Download Immunoflourescence in dermatopathology

Immunofluorescence in
Dermatopathology
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Antibody structure and
interactions. (Left) A cartoon of
an IgG antibody. Each oval
represents a 110-amino-acid
domain. The dark blue ovals
together represent a heavy-chain
polypeptide, the magenta ovals
represent a light-chain
polypeptide, and the yellow
rectangles represent disulfide
bonds between polypeptides.
(Right) An antigen (purple) and a
fluorophore-conjugated secondary
antibody demonstrate the binding
events between antigen
recognition and the fluorescent
signal
Labelers for immunoflourescence
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Immunological reactions that involve the antigenantibody binomial may be visualized or quantified
using different labelers for the antigen or the
antibody. Fluorochromes, enzymes, and radioactive
and electro-opaque compounds are among the
labelers most commonly used.
Fluorochromes are dyes that absorb radiation
(ultraviolet light), are excited by it and emit visible
light. To function as labelers, they need to contain
chemical groups capable of forming covalent bonds
with protein molecules, emitting high fluorescence in
the visible spectrum with a different coloration from
that emitted by tissues. They must have a relatively
simple conjugation, retention of the antibody activity
in the labeled protein, and stability of the fluorescent
conjugate obtained. One of the most used
fluorochromes is fluorescein isothiocyanate (FITC),
of green color, with absorption and emission peak
wavelengths of 490 l and 520 l, respectively.
Rhodamine, another agent used in DIF ( direct
immunoflourescence), of red color, has distinct
absorption and emission peak wavelengths (520
and 610 l). Epiluminescence and confocal
microscopy can be both used to read the results of
DIF.
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Confocal Microscope
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Epiluminescence and confocal microscopy can be both used to read the results of
DIF(direct immunoflourescence)
immunofluorescence studies are vital for the laboratory diagnosis of
autoimmune bullous dermatosis, but they are also important in the
investigation of other diseases, such as inflammatory dermatosis (lupus
erythematosus, lichen planus, porphyrias, vasculitis).
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. By direct immunoflourescence (DIF), presence of immune complexes in
the skin biopsy at various locations, e.g., at the dermoepidermal junction
(DEJ), upper dermal blood vessels, cytoid bodies, and intraepidermal
intercellular spaces, etc., helps us to arrive at a definite diagnosis. "Lupus
band test" (LBT) is most common pattern observed on DIF examination of
skin biopsies of patients suffering from connective tissue diseases . In
addition, DIF microscopy of the skin has also disclosed antibodies bound to
epidermal cell nuclei in several connective tissue disorders also known as in
vivo ANA (antinuclear antibody) phenomenon or epidermal nuclear staining
(ENS) which presents as keratinocyte nuclear fluorescence. Circulating
ANAs are commonly found in patients with connective tissue disease.
Site of biopsy
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The best site and evolution time of skin lesions to perform biopsy for direct immunofluorescence
examination (DIF) depend on the disease under investigation. Generally, the biopsy should have
an appropriate extension (4 mm punch) and depth that involves both the epidermis and dermis in
sufficient proportion. In addition, the sample will be better for analysis when fewer traumas are
involved in the procedure. Fluorochromes, enzymes, and radioactive and electro-opaque
compounds are among the labelers most commonly used.
The following sites are recommended for biopsy:
In autoimmune vesico-bullous dermatosis, the best site is the perilesional region;
In collagenosis, the biospy should be done in the active lesion in evolution (avoid recent lesions,
with less than 60 days);
In vasculitis, preference should be given to recent lesions with up to 24 hours of evolution.
After the procedure, the material can be immediately frozen in liquid nitrogen or placed in a proper
transport medium - Michel's medium.7 Michel's medium is composed of ammonium sulphate, Nethyl-maleimide, and magnesium sulphate in a citrate buffer, which allows the conservation of the
specimen for up to two weeks.
The specimen is then sectioned in a cryostat into 4-micron fragments. Primary anti-human
antibodies conjugated to FITC fluorescein (anti-IgA, anti-igG, anti-IgM, and anti-C3) are applied to
each section and the reading is done on fluorescence microscopy
The indirect immunoflourescence (IIF) technique employed in studies of circulating antibodies in
vesico- bullous dermatosis (VBD) uses the healthy epithelium as substrate. Substrates vary based
on the protocols of each laboratory: healthy human skin obtained from prepuce, breasts or eyelids
ideal ( site, good antigenicity), as a substitute for monkey esophagus
Direct immunoflourescence
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A. Epithelium:
I. Intercellular flourescence:
Pemphigus vulgaris, pemphigus folacious, pemphigus herpitiformis , Paraneoplastic pemphigus ,
IgA pemphigus ( two types: subcorneal pustular dermatosis,& intra-epidermal neutrophilic
dermatosis)
II. Flourescence of the nuclei of keratinocytes (in vivo ANF):
Lupus erythematosis, mixed connective tissue syndrome, overlap syndrome, vasculitis
B. Basement membrane zone:
Lupus erythematosus, Vasculitis, Lichen planus, porphyrias There are different fluorescence
patterns of the BMZ. The most frequent are linear, homogeneous, granulous and reticulate.
I.LINEAR IGG AND/OR C3 DEPOSITS IN THE BASEMENT MEMBRANE ZONE:
Bullous pemphigoid,pemphigoid gestationis or herpes gestationis
II. MULTIPLE LINEAR DEPOSITS (IGA, IGG, IGM AND/OR C3) IN THE BASEMENT
MEMBRANE ZONE: Epidermolysis bullosa acqusita, bullous systemic lupus eryhematosus
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III.MULTIPLE LINEAR DEPOSITS (IGA, IGG, IGM AND/OR C3) IN THE
BASEMENT MEMBRANE ZONE: Linear IgA bullous dermatosis
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C.DERMAL FLUORESCENCE:
Dermatitis herpitiformis,vasculitis, lichen planus, porphyrias, lupus erythematosus
INDIRECT IMMUNOFLUORESCENCE
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INDIRECT IMMUNOFLUORESCENCE:
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@ Intraepidermal bullous dermatosis
Pemphigus vulgaris, pemphigus folacious, paraneoplastic pemphigus, IgA pemphigus
@ Subepidermal bullous dermatosis
Bullous pemphigoid , Epidermolisis bullosa acquisita/ Bullous systemic lupus
erythematosus
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Salt-Split Skin
The salt split skin technique (SS) increased the sensitivity of detection of anti-BMZ
antibodies in subepidermal VBD (vesico bullous dermatosis) when compared with the
non-cleaved substrate (skin)
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Mucous membrane pemphigoid
Pemphigoid gestationis (PG) or herpes gestationis (HG)
Linear IgA bullous dermatosis
Dermatitis herpetiformis
Direct immunoflourescence
Pemphigus foliaceus
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Histology of pemphigus foliaceus includes loss of the stratum corneum, increased
prominence of the granular layer, or visible superficial epidermal separation with
blister formation . At higher magnification subtle acanthloysis and spongiosis can be
seen within the stratum granulosum, extending into the stratum corneum . This can
form separation within the superficial epidermis, or as mentioned above, lead to
complete loss of the stratum corneum. The prominent granular layer is seen as
hyperchromasia of the nuclei within dyskeratotic cells in this layer, similar to the
grains seen in Dariers disease .
In the dermis there is a predominantly superficial lymphocytic infiltrate with scattered
eosinophils . Neutrophils may be more common in the IgA subtype.
If there is clinical suspicion for pemphigus foliaceus but little to see on first inspection,
remember to assess the hair follicles, as early changes may be seen here.
The level of cleavage allows us to differentiate the two main forms of pemphigus:
pemphigus vulgaris and pemphigus foliaceus. In pemphigus vulgaris (PV), the
cleavage is suprabasal, whereas in pemphigus foliaceus (PF) it is intramalpighian.
Direct immunofluorescence reveals intercellular fluorescence, of linear pattern,
intraepidermal
Pemphigus foliaceus
Pemphigus foliaceus
Pemphigus foliaceus
Findings from direct immunofluorescence on classical pemphigus foliaceus (PF) and endemic
pemphigus foliaceus (EPF) show the same characteristics. IgG autoantibodies target desmoglein 1
(Dsg1), the main autoantigen in PF.
Pemphigus foliaceus
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Pemphigus foliaceus is an autoimmune disease,
which basically means that an individual's immune
systems starts reacting against his or her own
tissue.
The building block cells of the epidermis are called
keratinocytes. These cells are cemented together at
special sticky spots called desmosomes. In
pemphigus foliaceus autoantibodies bind to a
protein called desmoglein-1, which is found in
desmosomes in the keratinocytes near the top of
the epidermis. The result is the surface
keratinocytes separate from each other, and are
replaced by fluid: the blister. Because the blister is
very close to the surface of the skin the blisters
rupture easily. In most cases the autoantibodies are
immunoglobulin type G (IgG) but in IgA pemphigus
foliaceus the autoantibodies are type A (IgA).
Pemphigus foliaceus is sometimes provoked by sun
exposure.
Endemic pemphigus foliaceus occurs in South
America, where it is commonly known as Fogo
Selvagem. It appears to be set off by a virus
transmitted by an insect bite.
Pemphigus foliaceus
Pemphigus foliaceus
DIF: intercellular deposits of IgG and C3 are found throughout the epidermis in 100% of the cases of
active disease. Autoantibodies of the IgG class are also deposited in the oral squamous epithelium,
despite the absence of clinical lesions of EPF in the mucous membranes. IgG subclasses may be
employed, showing that in patients with active PF lesions the predominant IgG isotype is IgG4, in
contrast to IgG1, found more often in patients in remission.
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Pemphigus folaceus: linear intercellular, intraepithelial IgG
The bulla of pemphigus vulgaris is acantholytic and cells are found free in the cavity. Tipically,
acantholysis is suprabasal; therefore, the basal layer remains intact and forms the floor of the bulla.
Microscopic image of direct immunoflourescence using an anti-IgG antibody. The tissue
is skin from a patient with Pemphigus vulgaris. Note the intercellular IgG deposits in the
epidermis and the early intraepidermal vesicle caused by acantholysis
Autoimmune Blistering Diseases (ABDs) are a group of disorders associated with
autoantibodies that are directed against desmosomal structural proteins (Pemphigus) or
hemidesmosomal proteins (Bullous Pemphigoid and Epidermolysis Bullosa Acquisita).
MBL International offers ELISA kits for detection and monitoring of ABDs.
Direct immunofluorescence of a skin biopsy of pemphigus vulgaris
Direct immunofluorescence of a skin biopsy from a patient with pemphigus vulgaris revealing
deposition of IgG throughout the epidermis resulting in a chicken wire appearance
PEMPHIGUS VULGARIS
Consultant. 2013;53(3):168-176
PEMPHIGUS VULGARIS
PEMPHIGUS VULGARIS
Nikplosky sign (acantholytic cells) It is useful in differentiating pemphigus vulgaris (where it is present)
from bullous pemphigoid (where it is absent)
PEMPHIGUS VULGARIS (Lancet 354: 667, 1999) and the other blistering disorders
Consultant. 2013;53(3):168-176
Pemphigus herpetiformis of age of onset at 6 year
Dermatology Online Journal 17 (6): 10, 2O11
Pemphigus herpetiformis is a rare entity that combines the clinical features of dermatitis herpetiformis
with the immunologic and histological features of pemphigus
Direct immunofluorescence showed
positive intercellular IgG and C3 staining
throughout the epidermis and on the
dermo-epidermal junction
Erosive plaque on the arm in pemphigus herpetiformis
Pemphigus herpetiformis (PH) is a variant of PV or PF, where grouped pruriginous
papules and vesicles are clinically observed. It resembles dermatitis herpetiformis.
Findings from DIF are similar to those of PF or PV, that is, intraepithelial intercellular IgG
deposits
Pemphigoid gestationis
Direct immunofluorescence of peri-lesional skin, showing linear deposition of complement (C3)
along the BMZ
Paraneoplastic pemphigus
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Clinical, histological, and immunofluorescence
photographs of the paraneoplastic pemphigus
(PNP).
(a) The patient suffered from severe mucosal and
skin erosions.
(b) Patient's skin biopsy showed suprabasal
blistering and vacuolar degeneration.
(c) Direct immunofluorescence (DIF) of the patient's
skin showed IgG deposition on the surfaces of
keratinocytes.
(d) Indirect immunofluorescence of the patient's
IgG-stained rat bladder transitional epithelium
The disease affects the skin and mucous
membranes and is associated with neoplasms
(Castleman's disease, lymphomas, thymomas). It is
very similar to PV, but it shows diversity of
autoantigens (reactivity with desmoglein 3,
desmoplakins, and BMZ antigens).
DIF: Similar pattern to that of PV, but with
occasional homogeneous deposits of IgG and C3 in
the basement membrane zone .
One way to differentiate PNP from PV is to perform
indirect immunofluorescence (IIF) using as a
substrate mouse vesicle epithelial cells (simple nonstratified epithelium, transitional
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IgA pemphigus
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IgA pemphigus (IgAP) is a rare neutrophilic
acantholytic dermatosis. It is characterized by
intercellular intraepidermal IgA deposits on DIF . It
can be classified into two types: subcorneal
pustular dermatosis (SPD), whose autoantigen is
desmocollin 1 (Dsc1) and intraepidermal
neutrophilic dermatosis (IND)
Sub Corneal Pustular
Dermatosis Sneddon
Wilkinson Disease
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The cause of SPD is unknown. Cultures
of the pustules consistently do not reveal
bacterial growth. The role of trigger
mechanisms such as preceding or
concomitant infections, though
repeatedly discussed, has remained
speculative. Immunologic mechanisms
have been implicated in the
pathogenesis, and in a subset of
patients, whose disease clinically
resembled SPD, intraepidermal IgA
deposits have been detected. Some of
these patients also had circulating IgA
antibodies against the same sites within
the epidermis. Desmocollin 1 has been
described as an autoantigen in these
cases and the disease has been
classified as a rare pemphigus variant
(SPD-type IgA pemphigus). The
pathogenetic role of these antibodies is
still to be demonstrated
Direct immunofluorescence, reveals the immunoglobulin IgA along the basement membrane of the
epidermis in a linear pattern. Sometimes these IgA antibodies can be detected by a blood test (indirect
immunofluorescence). Research indicates the antibodies are directed against various basement
membrane components (target antigens
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Linear deposition of IgA
No deposition of IgG Direct immunofluorescence
No deposition of
IgG
Classification of vasculitis. ANCA indicates antineutrophil
cytoplasmic antibodies; IF, immunofluorescence
Classification of vasculitis. ANCA indicates antineutrophil cytoplasmic
antibodies; IF, immunofluorescence; IgA, immunoglobulin A.
Direct immunofluorescence of lupus erythematosus
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Chronic cutaneous lupus erythematosus
In chronic cutaneous lupus erythematosus (CCLE), the occurrence of immunoreactant deposits
varies between 60 and 90%. DIF often shows positivity in CCLE after the second month of the
disease. The site of the biopsy is extremely important: lesions in the trunk are generally negative,
while those in the cephalic portion, neck, and upper extremity show more than 80% of positivity.
IgG and IgM with homogeneous, granulous or reticulate pattern are the most frequent, and most
authors find greater positivity for IgM. DIF is usually negative in healthy skin.
Fluorescent cytoid bodies (IgA and IgM) are found in the papillary dermis and represent
degeneration of basal keratinocytes. They are not exclusive to LE, since they are frequently found
in lichen planus (LP) and other inflammatory dermatoses.
Subacute cutaneous lupus erythematosus (SCLE)
DIF findings are similar to those of CCLE, with positivity around 54 and 100% of the cases.
Nevertheless, fluorescence of the BMZ is often granulous and occasional fluorescence of the
nuclei of keratinocytes occurs - the in vivo ANF phenomenon.
Systemic lupus erythematosus
In systemic lupus erythematosus (SLE) immunoreactant deposits (lupus band test=LBT) 37 are
essential in the diagnosis and prognosis of the disease when associated with clinical findings and
serologic tests. As a diagnostic test, LBT is 60 to 90% sensitive in the photo exposed normal skin
of SLE patients, as compared with non-exposed areas (40-60%). The area currently
recommended is the deltoid area or dorsal portion of the forearm. As a prognostic test, LBT should
be performed in the non-exposed area of normal skin (gluteal region and flexor portion of the
forearm)..
Immunocomplex deposits involve various immunoglobulins, associated or not with C3. The most
frequent association is of IgG / IgM. Fluorescence can also occur in the dermal vessel walls,
annexes and in the nuclei of keratinocytes.
IgG deposits in systemic lupus erythematosus
• Microphotograph of a
histological section of human
skin prepared for direct
immunofluorescence using
an anti-IgG antibody. The skin
is from a patient with systemic
lupus erthematosus and shows
IgG deposit at two different
places: The first is a band-like
deposit along the epidermal
basement membrane ("lupus
band test" is positive). The
second is within the nuclei of
the epidermal cells (antinuclear antibodies).
IN VIVO ANTI-NUCLEAR FACTOR
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Antinuclear antibodies (ANAs, also
known as antinuclear factor or ANF)
are autoantibodies that bind to
contents of the cell nucleus. In normal
individuals, the immune system
produces antibodies to foreign proteins
(antigens) but not to human proteins
(autoantigens).
Immunoglobulin deposits, especially
IgG or complement (C3) in the nuclei
of keratinocytes , may appear in
autoimmune disorders, such as lupus
erythematosus, mixed connective
tissue disease (MCTD), overlap
syndrome, and vasculitis. This
phenomenon is called in
vivo antinuclear factor (ANF) and is of
unknown immunopathology. Seventyone percent of patients also show
circulating antinuclear antibodies. This
DIF pattern may be one of the first
pieces of evidence of autoimmune
disease, with positive predictive value
for collagenosis varying from 75% to
88%.
Direct immunofluorescence photomicrograph of skin biopsy
showing IgG reactive 2+ diffuse nuclear staining in epidermal cells
(ANA in vivo)
Anti-nuclear antibody
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There are many subtypes of ANAs such as anti- RO antibodies, anti –LA antibodies, anti – Sm
anti –antibodies, anti – nRNP antibodies, anti – Scl 7O antibodies, anti ds DNA antibodies, anti –
histone antibodies,, antibodies to nuclear pore complexes, anti –centromere antibodies & anti -sp
1OO antibodies. Each of these antibody subtypes binds to different proteins or protein complexes
within the nucleus. They are found in many disorders including autoimmunity, cancer & infection,
with different prevalence's of antibodies depending on the condition. This allows the use of ANAs
in the diagnosis of some autoimmune disorders, including systemic lupus
erythematosus,Sjogren’s syndrome, scleroderma, mixed connective tissue disease, polymyositis,,
dermatomyositis,, autoimmune hepatitis & drug induced
The ANA test detects the auto antibodies present in an individual's blood serum. The common
tests used for detecting and quantifying ANAs are indirect immunoflourescence & enzyme linked
imminosorbent assay indirect (ELISA). In immunofluorescence, the level of autoantibodies is
reported as a titer. This is the highest dilution of the serum at which autoantibodies are still
detectable. Positive autoantibody titers at a dilution equal to or greater than 1:160 are usually
considered as clinically significant. Positive titers of less than 1:160 are present in up to 20% of
the healthy population, especially the elderly. Although positive titers of 1:160 or higher are
strongly associated with autoimmune disorders, they are also found in 5% of healthy individuals.
Autoantibody screening is useful in the diagnosis of autoimmune disorders and monitoring levels
helps to predict the progression of disease A positive ANA test is seldom useful if other clinical or
laboratory data supporting a diagnosis are not present
Homogeneous immunoflourescence staining pattern of double stranded DNA antibodies on HEp-20-10 cells. Interphase cells show
homogeneous nuclear staining while mitotic cells show staining of the condensed chromosome regions
Anti-dsDNA
• Direct immunofluorescence
of lupus erythematosus
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In lupus erythematosus (LE), immunocomplexes
target the nuclear components of keratinocytes and
structures of the basement membrane zone. DIF
aids in the diagnostic confirmation of lupus
erythematosus, distinguishing it from other
diseases. IgG, IgM, IgA, and C3 deposits may
occur, in addition to other immunoreactants in the
BMZ. There are several deposit patterns in the
BMZ, such as: homogeneous, fibrillar, linear, and
granulous, which can be focal or continuous.
Fluorescent cytoid bodies can be observed in the
dermis in the dermo-epidermal junction with IgM or
IgA. Prevalence of immunoglobulins in the BMZ is
partly determined by age, localization and
morphology of the lesion, activity of the disease,
and treatment
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Anti-dsDNA antibodies are a group of
anti-nuclear antibodies and their target antigen is
double stranded DNA. Blood tests such as enzyme
linked immunosorbent assay (ELISA) and
immunofluorescence are routinely performed to
detect anti-dsDNA antibodies in diagnostic
laboratories. They are highly diagnostic of systemic
lupus erythematosus (SLE) and are implicated in
the pathogenesis of lupus nephritis
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dsDNA antibody. The variable regions (yellow) are
complementary to the dsDNA strands. These
antibodies are found commonly in the sera of
people with SLE
ANTINUCLEAR ANTIBODIES PATTERN
Role of direct immunofluorescence (DIF) in the diagnosis of lupus erythematosus (LE) and other connective tissue
diseases (CTD) is well-established. Deposition of various immunoreactants along the dermal-epidermal junction (DEJ)
is highly characteristic of LE. However, DEJ is not the only site of immunopathological changes in connective tissue
diseases. Immunoreactants may also be deposited in the epidermis (seen as epidermal nuclear staining or ENS) or in
the papillary dermis.
Anti-neutrophil cytoplasmic
antibodies (ANCAs) are a group of autoantibodies, mainly of the IgG type, against antigens in the
cytoplasm of neutrophol granulocytes & monocyte. They are detected as a blood test in a number of
autoimmune disorders, but are particularly associated with systemic vasculitis, so called ANCA-
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associated vasculitides
Perinuclear staining typical of p-ANCA
The granular, cytoplasmic
staining pattern of c-ANCA
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Immunofluorescence staining pattern of ANCA.in vasculitis Top left - PR3 antibodies
on ethanol-fixed neutrophils (c-ANCA pattern). Bottom left - PR3 antibodies on
formalin-fixed neutrophils(c-ANCA pattern). Top right - MPO antibodies on ethanolfixed neutrophils (p-ANCA pattern). Bottom right - MPO antibodies on formalin-fixed
neutrophils (c-ANCA pattern).(FITC conjugate
IgA deposits in Henoch Schonlein purpura
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VASCULITIS
In vasculitis, immunodeposits are often located in
the walls of postcapillary venules of the superficial
dermis, since the most frequent processes are
leukocytoclastic vascullitis (LCV) and HennochSchönlein purpura (HSP). The specimen should be
collected within the first 24 hours because
immunocomplexes are rapidly degrated.
DIF: In Hennoch-Schönlein purpura, IgA deposits of
granulous pattern predominate (75-100%) in the
vessel walls of the superficial dermis . In
leukocytoclastic vasculitis, deposits on the vascular
walls are predominantly constituted by C3, followed
by IgM and IgG, and they are fibrillar. In
cryoglobulinemias, C3 predominates and
sometimes IgM and IgA are observed in the
vessels. In collagenosis, the most frequently
observed deposits are of IgG, IgM, and C3
Microphotograph of a histological section of human
skin prepared for direct immunofluorescence
using an anti-IgA antibody. The skin is from a
patient with Henoch Schonlein purpura: IgA
deposits are found in the walls of small superficial
capillaries (yellow arrows). The pale wavy green
area on top is the epidermis, the bottom fibrous
area is the dermis.
Direct Immunofluorescence of Cutaneous Vasculitis
Fibrinogen blood vessels (vessels may also stain for IgG, IgM, IgA and C3; IgA vascular staining is
characteristic of Henoch Schönlein purpura
Direct Immunofluorescence of Lichen Planus
IgM scattered and clumped cytoids
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Lichen planus
DIF: presence of fluorescent cytoid bodies with IgM , and less frequently IgA and IgG. Granulous
IgM deposits may be found in the BMZ. However, findings do not indicate the diagnosis of lichen
planus because they can be associated with other conditions (LE, BP).
Lichen planus. Direct immunofluorescence examination of involved skin
Direct Immunofluorescence of Lichen Planus
C3 granular basement membrane zone
Direct Immunofluorescence of Porphyria
C3 granular and fibrinogen weak thick basement membrane zone and perivascular
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Porphyrias
Lesioned skin in porphyria (cutanea tarda, erythropoietic, variegate, coproporphyria) shows
homogeneous deposits of IgG, IgM (rare), C3, and IgA in the walls of dilated vessels in the
papillary dermis and throughout the BMZ. The frequency of such deposits in active lesions may
reach 100%, whereas in the normal skin of patients positivity is of 50%
A, Classic epidermolysis bullosa acquisita, showing scarring in areas of friction
and milium cysts. B, Subepidermal blister. Hematoxylin-eosin, original
magnification ×40. C, Scant inflammatory infiltrate Actas Dermosifiliogr. 2013;104:904
A, Inflammatory epidermolysis bullosa acquisita. B, Subepidermal blister.
Hematoxylin-eosin, original magnification ×40. C, Significant inflammatory
infiltrate with predominance of neutrophils and scant eosinophils Actas
Dermosifiliogr. 2013;104:904
Direct immunofluorescence showing the characteristic epidermolysis bullosa
acquisita pattern of deposition along the dermal-epidermal junction. A, Intense
linear immunoglobulin G deposition. B, Slight C3 deposition Actas Dermosifiliogr.
2013;104:904
Direct Immunofluorescence of Pemphigoid
IgG linear basement membrane zone (20x) and C3 linear basement membrane zone
Direct Immunofluorescence of Dermatitis Herpetiformis Skin Biopsy
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IgA granular basement membrane zone with
stippling in dermal papillae
• Dermatitis herpetiformis
• DIF is an important diagnostic
tool in DH, since deposits of
immunocomplexes (IgA) in the
dermal papillae diagnose the
gluten-sensitive disease.
• DIF: granulous, fibrillar or
dotted IGA deposits are found
in the dermal papillae . The IgA
subtype consists basically of
IgA1; IgA2 rarely occurs. Other
immunoglobulins and C3 may
be found in the dermal
papillae, but are rare.
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Abbreviations:
BMZ : basement membrane zone
BP : bullous pemphigoid
DIF : direct immunofluorescence
EBA : Epidermolysis bullosa
acquita
FITC : Fluorescein isothiocynate
HG : Herpes gestationalis
ICS : Inter cellular deposition
IF : Immunofluorescence
LAD : linear IgA disease
PNP : paraneoplastic pemphigus
UV : ultraviolet
PBS : phosphate buffered saline
PE : Pemphigus erythematosus
PF : Pemphigus foliaceous
PCT : Porphyria cutanea tarda
SLE : Systemic lupus
erythematosus
Differential diagnosis of Direct
immunoflourescence
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Differential diagnosis of direct immunofluorescence (DIF) depends
upon :
1. Primary site of immune deposition
2. Class of immune deposits
3. Number of immune deposits
4. If multiple, the identity of the most intense deposits is significant
5. Deposition in other sites besides the main sites.
Inter cellular deposition (ICS) pattern
ICS pattern results from binding of antibodies to desmosomal
proteins around keratinocyte cell surface and is characteristic of
pemphigus group. Parameters in further differential diagnosis to be
studied are:
a) Class of immunoglobulins deposited
b) Relative intensity of fluorescence in different levels of epidermis
c) Any other deposits besides ICS
IgG in the ICS
It is characteristic of all pemphigus except IgA pemphigus. C3 may
be present along with IgG.
IgG deposition in the ICS and BMZ
D/D:
(1) Pemphigus erythematosus (PE)
(2) Pemphigus foliaceous (PF)
(3) Paraneoplastic pemphigus (PNP)
Antibodies will be directed to BMZ also in these lesions, however
those of PNP are weak, diffuse and nonspecific.
IgA deposition in the ICS
This will be seen in IgA pemphigus only.
Clinical and histopathological D/D of IgA pemphigus will be PF and
subcorneal pustular dermatosis which will not exhibit IgA deposit.
BMZ deposition
It is characteristic of subepidermal bullous disease. The patterns to
be studied are
a) Class of immunoglobulins deposited
b) Number of immune deposits
c) Morphology of fluorescence like continuous, discontinuous, linear,
granular, homogenous.
d) Deposits at other sites
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Exclusive BMZ deposits
IgG and/ or C3 deposits
IgG and/or C3 or multiple immunoreactants can be seen in following
conditions.
IgG, C3/ both
(1) BP
(1) Mucosal pemphigoid
(2) Herpes gestationalis (HG)- C3 alone may be seen sometimes.
(3) Epidermolysis bullosa acquita (EBA)
(4) Bullous SLE
When C3 is more intense than IgG, it favors pemphigoid group.
Pattern in BP and HG is linear, wavy, tubular or granular.
Multiple deposits in BMZ favors
EBA and bullous SLE. The deposits are homogenous, thick and
broad.
IgA depositions at the BMZ
This is diagnostics of linear IgA disease (LAD)
Deposition at the BMZ and blood vessel walls
Homogenous deposition of multiple immunoreactants favors
- Porphyria cutanea tarda (PCT)
- Pseudo – PCT
- Erythropoeitic protoporphyria. Here usually IgG, IgA ± C3 deposit
are seen.
Papillary dermal deposition
Granular IgA, C3 in papillary dermis and BMZ is diagnostic of DH.
I have the question in the last power point a lot lately
Indirect Immunofluorescence
Indirect Immunofluorescence of Pemphigus Serum in Calcium Buffer
on Intact Human Skin
IgG cell surface (intercellular substance
Bullous Pemphigoid
Indirect Immunofluorescence of Pemphigoid Serum on Human Split
Skin Substrate
IgG epidermal pattern (roof of split
Bullous pemphigoid, indirect Immunofluorescence on salt-split skin substrate
Bullous Pemphigoid
Bullous pemphigoid
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Diagnostic pathway in bullous pemphigoid (BP)
Direct immunofluorescence (IF) microscopy of a
perilesional biopsy is the gold standard for the diagnosis
of BP and differentiates subepidermal blistering
autoimmune diseases from pemphigus. By indirect IF
microscopy on 1 M NaCl spilt human skin, BP patients'
sera are screened for anti-basal membrane zone (BMZ)
autoantibodies. Whereas sera from patients with
epidermolysis bullosa acquisita, anti-laminin 332 mucous
membrane pemphigoid, and anti-p200 pemphigoid label
the dermal side of the artificial split, sera of BP patients
bind to the blister roof. Anti-BP180 antibodies can be
detected by BP180 NC16A-specific enzyme-linked
immunosorbent assay (ELISA), Western blotting with
conditioned concentrated medium of cultured HaCaT
cells, which detects reactivity against LAD-1 (linear IgA
disease antigen 1) that corresponds to the cell-derived
ectodomain of BP180, and Western blotting with various
other recombinant fragments of BP180. Since four
different entities are associated with IgG antibodies to
BP180, the clinical phenotype determines the final
diagnosis. When no BP180 reactivity is found, sera are
assayed for BP230-specific antibodies that, only in
conjunction with a positive direct IF microscopy and
compatible clinical features support the diagnosis of BP.
In case of epidermal binding by indirect IF microscopy
and failure to detect IgG reactivity to both BP180 and
BP230, testing for antibodies against α6β4 integrin is
recommended (for example, by Western blotting of
keratinocyte extract) .
Bullous Pemphigoid
Indirect Immunofluorescence of Epidermolysis Bullosa Acquisita Serum
on Human Split Skin Substrate
IgG dermal pattern (floor of split)
Indirect immunofluorescence using 1.0 M sodium chloride-separated
skin. Antibodies are targeting the dermal side (floor) of the blister
Indirect Immunofluorescence of Linear IgA Bullous Dermatosis Serum
on Human Split Skin Biopsy
IgA epidermal pattern (roof of split
Pemphigoid gestationis
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Usually urticarial papules, also
blisters and rash
Usually resolves within weeks to
months after delivery
Tends to recur with subsequent
pregnancy
Associated with premature
delivery, small for gestational age
infants
Histology:Similar to bullous
pemphigoid - subepidermal blister,
with eosinophils in lumen
Marked edema in papillary dermis
Perivascular infiltrate consists of
lymphocytes, histiocytes and large
numbers of eosinophils
Eosinophilic spongiosis may be
seen
Pemphigoid gestationis
IgG stain
Differential diagnosis of INDIRECT
IMMUNOFLUORESCENCE f
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Indirect IF
This is tested in serum.
- ANA for Systemic lupus erythematosus (SLE)
- IgG anti-ICS antibodies – Pemphigus
- IgA anti-ICS antibodies – IgA pemphigus
- IgG anti BMZ - SLE, BP, HG, EBV, Bullous SLE
Diseases with immune deposits along the dermoepidermal junction
LE
Dermatomyositis
Systemic sclerosis
LCV
Rheumatoid arthritis
BP
HG
EBA
DH
Linear IgA bullous dermatosis
PCT
Pseudoporphyria
LP
Rosacea
Chronic active hepatitis
Primary biliary cirrhosis