterization of Netherlands).
circulating antibodies. Standard substrates for this technique
are monkey or rabbit esophagus as well as normal human
skin in which subepidermal splitting was induced by incubation
with 1 M NaCl solution (Figure 7) [3,123,131]. As in all pemphi- (a) (b)
goid diseases, antitype VII collagen antibodies bind along the
BMZ on the monkey and rabbit esophagus. By indirect IF
microscopy of human salt-split skin, antitype VII collagen anti-
bodies label the floor of the artificial blister (Figure 7) [138].
However, antibodies against laminin 332 and the p200 protein/
laminin γ1 also bind to the floor of the blister [3,127,139,140]. In
latter two diseases, however, direct IF microscopy reveals an
n-serrated pattern. Several studies have reported sensitivities of
human salt-split skin for the detection of serum autoantibodies
in EBA ranging from 57% to 100% with a mean of 64% in 191
Figure 9. Western blotting. (a) Western blot with dermal extract. 1, blood donor;
EBA sera [26,27,137,141–143]. 2, positive control; 3, EBA patient; arrow indicates the migration position of type
An elegant and relatively simple method for the detection VII collagen. (b) Western blot with recombinant NC1 domain of type VII collagen.
of antitype VII collagen antibodies by indirect IF microscopy is 1, blood donor; 2, positive control; 3, EBA patient; arrow indicates the migration
position of NC1 domain.
the use of type VII collagen-deficient skin from patient with
dystrophic epidermolysis bullosa. Incubation with EBA sera
with type VII collagen-specific antibodies will not show any
labeling of the BMZ, whereas on normal/salt-split human skin, bind to a 290-kDa protein which represents full-length type VII
as well as on laminin 332 and type XVII collagen-deficient skin, collagen (Figure 9) [52,53]. Moreover, type VII collagen has
linear fluorescence is seen at the BMZ (Figure 8) [144,145]. also been found in extracts of cultured human keratinocytes
and human amniotic membrane that consequently, were pro-
6.4.2. Western blotting posed as useful in the detection of serum antitype VII collagen
This semiquantitative method is based on cell-derived or antibodies by immunoblotting [147–149].
recombinant forms of type VII collagen that had been sub-
jected to SDS–PAGE, transferred onto a nitrocellulose mem- 6.4.3. Enzyme-linked immunosorbent assay
brane, and incubated with patient’s serum. Antitype VII In 1997, Chen and coworkers developed an ELISA based on
collagen antibodies bound to the nitrocellulose-attached anti- the recombinant NC1 domain of type VII collagen with a
gen can then be visualized. Extract of human dermis and the sensitivity of 100% demonstrating that the NC1 domain was
recombinant NC1 domain of type VII collagen are usually used the immunodominant stretch of type VII collagen [109]. This
[52,53,146]. In dermal extract, antitype VII collagen antibodies ELISA, however, was only available in this particular laboratory.
In 2011 and 2013, two ELISA systems became widely available
based on the combination of the recombinant NC1 and NC2
(a)
A (b)
B domains and the NC1 domain alone, respectively (MBL,
Nagoya, Japan; Euroimmun, Lübeck, Germany) [55,65]. These
assays revealed sensitivities of 93.8% and 94.5% and specifi-
cities of 98.1% and 98.7%, respectively, in large cohorts of 49
and 73 EBA sera [55,65]. Sera were, however, selected based
on the clinical picture, and the presence of circulating auto-
antibodies by indirect IF microscopy on salt-split human skin
and/or immunoblotting with cell-derived type VII collagen.
When these ELISA were probed with EBA sera diagnosed by
Figure 7. Indirect immunofluorescence microscopy. (a) Linear binding of IgG
along the basement membrane on monkey esophagus. (b) IgG locates to the serration pattern analysis of direct IF microscopy or direct
floor of the artificial blister in 1 M NaCl-split human skin. immunogold electron microscopy, much lower sensitivities of
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 163

53% and 30% are reported [137,150]. In EBA sera unreactive by Subsequently, this BIOCHIP® mosaic has been extended by
indirect IF microscopy on human salt-split skin, ELISA reactivity HEK293 cells expressing the recombinant NC1 domain of type
could only be detected in 12% and 23% [137,150]. VII collagen on their cell surface revealing a sensitivity and
Most recently, two multivariant ELISA systems became specificity of 91.8% and 99.8% (Figure 11) [65]. Other investi-
widely available (Euroimmun, MBL). These assays allow the gators also showed sensitivities of the type VII collagen-spe-
simultaneous testing of sera for IgG autoantibodies against cific BIOCHIP® comparable with the corresponding ELISA
the most relevant target antigens of autoimmune blistering [137,157]. As pointed out above, the sensitivity of detection
diseases including type VII collagen [151] (van Beek et al., assays for antitype VII collagen IgG may vary considerably
submitted). depending on the analyzed patient cohort with circulating
Compared to Western blotting, ELISA is significantly faster, autoantibodies reported to be only present in about 60%
more convenient, and better standardized. Furthermore, ELISA EBA patients [26].
allows quantitative measurement of antitype VII collagen IgG
and may, thus, be a valuable tool in the follow-up of EBA
patients during the course of their disease. In fact, ELISA 6.5. Diagnostic pathway
values were shown to correlate with disease activity in EBA
patients (Figure 10) [150,152]. Recently, an in-house ELISA with In a patient with a clinical picture compatible with EBA, the
a recombinant form of full-length type VII collagen was current diagnostic gold standard is the direct IF microscopy of
reported with a sensitivity of 65% [137]. a perilesional skin biopsy including serration pattern analysis.
When an u-serrated pattern is observed EBA can be diag-
nosed. Direct immunogold electron microscopy may have a
6.4.4. BIOCHIP® mosaic similar sensitivity, however, is a sophisticated procedure only
Indirect IF microscopy based on BIOCHIP® mosaics allows the available in few centers for the diagnosis of EBA. Even when
simultaneous detection of serum autoantibodies against var- diagnosis of EBA has been established by pattern analysis of
ious substrates in a miniature incubation field of only several direct IF or immunogold electron microscopy serum should be
millimeters within a normal laboratory slide (Euroimmun). In analyzed for autoantibodies against type VII collagen.
autoimmune blistering diseases, a BIOCHIP® mosaic contain- When the serration pattern cannot be determined serolo-
ing six substrates comprising monkey esophagus, salt-split gical analysis is favored. By indirect IF microscopy on human
skin, recombinant BP180 NC16A, and HEK293 cells transfected salt-split skin, EBA sera bind to the dermal side of the artificial
for expression of desmoglein 1, desmoglein 3, and BP230 is split. However, this method is not diagnostic since sera from
widely used in the dermatological community [136,153–156]. patients with anti-laminin 332 and anti-p200/laminin γ1

Figure 10. Correlation of serum levels of IgG antibodies against type VII collagen with disease severity. Serum autoantibody levels were detected by ELISA
(diamonds; Euroimmun, Lübeck, Germany), disease activity was measured by a clinical score (triangles; 3, >10 lesions; 2, 4–10 lesions; 1, 1–3 lesions; 0, no lesions).
Representative clinical pictures during the course of the disease are shown at the top.
164 A. VOROBYEV ET AL.

Clinical picture
compatible with
EBA

direct immunofluorescence (IF) microscopy


perilesional

IgG/IgA/C3 negative
along the DEJ

pattern analysis no AIBD

u-serrated1 n-serrated undetermined if in doubt


repeat
and
Figure 11. Indirect immunofluorescence on HEK 293 cells expressing the recom-
binant NC1 domain of type VII collagen on their cell surface. BP, MMP, LAD,
anti-p200
pemphigoid

pemphigoid also label the floor of the artificial split. indirect immunofluorescence microscopy
Serological diagnosis of EBA requires the detection of antitype on human salt-split skin
VII collagen autoantibodies. At present, the most convenient IgG/IgA at IgG/IgA at
negative
and standardized assays are widely available ELISA systems blister roof blister floor
and an indirect IF test based on the use of recombinant
fragments of type VII collagen (Euroimmun, MBL) [55,65]. In
addition, several in-house systems are in use such as immuno-
BP, MMP,
LAD
- type VII collagen-specific
ELISA or IF test2
blotting with various cell-derived or recombinant forms of 3
type VII collagen. These assays are less standardized but
BP, MMP, LAD,
+
have a role e.g. in detecting IgA antibodies against type VII anti-p200 Immunoblotting or ELISA with
pemphigoid6
collagen which cannot be achieved by the commercial assays. recombinant or cell-derived forms of
type VII collagen4
A highly reliable, however, also not widely available
approach, is the use of type VII collagen-deficient human - indirect IF microscopy on type VII
skin as substrate for indirect IF microscopy. If no circulating collagen-deficient skin4,5

antitype VII collagen antibodies are detectable and an unde-


fluorescence overlay antigen mapping
termined serration pattern is seen by direct IF microscopy, (FOAM)4
FOAM may be applied [4]. The suggested diagnostic proce- +
direct immunoelectron microscopy4
dure in EBA is schematically shown in Figure 12.
Epidermolysis bullosa acquisita (EBA)
7. Expert commentary
Figure 12. Diagnostic pathway for EBA.
Despite recent progress in our understanding of the patho- 1
even when the diagnosis of EBA can be made based on an u-serrated pattern, detection of
physiology of EBA and development of novel diagnostic meth- serum anti-type VII collagen antibodies is recommended; 2commercially available;
3
depending of availability; positivity in any of the 4 assays will allow diagnosis of EBA;
ods, diagnosis and management of EBA still remain 4
only available in specialized laboratories; 5from patients with dystrophic epidermolysis
challenging. This is due to the low incidence of EBA and the bullosa; 6bullous pemphigoid (BP), predominant IgG reactivity by direct and/or indirect IF
microscopy, no floor binding by indirect IF microscopy, and no predominant mucosal
related lack of prospective controlled clinical trials. Diagnosis involvement; mucous membrane pemphigoid (MMP), predominant mucosal involvement,
is complicated by the polymorphous clinical picture and the when floor binding by indirect IF microscopy, laminin 332 reactivity needs to be analyzed;
linear IgA diseases (LAD), predominant IgA reactivity by direct and/or indirect IF micro-
sophisticated diagnostic approach summarized in Figure 12. scopy; anti-p200 pemphigoid, reactivity with the p200 protein and/or laminin γ1 [4].
Most EBA patients are diagnosed and treated in specialized
centers experienced in the management of AIBDs.
analysis needs to be further promoted and underlined by
additional studies that are currently being performed. An
8. Five-year view international guideline of the diagnosis of EBA is being
Considerable progress has been made in the diagnosis of discussed in the community for some time and will be
EBA by the introduction of serration pattern diagnosis of finalized within the next 1 or 2 years. Due to the availability
direct IF microscopy and the availability of commercial test of robust mouse models of EBA, valuable data about the
systems for the detection of serum IgG against type VII pathophysiology of pemphigoid diseases and autoantibody-
collagen. While the serological tests are already widely mediated diseases will be generated that may also help to
used in the community, the concept of serration pattern better understand the human disorders including EBA.
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 165

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This work was supported by the Schleswig-Holstein Cluster of Excellence skin basement-membrane autoantigen in epidermolysis bullosa
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Groups ‘Modulation of Autoimmunity’ (GRK1727/1 and 2) and ‘Genes, •• First description of the autoantigen of epidermolysis bullosa
Environment and Inflammation’ (GRK1743/1). acquisita.
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Declaration of interest procollagen. J Clin Invest. 1988;81(3):683–687.
18. Borradori L, Sonnenberg A. Structure and function of hemidesmo-
E Schmidt has a scientific cooperation with Euroimmun. The authors have
somes: more than simple adhesion complexes. J Invest Dermatol.
no other relevant affiliations or financial involvement with any organiza-
1999;112(4):411–418.
tion or entity with a financial interest in or financial conflict with the
19. Bernard P, Vaillant L, Labeille B, et al. Incidence and distribution of
subject matter or materials discussed in the manuscript apart from those
subepidermal autoimmune bullous skin diseases in three French
disclosed.
regions. Bullous Diseases French Study Group. Arch Dermatol.
1995;131(1):48–52.
20. Bertram F, Brocker EB, Zillikens D, et al. Prospective analysis of the
ORCID incidence of autoimmune bullous disorders in Lower Franconia,
Ralf J. Ludwig http://orcid.org/0000-0002-1394-1737 Germany. J Dtsch Dermatol Ges. 2009;7(5):434–440.
21. Zillikens D, Wever S, Roth A, et al. Incidence of autoimmune sub-
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