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DOI: 10.5146/tjpath.2023.01607 |
Is it Possible to Obtain Immunofluorescence Data in Formalin-Fixed Paraffin-Embedded Skin Samples for the Diagnosis of Pemphigus Vulgaris and Bullous Pemphigoid |
Deniz ATES OZDEMIR, Ozay GOKOZ, Arzu SAGLAM |
Department of Pathology, Hacettepe University, Faculty of Medicine, ANKARA, TURKEY |
Keywords: Protease digestion, IgG, Pemphigus vulgaris, Bullous pemphigoid, C4d |
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Objective: The gold-standard method for assessment of autoimmune bullous disease is direct/indirect immunofluorescence (IF) examination
applied to fresh frozen tissue. Since the sensitivity of IF is greatly reduced in formalin-fixed paraffin-embedded (FFPE) tissues, IF cannot be
relied upon in these samples. However, immunohistochemistry with the C4d antibody is a promising marker used as a surrogate for immune
complex deposition, in nephropathology practice, and the paraffin IF method is also used as a “salvage” technique when fresh frozen tissue is not
available or lacks glomeruli. We aimed to investigate whether it is possible to obtain immunofluorescence data from FFPE tissues diagnosed with
bullous pemphigoid (BP) and pemphigus vulgaris (PV) and its relationship with inflammatory parameters in the skin.
Material and Methods: Eighty-nine in-house cases with both IgG and C3 positivity by routine immunofluorescence examination were included
in the study. Inflammation parameters were evaluated in hematoxylin-eosin sections. Immunofluorescence study with IgG protease digestion
and C4d immunohistochemistry were performed.
Results: Results of 83 biopsies were obtained by paraffin immunofluorescence with IgG. There were positive reactions in 28 (34%) of these 83
biopsies. Five of the 28 positive results belonged to BP (18%), and 23 were PV (82%). Ten positive results were on lesional skin (36%), and 18
(64%) were on non-lesional skin. In the immunohistochemical study with C4d, 84 biopsy results were obtained. There were positive reactions
in 34 (40.4%) of 84 biopsies. Of the 34 positive results, 12 belonged to BP (35.3%) and 22 to PV (64.7%). Again, 22 (64.7%) of 34 positive results
belonged to lesional skin, and 12 (35.3%) belonged to non-lesional skin. When both techniques were used together, 44 (54%) of 81 biopsies
yielded positive results for at least one of the two studies, while in 37 (46%), both tests showed negative results.
Conclusion: The sensitivity of both IgG and C4d was less than in the literature, especially in BP-diagnosed biopsies. Positive samples were mostly
PV. In conclusion, obtaining immunofluorescence data in FFPE samples is possible and is independent of the related skin being lesional or not,
however, negative results should not be relied upon. |
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Diagnosing an autoimmune bullous disease is a challenging
field of dermatology and dermatopathology. Light
microscopic findings are insufficient for accurate classification
and additional direct immunofluorescence (IF)
findings are necessary. According to the EADV (European
Academy of Dermatology and Venereology), a total of 2
skin biopsies are needed if an autoimmune bullous disease
is suspected: one from the lesional skin and another from
the perilesional intact/undamaged skin 1. The sample
taken from the lesional tissue should be sent to the pathology
laboratory in formaldehyde solution. The other biopsy
is taken for direct IF examination and should be delivered to the pathology laboratory fresh. Michel’s solution, which
is an expensive solution, can also be used as a transport
solution for tissues that cannot be sent immediately 2.
Paraffin tissue processing procedures are applied to the
sample sent in formaldehyde, and the sections are stained
with hematoxylin-eosin.
In recent years, it has been reported that paraffin immunofluorescence
(PIF) examination can be performed with
various special protocols in cases where immunofluorescence
examination cannot be performed or when there
is insufficient glomerulus in nephropathology practice;
this method has been recognized as a “salvage method” in
nephropathology3,4. In skin pathology, Valencia-Guer-rero et al.’s study in 2018 is the only study published on PIF
in autoimmune bullous diseases5. This study has demonstrated
that this technique is valuable, though less sensitive
than routine frozen examination. Another method that
indirectly indicates the presence of immune complexes in
formalin-fixed paraffin-embedded (FFPE) skin tissues is
immunohistochemical studies with C4d antibody, an indicator
of classical pathway-related complement activation.
Studies have shown that demonstration of C4d is successful
in aiding the diagnosis6-10.
In daily practice, when the histopathologist suspects autoimmune
bullous disease in hematoxylin-eosin sections,
disease classification cannot be made and a new biopsy is
requested. Furthermore, there can be rare instances where
paraffin processing is inadvertently applied to tissues sent
for DIF examination, or the lesional skin is biopsied, and
these may cause delays in diagnosis and possible morbidity.
In this study, we aimed to perform a PIF study with the
protease digestion method by using IgG and standard C4d
immunohistochemistry in formalin-fixed paraffin-embedded
lesional and non-lesional skin biopsies with pemphigus
vulgaris and bullous pemphigoid diagnoses, and to
assess the effect of inflammation parameters (since the
preference for biopsy is non-inflamed perilesional skin in
routine practice, we wanted to investigate the relationship
of inflammation parameters such as inflammatory cells,
fibrin, presence or absence of acantholysis, and dominant
inflammatory cell type). |
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Abstract
Introduction
Methods
Results
Disscussion
References
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The research was approved by the Institutional Research
Board (GO-1882 /25-9-2018). The research complied with
the Helsinki Declaration. A total of 89 in-house (37 nonlesional,
52 lesional) biopsies of 43 patients diagnosed with
pemphigus vulgaris and bullous pemphigoid based on
clinical-pathological correlation between 2014 and 2018
were included in the study. All cases had deposition of
IgG and C3 as demonstrated with routine direct immunofluorescent
microscopy applied to frozen sections. Three
unstained 5μ sections were obtained from the FFPE tissue
samples of these cases. One of the unstained slides was
stained with H&E to determine microscopic parameters.
These microscopic parameters were classified in regards to
the presence or absence of inflammation: whether inflammation
was present, its severity (assessed as mild, moderate,
and intense), and the dominant cell type (indeterminatedominant
cell undetectable, eosinophils, lymphocytes, and
neutrophils). Acantholysis and the presence or absence of
fibrin deposition were noted. The second unstained section
was used for the PIF study using the proteinase digestion method as detailed. Sections were kept in phosphate salt
buffer (PBS) for 10 minutes after deparaffinization and
rehydration. They were then treated with 0.05% proteinase
(Sigma cat # P8038) in PBS solution at 37°C for 5 minutes
for antigen expression. Then, the IgG antibody labeled with
FITC (fluorescent isothiocyanide) was dripped onto the
sections and kept in the dark for 45 minutes. Sections were
washed two times with PBS and covered with immunofluorescence-
specific mounting medium (Dako; cat. #S3023).
The last section was stained with C4d immunohistochemistry
in the Leica Bond Max (Shandon, Frankfurt, Germany)
autostainer with appropriate positive and negative
controls as detailed. After exposure to EDTA, sections were
incubated for 30 minutes with the primary antibody C4d
(404A-15, Rabbit polyclonal antibody, 1/200 dilution, Cell
Marque, Rocklyn, CA). 3-amino-9-ethylcarbazole (AECRed)
chromogen was used. Selective linear staining/deposition
along the dermo-epidermal junction was accepted as
positive for samples diagnosed with bullous pemphigoid,
and intraepidermal membranous staining/deposition was
accepted as positive for samples diagnosed with pemphigus
vulgaris (Figure 1). Two experienced dermatopathologists
(OG, DAO) performed fluorescent microscope evaluation.
Cases with discordant findings were re-evaluated jointly,
and a consensus was reached.
Click Here to Zoom |
Figure 1: A) In the biopsy of pemphigus vulgaris, an intraepidermal positive reaction with IgG via protease digestion is seen in the
lesioned skin. An intraepidermal positive reaction was observed in hair follicle epithelium (Orange arrow) (DIF-P, 100x). B) In a case
of bullous pemphigoid, positive reaction with IgG by protease digestion is seen along the basement membrane in the roof of the split
cavity (Orange arrows) (DIF-P, 100x) Autofluorescent cells are leukocytes. C) In a case of bullous pemphigoid, a positive reaction is seen
along the basement membrane with C4d (C4d, 100x). D) Positive reaction with C4d along the basement membrane in a case of bullous
pemphigoid (C4d, 100x) |
The Shapiro-Wilk test, histogram, box-line, and Q-Q graph
results were used to determine the conformity of continuous
variables (age) to normal distribution. The difference
between the two independent groups (age) was compared
with the Mann-Whitney U test for continuous variables.
The difference between groups was examined for categorical
variables, with the Chi-Square or Fisher Exact test (gender,
IgG, C4d positivity or negativity, presence or absence
of acantholysis, inflammatory cells, fibrin, predominant
cell type, lesional / non-lesional, PV/BP diagnosis). The
significance level was taken as 0.05 in all tests. Statistical
analyses were performed using IBM SPSS version 23 (IBM
Co. USA). |
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Abstract
Introduction
Methods
Results
Disscussion
References
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A total of 89 biopsies of 43 patients with positive IgG and C3
immunofluorescence results applied to frozen sections with
clinicopathological correlations were accessed from the
hospital’s patient file software. Of the 43 patients, 21 were
male, and 22 were female. Their mean age was 60.70 years
(youngest: 27, oldest: 91 years, standard deviation 18.79).
Twenty-four of the 43 patients were diagnosed with pemphigus
vulgaris (PV), and 19 were diagnosed with bullous
pemphigoid (BP). Forty-seven biopsies were from patients
diagnosed with PV and 42 with BP. Of the 89 biopsies, 52
were lesional, and 37 were non-lesional skin (Table I).
The dominant cell type of BP biopsies (42) was lymphocytes
in 6 (14%), eosinophils in 13 (31%), and neutrophils
in 4 (10%). In 19 (45%), the presence of a dominant cell was
not detected. Of the PV biopsies (47), the predominant cell
type was lymphocytes in 22 (47%), eosinophils in 4 (9%),
and neutrophils in 1 (2%). In 20 (42%), the presence of a
dominant cell was not detected.
The relationship between IgG and C4d results and the
histomorphologic parameters commonly seen in autoimmune
bullous diseases is shown in detail in Tables II, III,
and IV.
Click Here to Zoom |
Table II: The relationship between IgG results with histomorphologic parameters commonly seen in autoimmune bullous diseases |
Click Here to Zoom |
Table IV: The relationship between C4d results and histomorphologic parameters commonly seen in autoimmune bullous
diseases |
a) Results of Paraffin Immunofluorescence with IgG
Data were obtained from 83 of the 89 biopsies due to dropout
during the immunofluorescence study and the absence
of epidermis in the resulting sections. There was positive
staining in 28 (34%) of the 83 biopsies. Of the 28 specimens
positive with PIF IgG, 5 belonged to BP (18%) and 23 to
PV (82%). There were 10 (36%) that were lesional skin, 18
(64%) were non-lesional, 16 had inflammation, and 5 contained
fibrin (Tables II and III).
Amongst the 55 IgG-negative specimens, 32 were lesional,
22 had inflammation, 40 showed acantholysis, 25 contained
fibrin, and 34 (62%) were samples diagnosed as bullous
pemphigoid (Table II).
When specimens with positive and negative PIF IgG were
compared, statistically significant differences were obtained
with regard to the presence of acantholysis (present in 72%
of negative samples) and fibrin (no fibrin in 82% of positive
samples), and the diagnosis of the disease (82% of positive
samples, PV). (p values 0.018 - 0.013 - 0.000, respectively).
Age and biopsy localization did not yield significant results
in terms of positivity and negativity.
b) Results of Immunohistochemistry with C4d
Data were obtained from 84 of 89 biopsies due to dropout
during the immunofluorescence study and the absence
of epidermis in the resulting sections. There was positive
staining in 34 (40%) of 84 biopsies. Of the 34 specimens
positive for C4d, 12 were BP- (35%) and 22 (65%) PV-diagnosed
biopsies. There were 22 (65%) that were lesional, and
12 (35%) were non-lesional. Twenty samples had inflammation,
and 8 contained fibrin (Table IV).
Of the 50 C4d negative specimens, 27 were lesional, 28 had
inflammation, 14 showed acantholysis, 20 contained fibrin,
and 26 (52%) were samples diagnosed as bullous pemphigoid.
These results are summarized in Table III and Table
IV.
There was a statistically positive correlation between positive
C4d staining and the presence of acantholysis (p=0.04).
Age and biopsy localization did not yield significant results
regarding C4d positivity and negativity. |
Top
Abstract
Introduction
Methods
Results
Disscussion
References
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The paraffin immunofluorescence (PIF) method has been
used as a salvage technique in nephropathology for quite
some time 3,4. However, its use in skin pathology has
only been reported once 5. In this study, PIF for the diagnosis
of autoimmune bullous disease was performed on
samples representing non-lesional skin. In the same study,
14 of 18 BP cases (78%) and 3 of 7 pemphigus cases (43%)
were positive. In our study, IgG PIF was applied to lesional
and non-lesional skin. “Lesional skin” refers to skin with
a dermatologic lesion and microscopic findings specific
to the disease (PV/BP), such as inflammation and a split.
“Non-lesional skin” refers to tissue without a dermatologic
lesion (usually representing the perilesional area biopsied
for immunofluorescence evaluation) and microscopically
without features such as inflammation or a split. A positive
reaction was obtained in 5 of 39 BP biopsies (13%)
and 23 of 44 PV biopsies (53%). Cases diagnosed with PV
comprised most of our cases that showed IgG PIF positivity
(23/28, 82%), with cases of BP comprising the minority
(5/28 positive biopsy).
In contrast to the study mentioned above, false negative
results in our series were predominantly seen in BP-diagnosed
biopsies. It was observed that 10 (36%) of the 28 positive
results belonged to the lesional skin and 18 (64%) to
non-lesional skin. Although most positive biopsies belong
to non-lesional skin, there was no statistically significant
difference in PIF IgG positivity between lesional and nonlesional
skin or the presence or absence of inflammation
(Table I). Our findings highlight the possibility of demonstrating
IgG deposition with PIF in lesional skin biopsies.
Although the sensitivity is low, this finding opens up the
opportunity to diagnose bullous diseases from a single
biopsy in those patients where it is the histopathologist
who suspects an autoimmune bullous disease or the biopsy
of the accompanying non-lesional skin is mistreated or
harmed.
Regarding C4d immunohistochemistry, 12 out of 48 BP
biopsies were positive (25% of all BP-diagnosed biopsies).
We also found that 22 of 46 PV-diagnosed samples were
positive (48% of all PV-diagnosed biopsies). In the literature,
we usually found more promising results than ours
when we reviewed the positivity of C4d immunohistochemistry
in BP and pemphigus. In series with the number
of BP cases ranging between 4 and 30, the positivity rate
with C4d immunohistochemistry in BP varied between
77% and 90%{7-10. On the other hand, in the study by
Margo and Dyrsen, 4 of 17 BP cases (23%) had a low rate of
positive results, just like us6.
Our study is the series with the highest number of biopsies
since it included 42 BP biopsies. Regarding PV, C4d immunohistochemistry
results in FFPE tissues were obtained in
two series with 11 and 22 pemphigus cases6,8. In these
series, C4d immunohistochemistry positivity rates in FFPE
tissues were 82%6 and 77%8. Both of these studies
reached a higher positivity rate than ours. This difference
may be due to the variable levels of autoantibodies targeting
structural proteins and the associated difference in the
amount of immune complex deposition. Serologic titers of
the relevant autoantibodies need to be known and compared
to understand this. Also, intensity comparisons in
direct immunofluorescence studies are needed to reflect
the amount of immunocomplex deposition.
The melanin pigment content in keratinocytes is a factor
that complicates evaluation, especially in the intraepidermal
area. Unlike other studies, we performed C4d immunohistochemistry
with AEC (3-Amino-9-ethylcarbazole)
chromogen, which allowed for an accurate evaluation. The
presence of acantholysis showed a statistically significant
correlation with C4d positivity (72% of negative biopsies
did not have acantholysis). That may be because positive
biopsies are mostly PV-diagnosed samples (64%), and
acantholysis is a hallmark histologic feature of PV. It could
also be that complement activation is more prominent in
such cases. Statistically, whether the tissue was lesional or
non-lesional did not affect the result. Similar to PIF IgG,
it can be concluded that diagnosis of bullous diseases can
be made possible with C4d immunohistochemistry from a
single lesional biopsy, although it has low sensitivity.
Furthermore, if PIF IgG and immunohistochemical C4d
evaluation are used together in a biopsy, our findings suggest
that at least one can be positive in 54% of the biopsies.
Combined with the literature data, our study shows that
the positivity rate may be much lower.
Is performing C4d immunohistochemistry and/or IgG PIF
on formalin-fixed paraffin-embedded tissues worthwhile?
There is no straightforward answer to this question. When
performed together, 54% of the biopsies were positive in
our series. That is like flipping a coin. If we get a positive
biopsy, we save the patient from another punch biopsy.
The question may be whether we want to save the patient
from a new punch biopsy, which is the gold standard? If
it will cause morbidity (cosmetic problem, general impairment
in a patient that is not suitable for biopsy, pediatric
patient, delicate areas such as conjunctiva) or if the patient
does not allow a biopsy, the answer is yes, we do.
In summary, our findings suggest that PIF IgG can also
be used as a salvage technique in skin biopsies of patients
with bullous diseases, similar to kidney biopsies, and reveal
the fact that even biopsies of lesional skin can potentially
be diagnostically useful, a unique finding that has not
been previously mentioned in the literature. However, one
should remember that the absence of staining should not
be relied upon to rule out bullous disease.
Conflict of Interest
We have no conflicts of interest to disclose.
Acknowledgments
This project is supported by the Hacettepe University Research
Project Coordination Unit (Project number THD-2018-17447).
Ethical Approval
This project was approved by the Hacettepe University noninterventional
studies ethics committee with decision number
18/882-25.
Authorship Contributions
All authors contributed to the study.
Contributed to the concept, design, data collection, processing,
analysis, interpretation, literature search and writing of the study:
DAO, Contributed to the design, data collection, processing, analysis,
interpretation and approval of the study: OG, Contributed to the
study’s concept and design, writing and approval: AS. |
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Abstract
Introduction
Methods
Results
Discussion
References
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8. |
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Abstract
Introduction
Methods
Results
Discussion
References
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Copyright © 2023 The Author(s). This is an open-access article published by the Federation of Turkish Pathology Societies under the terms of the Creative Commons Attribution License that permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No use, distribution, or reproduction is permitted that does not comply with these terms. |
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