2016, Volume 32, Number 2, Page(s) 091-098
Utility of Direct Immunofluorescence Studies in Subclassification of Autoimmune Sub-Epidermal Bullous Diseases: A 2-Year Study in a Tertiary Care Hospital
Supriya JAIN1, Vijaya BASAVARAJ2, Manjunath GUBBANNA VIMALA2
1Department of Pathology Bharati Vidyapeeth Deemed University Medical College and Hospital, SANGLI, INDIA
2JSS Medical College (JSS University), MYSORE, INDIA
Keywords: Direct immunofluorescence, Bullous skin diseases, Bullous Pemphigoid, Dermatitis Herpetiformis
Sub-epidermal bullous disorders belong to immunobullous diseases which develop as a result of autoantibody action against
epidermal basement membrane proteins. Clinically, they are tense bullae and do not rupture easily. They are classified into various forms based
on histopathology and direct immunofluorescence patterns. This study was undertaken to assess the incidence of various sub-epidermal bullous
disorders and the utility of direct immunofluorescence in accurately classifying them, and to study the intensity and pattern of immunofluorescence
in various sub-epidermal bullous disorders
Material and Method: A 2-year study of 38 cases of sub-epidermal bullous disorders sent for direct immunofluorescence studies formed the
study group. The specimens were processed as per standard protocols. The clinical details were obtained from case files and requisition sent for
histopathological and direct immunofluorescence studies.
Results: Thirty-eight patients were diagnosed to have sub-epidermal bullous disorders over the period of 2 years. Twenty five cases of Bullous
Pemphigoid, 5 cases of Dermatitis Herpetiformis, 3 cases of Linear IgA Bullous disorder, 2 cases of Bullous Systemic Lupus Erythematoses
and 1 case each of Epidermolysis Bullosa Acquisita, Cicatricial Pemphigoid and Pemphigus Gestationis was diagnosed. Positive direct
immunofluorescence was seen in 91.3% of the cases.
Conclusion: Histopathology alone cannot classify sub-epidermal bullous disorders and direct immunofluorescence studies are mandatory in all of
them. Bullous Pemphigoid needs to be distinguished from Epidermolysis Bullosa Acquisita which requires Salt split direct immunofluorescence
studies. Dermatitis Herpetiformis, Bullous Systemic Lupus Erythematosus and Linear IgA Bullous disorder show more or less similar
histological picture with neutrophilic microabscess. Direct immunofluorescence studies help in the majority of cases but further testing such as
immunoblotting, immunoelectron microscopy or indirect immunofluorescence becomes essential in cases with overlapping features.
Sub-epidermal bullous disorders (SEBDs) are characterized
by autoantibodies specifically against the components
of the epidermal basement membrane zone1
embrace the following entities: Bullous Pemphigoid (BP),
Cicatricial Pemphigoid (CP), Epidermolysis Bullosa
Acquisita (EBA), Dermatitis Herpetiformis (DH), Linear
IgA Bullous Dermatoses (LABD) and Bullous Systemic
Lupus Erythematosus (SLE).
SEBDs are characterized clinically by the presence of
cutaneous and/or mucosal blisters on a hemorrhagic or
non-hemorrhagic base, associated with intense pruritus.
Histology shows all SEBD's characterized by sub-epidermal
split with inflammatory infiltrate in the bullous cavity.
Direct immunofluorescence (DIF) studies show deposition of immunoglobulins and/or complement at the basement
membrane zone, with the exception of DH, where the
deposits are seen in the dermal papillae.
The study was conducted during a 2-year period. Two 5
mm punch skin biopsies, one in 10% formalin and the
other in normal saline, were sent from patients with a
suspected clinical diagnosis of vesiculobullous disease
to the department of pathology for both histopathology
and DIF studies, respectively. Biopsy specimens sent in
formalin were processed and serial haematoxylin and eosin
sections were analyzed. The biopsy for DIF studies was sent
in normal saline and was processed as follows. Specimens
were embedded in a cryomatrix embedding medium
and snap-frozen at -400C until sectioned. Cryostat tissue sections of 4 microns were air-dried and washed thrice with
phosphate-buffered saline (PBS), pH 7.4, for 10 minutes
before being overlaid for 30 minutes with fluorescein
isothiocyanate-conjugated rabbit anti-human IgG, IgA,
IgM and C3. Sections were then incubated in a humidified
chamber at room temperature, washed thrice with PBS at
pH 7.4 for 10 minutes and mounted with glycerine before
viewing with a fluorescent microscope (Olympus BX-40).
A total of 75 cases of immunobullous diseases were
evaluated of which 38 cases were from the SEBD group
and were included in the study. The remaining 44 cases
belonged to the pemphigus group and were excluded from
the study. Clinical data provided along with the biopsy
specimen were documented separately. DIF patterns were
interpreted according to standard criteria.
Thirty-eight patients were diagnosed as SEBD over the
period of 2 years. Twenty five cases were of BP (65.7%),
5 cases were of DH (13.1%), 3 cases were of LABD (7.8%), 2
cases were of Bullous SLE (5.2%), and there was 1 case each
of EBA, CP and Pemphigus Gestationis (PG) (2.6%).
BP was the most common lesion among SEBDs followed
by DH, LABD and Bullous SLE. The clinical features
and histopathological findings are shown in Table I.
Histopathologically all the cases of BP except for three
cases showed a sub-epidermal bulla with fibrin and
inflammatory infiltrate in the bullous cavity. Three
cases showed an intraepidermal bulla with regenerative
epithelium on the floor. Eosinophils were the most common
inflammatory infiltrate seen followed by neutrophils.
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|Table I: The clinical and histopathological findings of sub-epidermal bullous diseases
The adjacent epidermis showed eosinophilic spongiosis in
8 cases. (Table I, Figure 1A-B). DIF studies showed linear
deposition of C3 along the dermo-epidermal junction in
all the cases (100%) and deposition of IgG in 91.8% of the
cases (Figure 1C-D). The fluorescence was appreciable at
lower magnification of the 10x objective in 66.6% of cases
and at 40x in the remaining 33.3% of the cases representing
a lower intensity of fluorescence.
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|Figure 1: Bullous pemphigoid: A) Scanner view showing sub-epidermal bulla with inflammatory infiltrate in bullous cavity (H&E; x40),
B) Low power view showing sub-epidermal bulla with eosinophils and exudates in bullous cavity (H&E; x100), C,D) Linear deposition
of C3 and IgG along the dermo-epidermal junction (IF; x200).
PG showed identical histopathological features as BP with
only C3 deposition along the dermoepidermal junction. CP
on DIF studies showed deposition of IgG and IgM along
the dermo-epidermal junction. The histopathology showed
fibrosis of the subepithelial region.
DH showed sub-epidermal clefting with presence of fibrin
and neutrophils. There was denudation of the epidermis
with collection of neutrophils and fibrin in the underlying papillary dermis. DIF studies showed deposition of IgA in
granular and fibrillary pattern in the dermal papillae.
LABD showed sub-epidermal bulla rich in neutrophils
and eosinophils (Figure 2A). DIF studies showed a linear
homogenous deposition of IgA along the basement
membrane zone. One case showed deposition of IgM along
with IgA (Figure 2B,C).
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|Figure 2: A) Linear IgA bullous dermatosis showing subepidermal
bulla rich in neutrophils, B,C) Immunofluorescence
showing linear homogenous deposition of IgA and IgM along the
basement membrane zone (IF; x200).
Bullous SLE showed epidermis displaying focal basal cell
vacuolar degeneration and a small sub-epidermal cleft was
seen with intense collection of neutrophils in the papillary
and mid dermis with karyorrhexis (Figure 3A). DIF studies
showed granular and linear deposits of IgG, IgA, IgM and
C3 along the dermoepidermal junction and around hair
follicle epithelium (Figure 3B-D).
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|Figure 3: A) Bullous systemic lupus erythematosus showing epidermis with focal basal cell vacuolar degeneration, small subepidermal
cleft with intense collection of neutrophils, in the papillary and mid dermis with karyorrhexis, B-D) Immunofluorescence showing
granular and linear deposits of IgG, IgA and IgM along the dermo-epidermal junction respectively (IF; x200).
EBA showed deposition of IgG and C3 along DEJ in
continous and linear pattern (Figure 4)
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|Figure 4: Epidermolysis bullosa acquisita showing deposition of
IgG along the floor of the bulla (IF; x200).
Bullous Pemphigoid usually occurs in elderly individuals
and presents with multiple tense bullae commonly on the
extremities and the trunk. Erosions in the oral cavity are
. BP has been reported in association
with diabetes, ulcerative colitis and vitiligo2
with diabetes was seen in 26% and with hypertension in
13% of our patients.
Histopathology of BP may show a regenerative epithelium
at the floor of the bulla making it difficult to discern the
anatomical level of split and give a false negative impression
of intraepidermal bulla. Such cases warrant DIF studies.
A biopsy from an older lesion causes such changes and
difficulty in interpretation of the biopsy and therefore a
biopsy from an early bulla is advised for histopathological
DIF studies in BP show linear deposition of C3 along the
dermo-epidermal junction in all the cases (100%) whereas
deposition of IgG was seen in 91.8% cases. Hence, C3 has
a higher sensitivity and specificity as compared to IgG.
This was in accordance to the other studies reported in the
PG is common in the second to third trimester of pregnancy
and presents with itchy plaques, papules and vesicles initially
on the abdomen and later involving the extremities. Cases
reported advocate the average age for the appearance of
lesions as around 21 week of gestation (2nd or 3rd trimester)
with the lesions typically beginning on the abdomen
around umbilicus as was noted in our case4. The etiology
is controversial but it is attributed to autoimmune reaction
against placental tissue5. A few studies have shown an
associated underlying hydatidiform mole or choriocarcinoma.
Hence it is essential to thoroughly evaluate for an
underlying cause5. There was no such underlying lesion
in our case. The patient had a history of similar lesions of
mild severity in the previous pregnancy, which resolved
after delivery, as conventional for PG. It usually recurs in
subsequent pregnancies, when it tends to be more severe.
PG on DIF shows linear deposits of C3 along the dermoepidermal
junction. Similar findings have been observed
by other authors2,3,6. DIF studies are also required to
differentiate pemphigoid gestationis from other cutaneous
lesions occurring in pregnancy, especially pruritic urticarial
papules and plaques of pregnancy, the latter showing
negative results on DIF6.
DH comprised the second most common lesion among
cases of SEBD. Patients present with intense itchy papulopruritic lesions on the extremities, abdomen and chest.
Intact bullae are rarely seen in DH because the lesions
are scratched due to intense pruritus and thus the vesicles
are replaced by excoriations. According to the literature,
mucous membrane involvement is seen infrequently7-9.
There was no mucous membrane involvement in any of
our cases. DH is known to be associated with celiac disease
and other auto-immune diseases. In our study, none of the
cases had a history of diarrhoea or any significant medical
history. We found a good number of patients with intact
bullae (66.6%). This finding, along with a lower prevalence
of intestinal symptoms, might reflect a different phenotype
which is more common in our population. A similar pattern
has also been noted in a study by Fuertes et al. in Spain10.
Histopathology usually showed a sub-epidermal clefting
with neutrophilic microabscess, In three of the cases, there
was no discernible sub-epidermal bulla due to denudation of the epidermis with collection of neutrophils and
fibrin in the underlying papillary dermis. The presence
of neutrophilic microabscess in the papillary dermis
authenticates the diagnosis (Table II). Although DH was
included in the clinical differential diagnosis of many
cases of SEBD, it was proved histopathologically only in 5
cases and DIF was positive only in two cases. This result
accounted the lowest compliance (66%) in our study. As has
been stated by Lebe et al., one of the reasons for discordance
could be the tendency of the clinicians to include DH as
differential diagnosis in all pruritic skin lesions11. DIF
studies show deposition of IgA in a granular and fibrillary
pattern in the dermal papillae. There were a high number
of false negatives and the reasons could be biopsy from
the involved skin, biopsy from an area that has never been
involved, the patient being on a long-term gluten free diet,
and pruritic papulo-vesicular eruptions of DH that evolved and disappeared rapidly9,11. In one of the largest series,
reported by Alonso et al. in 2007, granular deposits of IgA
at the dermal papillae was considered the hallmark of DH,
and this was reproduced in a study of the author who found
a sensitivity of 92.4%9. However, subsequent studies
and the present study have failed to reproduce the same12-15. In our study and few other studies, it was found
that DH had least concordance when clinical diagnosis
and histopathological diagnosis was considered with DIF
studies11,15. All these findings in the more recent studies
prod us to think whether deposition of IgA in the dermal papillae should be considered as a diagnostic hallmark
in DH14,15 or should the consideration be given to a
combination of clinical, histologic and immunologic and
more specific presence of IgA anti-endomysial antibodies?
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|Table II: Direct immunofluorescence findings in sub-epidermal bullous diseases
LABD was third in frequency among SEBDs. Two were
childhood type and one was adult type (16,17). The patient
present with vesicles of varying sizes, tense to flaccid and
hemorrhagic bullae. Hemorrhagic bullae have also been
reported by Rogers et al.18. Patients with childhood type
of LABD present with tense bullae and vesicles with erosions
and central crusting. Histopathology shows sub-epidermal
bulla (Table II). Eosinophils and neutrophils can be seen
in the bulla cavity and around blood vessels in the upper
dermis. The presence of eosinophils poses a diagnostic
dilemma between LABD and bullous pemphigoid16.
The clinical findings and DIF findings help in resolving the
diagnostic issue. DIF studies show deposition of IgA along
the dermoepidermal junction in a linear and homogenous
pattern. Deposition of IgG and IgM along the DEJ in
addition to deposition of IgA in linear fashion may also be
present as was seen in our case.
Chan et al. suggested that LABD and BP can usually be
differentiated based on the greater fluorescence intensity of
one of the antibodies, either IgA or IgG, in combination
with clinical and immunological features19. This
diagnostic dilemma further surges when there is additional
deposition of C3 along the DEJ along with IgA/IgG. Powell
et al. observed that irrespective of whether IgA or IgG is predominantly deposited, the clinical features, response to
treatment with dapsone, and prognosis of the childhood
disease remain the same20. Therefore, the classical cases
with predominant IgA deposition (linear IgA disease) as
well as mixed immune bullous disease (linear IgG / IgA
disease or linear IgA / IgG disease) seem to be practically
the same entity. The division of this clinically homogeneous
entity into the classical form and variants, based on the
immunopathology seems artificial, futile, and confusing.
Considering all these factors, indiscriminate use of terms
like linear IgA disease, linear IgA bullous dermatoses of
childhood, mixed immune bullous disease, linear IgG /
IgA disease and so on, should best be avoided in childhood
cases21. Haneef et al. proposed that all cases showing
the typical clinical picture of ‘cluster of jewels' or ‘string
of pearls' sign should be included under the broad term
‘chronic bullous disease of childhood,' irrespective of the
nature of the immune deposits, as the clinical behavior is
same and the patient responds well to the treatment21.
Bullous SLE is a rare cutaneous manifestation of systemic
lupus erythematosus (SLE). In 23% of patients with
SLE, cutaneous involvement is the initial manifestation.
Patients present with cutaneous blisters along with other
signs and symptoms of SLE. In a few cases including
ours, cutaneous bullae were initial manifestations of SLE,
and SLE presenting as a bullous eruption is indeed a rare
manifestation22. Histopathology shows sub-epidermal
clefting and DIF studies shows “full house” deposition of
immunoglobulins and C3 in granular and linear pattern
at the dermoepidermal junction and sometimes around
adnexal epithelium. DIF is essential in making the diagnosis
of bullous SLE.
EBA is another SEBD that merits mention as it is not
infrequent. Although a clinical history of mechanobullous
eruption aids in making the diagnosis, histopathology
and immunofluorescence similarity warrants a salt split
immunofluorescence study which shows deposition of
immunoreactants, C3 and IgG along the floor of the split
EBA and bullous SLE are sub-epidermal blisters that target
type VII collagen, but can look identical to BP on DIF. Saltsplit
DIF studies will distinguish between these entities.
The patterns of immunoreactant deposition in bullous SLE
can be granular, linear and rarely fibrillar23. The dermal
floor pattern of indirect immunofluorescence on salt-split
skin substrate is found in the sera of patients with both
bullous SLE and EBA24. Immunoelectron microscopy
and serological tests for bullous SLE become the gold
standard in distinguishing the two disorders in such cases. The clinical manifestations also help to distinguish between
Bullous SLE and EBA.
In conclusion, SEBDs present with tense bullae clinically
with an anatomical level of split in the sub-epidermal
location. Eosinophil rich infiltrates are characteristic of
bullous pemphigoid whereas neutrophilic rich infiltrates
are seen in DH and LABD. EBA usually shows a cell-poor
sub-epidermal bulla. BP of the cell-poor type and EBA
may be difficult to distinguish both histologically and on
DIF. A salt split skin IF helps in differentiating cell-poor
BP from an EBA. The nonavailability of salt split skin
immunofluorescence studies was the limitation of the study.
In a few cases with overlapping features, further testing
such as immunoblotting, immunoelectron microscope or
indirect immunofluorescence becomes essential.
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