2. CONTENTS
• Immunofluoresence – types , technique, indications
• Bullous lesions – classification & brief overview of
immunobullous disorder
• Antigen mapping in HEB
• Brief role of IF in other lesions
3. INTRODUCTION
• Immunofluorescence (IF) microscopy is a well established technique
used for the detection of a wide variety of antigens in tissues or on
cells in suspension
• Diagnostic immunopathology in dermatology started in 1963
with the description of the lupus band test (LBT),i.e. deposits of
immunoglobulins and complement at the dermo-epidermal junction
4. • In 1964, Beutner and Jordon used the indirect IF technique to
demonstrate antibodies in the sera of Pemphigus patients
• Since then, the use of IF has become routine in the
diagnosis of immunologically mediated diseases of the skin
the demonstration of circulating antibodies such as
autoantibodies
10. Direct Immunofluorescence (DIF)
One-step procedure for detecting in-
vivo deposition of immunoglobulins,
complement components and
fibrinogen in patient’s skin
It involves application of
fluoresceinated antibodies to a frozen
section of the patient’s skin
11. Indirect Immunofluorescence (IIF)
Two step procedure for demonstrating
circulating autoantibodies in a patient’s
serum
It is utilised to identify and titer circulating
autoantibodies in the patient’s serum
Patient’s serum is incubated with the
substrate(monkey esophagus) followed by
the application of fluoresceinated
antibodies
12. Antigen Mapping ( Modified IIF)
It is used in various forms of hereditary
epidermolysis bullosa
To determine the site of cleavage or
abnormalities in the distribution of
mutated structural proteins
13. Salt-split Technique( Modified IIF)
Artificially splitting of the skin with NaCl done
Afterwards IIF is performed
There are two types of salt-split technique (SST)
- direct and indirect
BMZ staining : “roof” ; “floor” ; “combined”
pattern
14. Immunofluorescence Technique
ANTIGEN
DIF – tissue sample
IIF - patient’s serum
incubated with
substrate
CONJUGATE
(antiserum)
CONVALENTLY LINKED TO
FLUOROCHROME
IF
IF technique involves viewing of
antigen–antibody complexes
under ultraviolet microscope
using corresponding antibodies
tagged to a fluorochrome
15. The most important factors are:
1. Preservation of substrate antigen
2. Antibody conjugate
3. Fluorescence microscopy system
4. Staining and incubation
Immunofluorescence Technique
17. Transportation of biopsy specimen
• Phosphate Buffered Saline (PBS)
• Michel’s Medium (MM ) :
ammonium sulfate, N ethylmaleimide,Potassium citrate buffer, magnesium
sulfate, distilled water
Can preserve specimen upto 6 months
pH 7- 7.2
Store at 4ºC
• Normal Saline - upto 24 hour
18. SUBSTRATE ANTIGEN(DIF)
• Skin Biopsy - either quick-frozen or placed in Michel’s transport medium
for later quick freezing
• Quick freezing by isopentane liquid nitrogen is the most widely used
method
• Unfixed cryostat section (2-5µm ) used
• These are mounted on a slide previouly coated with gelatin adhesive and
dried
• Frozen tissue blocks can be stored in air tight plastic bag in low
temperature cabinets at -70˚C or lower
19. SUBSTRATE ANTIGEN (IIF)
• Monkey esophagus antigen : substrate for pemphigus ,pemphigoid
antibody tests & herpes gestationis
• Rat urinary bladder epithelium : for the diagnosis of paraneoplastic
pemphigus
• Others - guinea pig lip and esophagus, rabbit lip and esophagus and
normal human skin (NHS)
• Human salt-split skin biopsies - to separate acquired autoimmune
subepidermal bullous disorders
20. • Sections of monkey esophagus prepared
• Used within 10 days to 2 weeks after killing and quick freezing
• Tissues and sections need to be stored at -70 °C to -80 °C
• Sections should be cut at 4 μm
21. • For antinuclear antibody (ANA) testing :
HEp-2 cell substrates - cultured monolayered cell preparations of
laryngeal SCC
available commercially prefixed on glass slides
• Tests for anti-DNA antibodies :
Crithidia luciliae as a substrate
nonpathogenic protozoan with DNA both in the nucleus and the
kinetoplast
22. • Antineutrophil cytoplasmic antibody (ANCA) testing
commercially prepared neutrophil preparations
can be prepared in the lab (in house ethanol fixed neutrophil
spots)
23. Handling of sera for indirect test
• About 3 ml of clotted blood used
• Hereditory EB- 2-5 ml EDTA blood for mutation analysis
• All sera should be refrigerated until tests are performed
• Repeated freezing and thawing should be avoided, since this causes a
rapid loss of antibody activity
• Positive and negative control sera must be frozen in aliquots of a size
adequate for single experiment
• EDTA blood should not be frozen
25. ANTIBODY CONJUGATE
• Two types
monospecific reagents used for direct staining of biopsies
human anti-whole-IgG conjugates used for the IIF test of sera
26. • Most commercial conjugates are supplied in a lyophilized form
• They require reconstitution with distilled water or in diluents
• The undiluted stock should be divided into volumes of 0.10–0.50 ml
and stored frozen (−20 °C) until ready to be diluted
• The diluted conjugates should be stored at 4 °C and not refrozen
28. • Compounds containing electrons which when irradiated with a light of a
particular wavelength achieve an unstable higher energetic state
• On returning to the ground state as a spontaneous process, they emit light
of a longer wavelength
• To function as labelers, they must possess chemical groups capable of
forming covalent bonds with protein molecules, which emit high
fluorescence in visible spectrum
FLUOROCHROME
34. ANTIGEN
MAPPING
Patient’s skin (4 micron) section & NHS/salt split skin on glass slide
Washed with PBS X 15 min & fan dried
Sections treated with panel of primary Ab against known BMZ
components ( type IV, XVII& VII collagen; laminin 322) & incubated at
room temp X 1 hr
Washed with PBS X 15 min & fan dried
Sections treated with secondary Ab ( anti – IgG mouse specific Abs )
conjugated with fluorescein dye
Washed with PBS after 1 hr , dried and mounted in buffred glycerol
and examined under fluorescent microscope
35. Direct SST - Patient’s skin &
Indirect SST - NHS/on glass slide
Artificially splitting of the skin at the level of
lamina lucida by incubating it in 1 M solution
of sodium chloride for 24 h
cryocut sections are prepared
IIF with patient’s serum is carried out
SALT SPLIT SKIN
TECHNIQUE
(SST)
37. DIF is diagnostic (A) Bullous diseases • Pemphigus(all forms)
• Pemphigoid(all forms)
• Herpes gestationis
• Dermatitis Herpetiformis
• Linear IgA bullous dermatosis
• Epidermolysis bullosa acquisita
(B) Connective tissue diseases DLE
SLE
DIF : highly characteristic &
some diagnostic value
(A) Vascular diseases Allergic vasculitis
HSP
Essential mixed cryoglobulinemia
Polyarteritis nodosa
(B) Other diseases Porphyria cutanea tarda
Other forms of porphyria
Lichen Planus
DIF : not diagnostic but only
suggestive
(A) Connective tissue diseases Mixed CTD
Systemic Sclerosis
Dermatomyositis
Psoriasis
38. Biopsy techniques
• A 3- to 4-mm punch biopsy is generally adequate
• In autoimmune blistering diseases (AIBD) ,an inflammed but unblistered
perilesional area is the ideal specimen
• Whenever possible biopsy a single fresh small blister including adjacent
clinically uninvolved skin (perilesional)
39. • For a large blister, biopsy edge of blister and adjacent uninvolved skin
(perilesional)
• Cut the perilesional end and send for IF studies and the remaining blister
for histology
• For uninvolved skin, a 3-mm punch biopsy is sufficient
40.
41.
42. SITE OF THE BIOPSY CHARACTERIZATION
Mucosa
Perilesional with apparently normal mucosa with intact
epithelium
Pemphigus or pemphigoid (Skin) Edge of lesion, second biopsy 3 mm from lesion
Pemphigus or pemphigoid
(Mouth)
1st biopsy 3mm from lesion, 2nd at edge
Vasculitis
PAN
Very fresh lesions
Deep skin biopsy
Dermatitis herpetiformis normal skin about 3 mm. from the lesion
Porphyria and pseudoporphyria dorsum of hand lesions as well as normal skin
Systemic Lupus erythematosus
both lesional and apparently normal skin in sun-exposed areas
DLE
lesional skin
Lichen planus Inflammed mucosa and/or skin
44. • Blister - fluid-filled cavity formed within or beneath the
epidermis
Vesicle - <0.5 cm
bulla- > 0.5 cm
45. • Blistering/vesicobullous disorders include several diseases may
be
hereditary in origin
immunologically mediated
secondary to infections
inflammatory process
46. APPROACH
• Clinical history & classical features
• Age of onset, family history & drug history
• Nature of bullae- flaccid /tense
• Bulla spread sign & Nikolsky sign : to look for acantholysis
• Tzanck smear : acantholytic cells ; eosinophils; neutophils
• Histopathology : level of split & type of cellular infiltrate
• Immunofluorescence : both direct & indirect for autoimmune bullous disorders
48. Nikolsky's sign
• Slight mechanical pressure (by rubbing) is exerted on the skin →
upper epidermal layer slips away from lower layer → separation
of epidermis → blistering
• Test is positive on previously unaffected skin
• present in Pemphigus vulgaris , toxic epidermal necrolysis, staphylococcal
scalded skin syndrome, bullous impetigo and Stevens-Johnson syndrome
• Not present in Bullous pemphigoid
• Bulla spread sign : refers to the extension of a blister to adjacent unblistered
skin when pressure is put on the top of the bulla
49. • Tzanck test
• Microscopic examination of scrapings from the
base of a lesion to look for Tzanck cells
• Tzanck cells (multinucleated giant cells) are
present in:
• Pemphigus vulgaris
• Herpes simplex type 1 (HSV-1) infection
• Varicella zoster virus infection
(chickenpox or shingles)
• Cytomegalovirus
HSV
PEMPHIGUS
VULGARIS
50. Mechanism of blister formation
• Spongiosis
ECF accumulation form
vesicle, bulla
52. Reticular/ballooning degeneration
intracellular odema lead to
keratinocyte rupture
remaining desmosomal
attachments often connect
ruptured keratinoctyic
membranes and cytoplasm to
intact keratinocytes
this gives epidermis an irregular
meshwork appearance
53. Cytolysis
disruption of keratinocytes
in normal epidermis : by high levels of
physical agents such as friction and heat
Friction leads to the shearing of
keratinocytes one from another and of the
keratinocytes themselves
Minimal friction may lead to cytolysis in
subjects whose keratinocytes do not have
normal structural matrix and desmosomes
65. PEMPHIGUS GROUP OF DISORDERS
• Autoimmune condition
• Characterised by vesicles and bullae due to antibody formation
against cell adhesion molecules of keratinocyte
68. • Desmoglein Type 1-
upper layer of epidermis throughout body
very few in mucosa
Ab Dsg1 – Pemphigus foliaceus, only skin involved
• Desmoglein type 2-
simple epithelia & basal epidermis
• Desmoglein Type 3-
mainly basal and suprabasal layers of mucosa
69.
70. P.vulgaris P.vegetans P. foliaceous Drug
induced P.
IgA P. PNP
Age
group
middle aged , elderly
&
rarely children
Rare variant of
P. Vulgaris
2 types
Neumann
Hallopeau
middle age
Variants
Pemphigus
erythematosus -
has LE features
Pemphigus
herpetiformis
resemble DH
penicillamin
e, captopril,
penicillin
derivatives
middle-aged &
elderly
two types:
Subcorneal pustular
dermatosis (SPD)
Intraepidermal
neutrophilic
dermatosis (IEN)
patients with underlying
malignancy : thymoma,
B –CLPD, sarcomas etc
Site oral mucosa, scalp,
midface, sternum,
groin, pressure points
intertriginous
areas
trunk, face, back
Mucosa spared
axilla and groin
Lesions flaccid intraepithelial
blisters , erosions,
ulcerations of skin and
mucous membrane
Neumann type:
vesicles/bulla
rupture to form
erosions
Later : verrucous
vegetations
Hallopeau type:
mild,present as
pustule: dry,
hyperkeratotic &
fissured
Well demarcated
crusting , scaly
exfoliative lesions
PE: erythematous
plaques and
patches in
butterfly
distribution
PH : severely
pruritic papulo-
vesicular lesions
nonspecific
morbilliform
or urticarial
eruption
pruritic pustular
eruption
flaccid pustules on
an erythematous
base in annular
arrangement
six variants –
Bullous pemphigoid like
Lichen Planus like
Erythema multiforme
like
Cicatricial pemphigoid
like
Pemphigus like
GVHD like
polymorphous eruption
71. P.Vulgaris P. Vegetans P. Foliaceous Drug induced P. IgA P. PNP
M/E • Earliest feature
spongiosis
(eosinophilic)
• suprabasal cleft
formation
• acantholytic cells
• Downward growth
of epidermal cells
into dermal papilla
give rise to villi
• Firmly attached
basal cells to the
basement
membrane shows
a tomb stone
appearance
• Neumann :
early lesions -similar
to P.vulagris
Older lesions:
acantholysis ±
papillomatosis of
dermal papilla
VEH
Eosinophils
• Hallopeau :
suprabasal
acantholysis with
acantholytic cells,
pustules &
eosinophilic abscess
Verrucous epidermal
hyperplasia
• acantholysis
within or
adjacent to
granular layer
• subcorneal
bulla with
dyskeratotic
granular
keratinocytes
• Older lesions
may show
hyperkeratosis
, acanthosis &
parakeratosis
. Early eruption
nonspecific,
spongiosis,
parakeratosis
variable dermal
infiltrate
. Well developed
lesions identical
to those of P.
foliaceus P.
vulgaris
eosinophilic
spongiosis
SPD type
subcorneal
vesico-
pustules/
pustules
with minimal
acantholysis
IEN type
Intraepi.
vesico-
pustules or
pustules
with variable
no.
neutrophils
• Suprabasal
acantholysis
• dyskeratotic
keratinocytes,
• variable epi.
necrosis,
• vacuolar
interface
dermatitis ±
lichenoid
inflammation
• Dermal
changes
include supf
PVLI
D/D Hailey-hailey disease
Transient acantholytic
dermatosis/grovers
disease
Pyoderma vegetans SSSS
SCPD
SPD type
Sneddon –
Wilkinson
Pustular
psoriasis
P. Vulgaris
72. IF P.Vulgaris P. Vegetans P.Foliaceous Drug induced
P.
IgA P. PNP
DIF lacelike IgG in
the squamous
intercellular
Substance
(95-100%)
C3 (50–100%)
squamous
intercellular IgG
(100%)
squamous ICS IgG (full
thickness)
superficial portion of
the epidermis (rarely)
PE : squamous ICS
IgG (75%) granular
deposition of IgM and
IgG
(i.e., a positive lupus
band test) at the DEJ
PH : ICS IgG upper
epidermis
squamous ICS
IgG (90%)
squamous ICS IgA
throughout the
epidermis IIF
results are positive
in fewer than 50%
of
reported cases. In
the (66). In the;
however, further
study is needed
squamous
ICS + immune
reactant deposition
at DEJ
DEJ : granular
complement
Linear deposition of
complement, IgG,
IgM and granular
deposition of
complement and IgG
IIF IgG autoantibody
against Dsg3 &
Dsg 1
(80-90%)
squamous ICS IgG Dsg
1 (80-90%)
PE :Antinuclear
antibodies (30-80%)
PH : IgG against Dsg1
circulating
squamous ICS
IgG antibodies
(70%)
SPD type : IgA
autoantibodies for
desmocollin1
IEN type : Ab for
desmoglein 1 or
desmoglein 3
(50%)
Circulating PNP
antibodies against
multiple antigens
plakin ,Dsg
bind squamous
keratinocytes of rat
bladder epithelium
73. Pemphigus vulgaris
A: intercellular edema with
eosinophilic spongiosis leading to
loss of intercellular bridges in the
lower epidermis
B:The suprabasal blister contains
acantholytic cells, neutrophils,
and eosinophils.
dermal papillae lined by a single
layer of basal keratinocytes, so-
called villi
C: row of tombstone
D: lacelike squamous
ICS deposition of IgG in the lower
epidermis (direct
immunofluorescence)
78. PEMPHIGOID GROUP
• Autoimmune disease
• The IgG antibodies bind to two main antigens at the basement
membrane :
BPAg1 (BP230 most commonly)
BPAg2 (BP180 less often)
• Activate complement starting an inflammatory cascade causing the
epidermis to separate from the dermis
80. BULLOUS PEMPHIGOID
• Most common subepidermal blister
• Occurs primarily in elderly
• tense bullae develop on normal or erythematous skin
• Oral lesions seen in 10-40%
• May occur in childhood – two types
1. Infantile BP – in 1st yr of life – in acral areas
2. Localised vulval BP- confined to vulva only
81. • Target antigens – BPAg1/230 kDa and BPAg2/180kDa
• Abs are – IgG4 and IgG1
• Abs to BPAg2 is more associated with oral lesions, less
responsive to steroids and poor prognosis
82. HISTOPATHOLOGY
• Unilocular subepidermal blister
• Cell rich type – blister develop on erythematous skin
eosinophils predominant cell in blister cavity and in dermis
• Cell poor type – blister develop on normal skin
scant perivascular lymphocytic infiltrate with few eosinophils
83. IMMUNOFLUORESCENCE
• DIF - linear , homogenous deposition of IgG/C3 along BM
• IIF - identifies IgG antibodies that react with BMZ (70%)
• Salt-split skin technique -
deposition found on the epidermal side of the blister
84.
85.
86.
87.
88.
89. PEMPHIGOID GESTATIONIS
• aka herpes gestationis
• Rare, pruritic, vesicobullous dermatosis of pregnancy and puerperium
• Occasionally seen in association with H. mole or Choriocarcinoma
• Onset is in 2nd or 3rd trimester
• Subside within several weeks of delivery
• IgG1 class Ab (pemphigoid gestationis factor) is formed against a
placental antigen
• This cross reacts with BP180 antigen of BM
90. HISTOPATHOLOGY
• Early lesion :
marked edema of papillary dermis
superficial and mid dermal perivascular infiltrate by lymphocytes,
eosinophils and histiocytes
• Established blister
subepidermal , contains similar inflammatory infiltrate within the
cavity
eosinophilic microabscesses may be formed in dermal papillae
• DIF – Linear pattern of C3/ IgG (30-40%) in BM zone
• Salt split skin – deposits in epidermal side
91.
92. Cicatricial Pemphigoid/Mucosal Pemphigoid
• Characterized by a chronic course, scarring, and predilection for mucosal
surfaces
• Most patients : elderly and male predilection seen
• Oral blisters are present (100%) ,ocular involvement (75%) & cutaneous
involvement in 33% or less
• The cutaneous lesions are of two types:
(a) an extensive eruption of bullae that heals without scarring
(b) areas of erythema mainly on the face and scalp in which bullae erupt
intermittently followed by atrophy and scarring
93. Histopathology
• Cutaneous lesions
Subepidermal blister develops
Neutrophils and lymphocytes predominate
Eosinophils may or may not be numerous
Lamellar fibrosis beneath the epidermis - hallmark
• Mucosal lesions
lichenoid lymphocytic infiltrate in which neutrophils or eosinophils or
both present
94.
95. IMMUNOFLUORESCENCE
• DIF : linear IgG and C3 at the squamous BMZ in lesional and
perilesional skin ( 80% of cases)
• IIF : salt-split human skin is used as substrate
IgG may be localized only to the roof or to the base of the induced
separation
96. LINEAR IgA BULLOUS DERMATOSES
• Subepidermal blistering disorder
• Two clinical variants –
Chronic bullous dermatosis of childhood
Adult IgA bullous dermatosis
• Target Ag – 97 kDa Ag (degradation products of 180kDa/BPAg2)
• Circulating IgA Abs are present in 70% of childhood disease and in
20% of adult cases
97. HISTOPATHOLGY & IMMUNOFLUORESCENCE
• Subepidermal blisters with neutrophil as predominant cell
• Indistinguishable from DH by presence of papillary microabscesses
and neutrophils in light microscope
• DIF – homogenous linear pattern of IgA deposition along BM zone of
non-lesional area
98.
99.
100. DERMATITIS HERPETIFORMIS
• Subepidermal blistering disorder – intensely pruritic papules and
vesicles – bullae are uncommon
• Ass. with high incidence of gluten sensitive enteropathy – in 90%
cases and also with internal cancers, esp intestinal lymphoma
• Sites- elbows, knees, shoulders, nape of neck
• Onset – early adult life
101. • Target Ag – tissue transglutaminase (tTG)
• IgA Abs formed in gut binds with skin transglutaminase(TG)
• In skin 6 TG isoenzymes are present
• Gluten free diet – reversal of villous atrophy and skin lesions
/also protective effect against development of lymphoma
102. (laminin and type IV collagen) – formation of blisters
enzymes released by these neutrophils destroy two BM components
CD11b on neutrophil cell surface – increase neutrophil function –
Also serum levels of IL-8 increases - increases the expression of
This complex activates complement – chemotaxis of neutrophils in papillary
dermis
Deposits mainly seen are IgA/TG3 aggregates in small blood vessels in
papillary dermis
103. HISTOPATHOLOGY
• Early lesions –
collection of neutrophils and occasional eosinophils at tips of dermal
papillae – papillary microabscesses
fibrin present at the tips of dermal papillae : necrotic appearance
• Older lesions –
subepidermal vesiculation occurs
initially multilocular blisters due to interpapillary ridges – but after
few days these attachments break down – formation of unilocular
blister
104. IMMUNOFLUORESCENCE
• DIF -
Granular / fibrillary/ thready deposits of IgA in dermal papillae of
perilesional and uninvolved skin
If DIF testing is negative – repeat test
• IIF –
• Circulating IgA antibodies that react against reticulin, smooth muscle
endomysium, the dietary antigen gluten, bovine serum albumin and
β-lactoglobin may be present
• detect anti endomysial antibodies (52-100%)
105.
106. EPIDERMOLYSIS BULLOSA ACQUISITA
• Non inflammatory subepidermal bullae develop in areas
subjected to minor trauma such as extensor surface of limbs
• Target antigen – EBA antigen 290 kDa type VII collagen – a
major component of anchoring fibrils
107. HISTOPATHOLOGY
• Classic form – non inflammatory subepidermal blisters
• Bullous pemphigoid like – inflammatory blisters – mostly
lymphocytes and neutrophils
• PAS stain- BM is split and most of the PAS positive material are
in blister roof
108. IMMUNOFLUORESCENCE
• DIF –
• linear deposition of IgG / C3/ C5 along BM zone
• A useful clue to the presence of Abs targeting type VII collagen
: presence of u-serrated pattern of linear IgG deposition
• Routine DIF cannot distinguish between EBA and bullous
pemphigoid
111. Hereditary Epidermolysis Bullosa
• EB is a genetically heterogeneous mechanobullous disorder
• defined by fragility of the skin and mucous membranes
• more than 30 subtypes described so far
• consequence of mutations in genes coding for proteins involved in
adhesion of epidermal keratinocytes to each other or to the
underlying dermis
112. There are four major forms of EB:
simplex, junctional, dystrophic and mixed
based on the level of cleavage at the
dermoepidermal BMZ
The level of cleavage occurs
within the epidermis : EB simplex(EBS)
lamina lucida : Junctional EB (JEB)
below the lamina densa : Dystrophic B (DEB)
Kindler syndrome (KS) can be in the basal
layer of keratinocytes, lamina lucida or
sublamina densa
113.
114. DIAGNOSIS
• Light microscopy is not very useful
• Confirmed by immunofluorescence mapping (IFM) and/or
electron microscopy
• Electron microscopy is very time consuming and expensive
• IFM : based on detection of structural proteins in the epidermis
or DEJ using specific monoclonal antibodies
123. All classes of immunoglobulins,
occasionally including IgE and
IgD, as well as the C3, C4,
C1q,MAC, properdin, and
deposits of fibrin can be
detected (LBT)
The immune deposits most
frequently present are IgM,
IgG, and C3, and sometimes
IgA
Disease Lesional
LBT(%)
Nonlesion
al LBT
(%)
Chronic DLE 60-90 0
Subacute
cutaneous LE
60-100 0
SLE 90-100 50-90
125. IIF
• Indirect immunofluorescence detects nuclear, homogeneous, rim,
speckled and nucleolar patterns
• The fluorescence ANA test can be regarded as a specific marker for
SLE in a rim pattern with a titer of 1:160 or higher
• It is indicative of the presence of anti–native or double-stranded (ds)
DNA antibodies
126. HEp-2 cells are used
as a substrate to
detect the antibodies
in human serum
Microscope slides
are coated with
HEp-2 cells and
the serum is
incubated with the
cells
If antibodies are
present; the
antibodies will bind
to the nucleus.
These can be visualised
by adding a fluorescent
tagged (usually FITC or
rhodopsin B) anti-
human antibody that
binds to the antibodies
127. IIFT Crithidia luciliae sensitive (anti-dsDNA)
• The haemoflagellate Crithidia luciliae is particularly suitable as a substrate
in IIFT
• It contains a highly dense mass of circular dsDNA in its large
mitochondrion
• BIOCHIPs coated with smears of Crithdia luciliae are incubated with
diluted patient samples
• In the case of positive reactions, specific antibodies bind to the antigens.
• In a second step, the attached antibodies (IgG) are stained with
fluorescein-labelled anti-human antibodies and made visible with the
fluorescence microscope
128.
129. Scleroderma, dermatomyositis and MCTDs
• DIF of lesional skin from patients with dermatomyositis and MCTDs
may yield similar fluorescence results as in DLE
• In diffuse scleroderma, the frequency of positive LBT consisting of
IgG or IgM class and/or in vivo ANA varies from 0 to 60%
130. Vasculitis
• DIF of skin biopsies taken from very fresh lesions display vascular
deposits of IgM, C3, fibrinogen, and sometimes IgG
• A negative result does not exclude vasculitis
• HSP lesions : characterized by predominant deposition of IgA in the
walls of upper dermal vessels
• Wegener’s granulomatosis : immune deposits can be detected in skin
biopsies with IgG (most common) immunoreactant deposited in and
around subepidermal blood vessels, but occasionally also along the
BMZ
132. IIF
ANCA associated small vessel
vasculitides (AASVV)
characterized by the presence of
circulating ANCA
Various ELISAs directed against
various ANCA specificities (including
PR3, MPO, lactoferrin, etc.) are
routinely available
133.
134. LICHEN PLANUS
• DIF
characterized by large, grouped and globular deposits of
immunoglobulins and complement, i.e. civatte bodies(CB)
• The most important findings on DIF favoring the diagnosis of
LP
any immunoreactant deposit at CBs plus shaggy fibrinogen
deposition, whether alone or combined with other immunoreactants
at DEJ
135. • In LP, CBs tend to be more numerous in number, form clusters
or groups of 10 or more in the papillary dermis
• This cluster formation on DIF may be useful in distinguishing LP
from LE (more linear arrangement)
• The positive yield of DIF in LP is in the range of 37–97%
136.
137. Porphyria Cutanea Tarda
• The blisters are subepidermal, with preservation of the dermal papillae in
the floor of the lesion (‘festooning’).
• Hyaline material : PAS positive and diastase resistant, present in the walls
of the small vessels in the upper dermis and sometimes in the basement
membrane of the epidermis
• Usually no inflammatory infiltrate
• DIF : Homogenous deposits of IgG in dermal vessel walls and in BMZ
(90–100%)
