IMMUNOFLUORESCENCE : TECHNIQUES AND PRESENTATION IN DIFFERENT DISEASES BY DR TAHIR HASSAAN

Anatomy of Epidermis and Dermoepidermal Junction

Epidermal Layers :

stratum corneum stratum granulosum stratum spinosum basal layer Basement membrane zone and Dermoepidermal junction Dermal papillae

Epidermal Target antigen

They are located in the desmosomes Desmosome complex contains o Desmoglein and desmocollin as

transmembranous component o Desmoplakin and plakoglobin as intracellular component

BMZ Target Antigens

Major adhesion complex is Hemidesmosome Intracellular

Keratin filaments ( k-5 n k-14) Plectin BPAG-1 (m.w 230) Sub epidermal BPAG-2 (m.w 180, type VII collagen) Integrins ( alpha-6, B-4) Laminin Type VII collagen

Immunofluorescence
Immunofluorescence is the labeling of antibodies or

antigens with fluorescent dyes.

This technique is used to analyze antigen, antibodies and

their location and distribution.

Immunofluorescent labeled tissue sections are studied using

a fluorescence microscope.
Fluorescein is a dye which emits greenish fluorescence under

UV light. It can be tagged to immunoglobulin molecules.

 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

Direct immunofluorescence

2. Indirect immunofluorescence:

Indirect test is a double-layer technique The unlabelled antibody is applied directly to the tissue substrate Treated with a Fluorochrome-conjugated antiimmunoglobulin serum

Immuno-mapping

 Advantage over Direct IF

Because several fluorescent anti- immunoglobulins

can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test.
However there is also increased risk of non specific

staining resulting in false positive results

PRINCIPLE OF IMMUNOFLUORESCENCE Protein or Protein containing compound is tagged by chemical combination of Protein and a Fluorochrome
FLUORESCENT MICROSCOPE FLUOROCHROME DYE

SPECIAL REQUIREMENTS FOR IF FLUORESCENT MICROSCOPE Exciter and Suppressor filters UV Light source High pressure mercury vapour lamp Iodine quartz lamp Xenon mercury lamp Cadmium lamp FLUOROCHROME DYE Fluorescein Iso Thiocyanate (FITC) Lissamine Rhodamine B 1-Dimethyl-Amino-Naphthalene-5-Sulfonic Acid (DANS) Tetramethyle Rhodamine

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.

PRESERVATION OF FLUORESCENCE
Dry the sections and mount with buffered glycerol. 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.

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

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

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

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 diseases antibodies to one or more components of basement membrane causing blistering lesions

EPIDERMAL ADHESION MOLECULES Cadherins E. Cad & desmosomal cad.

Desmosomal cad - desmogleins and desmocollins

Desmo cad linked to keratin Desmoplakin and plakoglobin

ACANTHOLYTIC LESIONS
Pemphigus Vulgaris Pemphigus Foliaceus PNP Desmoglein 3 &(1) Desmoglein 1 Desmoglein 3 , desmoplakin 1 & 2, periplakin, envoplakin, BPAG-1 ( 230) Desmocollin 1 Desmoglein 1 & ANA

IgA Pemphigus Pemphigus erythematosus

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SITE OF IMMUNE DEPOSITION FOR PEMPHIGUS GROUP Intercellular space (ICS) Pemphigus vulgaris -- IgG and / or C 3 ICS fluorescence uniformly in the epidermis
Pemphigus Foliaceus IgG ICS 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

PV

P FOLIACEUS

IgA PAMPHIGUS

PNP

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 :

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

BP

Linear IgA

SPLIT SKIN TECHNIQUE Splitting of basement membrane through lamina Lucida 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 in the floor

DIF USING SALT SPLIT SKIN To differentiate Pemphigoid from EBA, B SLE Extracellular domain of BP 180 antigen is in the outer 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

BP

EBA

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

DH

DIF IN CONNECTIVE TISSUE DISORDERS Confirms the diagnosis of LE Helps distinguish various sub types Helps in excluding PLE, and other cutaneous lymphoid infiltrates.

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

DLE

FACTS ABOUT DIF IN DLE Lesional skin -- DIF + in 60 to 94% Treated lesions are less positive At least 3wks should pass after stopping medication 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

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 or shaggy pattern
2.PAPILLARY DERMIS -cytoid bodies IgM, A

3.SUPERFICIAL DERMAL Blood vessels 4.EPIDERMAL CELL NUCLEI -IgG

SLE

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

DIF IN SCLE Look along DEJ and speckled cytoplasm of basal keratinocytes 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 cells anti Ro(SS-A) & anti La(SS B)

DIF IN MIXED CONNECTIVE TISSUE DISEASE MCTD shares features of SLE & SS Anti body to Ribonuclease sensitive component of extractable nuclear antigen U1 RNP Characteristic feature is Ig G immune deposits in epidermal 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 positivity therefore not diagnostic.

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 Systemic Sclerosis DIF is negative or nonspecific

Positive DIF pts have overlapping features of SLE or Dermatomyositis

Epidermal cell nuclear positivity may be seen like in MCTD DIF has little or no value in SS, Scleroderma and Morphea

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.

HSP

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

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

LP

DIF IN PEMPHIGUS AND PEMPHIGOID DISEASES SITE IgG IgM IgA C3

PEMPHIGUS IgA PEMPHIGUS P ERYTHEMATOSIS

ICS ICS ICS + BMZ ICS BMZ BMZ BMZ +

+/-

+/+

+/-

P N PEMPHIGUS

+ + + +

+/-

+ ++ ++ +

B PEMPHIGOID C PEMPHIGOID

DIF IN SUBEPIDERMAL BULLOUS DISEASES

DISEASES H GESTATIONIS EBA BULLOUS SLE L IgA DERMATOSIS CBDC PORPHYRIAS DH SITE BMZ BMZ BMZ BMZ BMZ BMZ +BV PAP DERMIS + + + IgG +/+ +60% +50% +33% +40% + + + +100% + +50% IgM IgA C3 ++

REASONS FOR FALSE NEGATIVE D.I.F IMPROPER SELECTION OF CASES FOR D.I.F.(SUBCLINICAL INFLAMMATION OR EARLY BULLA IN THE BIOPSY) IMPROPER TIMING OF SAMPLING THE TISSUE IMPROPER TECHNIQUE. LIMITED PANEL OF ANTISERA / WEAK ANTISERA

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