2013.4-3s.Immunohist

                                                                                                                            article in PDF  
Our Dermatol Online.  2013; 4(Suppl. 3): 585-595
DOI:.  10.7241/ourd.20134.148
Date of submission:  10.06.2013 / acceptance: 05.08.2013
Conflicts of interest: None
 

IMMUNOHISTOCHEMISTRY VERSUS IMMUNOFLUORESENCE IN THE DIAGNOSIS OF AUTOIMMUNE BLISTERING DISEASES

Ana Maria Abreu Velez1, Paul B. Googe, Jr.2, Michael S. Howard1

1Georgia Dermatopathology Associates, Atlanta, Georgia, USA
2Knoxville Dermatopathology Laboratory, Knoxville, Tennessee, USA
 

Corresponding author:  Ana Maria Abreu Velez, MD PhD    e-mail: abreuvelez@yahoo.com


 

Abstract
Introduction: In patients with autoimmune skin blistering diseases (ABDs), the diagnostic gold standard has classically been direct and indirect immunofluorescence (DIF and IIF), despite inherent technical problems of autofluorescence.
Aim: We sought to overcome autofluorescence issues and compare the reliability of immunofluorescence versus immunohistochemistry (IHC) staining in the diagnoses of these diseases.
Methods: We tested via IHC for anti-human IgG, IgM, IgA, IgD, IgE, Kappa light chains, Lambda light chains, Complement/C3c, Complement/C1q, Complement/C3d, albumin and fibrinogen in 30 patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF), and 30 control biopsies from the endemic area. We also tested archival biopsies from patients with ABDs whose diagnoses were made clinically, histopathologically and by DIF/IIF studies from 2 independent dermatopathology laboratories in the USA. Specifically, we tested 34 patients with bullous pemphigoid (BP), 18 with pemphigus vulgaris (PV), 8 with pemphigus foliaceus (PF), 14 with dermatitis herpetiformis (DH) and 30 control skin samples from plastic esthetic surgery reduction surgeries.
Results: The diagnostic correlation between IHC and DIF-IIF was almost 98% in most cases. IHC revealed evidence of autofluorescence around dermal blood vessels, dermal eccrine glands and neurovascular packages feeding skin appendices in ABDs; this autofluorescence may represent a non-specific immune response. Strong patterns of positivity were seen also in endothelial-mesenchymal cell junction-like structures, as well as between dermal fibrohistiocytic cells. In PV, we noted strong reactivity to neurovascular packages supplying sebaceous glands, as well as apocrine glands with edematous changes.
Conclusions: We suggest that IHC is as reliable as DIF or IIF for the diagnosis of ABDs; our findings further suggest that what has previously been considered DIF/IIF autofluorescence background may be of relevance in ABDs. Our discovery of reactivity against edematous dermal apocrine glands may be related to the fact that PV has a vegetant form, with lesions present in anatomic areas where these glands exist..
 
Key words:  autoimmune blistering skin diseases; autofluorescence; immunohistochemistry
 
Abbreviations and acronyms: Bullous pemphigoid (BP), immunohistochemistry (IHC), direct and indirect immunofluorescence (DIF, IIF), hematoxylin and eosin (H & E), basement membrane zone (BMZ), intercellular staining between keratinocytes (ICS), pemphigus vulgaris (PV), autoimmune blistering skin disease (ABD), fogo selvagem (FS), endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF), dermatitis herpetiformis (DH).

 

Practical learning:
• IHC may be as reliable as DIF or IIF for the diagnosis of ABDs. Running positive and negative controls is recommended, utilizing paraffin blocks of similar ages to the patient cases.
• IHC reveals that ABDs may present more antigenic molecules than are classically recognized.
• IHC cannot replace antibody titers, or salt split skin techniques in combination with IIF.
 
Introduction
The techniques of direct and indirect immunofluorescence (DIF and IIF) are of proven value in confirming the presence of immunoglobulins, complement, and fibrinogen; in turn, these findings contribute to the diagnosis of multiple autoimmune skin diseases [1-4]. In classic ABD immunofluoresence testing, a single fluorophore (fluorescein isothiocyanate; FITC) has been utilized, and it has been accepted that background autofluorescence exists [5]. Further, it is assumed that diagnostic DIF/IIF reactivity in ABD patients will vary depending on concomitant administration of therapeutic immunosupressor agents [6]. In addition, correlation of serum antibodies with disease severity in pemphigus and bullous pemphigoid (BP) via IIF is widely utilized [8,9]. Because correlating paraffin block biopsies are available in most dermatological services, we attempted to compare the diagnostic results obtained from DIF, IIF and immunohistochemistry (IHC) in several ABDs.
 
Material and Methods
Subjects of Study
We tested 30 biopsies from patients affected by endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF); diagnostic criteria were followed as previously described [8-10]. We also tested skin biopsies from 30 controls from the El Bagre EPF endemic area, and 30 additional control skin samples from cosmetic surgery patients in the USA, taken from the chest and/or abdomen. Biopsies were initially fixed in 10% buffered formalin, then embedded in paraffin and cut at 4 micron thicknesses. The tissue was then submitted for hematoxylin and eosin (H&E) and IHC staining. We also tested ABD cases from archival files of two private, board certified dermatopathology laboratories in the USA. Our patients were diagnosed clinically by the referring physicians, by H&E staining, and by DIF and IIF. We did not record the age of the biopsies, nor if the patients were taking immunosuppressive therapeutic medications at the time of the biopsy. We evaluated 34 biopsies from bullous pemphigoid (BP) patients, 4 from patients with pemphigus vulgaris (PV), 8 from patients with sporadic pemphigus foliaceus (PF), and 14 from patients with dermatitis herpetiformis (DH). For all of the El Bagre area patients and controls we obtained written consents, as well as Institutional Review Board permission. The archival biopsies were IRB exempt due to the lack of patient identifiers.
 
Quantificaton of staining intensity to obtain precise data on IHC parameters
We utilized the following algorithm: area of positive signal divided by the area studied. The staining intensity of these antibodies was also evaluated qualitatively by two independent observers. We utilized the following traditional categories to classify reactivity: intercellular staining between keratinocytes (ICS) and basement membrane staining (BMZ). In addition to these patterns, we added the following: upper dermal blood vessel perivascular staining (UVS), neurovascular staining around skin appendageal structures (NVS), endothelial-mesenchymal cell junction-like staining (EMCJ), dermal cell junction staining (DCS) and peritelocyte staining (ATS) [11]. Our IHC staining was performed as previously described before [7-10]. For IHC, all antibodies utilized were obtained from Dako(Carpinteria, California, USA). A summary of the antibodies utilized, their dilutions, catalogue numbers and methods of antigen retrieval show in Supplementary Table I.
 
Statistical methods
Differences in staining intensity and positivity were tested using a GraphPad Software statistical analysis system, and employing Student’s t-test. We considered a statistical significance to be present with p values of 0.05 or less, assuming a normal distribution of the samples.
 
Results
In most of the ABDs, our results followed established DIF and IIF patterns with statistical significance in comparison to controls run with similar markers (p values of less than 0.05). Our summarized results shown in Table I. In addition to the classical patterns of reactivity appreciated by DIF and IIF, multiple additional patterns of positivity were seen. The most common one featured positive staining around the upper dermal blood vessels in the ABD patients, again with statistical significance in comparison to controls (p = 0.0001). IgG, IgM, Complement/C3c, Complement/C1q and fibrinogen were the most common positive markers detected in this pattern. Most of the ABDs were positive in this pattern for more than 3 markers at a time. In BP, we also observed strong perivascular positivity in the intermediate and deep dermis with similar markers as those described above (Fig. 1-4). In BP, PV, El Bagre-EPF and PF, we noted strong positivity to neurovascular structures feeding skin appendageal structures, especially in eccrine glands and pilosebaceous units. In PV, this phenomenon was also observed in neurovascular packages supplying and surrounding dermal apocrine glands; on H&E review, these glands were also noted to be edematous with acantholysis-like features (p<0.05) (Fig. 1-4). We classified our findings as negative (-), weakly positive (+), positive (+++) and strongly positive (++++). Positivity was also observed in most ABDs in what seemed to be cell junction-like structures between endothelial cells in the dermis and the surrounding mesenchymal extracellular matrix (p<0.05) (Fig. 1-4). The degree of positivity varied depending of the size of the vessels and their relative deepness in the dermis (Tabl. I). In BP, such those that seems to be in the junctions between the dermal cells junctions with stronger positivity in the middle of the dermis. The normal controls did not show this staining, with exception of deposits of IgG, IgM and albumin in the dermis; however, this staining was weaker and without any specific pattern. In several active clinical cases of El Bagre-EPF, we also noted positive staining to some kind of cell junction-like structures in piloerector muscles with both IgG and IgM. Notably, the reactivity against the apocrine glands and the H&E alterations of edema and acanthoytic-like changes may be related with the fact that PV has a vegetant clinical form, with lesions anatomically present in areas where these glands predominate. In Table I and Figures 1 through 4, we summarize our primary results.
 
Antibody
BP n=34
PV n=14
PF n=4
DH n=10
El Bagre-EPF n=30
Controls
from
Endemic
arean= 30
Skin
plastic
Surgery
controls
n=15
IgA
Positive around some dermal
blood vessels. Some epidermal
keratinocytes showed some
cytoplasmic staining in several
biopsies (+).
Some positive cells debris inside
the blister. Some positive blood
vessels in the upper dermis and
some individual cells in the upper
dermis. Also positive on blood
vessels around the eccrine glands
and on vessels of the septae of
subcutaneous adipose tissue.
Some small dermal
bloodvessels positive.
Also, positive on some
small blood vesselsaround
eccrine sweat glands (+).
Positive below
subepidermal blisters,
mainly in the papillary
dermis (+++). Positive
in some linear areas in
the epidermal stratum
corneum (++). Also
positive in some areas of
the intracellular matrix
bundles (++). Some
positivity in neurovascular
packages feeding
sebaceous glands (++).
Some patients with positive staining around
upper and intermediate dermal blood vessels
(++).
Negative.
Negative.
IgG
BMZ linear positivity on roofs
and floors of the blisters, with
more positivity on the roofs
(34/34). Some areas of the
epidermis showed
pericytoplasmic staining in
keratinocytes (22/34).
Sometimes positive perinuclear
staining as well. Positive
staining also noted around
some small dermal blood
vessels. Some areas of the
papillary dermis extracellular
matrix and upper dermis
showed reactivity (+++).
Several fibroblastoid cells
positive through the entire
dermis. Many deep nerves
positive in the epineuria.
ICS, some upper and around
several neurovascular vessels.
Positive around some vessels
around the sweat glands upper
dermis and small vessels in the
septaes of the fatty tissue (++).
Positive some extracellualr matrix
fibers especially in the upper and
intermediate dermis. Positive also
some small vessels around the
sweat glands area (+).
ICS between epidermal
keratinocytes, mostly in
upper layers. Positive
staining of some small
upper dermal blood
vessels. Positive staining
around some dermal blood
vessels and eccrine glands
(++).
Similar distribution
as IgA; also in several
vessels.
Positive in several cases in epidermal stratum
corneum (++). Also, some intracytoplasmic
positive staining within epidermal
keratinocytes. Positive ICS, mainly in
epidermal stratum granulosum in acute and
relapsing cases. Positive in several cases on
upper dermal blood vessels (++). Chronic cases
demonstrated some positive staining against
mesenchymal-endothelial cell junction-like
structures in the dermis, as well as around some
dermal eccrine glands.
Negative.
Negative.
IgM
Some areas of ICS-like staining
and some areas of
pericytoplasmic staining
in epidermal keratinocytes
(21/34). Also, positive staining
around upper blood vessels and
the dermal extracellular matrix
(+++). The pattern of this
immunoglobulin is very similar
to that seen with IgG.
Some epidermal subcorneal
reactivity in several areas. Focal
epidermal ICS staining is noted
in several spots. Positive staining
also noted around several dermal
blood vessels in the dermis,
around some connective tissue and
some deep neurovascular tissue
around eccrine sweat glands,
sebaceous glands and adipose
septae (+++).
Positive ICS between
epidermal keratinocytes,
mostly in upper layers and
positive around some small
upper dermal blood
vessels. Also positive
around some blood vessels
around eccrine glands
(++). Positive around
sebaceous and eccrine
gland neurovascular
supplies.
Similar distribution as
with IgA. In some
biopsies, some
intercellular keratinocyte
staining was observed.
Also, some reinforcement
was noted around hair
follicles.
Some cases displayed spotty positive staining
in the epidermal corneal layer. Some epidermal
keratinocytic ICS in several cases (++). Some
epidermal keratinocyte spotty pericytoplasmic
positive staining in in several areas of the
epidermis, and some BMZ staining. In most
chronic cases, positive staining in a band-like
distribution in the upper dermis and/or
intermediate dermis, including on blood vessels
(++). Reinforcement of the mesenchymalendothelial
cell junction-like structures and
cells and telocyte- like structures also seen.
Negative
Negative
IgE
Linear on both floors and roofs of blisters in many cases
(23/34). Positive (+++)
intracytoplasmic, perinuclear
staining in some epidermal
keratinocytes.(26/34). Positive
staining on some individual
cells in dermis, and focally
around upper dermal blood vessels (++).
Positive nuclear and focal
cytoplasmic stain ing in epidermal
keratinocytes. Also, positive on
several large cells in the upper
dermal inflammatory infiltrate.
Positive staining of some vessels
around eccrine glands. (++).
Positive nuclear and focal
cytoplasmic staining in
epidermal keratinocytes
Also positive staining on
several large cells in the
upper dermal inflammatory
infiltrate. Positive staining
of some vessels around the
eccrine glands (++).
Most cases were negative.
Some positive staining in the
upper dermal inflammatory
infiltrate. Two cases were
positive in sebaceous glands,
in plasmacytoid cells in the
dermis and around eccrine
glands.
Several cases, both acute and chronic,
displayed positive staining on individual
cells, mainly around the upper dermal
blood vessels (++). Mesenchymalendothelial
cell junction-like
structures and telocyte-like structure
positive staining was also noted, as well
as around some dermal eccrine glands.
Negative.
Negative.
IgD
Positive linear staining in
several biopsies along the
BMZ, mostly on blister floors.
Positive on several
fibroblastoid cells in the
dermis. Positive around several
dermal blood vessels; superficial,
intermediate and deep
(+++) (20/34).
Some positive staining on
individual large cells in the upper
dermal perivascular inflammatory
infiltrate, and on some of the upper
dermal blood vessels (+). Some
epidermal ICS positivity and some
positive staining on cells inside the
blisters.
Positive in several upper
dermal small blood
vessels, and on some blood
vessels around eccrine
glands. Some scattered
staining around very
actively inflammatory
epidermal blisters (+).
Several cases followed the
same pattern as IgA,
including the positivity in the
dermal papillae, and blood
vessels. Two cases were
positive in the sebaceous
glands, in plasmacytoid cells
in the dermis and around the
eccrine glands.
Positive staining pattern followed the
distribution of the stronger
immunoglobulins, including some
epidermal keratinocytic intracytoplasmic
positive staining, and some staining on
upper dermal blood vessels. In some
cases, some spotty positivity along the
BMZ and some staining around selected
eccrine gland ducts.
Negative.
Negative.
Complement/C3c
Positive linear deposits at
blister splits, primarily on
blister roofs but also some on
blister floors (+++) in 34/34
cases. Also around several
small and large dermal
neurovascular packages (+++).
Positive staining also present
around eccrine ducts and BMZ
of eccrine ducts, as well as on
blood vessels around the hair
follicles (++). Some reactivity
also seen in the upper
epidermal corneal layer (+).
Epidermal ICS, and staining on
some upper dermal blood vessels
and several dermal neurovascular
packages. Some positivity in focal
areas of the epidermal corneal
layer. Also positive staining on
some fibroblastoid cells in the
dermis. Positive focal staining on
BMZs of the sebaceous glands
and on their neurovascular supply
packages. Positive staining on
some small blood vessels in the
deep connective tissue (++).
Positive staining on several
blood vessels in the upper
and intermediate dermal
plexus. Positive in some
small blood vessels in the
deep connective tissue.
Positive around
neurovascular packages
of dermal sebaceous and
eccrine glands (++).
Positive deposits in the upper
and lower dermal tissue
(+++). Positivity also noted
in the epidermal corneal
layer. Some epidermal
keratinocyte ICS and/or
cytoplasmic staining.
Staining in the extracellular
matrix and around eccrine
glands and ducts. Multiple
fibroblastoid cells were
positive in the dermis.
Some spotty positive staining on the
epidermal corneal layer. Some
epidermal positive ICS in several cases
(++). BMZ staining also in several cases
(++). Positive staining in upper dermis
and on neurovascular packages of all skin
appendageal structures (++). Positive
staining in multiple cases on
mesenchymal-endothelial cell
junction-like structures, and on
telocyte-like structures.
Negative
Negative
Complement/
C3d
Positive around several small
and large dermal blood
vessels. Positive linear BMZ
staining on blister floors and roofs (+++)
Positive around several upper
dermal blood vessels. Some
epidermal ICS, positive in focal
areas (++). Positive staining inside
the blisters. Positive staining
around neurovascular supplies of
pilosebaceous glands units. Some
extracellular matrix staining,
positive in the intermediate
dermis.
Positive around several
upper dermal blood
vessels. Some epidermal
ICS, positive in few areas
(++). Positive staining
around dermal sebaceous
and eccrine gland
neurovascular supplies.
In some biopsies, staining
followed the pattern of
positivity of IgA, although
with weaker intensity.
Positive in the majority of the cases in
most vessels in dermis.
Negative
Negative
Complement/
C1q
Positive linear BMZ staining
on both sides of the blisters,
but primarily on blister roofs.
Positive staining on focal
extracellular matrix fibers, in
bundles in the upper,
intermediate and deep dermis
(++). Also positive dermal
staining on the upper
neurovascular plexus (++).
Some focal areas of the
epidermis with cytoplasmic
staining of the keratinocytes.
Positive staining on blister floors,
and some inside blisters. Some
staining in the upper dermal
extracellular matrix. Positive staining
on dermal blood vessels, sebaceous
glands neurovascular supply
packages (++). Several BMZs of hair
follicles and sebaceous glands were
also patchy positive. Positive
staining around dermal eccrine
glands and ducts.
Positive staining around
pilosebaceous units (++). Positive
staining around several upper
dermal blood vessels. Some focal
epidermal ICS positive staining
(++). Positive staining around dermal
sebaceous and eccrine gland
neurovascular supplies.
Positive staining at the
subepidermal blister floor, and
in the papillary dermal areas
(+++). Some focal epidermal
corneal layer reactivity. The
dermal extracellular matrix was
very reactive, accentuated in
bundled groups (+++). Positive
staining around some dermal
neurovascular packages. In
summary, the staining pattern
followed a similar pattern to
that of IgA.
Positive several extracellular
matrix fibers, mostly
intermediate ones. Positive
cells-junction like of the
piloerector muscle. Positive
around sweat glands vessels.
Negative
Negative
Kappa light
chains
Positive staining on blister
floors and roofs, but more
prominent on the floors (++).
Positive staining in most areas
of the dermis (++).
Positive epidermal ICS, on upper
dermal blood vessels and around
eccrine ducts (++).
Positive staining in the epidermal
corneal layer. Positive
epidermal ICS, and positive
staining on upper dermal blood
vessels and around dermal eccrine
ducts (++). Positive staining
around the sebaceous and sweat
gland neurovascular supplies.
Similar distribution as IgA.
Follows same pattern than IgG
and IgM combined.
Negative.
Negative.
Lambda light
chains
Positive staining on blister
floors and roofs, but more
prominent on the floors (++).
Positive staining in most areas
of the dermis (++).
Positive epidermal ICS, on upper
dermal blood vessels and around
eccrine ducts. (++).
Positive in the epidermal corneal
layer. Positive epidermal ICS, and
positive staining on upper dermal
blood vessels and around dermal
eccrine ducts (++). Positive
staining around the sebaceous
and sweat gland neurovascular
supplies.
Similar distribution as IgA.
Follows same pattern than IgG
and IgM combined.
Negative
Negative
Fibrinogen
Positive staining in the
epidermal corneal layer, ICS in
epidermal stratum spinosum,
on upper and intermediate
dermal blood vessels and
around dermal eccrine glands
Positive, strong band-like
staining throughout the
papillary dermis (+++). Also,
some positivity in the deep
dermal extracellular matrix.
Positive staining around small
dermal blood vessels, including
those associated with eccrine
glands. Essentially, very similar
to IgG.
Positive epidermal ICS in stratum
spinosum. Some positive staining
inside disease blisters. Positive
staining on several small blood
vessels in the dermis, and around
some dermal connective tissues.
Positive staining in the epidermal
subcorneal area. Positive staining
on some deep dermal large nerves
around the eccrine glands Positive
strong band-like staining in the
dermal extracellular matrix in several
areas, and on several dermal neurovascular
plexus structures (++) and
deep dermal, small blood vessels.
Positive staining around dermal
eccrine glands. Positive staining also
noted around some subcutaneous
adipose tissue septae. Positive
staining on the BMZs in some areas
of the sebaceous glands.
Positive staining in the
epidermal corneal layer,
epidermal ICS, on upper and
intermediate dermal blood
vessels and around dermal
eccrine glands Positive strong
band-like staining throughout
the papillary dermis (+++).
Positive staining around
dermal sebaceous and eccrine
gland neurovascular supplies.
Similar positive staining
distribution as IgA. Positive
staining on neurovascular
supply structures of dermal
sebaceous glands (++).
Some spotty positive staining
on the epidermal corneal layer
in several cases (+). Some BMZ
staining. Positive staining on upper
dermal blood vessels (++). Positive
staining on dermal mesenchymalendothelial
cell junction-like
structures and telocyte-like
structures. Positive staining on cell-
-junction like structures in dermal
piloerector muscles. Positive staining
around dermal blood vessels
supplying dermal eccrine glands
Negative
Negative
Albumin
Positive, strong band-like
staining noted in the papillary
dermis and around dermal
vessels and eccrine glands
(++++). Also, positive staining
on the deep dermal
extracellular matrix.
Positive staining on the epidermal
corneal layer. Positive epidermal
ICS, and upper and intermediate
dermal blood vessel staining and
around dermal eccrine glands and
one perieccrine large nerve.
Positive, strong band-like staining
throughout the papillary and
intermediate dermis, suggesting a
compartmentalization of the overall
immune response (+++).
Positive staining on the
epidermal corneal layer.
Positive epidermal ICS, and
positive staining on upper and
intermediate dermal blood
vessels and around dermal
eccrine glands Positive, strong
band-like staining throughout
the papillary dermis (++++).
Positive staining around
dermal sebaceous and sweat
gland neurovascular supplies.
Positive strong band-like
staining in the papillary
dermis (+++). Positive
staining in some areas of the
epidermal corneal layer, and
on the dermal extracellular
matrix (+++). Some trace
staining in the epidermis
between keratinocytes, and
some keratinocytic cytoplasmic
staining. Followed a
similar pattern as IgA, but
with stronger positivity.
Followed the same pattern as
fibrinogen
Non- specific
staining
was present
in the dermis.
Negative.

Table I. Cell populations and markers in lesional skin from multiple autoimmune skin diseases.

Cat No.
Antibodies (all from Dako)
Working dilution
Retrieval method
(High, Low or Proteinase K(PK))
AR0423
Polyclonal rabbit anti-human IgG
Flex ready to use
High
IR153
Polyclonal rabbit anti-human IgM
Flex ready to use
High
IR510
Polyclonal rabbit anti-human IgA.
Flex ready to use
High
IR517
Polyclonal rabbit anti-human IgD
Flex ready to use
Low
A0094
Polyclonal rabbit anti-human IgE
1:800
High
A0062
Polyclonal rabbit anti-human Complement/C3c
1:1000
None
A0063
Polyclonal rabbit anti-human Complement/C3d
1:400.
PK
A0136
Polyclonal rabbit Complement /C1q
1:100.
PK
A0001
Polyclonal rabbit anti-human albumin.
1:13,000
None
A0080
Polyclonal rabbit anti-human fibrinogen
1:1000
PK
IR506
Polyclonal rabbit anti-human kappa light chains
Flex ready to use
High
IR507
Polyclonal rabbit anti-human lambda light chains
Flex ready to use
High

               Table I – Supplemental. Antibodies utilized, with their respective working parameters.

 
 
 
 
Figure 1. a. Classic H&E staining of a DH case, demonstrating a subepidermal blister (black arrow). b IHC, demonstrating positive staining with anti-human IgA antibodies in the blister, (brown staining; red arrow), and weaker, punctate staining in the upper dermis (brown staining; blue arrow). c. DIF of the same patient as in a and b, utilizing FITC conjugated anti-human IgA and showing positive “snow on the mountains” staining in the blister and around upper dermal blood vessels (green staining; yellow arrow); epidermal keratinocyte nuclei were counterstained with Dapi (light blue). d. Classic H&E image of a BP case; note the
eosinophils within the upper dermis, subjacent to a disease blister (red arrow). e. Case of BP, with IHC positive linear staining for Complement/C3 at the BMZ (dark staining; red arrows). f. Same BP case as in e, with positive linear IHC staining for IgM at the BMZ. g. BP case, with positive punctate IHC staining for IgE below a disease blister in the dermis(brown staining; red arrow). h. BP case, with positive linear IHC staining for IgG on both sides of a disease blister (brown staining; red arrows). i. The same BP case as in h, highlighting DIF positive staining of FITC conjugated IgG on both sides of a disease blister (green-yellow staining; white arrows). j. A classic case of PV, with classic H&E “tombstone” acantholytic keratinocytes along the epidermal basaloid layer (red arrow). k. A case of PV, demonstrating a positive IHC “chicken wire” pattern of ICS with IgG between epidermal keratinocytes (brown staining; yellow arrow). l. Positive IHC staining for IgG in an epidermal blister in a case of PF (brown staining; black arrow). Please also note the focal epidermal corneal staining.
Figure 2. Atypical IHC staining patterns seen in ABDs. a. Complement/C3c positive IHC staining in the central dermis in a patient with BP, in ATS and/or EMCJ patterns (black arrows). b. Positive IHC staining for Complement/C3d in a BP patient, highlighting positive staining in several upper and intermediate dermal blood vessels (brown staining; black arrows). c. Many BP cases also demonstrated positive IHC IgD staining, in both ATS and EMCJ patterns (brown staining; blue arrows). d. Many BP cases displayed positive, punctate IHC staining for IgD along the BMZ(brown staining; red arrows), but also in the dermis in an EMCJ pattern (brown staining; green arrows). e. BP case, displaying positive IHC staining in a mesenchymal-endothelial junction pattern (MES) with anti-human IgD (brown staining; red arrows). f. In a BP patient
biopsy, we utilized IHC vimentin staining to highlight compartmentalization of the inflammatory process. Note the positive linear staining at the BMZ in the floor of a disease blister (brown staining; red arrow), as well as the inflammation and structural reorganization of dermal blood vessels (brown staining; black arrows).
Figure 3. a. A PV case, demonstrating positive IHC staining for fibrinogen (note the strong dark brown staining inside an epidermal blister). In addition, note the positive staining around upper dermal inflamed blood vessels, and some parts of the extracellular matrix; this staining pattern is commonly seen in immunofluorescence and has been traditionally interpreted as autofluorescence of the vessels and dermal matrix fibers. Here, IHC is not a fluorescent method, and the observation of the identical pattern raises the possibility that the DIF staining may be due to real antigenicity not previously characterized. b. Same case of PV, highlighting fibrinogen reactivity in the blister (brown staining; blue arrow); also note the positive
staining in the upper dermal neurovascular plexus (brown staining; black arrows). c. In a PF case, we noted
positive IHC staining for fibrinogen in both subepidermal and subcorneal blister areas (brown staining; blue
arrows), and additional staining around upper dermal blood vessels(brown staining; red arrows). d. In most
ABDs, strong positive IHC staining was noted to small and intermediate sized dermal blood vessels, in this case with IgG (brown staining; blue arrow). e. Positive IHC staining for IgG around the neurovascular supply
package of a sebaceous gland in a PV case (brown staining; blue arrows). f. Most ABDs also stained positive
via IHC for Complement/C3, Complement/C3d, fibrinogen and sometimes IgG around dermal eccrine
glands and their ducts; an example of a positive stain is illustrated (brown staining; blue arrows). g. Also, in most ABDs the neurovascular packages supplying skin appendageal structures showed positive staining with
the markers documented in f. Sometimes, deep nerves also stained positive, as in this case of PV with fibrinogen (brown staining; blue arrow). h. Positive IHC staining with IgG in a BP patient, directed against either telocytes and/or the EMCJ (brown staining; blue arrow). i. A PV case, staining positive via IHC in a hair follicle for Complement/C3c (brown staining; red arrow). Note also some adjacent blood vessels with positive staining (brown staining; black arrow).
Figure 4. a. A PV case, demonstrating positive IHC staining with an ICS pattern and anti-Complement/C3c antibody between keratinocytes (brown staining; red arrow) as well as around upper dermal blood
vessels (brown staining; black arrow)(400X). b. Same case as in a, demonstrating positive staining with Complment/ C3c to neurovascular packages feeding a sebaceous gland (brown staining; black arrow)
(400X). c. A BP case, demonstrating positive staining with linear deposits of complement/C3c around a subepidermal blister (brown staining; black arrow) and also in the upper dermis (brown staining; black arrow) (200X). d. A PV case, demonstrating positive staining with IgA against small vessels in the upper dermis brown stain (brown staining; black arrows)(400x). e. A PV case H&E, demonstrating edematous and
acantholytic-like changes in apocrine gland cells (400X)(black arrow). f. A PV case, demonstrating positive IHC staining of a neurovascular package around an apocrine glands using anti-human fibrinogen (400X).
Discussion
DIF and IIF have been classically used for the diagnostic of ABDs; the salt split skin IIF technique was also developed to help to differentiate ABDs. IIF has been the gold standard to determine autoantibody titers that correlate with disease severity, further validated by ELISA testing [1-5]. DIF and IIF of the skin detect several autofluorescence molecules, including molecules in the extracellular matrix, blood vessels, and pigments like lipofucsin, melanin, collagen, indolamine, tryptophan, tyrosine, pyridoxine, folic acid, retinol, collagen, cholecalciferol, riboflavin and NAD(P)H [12]. The majority of these molecules fluoresce in the same UV range as FITC. In contradistinction, IHC techniques often recognize well established internal positive control staining patterns on recognized anatomic structures. In our study, we were able to see an “edge effect” in DIF and IIF of non-specific reinforced staining around the edges of the biopsies. We were also able to determine that all the skin biopsies should be run with a control biopsy of similar age as the biopsy to be tested, especially when using older archival biopsies. Besides the classic staining patterns seen in DIF and IIF and detected by IHC with similar specificity and sensitivity (such ICS and/or BMZ staining), we also observed other patterns that we describe as non-classic patterns. These patterns include positivity to the vessels of the dermal neurovascular packages feeding skin appendageal structures, and positivity to junctions between endothelial cells and the surrounding mesenchymal tissue. Other patterns included positivity to cell junctions within the dermis, and a pattern that we described as a telocyte-like pattern [11]. We didn’t find these patterns in the controls, with the exception of some even, nonspecific staining of IgG and IgM within the dermis. Other authors have reported studies utilizing IHCs in ABD diagnosis, with similar results to ours in the classical patterns [14-17]. Our non-classic patterns of positivity have been also documented in DIF and IIF, especially when using multicolor and confocal microscopy (not routinely utilized in most immnodermatopathology laboratories). In theory and based on other studies, these autofluoresence molecules should not be detected by IHC [10,17-21]. As noted, besides the classical patterns of reactivity we noticed additional patterns such as UVS. The increased reactivity of dermal blood vessels and/ or molecules involved in the transit of inflammatory markers through the dermal blood vessels has been previously shown to possibly play roles in ABDs [19-21]. The reactivity of patients with ABDs to dermal blood vessels and/or endothelial cells with strong inflammatory and immune activation markers, including autoantigens to plakophilins 3 and 4 (present in dermal blood vessels) has been also previously reported [23-29]. Some authors have shown DH to have evidence of endothelial cell activation in the skin, and systemic manifestations of the ongoing inflammation associated with the mucosal immune response. Endothelial cell activation may play a critical role in the development of skin lesions in patients with DH [27]. Other authors investigated the expression of vascular permeability factor (VPF), that plays an important role in increased vascular permeability and angiogenesis in three bullous diseases; these diseases feature subepidermal blister formation characterized by hyperpermeable dermal microvessels and pronounced papillary dermal edema [28]. The expression of VPF mRNA was strongly up-regulated in the lesional epidermis of BP (n = 3), erythema multiforme (n = 3), and DH (n = 4) as detected by in situ hybridization studies. Epidermal labeling was particularly pronounced over blisters, but strong expression was also noted in areas of the epidermis adjacent to dermal inflammatory infiltrates distant from the blisters. Moreover, the VPF receptors were upregulated in endothelial cells in superficial dermal microvessels. High levels of VPF were detected in blister fluids obtained from patients with BP. The findings described by these authors strongly suggest that VPF plays an important role in the induction of increased microvascular permeability in bullous diseases; leading to papillary edema, fibrin deposition and blister formation in these disorders [28]. Therefore, the positive staining to dermal blood vessels and neurovascular supply packages may be detecting secondary antigens. Previous authors also reported that soluble E-selectin (sE-selectin), an isoform of the cell membrane protein E-selectin (an adhesion molecule synthesized only by endothelial cells) is significantly increased in sera of patients with BP and PV [29]. Other authors recently investigated endothelial cell activation via gene profiling in patients affected by DH (e.g. SELL, SELE genes) that code for cell surface components, specifically members of a family of adhesion/homing receptors that play important roles in lymphocyte-endothelial cell interactions. The gene activation was increased, as well as neutrophil extravasation. The SELL/SELE coded molecules are composed of multiple domains: one homologous to lectins, one to epidermal growth factor, and two to the consensus repeat units found in Complement C3/C4-binding proteins [30]. Our findings of positive reactivity in dermal blood vessels, especially in the upper dermis and neurovascular skin appendices are suggestive of possible antigenicity for these structures in ABDs. Finally, recent studies have shown positive reactivity to eccrine and sebaceous glands in ABDs utilizing IIF and DIF [31-35]. In regard to the positive patterns seen in the dermis(possibly associated with cell junctions and/or telocytes), we were able to recently demonstrate activation of both multiple proteases and protease inhibitors following the same patterns of positivity we are describing here [36]. Electron microscopy studies using antibodies and colocalization will help to see if these non-classical patterns of reactivity seen in ABDs are artifacts, or possibly associated with pathological effects in these diseases.
 
Acknowledgements
Jonathan S. Jones, HT (ASCP) at Georgia Dermatopathology Associates for excellent technical assistance.
 
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