immunoflorescence in dermatology

7/30/2019 immunoflorescence in dermatology

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BY DR TAHIR HASSAAN

IMMUNOFLUORESCENCE :TECHNIQUES ANDPRESENTATION IN DIFFERENT

DISEASES


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Anatomy of Epidermis and

Dermoepidermal Junction Epidermal Layers :

stratum corneum

stratum granulosumstratum spinosum

basal layer

Basement membrane zone andDermoepidermal junction

Dermal papillae


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Epidermal Target antigen

They are located in the desmosomes

Desmosome complex contains

o Desmoglein and desmocollin astransmembranous component

o Desmoplakin and plakoglobin as intracellular

component


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BMZ Target Antigens

Major adhesion complex is Hemidesmosome

Intracellular

Keratin filaments ( k-5 n k-14)

PlectinBPAG-1 (m.w 230)

Sub epidermal

BPAG-2 (m.w 180, type VII collagen)

Integrins ( alpha-6, B-4)

Laminin

Type VII collagen


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Immunofluorescence

Immunofluorescence is the labeling of antibodies or

antigens with fluorescent dyes.

This technique is used to analyze antigen, antibodies andtheir location and distribution.

Immunofluorescent labeled tissue sections are studied usinga fluorescence microscope.

Fluorescein is a dye which emits greenish fluorescence underUV light. It can be tagged to immunoglobulin molecules.


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There are two ways of doing IF staining

Direct immunofluorescence Indirect immunofluorescence

1. Direct immunofluorescence

Ag is fixed on the slide

Fluorescein labeled Abs are layered over it

Slide is washed to remove unattached Abs

Examine under UV light in an fluorescent microscope

The site where the Ab attaches to its specific Ag will show

apple green fluorescence Use: Direct detection of Pathogens or their Ags in tissues or

in pathological samples


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Direct immunofluorescence


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2. Indirect immunofluorescence:

Indirect test is a double-layer technique

The unlabelled antibody is applied directly tothe tissue substrate

Treated with a Fluorochrome-conjugated anti-immunoglobulin serum

Immuno-mapping


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Advantage over Direct IF Because several fluorescent anti- immunoglobulins

can bind to each antibody present in the first layer, thefluorescence is brighter than the direct test.

However there is also increased risk of non specificstaining resulting in false positive results


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PRINCIPLE OF IMMUNOFLUORESCENCE

Protein or Protein containing compound

is tagged by chemical combination of

Protein and a Fluorochrome

FLUORESCENT MICROSCOPE

FLUOROCHROME DYE


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SPECIAL REQUIREMENTS FOR IF FLUORESCENT MICROSCOPE

Exciter and Suppressor filtersUV Light source

High pressure mercury vapour lampIodine quartz lamp

Xenon mercury lampCadmium lamp

FLUOROCHROME DYE Fluorescein

Iso Thiocyanate (FITC)

Lissamine Rhodamine B1-Dimethyl-Amino-Naphthalene-5-Sulfonic Acid

(DANS)

Tetramethyle Rhodamine


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SKIN BIOPSY FOR DIF

Choice of biopsy is very important.

Biopsy of lesion is not always satisfactory.

Clinically unaffected and immediately

perilesional skin.

Lesional, uninvolved sun exposed,

uninvolved non sun exposed skin.


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PRESERVATION OF FLUORESCENCE

Dry the sections and mount with bufferedglycerol.

View under Fluorescence microscope.

Store slides at 40 C to view later.

Add antifading agents, sodium azide.

Fluorescence remains - 12mths in 92%- 16mths in 49%

- 20mths in 28%

Ideally store for about 11mths.


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MICROSCOPIC EXAMINATION Look for the following:

Site of deposition of immune reactants

Class of immunoglobulin

Identify the most intense staining

Other deposits in other sites


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PATTERN OF IMMUNE STAINING

Uniform or limited to portions of epidermis.

Linear, wavy, tubular, granular or homogenous.

Continuous or discontinuous.

Thick and granular.

Deposits in dermis


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SITES TO EXAMINE

Inter cellular space (ICS)

Basement membrane zone (BMZ)

Dermoepidermal junction(DEJ)

Roof and floor of a salt split skin (lamina Lucida)

Papillary dermis

Blood vessels


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Auto immune Bullous Diseases

The pemphigus group

antibodies to adhesion molecules, desmosomal

proteins of the epidermis, causing functional

interference and acantholysis

The Sub epidermal Bullous diseasesantibodies to one or more components of

basement membrane causing blistering

lesions


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EPIDERMAL ADHESION MOLECULES

Cadherins E. Cad & desmosomal cad.

Desmosomal cad - desmogleins and desmocollins

Desmo cad linked to keratin Desmoplakin andplakoglobin


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Pemphigus Vulgaris

Pemphigus Foliaceus

PNP

IgA Pemphigus

Pemphigus

erythematosus

Desmoglein 3 &(1)

Desmoglein 1

Desmoglein 3 , desmoplakin1 & 2, periplakin, envoplakin,BPAG-1 ( 230)

Desmocollin 1

Desmoglein 1 & ANA

ACANTHOLYTIC LESIONS


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12SITE OF IMMUNE DEPOSITION FOR

PEMPHIGUS GROUP

Intercellular space (ICS)

Pemphigus vulgaris -- IgG and / or C 3ICS fluorescence uniformly in the epidermis

Pemphigus Foliaceus

IgGICS fluorescence mostly in upper epidermis

Ig A Pemphigus Ig A

ICS and BMZ

PNP (ICS) Ig G +/- C 3 & (BMZ) C 3 + Ig G

PE -- Ig G


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PV


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P FOLIACEUS


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IgA PAMPHIGUS


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PNP


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THE SUBEPIDERMAL BULLOUS DISORDERS

Classification - based on inflammatory cells

Sub epidermal blister with little inflammation :EBA & VARIANTS , PCT , TEN , Bullous Drug

Erruption

Sub epidermal blister with Lymphocytes :

PNP and LP Pemphigoides

Sub epidermal blister with Eosinophils :BP , PG , Drugs , Insect bite.

Sub epidermal blister with Neutrophils :


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DIF IN SUBEPIDERMAL BULLOUS DISEASE, BMZ

IgG +/- C3 BP , CP , HG , EBA , B-SLE

If C3 is more intense than IgG BP,HG IgG is more intense than C 3 EBA, B SLE IgG and C3 in mucus membrane than skin - MMP

MULTIPLE DEPOSITS IN BMZ - EBA , B SLE

IgG more intense than c3 , IgA & IgM Both have antibodies to type VII Collagen

Differentiate by clinical & serological features

IgA at BMZ

Linear IgA disease


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BP


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Linear IgA


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SPLIT SKIN TECHNIQUE

Splitting of basement membrane through laminaLucida

Suction blister creation or use sodium chloride ,

trypsin and PBS

Incubate the skin with 1M normal saline at 40c

for 72 hrs

Hemi desmosomes and upper lamina lucida in

the roof Lower lamina lucida and sub-lamina densa inthe floor


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DIF USING SALT SPLIT SKIN

To differentiate Pemphigoid from EBA,

B SLE

Extracellular domain of BP 180 antigen is in theouter portion of lamina lucida(roof).

EBA antigen, type VII collagen is in sub lamina

densa (floor).

DIF on salt split is done only if IIF is -ve


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BP


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EBA


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DIF IN SUBEPIDERMAL BULLOUS DISEASE

DEPOSITS IN BMZ AND BLOOD VESSELS

All types of porphyrias ( PCT).

The reactants are IgG, IgA and c3 In erythropoietic protoporphyrias ,diffuses.

DEPOSITS IN BMZ & PAPILLARY DERMIS

Ig A & c3 granular deposits in DH

PPV is 100% in a well chosen biopsy


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DH


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DIF IN CONNECTIVE TISSUE DISORDERS

Confirms the diagnosis of LE

Helps distinguish various sub types

Helps in excluding PLE, and other cutaneouslymphoid infiltrates.


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DIF IN DLE

Look at DEJ and Papillary dermis

IgG & IgM most frequently in DEJ

IgM & IgA are seen in cytoid bodies

Pattern can be linear, granular or shaggy


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DLE


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FACTS ABOUT DIF IN DLE

Lesional skin -- DIF + in 60 to 94%

Treated lesions are less positive

At least 3wks should pass after stoppingmedication to check for DIF

Lesion 1 month old positivity is 33% Lesion 1yr old positivity is 82%

Best biopsy for DIF should be oldest,

untreated and habitually non exposed skin


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DIF IN SLE

Look at these 4 sites

1. DEJ -- characteristic site, Lupus Band test,Diagnostic finding in Lesional and/or non Lesional

skin, Ig -G, M, A,& C3.with linear, granular orshaggy pattern

2.PAPILLARY DERMIS -- cytoid bodies IgM, A

3.SUPERFICIAL DERMAL Blood vessels

4.EPIDERMAL CELL NUCLEI -- IgG


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SLE


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FACTS OF DIF IN SLE

POSITIVE PREDICTIVE VALUE 64%

NEGATIVE PREDICTIVE VALUE 98%

S.L.E SPECIFIC LESIONAL SKIN D.I.F + 50-100%

NON-LESIONAL, SUN EXPOSED SKIN D.I.F + 73-90%

NON-LESIONAL NOT SUN EXPOSED D.I.F + 68-92%

NON-LESIONAL BUTTOCK SKIN D.I.F + 26-92%

FACIAL SKIN MORE OFTEN + THAN TRUNCAL SKIN


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DIF IN SCLE

Look along DEJ and speckled cytoplasm of basalkeratinocytes

DIF is positive in 18 to 100% of non lesional skin

and in 54 to 100% in lesional skin.

Ig G & Ig M in DEJ, Ig M & Ig A in cytoid bodies

Pattern resembles DLE

Unique to SCLE granular fluorescence through

out the nucleus and cytoplasm of basal cellsanti Ro(SS-A) & anti La(SS B)


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DIF IN MIXED CONNECTIVE TISSUE DISEASE

MCTD shares features of SLE & SS

Anti body to Ribonuclease sensitive component ofextractable nuclear antigen U1 RNP

Characteristic feature is Ig G immune deposits inepidermal cell nucleus and rarely DEJ Positivity range 46 to 100%in nuclei

Positivity in DEJ is 15%

D/d SLE and SS can also show nuclear positivitytherefore not diagnostic.


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DIF IN DERMATOMYOSITIS

Clinical features are usually characteristic

Occasionally indistinguishable from SLE

Histological features resembles SLE or SCLE

Muscle enzyme chemistry helps, Ig A in muscle.

DIF occasionally helps with c 5-9 (MAC) in BV.

DIF IN OTHER CT DISEASES


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DIF IN OTHER CT DISEASES

Systemic Sclerosis DIF is negative or nonspecific

Positive DIF pts have overlapping features of SLEor Dermatomyositis

Epidermal cell nuclear positivity may be seen like

in MCTD

DIF has little or no value in SS, Scleroderma andMorphea


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DIF IN VASCULITIS

LEUKOCYTOKLASTIC VASCULITIS:

Early lesion fibrinogen, C 3, Ig M.

Established lesion - albumin, fibrinogen & Ig G.Late lesion fibrinogen and C 3.

HENOCH SCHONLEIN PURPURA:

Diagnostic is Ig A , not the same as Ig A vasculitis.


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HSP


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DIF IN VASCULITIS

URTICARIAL VASCULITIS :

post capillary venule Ig G & C3

CUTANEOUS PAN:Ig M & C3 in vessel wall.

PITYRIASIS & PLEVA:

Ig M & C3 in BV in papillary dermis and BMZ

AVOID LESIONS FROM LOWER LIMB

NEGATIVE DIF DOES NOT RULE OUT VASCULITIS


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DIF IN LICHEN PLANUS

Histology is diagnostic

DIF is not usually required DIF helps in mucosal LP

DIF helps to differentiate LP & LE Deposits in cytoid bodies (clustered), DEJ Ig M and fibrinogen are most frequent

Cytoid bodies in large numbers, in groups,

larger, highly intense and multiple foci


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LP

DIF IN PEMPHIGUS AND PEMPHIGOID


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DIF IN PEMPHIGUS AND PEMPHIGOID

DISEASES SITE IgG IgM IgA C3

PEMPHIGUS ICS + +/- +/- +/-

IgA PEMPHIGUS ICS +

P ERYTHEMATOSIS ICS + BMZ +

P N PEMPHIGUS ICSBMZ

++ +/-

+++

B PEMPHIGOID BMZ + + +

C PEMPHIGOID BMZ + +


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DIF IN SUBEPIDERMAL BULLOUS

DISEASES

DISEASES SITE IgG IgM IgA C3

H GESTATIONIS BMZ +/- ++

EBA BMZ + +50% +33%

BULLOUS SLE BMZ +60% +40%

L IgA DERMATOSIS BMZ +

CBDC BMZ +

PORPHYRIAS BMZ +BV + + +

DH PAP DERMIS + + +100% +50%

REASONS FOR FALSE NEGATIVE D I F


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REASONS FOR FALSE NEGATIVE D.I.F

IMPROPER SELECTION OF CASES FOR

D.I.F.(SUBCLINICAL INFLAMMATION OR EARLYBULLA IN THE BIOPSY)

IMPROPER TIMING OF SAMPLING THE TISSUE

IMPROPER TECHNIQUE.

LIMITED PANEL OF ANTISERA / WEAK ANTISERA


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THANK YOU

immunoflorescence in dermatology

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