Bullous Lesions Of Skin & Role Of Immunoflourescence

INTRODUCTION
Blister
Fluid filled cavity formed within or beneath epidermis. Fluid consists of tissue fluid & plasma. Variable component of inflammatory cells Vesicles blisters < 0.5 cm Bullae blisters > 0.5 cm

Approach to bullous lesions

1. Separation plane
Subcorneal beneath the Stratum Corneum Intraepidermal within the epidermis Suprabasal above the basal layer Subepidermal - beneath the epidermis

2. Mechanism of blister formation 3. Type of inflammatory infiltrate

Eg : Porphyria - absent Bullous pemphigoid -- Eos Dermatitis herpetiformis -- neutro Erythema multiforme -- lym

Mechanism of blister formation

Spongiosis Intracellular epidermal oedema separation of keratinocytes Acantholysis Loss of appropriate keratinocyte cell cell contact Reticular Degeneration Balloon degeneration with secondary rupture of keratinocytes Cytolysis Disruption of keratinocytes by physical agents like friction , heat BMZ Disruption D/t primary structural deficiencies & immunological damage

PROBLEMS
1. Separation plane may change as blisters age 2. Microscopic slit like spaces seen in clefting ds.darriers, hailey-hailey 3. Mechanism cannot be assessed on HPE 4. Many subepidermal blisters on HPE strikingly mimic each other (EBA & BP) 5. Cell type infiltrating the lesion may change as lesions age & post therapy

 Bullous diseases often have overlap in the clinical and histologic findings Accurate diagnosis of bullous diseases of the skin requires evaluation of clinical, histologic, and IF findings IF aids in the diagnosis of autoimmune bullous diseases IF findings correlate with disease activity and also carry prognostic significance

Types of Immunofluorescence Techniques

Direct immunofluorescence (DIF)
Indirect immunofluorescence (IIF)
Salt-split technique

PRINCIPLE
Antigen

Antibody

Rabbit antihuman immunoglobulin tagged with FITC

DIF
3 mm biopsy 5 sections using cryostat

Washed in PBS

Washed in PBS for 30 min

Stained with antihuman FITC-conjugated Antibodies with defined specificities for 1 hr

Mounted in buffered glycerol

Examined under fluorescence microscope

IIF
2 step procedure Step 1:

Role in confirming diagnosis Titers correlate directly with clinical disease activity and may be used to follow progress of disease and response to therapy

Diluted with PBS in serial titre (1:10 & 1:80)

incubated with a suitable substrate for 1 hour

Step2:

Stained with FITC, as in DIF

Ideal substrateMonkey Esophagus-PV Guinea pig lip-PE-PF Rat Bladder -PNP

Salt-split technique (BP Vs EBA)

This method relies on splitting normal skin through the lamina lucida (weakest part of BMZ) also exposes the

epitopes on which ab are deposited enhanced sensitivity

of DIF Direct and indirect Specimen is incubated in 1M NaCl for 24-72 hrs and gently teased using a forceps

Sections are cut and stained as for DIF/IIF

Site of biopsy

The immune deposits are partially or completely degraded in inflamed or blistered skin, and DIF may be falsely negative

Normal skin
BP, CP, LAD,EBA, PG

Pemphigus,

DH

INTRA-EPIDERMAL BULLOUS DISEASE

Pemphigus

DG & DC targets for abs

DG 1- Upper layers of epidermis (PF) DG 3 - Deeper layers of epidermis & mucous membranes (PV)

PEMPHIGUS VULGARIS
Older individuals Large & flaccid bullae on a normal or erythematous base Break easily & leave denuded areas Characteristically involve oral mucosa, scalp, midface, trunk and pressure points. Nikolskys sign - application of pressure to a blister leads to its extension.

PEMPHIGUS VULGARIS

Eosinophilic Spongiosis in the lower epidermis Suprabasal bulla Acantholytic cells

Basalkeratinocytes separated from one another remain firmly attached to the dermis. ( row of tombstones )

Blister contains acantholytic cells, neutrophils, eosinophils

Ag- Desmoglein 3 Ab- IgG & C3 DIF-ICS staining in lower epidermal layers IIF- using monkey esophagus: binding of IgG antibodies to the epithelial cell surface

Pemphigus vulgaris
Type of lesion

Hailey-Hailey Dariers Disease Disease Intraepidermal Suprabasal bullae clefts

Grovers Disease Suprabasal epidermal acantholysis Spongiotic No No Eosinophilic

Intraepidermal bullae

Adjacent epidermis Adnexal involvement Corps ronds & grains Dermal inflammation

Intact Yes No Mononuclear, eosinophilic

Disintegrating No No Mononuclear

Intact Yes Yes Mononuclear

IF

Positive

Negative

Negative

Negative

PEMPHIGUS FOLIACEUS
Middle aged Flaccid bullae on erythematous base Bullae break easily leaving behind shallow erosion Erythema, oozing & crusting present Oral lesions do not occur Nikolsky sign +ve

Subcorneal blister with acantholytic Cells & neutrophils

Subcorneal bulla with few acantholytic keratinocytes & dyskeratotic granular keratinocytes

Ag- desmoglein 1 Ab- IgG & C3 DIF- ICS staining in upper epidermal layers

D/D :
SSSS : IF helpful, small no. of acantholytic cells in SSSS also Impetigo : P .F -> superinfection , IF helps Subcorneal pustular dermatoses : Main lesion is Pustules

PEMPHIGUS ERYTHEMATOSUS
Erythematous plaques & patches in butterfly fashion
Frequent erosion of blister roof, acantholytic layer ICS+BMZ (IgG+C3) Granular deposition of IgM &IgG at dermo-epidermal junction(+ve lupus band test)

Paraneoplastic Pemphigus
Autoimmune vesiculobullous & mucocutaneous disease Associated with underlying malignancy (MC- NHL, CLL) Polymorphic cutaneous lesions with involvement of multiple organs

 Variable features Erythema multiforme like, ( cell poor interphase dermatitis ) Lichen planus like, (cell rich lichenoid dermatitis)

Pem vulg like ( suprabasal bullae )

Pemphigoid like pattern Suprabasal acantholysis

 Ag -Desmoplakin +230KD, Bullous pemphigoid ag Ab- IgG + C3 DIF- Linear/granular deposits BMZ+ ICS It is not unusual for deposition in PNP to be faint or appear Antibodies bind to columnar, transitional & simple epithelia in addition to stratified squamous epithelia So, urinary bladder can be used as a substrate for IIF- DEFINITIVE DIAGNOSTIC TEST

Ig A pemphigus
Middle aged & elderly Pruritic pustular eruptions ,annular arrangement Mild leucocytosis , eosinophilia & Ig A kappa paraproteinemia M.C sites axilla & groin

IF - Ig A deposition in the squamous intercellular subs through out the epidermis IEN Type
Intraepi vesiculopustules / pustules with small no . of neutro

SPD Type
subcorneal vesiculopustules with num neutrophils

EPIDERMIS/ICS

IgG IgG+BMZ

IgA

Superficial epidermis

Lower epidermis

Paraneoplastic pemphigus (FAINT)

IgA pemphigus

P.Foliaceous

P.Vulgaris

Pemphigus erythematosus

SUBEPIDERMAL BULLOUS DISEASES

Basal keratinocyte, hemidesmosome

Delicate anchoring fibres connecting hd in basal keratinocytes to L.Densa

BP Ag2

Type 4 collagenStrength ANCHORING FIBRIL- type 7 collagen

BULLOUS PEMPHIGOID
Elderly Multiple tense bullae on normal or erythematous skin Erythematous macules,urticarial plaques & crusted erosions Chronic subepidermal blistering Oral lesions in 1/3 cases Nikolsky sign negative Trunk, extremities & intertriginous areas

Pathogenesis:
Pemphigoid antibodies binds to 2 antigens , BPAg1 & BPAg2. Activation of complement cascade and release of inflammatory mediators Lamina Lucida separation due to injury to keratinocytes, disruption of hemidesmosomes & proteolysis

Ag BP 180 & 230 (hemidesmosome & lamina

Cell rich blister Eosinophil rich infiltrate

lucida), DIF- linear IgG +C3 with active disease- pemphigoid band, IIF- Human skin

Bullous Pemphigoid Tense bullae Harder to rupture Nikolsky sign ve Oral involvement 33%

Pemphigus Vulgaris Flaccid bullae Easy to rupture Nikolsky sign +ve Oral involvement 95%

HPE: Subepidermal bullae No acantholysis IF: Linear IgG at dermo epidermal junction

HPE: Intraepidermal bullae Acantholysis seen IF: Intercellular IgG

HERPES GESTATIONIS
1 in 50,00 pregnancies

2nd/3rd trimester of pregnancy

Papules or urticarial plaques localized to periumbilical region which spread to trunk & extremities Subside within several days or weeks of delivery

Focal necrosis of basal keratinocytes -> subepidermal blister papillary dermal edema Perivascular lym / eos infil Spongiosis+/-

Ag-BP 230, BP 180 Ab-C3 DIF-Deposition of complement in linear pattern along BMZ Deposition of C3 with significantly higher intensity than IgG strongly favours BPand HG.

CICATRICIAL PEMPHIGOID
Chronic course , scarring & prediliction for mucosal surfaces Elderly, males Oral blisters -- in all cases Scarring commonest in conj Face, neck, Upper trunk - usual cutaneous sites Associations- Thymoma, Ca. Pancreas, Ca.Stomach, Ca.Lung, RA, SLE and ingestion of Practolol and Clonidine.

Pathogenesis: Autoantigens Include1. Epiligrin or Laminin 5 2. 4 subunit of 64 Integrin 3. BPAg2 & BPAg1 All antigens occur within the Lamina lucida. Autoantibodies target multiple extracellular (C-terminal) domain of BP 180.

DIF : linear Ig G & C3 in lesional & perilesional skin at dermo epidermal junction

LINEAR IgA BULLOUS DERMATOSES

Group of bullous disorders Mediated by Ig A antibodies & Differ in specificities for epidermal BMZ Ags

1. 2. 3.

3 variants Adult linear IgA bullous dermatitis Chronic bullous dermatoses of childhood Drug-associated Linear IgA bullous dermatoses

Adult linear IgA bullous dermatoses Over 40 yrs, females vesicles & bullae as in DH ocular & oral lesions - 50 % Palmar & plantar bullae Associations:Sarcoidosis,Ulcerative colitis,RA ,Lymphoma, Immune complex GN,CRF & Psoriasis.

Adult linear IgA bullous dermatoses

Uniform neutrophil infilt along D.E junction Papillary microabscesses - less common Eosinophils predominant in drug related cases

Adult linear IgA bullous dermatoses

DIF: Linear IgA deposition along the basement membrane zone in perilesional skin

Pathogenesis: Antigen in lamina lucida type is 97kd protein (Lacidin) or 120kd protein (LAD-1)

Dermatitis Herpetiformis
Intensely pruritic, chronic recurrent dermatitis, slight male predilection

Young middle aged

Symmetrical grouped erythematous papulovesicles on extensor surfaces Associated with gluten-sensitive enteropathy

Dermal papillary microabscesses

Ag- epidermal transglutaminase Ab- IgA DIF- Granular deposition of IgA and C3 in the papillary dermis and along the BMZ is diagnostic of DH

Bullous SLE
Vesicles & bullae may develop in patients with SLE
IgG & C3 deposition at BMZ - If the intensity of IgG deposition at the BMZ is significantly higher than that of C3, EBA and bullous SLE are more likely than pemphigoid

EPIDERMOLYSIS BULLOSA ACQUISITA

Mid-adult life, Chronic course Non-inflammatory bullae on acral area Antibodies to type VII collagen
Classic form or noninflammatory type Subepidermal blister Fibrosis & milia formation BPlike presentation: Inflammatory sub epidermal blisters, Neutrophils & eosinophils in the blister cavity

DIF-Intense IgG band at BMZ; other components are less intense than IgG

DIF using salt-split specimens

The combination of clinical, HP and IF findings may occasionally be inconclusive in the differentiation between the pemphigoid group of diseases

Roof binding

Floor binding

Deposition at the BMZ and blood vessel walls

Homogeneous deposition of immunoreactants (usually multiple) within superficial dermal blood vessel walls, in addition to BMZ deposition, is characteristic of PCT The most frequent immunoreactants are IgG and IgA

BMZ

+ VESSEL (IgG& A) PORPHYRIA

IgG/C3 Linear, wavy,tubular & granular

Multiple immunoreactants

IgA

Homogenous thick & broad

Granular linear

C3>IgG

IgG>C3 EBA BULLOUS SLE

BP HG CP

DERMATITIS HERPITIFORMIS

IgA bullous ds.

Histopathology Suprabasal

Table III. Algorithm for the diagnosis of autoimmune bullous diseases DIF IIF Diagnosis 1. IgG C3 at ICS IgG at ICS, monkey PV esophagus 2. IgG C3 at ICS + BMZ IgG at ICS, rat bladder PNP
1. IgA at ICS 2. IgG C3 at ICS IgA at ICS IgG at ICS IgA pemphigus PF

Subcorneal

Subepidermal noninflammatory

1. IgG, C3 IgM, IgA at BMZ

1. Dermal side of SSS 2. Epidermal side of SSS

EBA BP PCT, pseudo-PCT BP, HG, mucosal pemphigoid DH

Subepidermal with eosinophil-rich infiltrate Subepidermal with neutrophil-rich infiltrate

2. IgG, IgA IgM, C3 in blood vessel walls C3, IgG at BMZ 1. Granular IgA in dermal papillae and BMZ 2. Linear IgA C3, BMZ 3. IgG, IgM, C3, IgA, fibrinogen

Negative Epidermal side of SSS Negative on epithelium (+antiendomysial antibodies)

IgA at BMZ 1. Dermal side of SSS 2. Dermal side of SSS and positive lupus serology EBA, rare antiepiligrin disease Bullous SLE

LAD

References
Levers Mckee Immunofluorescence in dermatology: J Am Acad Dermatol 2001;45:803-22