Guidelines

DOI: 10.1111/ddg.12612

S2k guideline for the diagnosis of

pemphigus vulgaris/foliaceus and
bullous pemphigoid

Enno Schmidt1, Matthias Goebeler2, Michael Hertl3, Miklós Sárdy4,

Cassian Sitaru5, Rüdiger Eming3, Silke C. Hofmann6, Nicolas
Hunzelmann7, Johannes S. Kern5, Harald Kramer8, Hans-Dieter
Orzechowski9, Christiane Pfeiffer 10, Volker Schuster 11, Birte
Sporbeck12, Michael Sticherling13, Margitta Worm14, Detlef
Zillikens1, Alexander Nast12

(1) Department of Dermatology, Allergology, and Venereology, University of Lübeck,

Lübeck, Germany
(2) Department of Dermatology, Venereology, and Allergology, University Hospital
Würzburg, Würzburg, Germany
(3) Department of Dermatology and Allergology, University Hospital Marburg,
­Marburg, Germany
(4) Department of Dermatology, Venereology, and Allergology, University Hospital
Munich (LMU), Munich, Germany
(5) Department of Dermatology and Venereology, Albert-Ludwigs University
­Freiburg, Freiburg, ­Germany
(6) Center for Dermatology, Allergology, and Dermatosurgery, Helios Hospital
­Wuppertal, Wuppertal, Germany
(7) Department of Dermatology and Venereology, University of Cologne, Cologne,
Germany
(8) Dermatologist in Private Practice, Fulda, Germany
(9) Institute of Clinical Pharmacology and Toxicology, Charité - University Hospital
Berlin, Berlin, Germany
(10) Department of Dermatology and Allergology, University Hospital Ulm, Ulm,
Germany
(11) Pediatric Immunology and Rheumatology, University Hospital and Outpatient
Clinical for Children and Adolescents, Leipzig, Germany
(12) Division of Evidence Based Medicine (dEBM), Department of Dermatology,
­Charité – University Hospital Berlin, Berlin, Germany
(13) Department of Dermatology, University Hospital Erlangen, Erlangen, Germany
(14) Center for Allergy, Department of Dermatology, Charité – University Hospital
Berlin, Berlin, Germany

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 Guidelines  Diagnosis of pemphigus and bullous pemphigoid

Methodology Grades of recommendation

The methodology of this S2k guideline follows the specifi- Uniform wording was used in order to standardize the guide-
cations issued by the Association of the Scientific Medical line recommendations. The following grades are used:
Societies in Germany (AWMF)] [1]. The development level
S2k was chosen. Using a structured nominal group process Grade of recommendation Syntax
(consensus conferences), the guideline was developed by a re- Strong recommendation Is recommended
presentative interdisciplinary expert group.
Recommendation May be recommended
For expert nomination, selection of interventions, con-
sensus process, review procedures, implementation, and Recommendation pending May be considered
statement of conflicts of interest, refer to the methodology Negative recommendation Is not recommended
report (online appendix or www.awmf.org). The present gui-
deline is valid until Dec 31, 2018.

Clinical differential diagnosis of bullous skin lesions

Disease Features
Hereditary Epidermolysis bullosa simplex group Onset at birth or in early childhood; clinical features
­diseases Junctional epidermolysis bullosa group ­according to the genetic defect
Dystrophic epidermolysis bullosa group DIF and IIF negative
Ichthyosis bullosa of Siemens
Porphyria cutanea tarda Band-like deposits of IgG and – less commonly –
C3, IgA, and IgM at the dermal-epidermal junction and
around the blood vessels of the papillary dermis; UV
light-exposed areas
Congenital erythropoietic protoporphyria Measurement of porphyrins in stool, urine, and/or
Hepatoerythropoietic porphyria blood
Incontinentia pigmenti
Autoimmune Pemphigus direct and indirect IF microscopy usually positive
­bullous Pemphigoid/ linear IgA bullous dermatosis Direct IF usually and indirect IF microscopy often
­dermatoses ­positive
Epidermolysis bullosa acquisita Direct IF usually and indirect IF microscopy often
­positive
Dermatitis herpetiformis Direct IF often and indirect IF microscopy positive in
approximately 50 % of cases
Infectious diseases Impetigo Microbiology (Strep., Staph.), other signs of
­inflammation; direct and indirect IF microscopy
­negative
Staphylococcal scalded skin syndrome Usually circumscribed epidermolysis, histology; direct
and indirect IF microscopy negative
Bullous erysipelas Clinical and serological inflammatory parameters;
­direct and indirect IF microscopy negative
Herpes simplex Herpes simplex virus found in blister fluid
Varicella Clinical features, general symptoms; varizella zoster
Herpes zoster virus found at the base of the blister
Hand-foot-and-mouth disease Clinical features, serology

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Guidelines  Diagnosis of pemphigus and bullous pemphigoid

Disease Features
Immunological Bullous systemic lupus erythematosus Direct and indirect IF microscopy positive (anti-collagen
diseases VII IgG), ANA positive; other SLE criteria positive
Erosive lichen planus Direct IF microscopy subepidermal cytoid ­bodies,
i­ndirect IF microscopy negative; c­ utaneous
­involvement
Erythema multiforme (EM) Patient history; direct and indirect IF microscopy
occasionally positive in the major form: ICS pattern
(anti- ­desmoplakin antibodies)
Bullous drug eruption (SJS, TEN) EM-like appearance or extensive epidermolysis,
­histology; direct and indirect IF microscopy negative
Subcorneal pustulosis Leukocytosis, serological signs of inflammation,
(Sneddon-Wilkinson ­disease) direct and indirect IF microscopy negative
Papular acrodermatitis of childhood Typical distribution pattern, exclusion of other causes
(Gianotti-Crosti syndrome)
Other diseases Bullosis diabeticorum Glucose in serum/urine, direct and indirect IF
­microscopy negative
Traumatic/toxic blistering History, direct and indirect IF microscopy negative
Bullous insect bite reactions History, direct and indirect IF microscopy negative
Erosive acantholytic actinic Signs of chronic light damage, direct and indirect IF
keratosis microscopy negative
Artifacts
IF, immunofluorescence

Diagnosis of pemphigus vulgaris/­ The incidence of pemphigus shows marked variations

between different populations. It ranges from 0.6 and
pemphigus foliaceus
0.76 per million per year in Switzerland and Finland to 4.0,
The diagnosis of pemphigus vulgaris (PV)/pemphigus folia- 8.0, and 10.0 in Romania, Greece, and Iran [2–6]. The hig-
ceus (PF) is based on patient history, physical examination, hest incidence is found in the Jewish population, at 16.1 and
and laboratory tests. 32 per million per year [7, 8]. In Germany, it has been deter-
mined to be 1.5 per million per year. Here, the incidence of
History PV in the population with Southern European roots was nine
times higher than in individuals of German background [9].
History in pemphigus vulgaris/foliaceus
Physical examination
It is recommended to ask about the following points:
 Time of first onset of the lesions Physical examination in pemphigus vulgaris/foliaceus
 Pain (where?, when?)
The following is recommended in addition to the general
 Stomatitis, dysphagia, hoarseness
physical examination:
 Conjunctivitis
 Inspection of the oral cavity, nostrils, genitals, perianal
 Epistaxis
region, and nails
 Dysuria
 Positive Nikolsky’s sign: exertion of tangential pressure
 Weight loss
by rubbing erythematous skin with a gloved thumb.
  Medication history: especially drugs that have been
The test is positive when the epidermis can be separa-
associated with the induction of PV/PF such as penicill-
ted (dislodged).
amine, ACE inhibitors, pyrazolone derivatives, cepha-
losporins, and rifampicin
Pemphigus vulgaris is virtually always associated with
 Ethnic background
mucosal lesions, most commonly in the oral cavity. In about

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 Guidelines  Diagnosis of pemphigus and bullous pemphigoid
one-half of PV patients, erosions or blisters are also found on
the skin (mucocutaneous form). Pemphigus foliaceus is never as-
sociated with mucosal lesions and therefore cannot be ­clinically
mistaken for PV (or paraneoplastic pemphigus) [10, 11].
Two scoring systems are available for PV/PF, ABSIS and
PDAI [12, 13]. These are currently being validated in pro-
spective studies. Pemphigus-specific disease stages such as
disease control, consolidation phase as well as complete re-
mission with and without therapy have been defined on the
basis of an international consensus statement [14].
Determination of disease activity
 Quantification of disease extent and activity by
means of a clinical score (ABSIS and/or PDAI) may be
considered.
Further examinations
 In case of dysphagia or hoarseness, examination by an
ENT specialist or gastroenterologist is recommended.
Basic workup
Basic workup in case of clinical suspicion of pemphigus
vulgaris/foliaceus
Taking into account the suspected clinical diagnosis, the follo-
wing diagnostic measures are recommended as basic workup:
 Direct immunofluorescence microscopy
 Histopathological analysis
 Immunoserology (indirect IF microscopy, ELISA)
Direct immunofluorescence (IF microscopy)
In case of appropriate clinical features, PV/PF may already be
confirmed by positive direct IF microscopy.
Biopsy site: the perilesional biopsy site is crucial, as
biopsying a blister can lead to false positive (Ig/C3 is deposited
nonspecifically) or false negative reactions (Ig/C3 is degraded
proteolytically). From a diagnostic point of view, preferenti-
ally using a certain region of the body is not recommended.
Biopsy site for direct IF microscopy in pemphigus
­vulgaris/foliaceus
 It is recommended to take a 4 mm punch biopsy from
­ erilesional skin or mucosa, i.e. within a 1 cm radius
p pemphigus vulgaris/foliaceus
from a blister or erosion.
Transport/storage
If the biopsy is stored in 4 % formaldehyde (10 % formalin)
solution, the antibody structure is destroyed and performing
direct IF microscopy is no longer useful due to false negative
results.
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716
Storage for direct IF microscopy in pemphigus vulgaris/
foliaceus
 It is recommended to store biopsy specimens in
isotonic NaCl solution or Michel’s medium for a maxi-
mum of 72 hours or to promptly transfer it into liquid
nitrogen (within 15 minutes).
Assessment
In the presence of corresponding clinical features, the detection
of intercellular IgG and/or C3 deposits in the epidermis/epit-
helium confirms the diagnosis of pemphigus. In PV/PF, these
IgG/C3 deposits are primarily found in the lower half or within
the entire epithelium, whereas in pemphigus foliaceus, IgG/C3
is usually deposited in the superficial epidermis. Unequivocal
differentiation of PV and pemphigus foliaceus is not possible
through direct IF microscopy. In paraneoplastic pemphigus,
the intercellular IgG/C3 deposits may be combined with linear
staining of the dermoepidermal junction [11, 15, 16].
Histopathology
The histopathology of a lesional skin or mucosal biopsy is
not diagnostic for PV/PF. However, in the presence of corres-
ponding clinical features, suprabasal cleavage and acantholy-
sis are highly indicative of PV/PF.
Biopsy site for histopathology in pemphigus vulgaris/
foliaceus
It is
  recommended to completely biopsy a small intact
blister.
 If this is not possible, it is recommended to take the biopsy
in such a way that it also contains a small amount of perile-
sional skin (approximately ¼ of the biopsy) to prevent the
blister roof from floating off during processing.
 Preferentially using a certain region of the body for
biopsy is not recommended.
Storage and transport
 A standardized 4 % formaldehyde (10 % formalin)
­solution is recommended for storage and transport.
Serology
Serological workup in case of clinical suspicion of
It is recommended to perform the following serological
tests in every patient with suspected PV/PF:
 Indirect IF microscopy on monkey esophagus / on cells
expressing desmoglein 3 and 1
 Desmoglein 3 ELISA
 Desmoglein 1 ELISA
Guidelines  Diagnosis of pemphigus and bullous pemphigoid
Indirect immunofluorescence on monkey esophagus
In case of suspected PV/PF, monkey esophagus is the
most sensitive tissue for screening for serum autoantibo-
dies by indirect IF. Sensitivities between 86 % and 100
% have been described [17–21]. In PF, the sensitivity is
somewhat lower than in PV. The specificity has been re-
ported to be 100 % [22]. On the other hand, antibodies
against blood group antigens A and B, which are present
in approximately 10 % of sera from healthy blood do-
nors, show a virtually identical pattern to pemphigus au-
toantibodies [23–25]. By adding soluble A/B antigen or
erythrocytes from an AB-positive donor, such undesirab-
le intercellular reactivity may be eliminated in most
sera [25].
Test systems using recombinant desmoglein 3 and
­desmoglein  1
Two commercial ELISA systems using the ectodomains of
desmoglein 1 and 3 are currently available (Euroimmun, Lü-
beck, Germany; MBL, Nagoya, Japan) [26, 27]. In addition,
a commercial IF test (BIOCHIP ® Mosaik; Euroimmun) with
similar sensitivities and specificities is available, in which the
two ectodomains are expressed on the surface of a human cell
line (Euroimmun) [28–30]. In general, serum autoantibodies
against desmoglein 3 are found in PV, whereas PF is charac-
terized by desmoglein 1 autoantibodies. In a meta-analysis
of 1,058 PV patients, the sensitivity was 97 % (95 % con-
fidence interval, 95–98  %) and the specificity 98 % (95 %
confidence interval, 98–99 %) [31].
The sensitivity of the anti-desmoglein 1 ELISA is 96 %
in PF and about 50 % in PV. The specificity has been deter-
mined to be 99 % [26, 27].
It has clearly been shown that the clinical pemphigus
phenotype usually correlates with the autoantibody spe-
cificity: PV patients with exclusively mucosal lesions have
antibodies against desmoglein 3 but not against desmo-
glein 1, whereas PV patients with mucosal and cutaneous
lesions have antibodies against both desmoglein 3 and
desmoglein 1 [32–35]. Individual pemphigus patients with
exclusive anti-desmoglein 3 reactivity and lesions on the
scalp or nose have been described. Pemphigus foliaceus pa-
tients have no mucosal lesions and exclusively react with
desmoglein 1.
Because of its higher specificity and lower examiner de-
pendence, the anti-desmoglein 3 and anti-desmoglein 1 ELI-
SA is superior to indirect IF on monkey esophagus [22, 26,
36–38].
In many patients, anti-desmoglein 3 and anti-desmo-
glein 1 ELISA reactivity is above the upper threshold values.
Accurate measurement of ELISA reactivity is important in
order to obtain a baseline value for monitoring in the course
of the disease [39].
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
Western blot tests in the diagnosis of pemphigus
vulgaris/foliaceus
 The use of Western blotting to detect
autoantibodies against desmoglein 3 and
desmoglein 1 is not recommended in the routine
­workup of PV/PF.
Difficult constellations of findings / necessary
criteria for diagnosis
Clinical picture
Virtually all patients with PV exhibit erosions of the ­mucous
membranes. The oral mucosa is nearly always affected (appro-
ximately 80 %), less commonly the nasal mucosa, pharynx,
and genital mucosa. The conjunctiva, larynx, ­esophagus, and
perianal region are rarely involved. About half of the pati-
ents also show skin lesions at the time of diagnosis. Here,
flaccid blisters or erosions are typical; ­Nikolsky’s sign is
positive.
Necessary criteria for the diagnosis of pemphigus
­vulgaris/foliaceus
In case of the following constellations, it is recommended
to make the diagnosis of PV/PF:
 Compatible clinical picture and positive direct IF
microscopy
 Appropriate clinical picture and reactivity with
­desmoglein 1 or 3 by ELISA / in transfected cells
 Compatible clinical picture and corresponding
­histopathology and positive indirect IF microscopy on
monkey esophagus sections
In case of positive direct IF microscopy and compatible
clinical findings, the detection of circulating autoantibo-
dies is not absolutely essential for the diagnosis of PV/PF,
but may be important for monitoring in the course of the
disease.
Unclear constellations of findings
 It is recommended to repeat the immune workup (di-
rect IF microscopy and serology), if clinical suspicion
persists, direct IF microscopy is negative, and the cons-
tellation of findings is unclear.
Non-anti-desmoglein antibodies
More than 50 non-desmoglein target antigens have been de-
scribed in PV/PF, including acetylcholine receptors, pempha-
xin (annexin 9), plakoglobin, E-cadherin, desmoplakin, and
desmocollins [40–45].
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 Guidelines  Diagnosis of pemphigus and bullous pemphigoid

Further diagnostic measures Exclusion of paraneoplastic pemphigus

 In case of positive direct IF microscopy and no  In case of atypical clinical features (i.e.
­ etection of desmoglein 1 and 3-specific antibo-
d refractory ­p atients, progressive stomatitis)
dies, ­testing for anti-desmocollin antibodies may be or combined reactivity of IgG/C3 in the
­recommended. epithelium and at the dermoepidermal junction
by direct IF microscopy or a known neoplasm,
exclusion of paraneoplastic pemphigus is
Other diagnostic measures ­r ecommended.
 In case of negative direct IF microscopy and no
­ etection of desmoglein 1 and 3-specific antibodies,
d
Exclusion of paraneoplastic pemphigus
testing for non-desmoglein-specific antibodies is not
recommended. The following serological test systems may be recommen-
ded to exclude paraneoplastic pemphigus:
 Indirect IF microscopy on bladder epithelium (e.g. rat
Apart from PV, direct IF microscopy with intercellular
or monkey)
IgG and/or C3 deposits in the epithelium and serum antibo-
Alternatively or in addition:
dies against desmoglein 3 are also found in paraneoplastic
 Anti-envoplakin ELISA
pemphigus.
 Immunoblot with extract of cultured human
­keratinocytes
Paraneoplastic pemphigus
 Immunoprecipitation with extract of cultured human
Diagnostic criteria for paraneoplastic pemphigus are [46]–
keratinocytes
[49]:
Clinical features: severe stomatitis, lesions on other tran-
sitional mucous membranes, flaccid or tense blisters, lichen Extended workup / search for the cause
planus-like plaques, TEN-like epidermolysis, bronchiolitis
obliterans. A temporal association between the intake of penicillami-
Histopathology: suprabasal cleavage, acantholysis, ne, ACE inhibitors, pyrazolone derivatives, cephalosporins,
­lichenoid interface dermatitis with keratinocytic necrosis. and rifampicin has repeatedly been described in single case
Direct IF microscopy: intercellular/reticular IgG depo- ­reports [55–57]. The precise temporal and causal relation
sits at the epithelium or intercellular IgG in the epithelium between the intake of these drugs and the induction of PV/
and IgG/C3 at the basement membrane zone. PF is unclear.
Indirect IF microscopy on monkey esophagus sections:
intercellular IgG deposits in the epithelium.
Indirect IF microscopy on monkey/rat bladder sections: Search for the cause in pemphigus vulgaris/foliaceus
intercellular IgG deposits in the epithelium.  Following the diagnosis of PV/PF, it is recommended to
Detection of autoantibodies against envoplakin, peripla- discontinue penicillamine and rifampicin.
kin, desmoplakin, plectin, BP230, α2-macroglobulin-like 1,
desmoglein 1, desmoglein 3, and desmocollin.
Associated neoplasms: most commonly non-Hodgkin’s
lymphoma (especially chronic lymphocytic leukemia), which Search for the cause in pemphigus vulgaris/foliaceus
is the underlying disease in just under two-thirds of PNP pa-  Discontinuing or switching drugs (especially ACE in-
tients, followed by thymoma and Waldenström’s macroglo- hibitors, pyrazolone derivatives, and cephalosporins)
bulinemia. may be considered in treatment-refractory patients
Detection of a neoplasm is obligatory for the diagnosis or if there is a temporal association between the
[46–49]. The most characteristic autoantibodies for paraneo- onset of pemphigus lesions and the intake of these
plastic pemphigus are directed against envoplakin [50, 51], drugs.
periplakin [51, 52] and α2-macroglobulin-like 1 [53]. Auto-
antibodies against desmoglein 3 are also found in nearly all
patients [54]. Search for the cause in pemphigus vulgaris/foliaceus
A commercial ELISA for the detection of autoantibodies
 Further search for the cause in PV/PF is not
against envoplakin is available (Euroimmun, Lübeck, Ger-
­recommended.
many; sensitivity: 81 %, specificity 98.8 %) [51].

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Guidelines  Diagnosis of pemphigus and bullous pemphigoid

Disease course monitoring Diagnosis of bullous pemphigoid

Various studies have shown that indirect IF microscopy The diagnosis of bullous pemphigoid (BP) is based on the
­titers on monkey esophagus correlate with disease activity patient’s history, physical examination, and laboratory tests.
in most PV/PF patients [58]. Moreover, in many PV patients,
anti-desmoglein 3 ELISA levels also correlate with the extent History
of mucosal lesions, and, in most PF patients, anti-desmo-
glein 1 ELISA levels correlate with the extent of cutaneous History
lesions [27, 37, 59–61]. Thus, desmoglein 1 (and 3) ELISA
It is recommended to ask about the following points:
levels and indirect IF microscopy titers on monkey esopha-
 Pruritus
gus may be used when deciding on the discontinuation of
 Time of first onset of lesions
treatment: persistent high desmoglein 1 ELISA levels have a
 Neurological and psychiatric diseases
positive predictive value for cutaneous recurrences. Persistent
 Hematological or oncological diseases, diabetes melli-
high desmoglein 3 ELISA values do not always correlate with
tus, primary or secondary immune deficiencies
­mucosal recurrences.
  Medication history: drugs that have been associated
with the induction of BP such as spironolactone, phe-
Monitoring circulating autoantibody titers in nothiazines with an aliphatic side chain, and loop diu-
pemphigus vulgaris/foliaceus retics (especially furosemide) [62–64].
 In the course of the disease, it may be
recommended to measure serum desmoglein 1 and/
About one-third to one-half of BP patients shows neuro-
or 3 autoantibody levels by ELISA, and, in addition to
logical and/or psychiatric disorders. A clear association has
clinical disease a
­ ctivity, to use them for therapeutic
been described with dementia, Parkinson’s disease, stroke,
decisions.
epilepsy, and multiple sclerosis [62, 65–71].
Associated hematological and oncological diseases
Serological monitoring at intervals shorter than four and immune deficiencies are important for the choice of
weeks does not appear useful. In clinically stable disease, ­treatment.
measurement of circulating autoantibodies is of particular
relevance. An increase in autoantibody levels would then, for Physical examination
example, argue against further reduction of the immunosup-
pressive medication. Physical examination
At some centers, negative direct IF microscopy is a prere-
The following is recommended in addition to the general
quisite for discontinuing treatment.
physical examination:
 Inspection of the conjunctivae, oral cavity, nostrils,
­genitals, perianal region
Direct immunofluorescence prior to the discontinua-
  Negative Nikolsky’s sign: exertion of tangential
tion of immunosuppressive medication in pemphigus
­pressure by rubbing erythematous skin with a gloved
vulgaris/foliaceus
thumb does not allow dislodging of the epidermis
 Prior to the discontinuation of immunosuppressive me-
in BP.
dication in PV/PF, direct IF microscopy of a cutaneous
or mucosal biopsy may be considered.
Determination of disease activity

Follow-up intervals  Determination of disease extent and activity by means

Depending on the disease course and the patient’s general
condition, the following intervals may be recommended at
the end of the consolidation phase, possibly alternating with
physicians close to the patient’s home:
 Every two weeks until disease activity is controlled,
 Consolidation phase until complete remission/minimal
therapy is achieved: every 1–3 months,
 Complete remission on therapy: every 3–6 months.
of a score (BPDAI) may be considered.
For BP, a clinical score (BPDAI) and BP-specific disea-
se stages such as disease control, consolidation phase, and
complete remission with and without therapy have been
defined on the basis of an international consensus paper
[72]. The BPDAI is currently being validated in prospective
studies.

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 Guidelines  Diagnosis of pemphigus and bullous pemphigoid
Basic workup
Basic workup in case of clinical suspicion of bullous
pemphigoid
Taking into account the suspected clinical diagnosis, the
following diagnostic measures are recommended as basic
workup:
 Direct IF microscopy
 Histopathological analysis
 Immunoserology (indirect IF microscopy, ELISA)
Direct immunofluorescence (IF microscopy)
Biopsy site
Biopsy site for direct IF in bullous pemphigoid
 It is recommended to take a biopsy (preferably a 4 mm
punch biopsy) from perilesional skin or mucosa, i.e.
within a 1 cm radius from a blister or erosion.
The perilesional biopsy site is crucial, as biopsying a blis-
ter can lead to false positive (Ig/C3 is deposited nonspecifical-
ly) or false negative reactions (Ig/C3 is degraded proteolyti-
cally). From a diagnostic point of view, preferentially using a
certain region of the body is not recommended.
Transport/storage
Storage for direct IF microscopy in bullous pemphigoid
 It is recommended to store biopsy specimens in iso-
tonic saline or Michel’s medium for a maximum of
72 hours or to promptly transfer it into liquid nitrogen
(within 15 minutes).
If the biopsy is stored in 4 % formaldehyde (10 % for-
malin) solution, the antibody structure is destroyed and per-
forming direct IF microscopy is no longer useful due to false
negative results.
Assessment
In the presence of corresponding clinical features, the detec-
tion of linear IgG and/or C3 deposits at the dermo-epidermal
junction already allows for the diagnosis of an autoimmune
subepidermal blistering dermatosis [73]. However, BP can-
not be unequivocally differentiated from other autoimmune
subepidermal blistering dermatoses on direct IF microscopy.
Exceptions include linear IgA bullous dermatosis and derma-
titis herpetiformis, which can be diagnosed by the predomi-
nance of IgA deposits. Bullous pemphigoid also occasionally
shows linear IgA deposits, but they are – by definition – less
pronounced than the IgG deposits.
At 600-fold magnification, it can be seen that the "line-
ar" IgG deposits at the dermal-epidermal junction are slightly
wavy. In epidermolysis bullosa acquisita a "U pattern" is
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720
observed, that is, the small arches are open towards the top.
In all other autoimmune subepidermal blistering dermatoses,
including BP, an "N pattern" is seen, with the arches closed
towards the top [74, 75].
For a definitive diagnosis of BP, serological tests are
­required (see below).
Direct IF microscopy using split skin
The split within the biopsy following incubation with 1 M
saline solution allows for further differentiation of autoanti-
body specificity. If autoantibodies against BP180, BP230, or
α6β4-integrin are bound in the skin, these become visible at
the roof of the split. Antibodies against laminin 332, p200 an-
tigen, laminin γ1, and type VII collagen are deposited at the
floor of the blister [76]. This test is especially useful if no ser-
um is available or no circulating autoantibodies are detectable.
Histopathology
The histopathology of a lesional skin biopsy is not diagnostic
for BP. Definitive differentiation from mucous membrane pem-
phigoid, anti-p200 pemphigoid, and the inflammatory vari-
ant of epidermolysis bullosa acquisita is not possible [77, 78].
Subepidermal cleavage and a lymphocytic inflammato-
ry ­infiltrate with the addition of eosinophils are, however,
­t ypical for BP.
Biopsy site for histopathology in bullous pemphigoid
  It is recommended to completely biopsy a small intact
blister.
 If this is not possible, it is recommended to take the
biopsy in such a way that it contains an equal amount
of lesional and perilesional skin to prevent the blister
roof from floating off during processing.
 Preferentially using a certain region of the body for
biopsy is not recommended.
Storage and transport
 A standardized 4 % formaldehyde (10 % formalin)
­solution is recommended for storage and transport.
Serology
Serological workup in case of clinical suspicion of
bullous pemphigoid
Taking into account the suspected clinical diagnosis, the
following serological tests, preferably in the following or-
der, are recommended:
 Indirect IF microscopy on human split skin
 Indirect IF microscopy on monkey esophagus
 BP180 NC16A (ELISA/indirect IF microscopy)
 BP230 (ELISA/indirect IF microscopy)
Guidelines  Diagnosis of pemphigus and bullous pemphigoid
Indirect IF microscopy on human split skin
In case of suspected BP, normal human skin, split by 1 M
saline solution, is a sensitive tissue for screening for serum
autoantibodies using indirect IF microscopy. IgG (in some
patients, more weakly also IgA) antibodies typically bind to
the roof of the artificial blister [11]. Sensitivities between 73
% and 96 % have been described [79–85].
Specificities of 100 % (n  =  488) and 98.2 % (healthy
blood donors, n  =  50; patients with noninflammatory skin
­diseases >70 years of age, n  =  93; patients with chronic pruri-
tic dermatoses, n  =  79) have recently been reported [85, 86].
It is recommended to perform indirect IF microscopy on
human split skin in every patient with clinically suspected BP.
Indirect IF microscopy on monkey esophagus and rabbit
esophagus
If human split skin is not available, performance of indirect
IF microscopy on monkey or rabbit esophagus may be recom-
mended. When using these substrates, the sensitivity of 60–70
% in BP is below that of indirect IF microscopy with human
split skin [84, 85, 87, 88].
Indirect IF microscopy on split skin or monkey/rabbit
esophagus also allows for the detection of autoantibodies
against other target antigens of the dermo-epidermal junction
such as laminin 332, α6β4 integrin, p200 antigen/laminin γ1,
and type VII collagen as well as against proteins not yet cha-
racterized at the molecular level [89].
Test systems using recombinant BP180
The 16th non-collagenous domain (NC16A) of BP180 is the
immunodominant region of BP180 in BP [90, 91]. For the
diagnosis of BP, testing for anti-BP180-NC16A antibodies
should be performed in every patient with clinically suspected
BP. Three commercial systems with similar sensitivities and
­specificities are available: two anti-BP180-NC16A ELISAs [92,
93] (Euroimmun, Lübeck, Germany; MBL, Nagoya, ­Japan)
and one indirect IF test using recombinant BP180-NC16A
tetrapeptide (BIOCHIP ® Mosaik; Euroimmun) [28–30].
In a meta-analysis with 583 BP patients, the sensitivity of the
BP180-NC16A ELISA was 87 % (95 % confidence interval,
85–89  %) and the specificity was 98 % (95 % confidence
interval, 98–99 %) [31].
In many patients, the reactivity in the anti-BP180 ELISA
is above the upper threshold values. Accurate measurement
of ELISA reactivity is important in order to obtain a baseline
value for monitoring in the course of the disease.
Test systems using recombinant BP230
Three commercial systems with similar sensitivities and speci-
ficities are available: two anti-BP230 ELISAs [92, 93] (Euroim-
mun, Lübeck, Germany; MBL, Nagoya, Japan) and one indirect
IF test using recombinant C-terminal fragments of BP230
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
(BIOCHIP® Mosaik; Euroimmun) [28–30]. MBL additionally
uses an N-terminal fragment. The sensitivities of the BP230-­
specific assay are between 50 % and 72 % in BP [94–98], with
specificities ranging from 95.8 % to 99.5 % [94, 95, 99].
Combined use of anti-BP180 and anti-BP230 ELISA
Combined use of both ELISAs has yielded sensitivities bet-
ween 87 % and 93 % in BP [95, 96, 98].
In case of clinical suspicion of BP, it is recommended to
perform the anti-BP230 ELISA/IF test, if the anti-BP180-
NC16A assay is negative.
The majority of BP patients also have elevated total IgE
serum levels and/or peripheral eosinophilia.
Difficult constellations of findings / necessary
criteria for diagnosis
Clinical picture
The clinical picture of BP can be very diverse. Typically, there
are tense blisters filled with serous and hemorrhagic fluid on
erythematous or clinically unaffected skin. Urticarial lesions,
erythematous plaques, and erythema are likewise characteri-
stic [89]. After mechanical irritation, erosions and yellow or
hemorrhagic crusts develop. Nearly all patients are affected
by intense pruritus. Predilection sites include the flexor as-
pects of the extremities and also the trunk. Mucous mem-
branes are involved in 10–20 % of patients, especially the
oral mucosa [100–103]. If there is predominant mucosal in-
volvement, the diagnosis of mucous membrane pemphigoid is
appropriate [104].
Over weeks or months, the classic bullous stage is usual-
ly preceded by a prodromal stage with nonspecific skin le-
sions such as eczema and urticarial erythema (premonitory
stage); here, too, severe pruritus is already present.
Different clinical variants of BP occurring in about 20 %
of BP patients have been described [100]. Bullous pemphigoid
may clinically present as eczema, nodular prurigo, subacu-
te prurigo simplex, erythroderma, ecthyma gangrenosum,
or intertrigo as well as in the form of erythematous papu-
les, vegetating lesions, or small vesicles [103]. Some of these
forms subsequently evolve into the classic bullous phenotype.
Necessary criteria for the diagnosis of bullous
pemphigoid
In case of the following constellations, it is recommended
to make the diagnosis of bullous pemphigoid:
 Compatible clinical picture and positive direct
IF microscopy and reactivity with BP180* and/or BP230*
 Compatible clinical picture and positive direct
IF microscopy and epidermal binding of IgG in indirect
IF microscopy on split skin
721
 Guidelines  Diagnosis of pemphigus and bullous pemphigoid
 Clinical picture with tense blisters and epidermal binding
of IgG in indirect IF microscopy on split skin or monkey
esophagus and reactivity with BP180* and/or BP230*
 Clinical picture with tense blisters and corresponding
histopathology and epidermal binding of IgG in indi-
rect IF microscopy on split skin
 Compatible clinical picture and corresponding histo-
pathology (subepidermal cleavage) and reactivity with
BP180*
 Clinical picture with tense blisters and pronounced
­reactivity with BP180* (e.g. > 3 times the lower detecti-
on threshold in a commercial ELISA)
*in commercial assays (ELISA or indirect IF microscopy)
Negative direct immunofluorescence microscopy
 It is recommended to repeat the immune workup
(­direct IF microscopy and serology), if clinical suspicion
persists, direct IF microscopy is negative, and the cons-
tellation of findings is unclear.
Further serological tests
In about 10 % of BP sera, no IgG antibodies can be detec-
ted using commercially available anti-BP180 and anti-BP230
ELISA. By combining different detection systems (Western
blot and ELISA) using various recombinant and/or cellular
fragments of BP180 and/or BP230, circulating autoantibo-
dies can be detected in all BP patients [105, 106]. However,
these test systems are only available as in-house assays at
specialized laboratories [107]. The published sensitivities
and especially specificities are not sufficient in all tests to
be able to be used in routine diagnostics. Alternatively, ano-
ther autoimmune subepidermal blistering dermatosis such as
­anti-p200/laminin γ1 pemphigoid or epidermolysis bullosa
acquisita need to be considered.
Possible further workup
 In case of positive direct IF and in the absence of
r­ eactivity to BP180 NC16A and BP230, it may be recom-
mended to perform Western blot and/or ELISA tests to
detect other autoantibodies, e.g. using other domains
of BP180 and BP230 or laminin 332, p200 protein/­
laminin γ1, or type VII collagen*.
* Two commercial test systems using type VII collagen are
available (MBL, Euroimmun) [108, 109].
In this situation, other tests are possible:
 Splitting of the biopsy using 1 M saline solution: in case
of epidermal binding of IgG/C3, BP may be diagnosed
(see also above).
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
722
 Electron microscopy (lesional): in case of split formation
in the lamina lucida, BP may be diagnosed.
 Immunoelectron microscopy (perilesional): in case of Ig
deposition in the lamina lucida, BP may be diagnosed.
Extended workup / search for the cause
Search for the cause in bullous pemphigoid
 Discontinuing or switching spironolactone, phenothi-
azines with an aliphatic side chain, and loop diuretics
may be considered in treatment-refractory patients
or if there is a temporal association between the ­onset
of clinical lesions/pruritus and the intake of these
drugs.
In numerous case reports, the concurrence of malignant
tumors and BP has been reported. However, in three studies
with more than 6,400 BP patients, only a weak association
with gastric cancer was found in Japanese patients [110–112].
Recently, in a study with more than 1,700 BP patients and
10,000 age- and sex-matched controls, a clear associatio
nwith hematological malignancies (lymphomas and myeloic
leucemia) has been described [113].
Search for the cause in bullous pemphigoid
 A tumor search solely on the basis of the diagnosis of
BP is not recommended.
Disease course monitoring
Monitoring serum autoantibody levels in bullous
pemphigoid
 In the course of the disease – depending on changes
in the clinical picture – it may be recommended to
measure serum autoantibodies against BP180 NC16A
(and also against BP230 in case of baseline positivity)
by ELISA.
It has clearly been shown that, in nearly all BP patients,
disease activity correlates with serum levels of anti-BP180-
NC16A antibodies, but not with indirect IF titers on mon-
key esophagus and human split skin, which correlate with
anti-BP230 IgG reactivity [85, 92, 93, 106, 114–116]. Data
on the correlation of disease activity with anti-BP230 serum
antibody levels is inconsistent. Most studies have been unable
to show a clear association [94, 106, 117]. Immunoserologi-
cal monitoring of autoantibody titers (BP180, BP230 IgG)
should not be performed more often than every 2–3 months,
except in case of recurrence.
Guidelines  Diagnosis of pemphigus and bullous pemphigoid
Correspondence to
Enno Schmidt, MD, PhD
Department of Dermatology
Lübeck Institute of Experimental Dermatology (LIED)
University of Lübeck
Ratzeburger Allee 160
D-23562 Lübeck, Germany
E-mail: enno.schmidt@uksh.de
References
1 Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen
Fachgesellschaften (AWMF) - Ständige Komission Leitlinien.
AWMF-Regelwerk “Leitlinien”. 1. Auflage Edition, 2012.
2 Hietanen J, Salo OP. Pemphigus: an epidemiological study of
patients treated in Finnish hospitals between 1969 and 1978.
Acta Derm Venereol. 1982; 62: 491–6.
3 Marazza G, Pham HC, Scharer L, Pedrazzetti PP, Hunziker T,
Trueb RM, Hohl D, Itin P, Lautenschlager S, Naldi L, Borradori L.
Incidence of bullous pemphigoid and pemphigus in Switzer-
land: a 2-year prospective study. Br J Dermatol. 2009; 161: 861–8.
4 Michailidou EZ, Belazi MA, Markopoulos AK, Tsatsos MI, Mourel-
lou ON, Antoniades DZ. Epidemiologic survey of pemphigus
vulgaris with oral manifestations in northern Greece: retrospec-
tive study of 129 patients. Int J Dermatol. 2007; 46: 356–61.
5 Chams-Davatchi C, Valikhani M, Daneshpazhooh M, Esmaili
N, Balighi K, Hallaji Z, Barzegari M, Akhiani M, Ghodsi Z,
­Mortazavi H, Naraghi Z. Pemphigus: analysis of 1209 cases.
Int J Dermatol. 2005; 44: 470–6.
6 Baican A, Baican C, Chiriac G, Chiriac MT, Macovei V, Zillikens
D, Ciuce D, Sitaru C. Pemphigus vulgaris is the most common
autoimmune bullous disease in Northwestern Romania. Int J
Dermatol. 2010; 49: 768–74.
7 Pisanti S, Sharav Y, Kaufman E, Posner LN. Pemphigus vulgaris:
incidence in Jews of different ethnic groups, according to age,
sex, and initial lesion. Oral Surg Oral Med Oral Pathol. 1974;
38: 382–7.
8 Simon DG, Krutchkoff D, Kaslow RA, Zarbo R. Pemphigus in
Hartford County, Connecticut, from 1972 to 1977. Arch Derma-
tol. 1980; 116: 1035–7.
9 Hahn-Ristic K, Rzany B, Amagai M, Brocker EB, Zillikens
D. Increased incidence of pemphigus vulgaris in southern
­Europeans living in Germany compared with native Germans.
J Eur Acad Dermatol Venereol. 2002; 16: 68–71.
10 Kneisel A, Hertl M. Autoimmune bullous skin diseases. Part 1:
Clinical manifestations. J Dtsch Dermatol Ges. 2011; 9: 844–56;
quiz 57.
11 Schmidt E, Zillikens D. The diagnosis and treatment of
­autoimmune blistering skin diseases. Dtsch Arztebl Int. 2011;
108: 399–405.
12 Pfutze M, Eming R, Kneisel A, Kuhlmann U, Hoyer J, Hertl M.
Clinical and immunological follow-up of pemphigus patients
on adjuvant treatment with immunoadsorption or rituximab.
Dermatology. 2009; 218: 237–45.
13 Rosenbach M, Murrell DF, Bystryn JC, Dulay S, Dick S,
­Fakharzadeh S, Hall R, Korman NJ, Lin J, Okawa J, Pandya AG,
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
Payne AS, Rose M, Rubenstein D, Woodley D, Vittorio C, Werth
BB, Williams EA, Taylor L, Troxel AB, Werth VP. ­Reliability and
convergent validity of two outcome instruments for pemphi-
gus. J Invest Dermatol. 2009; 129: 2404–10.
14 Murrell DF, Dick S, Ahmed AR, Amagai M, Barnadas MA,
­Borradori L, Bystryn JC, Cianchini G, Diaz L, Fivenson D, Hall
R, Harman KE, Hashimoto T, Hertl M, Hunzelmann N, Iranzo P,
Joly P, Jonkman MF, Kitajima Y, Korman NJ, Martin LK, Mimou-
ni D, Pandya AG, Payne AS, Rubenstein D, Shimizu H, Sinha
AA, Sirois D, Zillikens D, Werth VP. Consensus statement on
definitions of disease, end points, and therapeutic response
for pemphigus. J Am Acad Dermatol. 2008; 58: 1043–6.
15 Anhalt GJ, Kim SC, Stanley JR, Korman NJ, Jabs DA, Kory M,
Izumi H, Ratrie H, 3rd, Mutasim D, Ariss-Abdo L et al. Paraneo-
plastic pemphigus. An autoimmune mucocutaneous disease
associated with neoplasia. N Engl J Med. 1990; 323: 1729–35.
16 Kneisel A, Hertl M. Autoimmune bullous skin diseases. Part 2:
diagnosis and therapy. J Dtsch Dermatol Ges. 2011; 9: 927–47.
17 Sabolinski ML, Beutner EH, Krasny S, Kumar V, Huang J,
­Chorzelski TP, Sampaio S, Bystryn JC. Substrate specificity of
anti-epithelial antibodies of pemphigus vulgaris and pemphi-
gus foliaceus sera in immunofluorescence tests on monkey
and guinea pig esophagus sections. J Invest Dermatol. 1987;
88: 545–9.
18 Jiao D, Bystryn JC. Sensitivity of indirect immunofluorescence,
substrate specificity, and immunoblotting in the diagnosis of
pemphigus. J Am Acad Dermatol. 1997; 37: 211–6.
19 Jarzabek-Chorzelska M, Strasz-Kolacinska Z, Sulej J, Jablonska
S. The use of two substrates for indirect immunofluorescence
in the diagnosis of pemphigus. Br J Dermatol. 2001; 145:
178–82.
20 Harman KE, Gratian MJ, Bhogal BS, Challacombe SJ, Black
MM. The use of two substrates to improve the sensitivity of
indirect immunofluorescence in the diagnosis of pemphigus.
Br J Dermatol. 2000; 142: 1135–9.
21 Hahn K, Kippes W, Amagai M, Rzany B, Brocker EB, Zillikens
D. [Clinical aspects and immunopathology in 48 patients with
pemphigus]. Der Hautarzt; Zeitschrift fur Dermatologie, Ven-
erologie, und verwandte Gebiete. 2000; 51: 670–7.
22 Zagorodniuk I, Weltfriend S, Shtruminger L, Sprecher E,
Kogan O, Pollack S, Bergman R. A comparison of anti-des-
moglein antibodies and indirect immunofluorescence in the
serodiagnosis of pemphigus vulgaris. International journal of
dermatology. 2005; 44: 541–4.
23 Ahmed AR, Workman S. Anti-intercellular substance
­antibodies. Presence in serum samples of 14 patients without
pemphigus. Arch Dermatol. 1983; 119: 17–21.
24 Goldblatt F, Gordon TP. Antibodies to blood group antigens
mimic pemphigus staining patterns: a useful reminder.
­Autoimmunity. 2002; 35: 93–6.
25 Lee FJ, Silvestrini R, Fulcher DA. False-positive intercellular
­cement substance antibodies due to group A/B red cell anti-
bodies: frequency and approach. Pathology. 2010; 42: 574–7.
26 Ishii K, Amagai M, Hall RP, Hashimoto T, Takayanagi A, Gamou
S, Shimizu N, Nishikawa T. Characterization of autoantibodies
in pemphigus using antigen-specific enzyme-linked immu-
nosorbent assays with baculovirus-expressed recombinant
desmogleins. J Immunol. 1997; 159: 2010–7.
723
 Guidelines  Diagnosis of pemphigus and bullous pemphigoid
27 Schmidt E, Dahnrich C, Rosemann A, Probst C, Komorowski L,
Saschenbrecker S, Schlumberger W, Stocker W, Hashimoto T,
Brocker EB, Recke A, Rose C, Zillikens D. Novel ELISA systems
for antibodies to desmoglein 1 and 3: correlation of disease
­activity with serum autoantibody levels in individual pemphi-
gus patients. Exp Dermatol. 2010; 19: 458–63.
28 van Beek N, Rentzsch K, Probst C, Komorowski L, Kasperkie-
wicz M, Fechner K, Bloecker IM, Zillikens D, Stocker W,
Schmidt E. Serological diagnosis of autoimmune bullous skin
diseases: prospective comparison of the BIOCHIP mosaic-
based indirect immunofluorescence technique with the con-
ventional multi-step single test strategy. Orphanet journal of
rare diseases. 2012; 7: 49.
29 Tampoia M, Zucano A, Villalta D, Antico A, Bizzaro N. Anti-skin
specific autoantibodies detected by a new immunofluores-
cence multiplex biochip method in patients with autoimmune
bullous diseases. Dermatology. 2012; 225: 37–44.
30 Zarian H, Saponeri A, Michelotto A, Zattra E, Belloni-Fortina A,
Alaibac M. Biochip technology for the serological diagnosis of
bullous pemphigoid. ISRN Dermatol. 2012; 2012: 237802.
31 Tampoia M, Giavarina D, Di Giorgio C, Bizzaro N. Diagnostic
accuracy of enzyme-linked immunosorbent assays (ELISA) to
detect anti-skin autoantibodies in autoimmune blistering skin
diseases: a systematic review and meta-analysis. Autoimmun
Rev. 2012; 12: 121–6.
32 Muller R, Svoboda V, Wenzel E, Muller HH, Hertl M. IgG
against extracellular subdomains of desmoglein 3 relates
to clinical phenotype of pemphigus vulgaris. Experimental
­dermatology. 2008; 17: 35–43.
33 Amagai M, Tsunoda K, Zillikens D, Nagai T, Nishikawa T.
The clinical phenotype of pemphigus is defined by the anti-
desmoglein autoantibody profile. J Am Acad Dermatol. 1999;
40: 167–70.
34 Ding X, Aoki V, Mascaro JM, Jr., Lopez-Swiderski A, Diaz LA,
Fairley JA. Mucosal and mucocutaneous (generalized) pem-
phigus vulgaris show distinct autoantibody profiles. J Invest
Dermatol. 1997; 109: 592–6.
35 Mahoney MG, Wang Z, Rothenberger K, Koch PJ, Amagai
M, Stanley JR. Explanations for the clinical and microscopic
­localization of lesions in pemphigus foliaceus and vulgaris. J
Clin Invest. 1999; 103: 461–8.
36 Harman KE, Gratian MJ, Seed PT, Bhogal BS, Challacombe SJ,
Black MM. Diagnosis of pemphigus by ELISA: a critical evalua-
tion of two ELISAs for the detection of antibodies to the major
pemphigus antigens, desmoglein 1 and 3. Clin Exp Dermatol.
2000; 25: 236–40.
37 Lenz P, Amagai M, Volc-Platzer B, Stingl G, Kirnbauer R.
­Desmoglein 3-ELISA: a pemphigus vulgaris-specific diagnostic
tool. Arch Dermatol. 1999; 135: 143–8.
38 Ng PP, Thng ST, Mohamed K, Tan SH. Comparison of
­desmoglein ELISA and indirect immunofluorescence using
two substrates (monkey oesophagus and normal human skin)
in the diagnosis of pemphigus. Australas J Dermatol. 2005; 46:
239–41.
39 Bystryn JC, Akman A, Jiao D. Limitations in enzyme-linked
immunosorbent assays for antibodies against desmogleins 1
and 3 in patients with pemphigus. Arch Dermatol. 2002; 138:
1252–3.
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
724
40 Nguyen VT, Ndoye A, Grando SA. Pemphigus vulgaris
­antibody identifies pemphaxin. A novel keratinocyte annexin-
like molecule binding acetylcholine. J Biol Chem. 2000; 275:
29466–76.
41 Nguyen VT, Ndoye A, Grando SA. Novel human alpha9
­acetylcholine receptor regulating keratinocyte adhesion is
targeted by Pemphigus vulgaris autoimmunity. Am J Pathol.
2000; 157: 1377–91.
42 Cozzani E, Dal Bello MG, Mastrogiacomo A, Drosera M, Parodi
A. Antidesmoplakin antibodies in pemphigus vulgaris. Br J
Dermatol. 2006; 154: 624–8.
43 Evangelista F, Dasher DA, Diaz LA, Prisayanh PS, Li N.
E-cadherin is an additional immunological target for
­pemphigus autoantibodies. J Invest Dermatol. 2008; 128:
1710–8.
44 Grando SA. Pemphigus autoimmunity: hypotheses and
­realities. Autoimmunity. 2012; 45: 7–35.
45 Muller R, Heber B, Hashimoto T, Messer G, Mullegger R,
­Niedermeier A, Hertl M. Autoantibodies against desmocollins
in European patients with pemphigus. Clinical and experi-
mental dermatology. 2009; 34: 898–903.
46 Anhalt GJ. Paraneoplastic pemphigus. J Investig Dermatol
Symp Proc. 2004; 9: 29–33.
47 Joly P, Richard C, Gilbert D, Courville P, Chosidow O, Roujeau
JC, Beylot-Barry M, D’Incan M, Martel P, Lauret P, Tron F.
­Sensitivity and specificity of clinical, histologic, and immuno-
logic features in the diagnosis of paraneoplastic pemphigus. J
Am Acad Dermatol. 2000; 43: 619–26.
48 Zhu X, Zhang B. Paraneoplastic pemphigus. J Dermatol. 2007;
34: 503–11.
49 Zimmermann J, Bahmer F, Rose C, Zillikens D, Schmidt E.
Clinical and immunopathological spectrum of paraneoplastic
pemphigus. J Dtsch Dermatol Ges. 2010; 8: 598–605.
50 Kim SC, Kwon YD, Lee IJ, Chang SN, Lee TG. cDNA cloning of
the 210-kDa paraneoplastic pemphigus antigen reveals that
envoplakin is a component of the antigen complex. J Invest
Dermatol. 1997; 109: 365–9.
51 Probst C, Schlumberger W, Stocker W, Recke A, Schmidt E,
Hashimoto T, Zhu XJ, Zillikens D, Komorowski L. Develop-
ment of ELISA for the specific determination of autoantibodies
against envoplakin and periplakin in paraneoplastic pemphi-
gus. Clin Chim Acta. 2009; 410: 13–8.
52 Mahoney MG, Aho S, Uitto J, Stanley JR. The members of the
plakin family of proteins recognized by paraneoplastic pem-
phigus antibodies include periplakin. J Invest Dermatol. 1998;
111: 308–13.
53 Schepens I, Jaunin F, Begre N, Laderach U, Marcus K,
­Hashimoto T, Favre B, Borradori L. The protease inhibitor
alpha-2-macroglobulin-like-1 is the p170 antigen recognized
by paraneoplastic pemphigus autoantibodies in human.
PLoS One. 2010; 5: e12250.
54 Amagai M, Nishikawa T, Nousari HC, Anhalt GJ, Hashimoto
T. Antibodies against desmoglein 3 (pemphigus vulgaris
­antigen) are present in sera from patients with paraneoplastic
pemphigus and cause acantholysis in vivo in neonatal mice.
J Clin Invest. 1998; 102: 775–82.
55 Ruocco V, Pisani M. Induced pemphigus. Arch Dermatol Res.
1982; 274: 123–40.
Guidelines  Diagnosis of pemphigus and bullous pemphigoid
56 Wolf R, Tamir A, Brenner S. Drug-induced versus drug-­
triggered pemphigus. Dermatologica. 1991; 182: 207–10.
57 Brenner S, Bialy-Golan A, Ruocco V. Drug-induced
­pemphigus. Clin Dermatol. 1998; 16: 393–7.
58 Fitzpatrick RE, Newcomer VD. The correlation of disease
­activity and antibody titers in pemphigus. Arch Dermatol.
1980; 116: 285–90.
59 Abasq C, Mouquet H, Gilbert D, Tron F, Grassi V, Musette P,
Joly P. ELISA testing of anti-desmoglein 1 and 3 antibodies in
the management of pemphigus. Arch Dermatol. 2009; 145:
529–35.
60 Cheng SW, Kobayashi M, Kinoshita-Kuroda K, Tanikawa A,
Amagai M, Nishikawa T. Monitoring disease activity in pem-
phigus with enzyme-linked immunosorbent assay using
recombinant desmogleins 1 and 3. Br J Dermatol. 2002; 147:
261–5.
61 Harman KE, Seed PT, Gratian MJ, Bhogal BS, Challacombe SJ,
Black MM. The severity of cutaneous and oral pemphigus is
related to desmoglein 1 and 3 antibody levels. Br J Dermatol.
2001; 144: 775–80.
62 Bastuji-Garin S, Joly P, Lemordant P, Sparsa A, Bedane C,
­Delaporte E, Roujeau JC, Bernard P, Guillaume JC, Ingen-
Housz-Oro S, Maillard H, Pauwels C, Picard-Dahan C, Dutronc
Y, Richard MA, French Study Group for Bullous D. Risk factors
for bullous pemphigoid in the elderly: a prospective case-
control study. The Journal of investigative dermatology. 2011;
131: 637–43.
63 Bastuji-Garin S, Joly P, Picard-Dahan C, Bernard P, Vaillant L,
Pauwels C, Salagnac V, Lok C, Roujeau JC. Drugs associated
with bullous pemphigoid. A case-control study. Arch Derma-
tol. 1996; 132: 272–6.
64 Lloyd-Lavery A, Chi CC, Wojnarowska F, Taghipour K.
The a­ ssociations between bullous pemphigoid and drug
use: a UK case-control study. JAMA Dermatol. 2013; 149:
58–62.
65 Chen YJ, Wu CY, Lin MW, Chen TJ, Liao KK, Chen YC, Hwang
CY, Chu SY, Chen CC, Lee DD, Chang YT, Wang WJ, Liu HN.
Comorbidity profiles among patients with bullous pemphi-
goid: a nationwide population-based study. Br J Dermatol.
2011; 165: 593–9.
66 Cordel N, Chosidow O, Hellot MF, Delaporte E, Lok C, ­Vaillant
L, Bernard P, D’Incan M, Roujeau JC, Joly P. Neurological
­disorders in patients with bullous pemphigoid. Dermatology.
2007; 215: 187–91.
67 Cortes B, Marazza G, Naldi L, Combescure C, Borradori L.
Mortality of bullous pemphigoid in Switzerland: a prospective
study. Br J Dermatol. 2011; 165: 368–74.
68 Jedlickova H, Hlubinka M, Pavlik T, Semradova V, Budinska E,
Vlasin Z. Bullous pemphigoid and internal diseases - A case-
control study. Eur J Dermatol. 2010; 20: 96–101.
69 Kulthanan K, Chularojanamontri L, Tuchinda P, Sirikudta W,
Pinkaew S. Prevalence and clinical features of Thai patients
with bullous pemphigoid. Asian Pac J Allergy Immunol. 2011;
29: 66–72.
70 Langan SM, Groves RW, West J. The relationship between
neurological disease and bullous pemphigoid: a population-
based case-control study. The Journal of investigative derma-
tology. 2011; 131: 631–6.
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
71 Taghipour K, Chi CC, Vincent A, Groves RW, Venning V,
­ ojnarowska F. The association of bullous pemphigoid with
W
cerebrovascular disease and dementia: a case-control study.
Arch Dermatol. 2010; 146: 1251–4.
72 Murrell DF, Daniel BS, Joly P, Borradori L, Amagai M, Hashi-
moto T, Caux F, Marinovic B, Sinha AA, Hertl M, Bernard P,
Sirois D, Cianchini G, Fairley JA, Jonkman MF, Pandya AG,
Rubenstein D, Zillikens D, Payne AS, Woodley D, Zambruno G,
Aoki V, Pincelli C, Diaz L, Hall RP, Meurer M, Mascaro JM, Jr.,
Schmidt E, Shimizu H, Zone J, Swerlick R, Mimouni D, Culton
D, Lipozencic J, Bince B, Grando SA, Bystryn JC, Werth VP. Def-
initions and outcome measures for bullous pemphigoid: rec-
ommendations by an international panel of experts. Journal of
the American Academy of Dermatology. 2012; 66: 479–85.
73 Schmidt E, Zillikens D. Modern diagnosis of autoimmune blis-
tering skin diseases. Autoimmunity reviews. 2010; 10: 84–9.
74 Terra JB, Pas HH, Hertl M, Dikkers FG, Kamminga N, Jonk-
man MF. Immunofluorescence serration pattern analysis as
a ­diagnostic criterion in antilaminin-332 mucous membrane
pemphigoid: immunopathological findings and clinical expe-
rience in 10 Dutch patients. Br J Dermatol. 2011; 165: 815–22.
75 Vodegel RM, Jonkman MF, Pas HH, de Jong MC. U-serrated
immunodeposition pattern differentiates type VII collagen
targeting bullous diseases from other subepidermal bullous
autoimmune diseases. Br J Dermatol. 2004; 151: 112–8.
76 Gammon WR, Kowalewski C, Chorzelski TP, Kumar V, Brigga-
man RA, Beutner EH. Direct immunofluorescence studies of
sodium chloride-separated skin in the differential diagnosis of
bullous pemphigoid and epidermolysis bullosa acquisita. J Am
Acad Dermatol. 1990; 22: 664–70.
77 Rose C, Schmidt E, Kerstan A, Thoma-Uszynski S, Wesselmann
U, Kasbohrer U, Zillikens D, Shimanovich I. Histopathology of
anti-laminin 5 mucous membrane pemphigoid. Journal of the
American Academy of Dermatology. 2009; 61: 433–40.
78 Rose C, Weyers W, Denisjuk N, Hillen U, Zillikens D, Shimanov-
ich I. Histopathology of anti-p200 pemphigoid. Am J Derma-
topathol. 2007; 29: 119–24.
79 Gammon WR, Briggaman RA, Inman AO, 3rd, Queen LL,
Wheeler CE. Differentiating anti-lamina lucida and anti-
sublamina densa anti-BMZ antibodies by indirect immuno-
fluorescence on 1.0 M sodium chloride-separated skin. J Invest
Dermatol. 1984; 82: 139–44.
80 Kelly SE, Wojnarowska F. The use of chemically split tissue in
the detection of circulating anti-basement membrane zone
antibodies in bullous pemphigoid and cicatricial pemphigoid.
Br J Dermatol. 1988; 118: 31–40.
81 Ghohestani R, Kanitakis J, Nicolas JF, Cozzani E, Claudy A. Com-
parative sensitivity of indirect immunofluorescence to immunob-
lot assay for the detection of circulating antibodies to bullous
pemphigoid antigens 1 and 2. Br J Dermatol. 1996; 135: 74–9.
82 Ghohestani RF, Nicolas JF, Rousselle P, Claudy AL. Diagnostic
value of indirect immunofluorescence on sodium chloride-
split skin in differential diagnosis of subepidermal autoim-
mune bullous dermatoses. Arch Dermatol. 1997; 133: 1102–7.
83 Chan YC, Sun YJ, Ng PP, Tan SH. Comparison of immunofluo-
rescence microscopy, immunoblotting and enzyme-linked
immunosorbent assay methods in the laboratory diagnosis of
bullous pemphigoid. Clin Exp Dermatol. 2003; 28: 651–6.
725
 Guidelines  Diagnosis of pemphigus and bullous pemphigoid
84 Kippes W, Schmidt E, Roth A, Rzany B, Brocker EB, Zillikens D.
[Immunopathologic changes in 115 patients with bullous pem-
phigoid]. Hautarzt. 1999; 50: 866–72.
85 Sardy M, Kostaki D, Varga R, Peris K, Ruzicka T. Comparative
study of direct and indirect immunofluorescence and of bul-
lous pemphigoid 180 and 230 enzyme-linked immunosorbent
assays for diagnosis of bullous pemphigoid. Journal of the
American Academy of Dermatology. 2013; 69: 748–53.
86 van Beek N, Dohse A, Riechert F, Krull V, Recke A, Zillikens
D, Schmidt E. Serum autoantibodies against the dermal-
epidermal junction in patients with chronic pruritic disorders,
elderly individuals and blood donors prospectively recruited.
Br J Dermatol. 2014; 170: 943–7.
87 Jordon RE, Beutner EH, Witebsky E, Blumental G, Hale WL,
Lever WF. Basement zone antibodies in bullous pemphigoid.
Jama. 1967; 200: 751–6.
88 Beutner EH, Jordon RE, Chorzelski TP. The immunopathology
of pemphigus and bullous pemphigoid. J Invest Dermatol.
1968; 51: 63–80.
89 Schmidt E, Zillikens D. Pemphigoid diseases. Lancet. 2013; 381:
320–32.
90 Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Liu Z, Diaz
LA. Bullous pemphigoid and herpes gestationis autoantibod-
ies recognize a common non-collagenous site on the BP180
ectodomain. J Immunol. 1993; 151: 5742–50.
91 Zillikens D, Rose PA, Balding SD, Liu Z, Olague-Marchan M,
Diaz LA, Giudice GJ. Tight clustering of extracellular BP180
epitopes recognized by bullous pemphigoid autoantibodies.
J Invest Dermatol. 1997; 109: 573–9.
92 Kobayashi M, Amagai M, Kuroda-Kinoshita K, Hashimoto T, Shi-
rakata Y, Hashimoto K, Nishikawa T. BP180 ELISA using b ­ acterial
recombinant NC16a protein as a diagnostic and monitoring
tool for bullous pemphigoid. J Dermatol Sci. 2002; 30: 224–32.
93 Sitaru C, Dahnrich C, Probst C, Komorowski L, Blocker I,
Schmidt E, Schlumberger W, Rose C, Stocker W, Zillikens D.
Enzyme-linked immunosorbent assay using multimers of
the 16th non-collagenous domain of the BP180 antigen for
­sensitive and specific detection of pemphigoid autoantibod-
ies. Exp Dermatol. 2007; 16: 770–7.
94 Yoshida M, Hamada T, Amagai M, Hashimoto K, Uehara R,
­Yamaguchi K, Imamura K, Okamoto E, Yasumoto S, Hashimoto
T. Enzyme-linked immunosorbent assay using bacterial re-
combinant proteins of human BP230 as a diagnostic tool for
bullous pemphigoid. J Dermatol Sci. 2006; 41: 21–30.
95 Blocker IM, Dahnrich C, Probst C, Komorowski L, Saschen-
brecker S, Schlumberger W, Stocker W, Zillikens D, Schmidt E.
Epitope mapping of BP230 leading to a novel enzyme-linked
immunosorbent assay for autoantibodies in bullous pemphi-
goid. Br J Dermatol. 2012; 166: 964–70.
96 Charneux J, Lorin J, Vitry F, Antonicelli F, Reguiai Z, Barbe C,
Tabary T, Grange F, Bernard P. Usefulness of BP230 and BP180-
NC16a enzyme-linked immunosorbent assays in the initial
diagnosis of bullous pemphigoid: a retrospective study of 138
patients. Arch Dermatol. 2011; 147: 286–91.
97 Tampoia M, Lattanzi V, Zucano A, Villalta D, Filotico R, Fon-
tana A, Vena GA, Di Serio F. Evaluation of a new ELISA assay
for detection of BP230 autoantibodies in bullous pemphigoid.
Ann N Y Acad Sci. 2009; 1173: 15–20.
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
726
98 Roussel A, Benichou J, Randriamanantany ZA, Gilbert D,
Drenovska K, Houivet E, Tron F, Joly P. Enzyme-linked immu-
nosorbent assay for the combination of bullous pemphigoid
antigens 1 and 2 in the diagnosis of bullous pemphigoid. Arch
Dermatol. 2011; 147: 293–8.
99 Wieland CN, Comfere NI, Gibson LE, Weaver AL, Krause PK,
Murray JA. Anti-bullous pemphigoid 180 and 230 antibodies
in a sample of unaffected subjects. Arch Dermatol. 2010; 146:
21–5.
100 della Torre R, Combescure C, Cortes B, Marazza G, Beltrami-
nelli H, Naldi L, Borradori L. Clinical presentation and diag-
nostic delay in bullous pemphigoid: a prospective nationwide
cohort. Br J Dermatol. 2012; 167: 1111–7.
101 Di Zenzo G, Marazza G, Borradori L. Bullous pemphigoid:
physiopathology, clinical features and management. Adv
­Dermatol. 2007; 23: 257–88.
102 Schmidt E, della Torre R, Borradori L. Clinical features and
practical diagnosis of bullous pemphigoid. Dermatol Clin.
2011; 29: 427–38, viii-ix.
103 Korman N. Bullous pemphigoid. J Am Acad Dermatol. 1987;
16: 907–24.
104 Chan LS, Ahmed AR, Anhalt GJ, Bernauer W, Cooper KD, Elder
MJ, Fine JD, Foster CS, Ghohestani R, Hashimoto T, Hoang-
Xuan T, Kirtschig G, Korman NJ, Lightman S, Lozada-Nur F,
Marinkovich MP, Mondino BJ, Prost-Squarcioni C, Rogers
RS, 3rd, Setterfield JF, West DP, Wojnarowska F, Woodley DT,
Yancey KB, Zillikens D, Zone JJ. The first international consen-
sus on mucous membrane pemphigoid: definition, diagnostic
criteria, pathogenic factors, medical treatment, and prognos-
tic indicators. Arch Dermatol. 2002; 138: 370–9.
105 Di Zenzo G, Calabresi V, Grosso F, Caproni M, Ruffelli M,
Zambruno G. The intracellular and extracellular domains of
BP180 antigen comprise novel epitopes targeted by pemphi-
goid gestationis autoantibodies. J Invest Dermatol. 2007; 127:
864–73.
106 Di Zenzo G, Thoma-Uszynski S, Fontao L, Calabresi V, Hof-
mann SC, Hellmark T, Sebbag N, Pedicelli C, Sera F, Lacour JP,
Wieslander J, Bruckner-Tuderman L, Borradori L, Zambruno
G, Hertl M. Multicenter prospective study of the humoral
autoimmune response in bullous pemphigoid. Clin Immunol.
2008; 128: 415–26.
107 van Beek N, Knuth-Rehr D, Altmeyer P, Assaf C, Babilas P,
­Bayerl C, Benoit S, Dippel E, Effendy I, Eming R, Fischer M,
Glaenz T, Glaser R, Goebeler M, Gollnick H, Gotze S, Gross
G, Hadaschik E, Herbst R, Hermes B, Homey B, Hunzelmann
N, Junger M, Kapp A, Kern JS, Korber A, Luger T, Mechtel
D, Megahed M, Moll I, Peters KP, Pfeiffer C, Ring J, Rocken
M, Sardy M, Seitz CS, Stadler R, Steinbrink K, Sticherling M,
Szeimies RM, Tronnier M, Ulrich J, Vogt T, Wagner N, Welzel J,
Wenzel J, Wozel G, Zouboulis CC, Zillikens D, Schmidt E. Diag-
nostics of autoimmune bullous diseases in German dermatol-
ogy departments. J Dtsch Dermatol Ges. 2012; 10: 492–9.
108 Komorowski L, Muller R, Vorobyev A, Probst C, Recke A,
­Jonkman MF, Hashimoto T, Kim SC, Groves R, Ludwig RJ,
­Zillikens D, Stocker W, Schmidt E. Sensitive and specific as-
says for routine serological diagnosis of epidermolysis bullosa
acquisita. Journal of the American Academy of Dermatology.
2013; 68: e89-95.
Guidelines  Diagnosis of pemphigus and bullous pemphigoid
109 Saleh MA, Ishii K, Kim YJ, Murakami A, Ishii N, Hashimoto T,
Schmidt E, Zillikens D, Shirakata Y, Hashimoto K, Kitajima Y,
Amagai M. 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. J Dermatol
Sci. 2011; 62: 169–75.
110 Lindelof B, Islam N, Eklund G, Arfors L. Pemphigoid and can-
cer. Arch Dermatol. 1990; 126: 66–8.
111 Ogawa H, Sakuma M, Morioka S, Kitamura K, Sasai Y, Imamura S,
Inaba Y. The incidence of internal malignancies in pemphigus
and bullous pemphigoid in Japan. J Dermatol Sci. 1995; 9: 136–41.
112 Ong E, Goldacre R, Hoang U, Sinclair R, Goldacre M. Associa-
tions between bullous pemphigoid and primary malignant
cancers: an English national record linkage study, 1999–2011.
Arch Dermatol Res. 2014; 306: 75–80.
113 Schulze F, Neumann K, Recke A, Zillikens D, Linder R, Schmidt
E. Malignancies in pemphigus and pemphigoid diseases.
J Invest Dermatol. 2015; 135: 1445–7.
© 2015 The Authors | Journal compilation © Blackwell Verlag GmbH, Berlin | JDDG | 1610-0379/2015/1307
114 Amo Y, Ohkawa T, Tatsuta M, Hamada Y, Fujimura T, Katsuoka
K, Hashimoto T. Clinical significance of enzyme-linked immu-
nosorbent assay for the detection of circulating anti-BP180 au-
toantibodies in patients with bullous pemphigoid. J Dermatol
Sci. 2001; 26: 14–8.
115 Schmidt E, Obe K, Brocker EB, Zillikens D. Serum levels of
autoantibodies to BP180 correlate with disease activity in
patients with bullous pemphigoid. Arch Dermatol. 2000; 136:
174–8.
116 Pas HH, de Jong MC, Jonkman MF, Heeres K, Slijper-Pal IJ, van
der Meer JB. Bullous pemphigoid: serum antibody titre and
antigen specificity. Exp Dermatol. 1995; 4: 372–6.
117 Di Zenzo G, Thoma-Uszynski S, Calabresi V, Fontao L,
­Hofmann SC, Lacour JP, Sera F, Bruckner-Tuderman L,
­Z ambruno G, Borradori L, Hertl M. Demonstration of epitope-
spreading phenomena in bullous pemphigoid: results of a
prospective multicenter study. J Invest Dermatol. 2011; 131:
2271–80.
727