Immunofluorescence and skin biopsies

Dr Claire Murray

For IMF

Normal Skin

Perilesional skin

Lesion

For histology

Procedure for biopsy

Ellipse incisional biopsy helps preserve an intact blister. Punch biopsies are more likely to disrupt the roof

Direct Immunoflorescence

• Performed on lesional or perilesional tissue from skin, mucosa or conjunctiva

• Detects in vivo deposition of:– Immunoglobulins (IgA, IgG, IgM)– Complement proteins (C1, C3)– Fibrinogen

• Used for– Autoimmune blistering disorders– Connective tissue disease (SLE, DM)– Vasculitis

Direct Immunoflorescence

• Uses single primary antibody chemically linked to a fluorophore

• Fluorophore = a fluorsecent chemical compound that can re-emit light upon light excitation.

• Antibodies are directly applied to the lesional/perilesional tissue

Indirect Immunofluorescence

• Patient’s serum is tested for antibodies directed towards a defined antigen

• A double layer technique– Primary antibody (within serum) binds to the target antigen

on tissue (monkey oesophagus or similar)– Secondary antibody carrying the fluorophore binds to the

primary antibody as fluorescent label

• Multiple secondary antibodies can bind to single primary antibody providing signal amplification

Where to take the biopsy for DIMF?

• Blistering disorders – perilesional skin– Perilesional skin = normal skin immediately adjacent to a lesion– Immune deposits are degraded in inflamed or blistered skin which

can result in false negative DIF– avoid an ulcer or an area where the epidermis is disrupted– Avoid active lesions

• Connective tissue diseases – lesional skin– For SLE lupus band test x 2 biopsies of lesional and non-sun exposed

normal skin (buttock or inner thigh)– Avoid old lesions and facial lesions

• Vasculitis – lesional skin

How to transport the biopsy material?

• Rinse biopsy in saline• Place in saline soaked gauze• Send unfixed in Michels transport medium– does not fix the tissue– maintains isotonticity and pH of the tissue for around 2

days– stabilises proteins in tissues to allow preservation of

antigenicity and use of immunofluroescence.• Keep in fridge overnight (do not freeze in

uncontrolled manner)

Fluorescent Microscopy

• Tissue sample acts as light source

• Microscope emits high intensity, excitation light

• Fluorophores illuminated by the excitation light (UV light)

• Flurophores emit longer lower energy wavelength light (fluorescent light)

• Fluorescent light is separated from surrounding radiation by filters

• Filters only allow light with same wavelength as fluorescing material through

• The low energy light can be seen against a dark background

• Slides stored in fridge to reduce degradation of immunofluorescence

• Photobleaching (fading) of slides occurs when over exposed to the high intensity light

• Photobleaching can be reduced by reducing the insity of the light or the duration of time the tissue is exposed to the light

Bullous pemphigoid

• Most common subepidermal autoimmune bullous disorder

• Typically affects elderly• Common sites are lower

abdomen, groins, legs and arms

Bullous pemphigoid

• Unilocular, subepidermal blister

• Roof attenuated or normal in early lesions. May become necrotic in large or older lesions

• Blister contents: fibrin, inflammatory cells

Bullous pemphigoid

• Inflammatory (cell rich) blister– Predominant eosinophils– Variable neutrophils and

lymphocytes

• Non-inflammatory (cell poor) blister– Sparse inflammatory

cells– Can be appearance in

very early lesions

Bullous pemphigoid

• Festooning of dermal papillae = preservation of outline dermal papillae

• Severe dermal oedema• Perivascular eosinophils

and histiocytes• Eosinophilic spongiosis

in adjacent epidermis

Bullous pemphigoidHomogenous, linear deposition of IgG and/or C3 along the dermo-epidermal junction

Differential Diagnosis of BPBullous Pemphigoid

Epidermolysis Bullosa

Bullous SLE

Direct IMF Linear IgG and C3 Linear IgG and C3 Linear IgG and C3

Indirect IML IgG antibodies75 – 80%

IgG antibodies 25 – 50%

IgG antibodies 60%

Salt-split skin Roof Floor Floor

Split skin immunofluorescence

• A modified indirect IMF technique

• Normal skin is split to create artificial blister cavity

• Split achieved by immersing in saline

• serum applied to split skin• Antibodies localised to

roof or floor of blister

IgG localised to roof in bullous pemphigoid

Epidermolysis Bullosa

• Group of non-inflammatory skin disorders characterised by development of blisters following minor trauma

• Autosomal dominant or recessive inheritance• Presentation varies depending on the class of

the disease

Epidermolysis Bullosa Acquisita

• Non-inherited variant• Onset in mid-life• Development of non-

inflammatory bullae after minor trauma

• Extensor surfaces of limbs most affected

Epidermolysis Bullosa Acquisita

• Sub-epidermal bulla with fibrin

• Scanty inflammatory cells• Intact roof of blister• Variable inflammatory

infiltrate in dermis• PAS stain demonstrates

the level of split within the BM with most in the roof

Epidermolysis Bullosa Acquisita

Direct IMF: intense deposition of IgG and faint C3 along dermoepidermal junction

Epidermolysis Bullosa Acquisita

Salt-split skin IMF: antibodies bind to the floor of the blister

Lupus Band Test for Lupus erythematosus

• Direct IMF performed on lesional and non-lesional sun-protected skin

• Band-like deposition of IgG, IgM & C3 at dermoepidermal junction in lesional skin

• Epidermal nuclear IgG in small percentage

Lupus Band Test for Lupus erythematosus

• False positive lupus band test in 30% of sun-exposed skin biopsies from unaffected patients

• Negative IMF can occur in early lesions, treated lesions, lesions from the trunk, when in remission.

Bullous SLE

• A rare variant of SLE• Subepidermal blisters• Neutrophils in the papillary

dermis• Lymphocytes around vessels

in the superficial plexus• Linear or mixed

linear/granular deposition of IgG and less commonly IgA and/or IgM along dermoepidermal junction

Bullous SLE

• Linear or mixed linear/granular deposition of IgG and less commonly IgA and/or IgM along dermoepidermal junction

• Salt-split IDIMF shows deposition along floor of blister

Porphyria Cutanea Tarda

• Rare inherited or acquired disease (liver disease

• Defect in enzyme uroprophyrinogen decarboxylase involved in synthesis of haem pathway resulting in accumulation of porphyrins

• Blisters arise on sun-exposed sites

Porphyria Cutanea Tarda

• Subepidermal blister• Cell-poor• Festooning of dermal

papillae• Deposition of hyaline

material in BM and around dermal vessels

• ‘caterpillar bodies’ – hyaline material within epidermis that stains with PAS

Porphyria Cutanea Tarda: DIMF

• IgG, IgM and C3 outline vessels in the papillary dermis ‘doughnut’ distribution

• Linear deposition of IgG, IgM and C3 at the dermoepidermal junction.

Dermatitis Herpetiformis

• Associated with coeliac disease

• Affect all ages• Lesions on posterior

scalp, back, buttocks, backs of arms and legs

• Intensely pruritis, widespread, papulovesicular erruption

Dermatitis Herpetiformis

• Neutrophilic abscesses within the dermal papillae in early lesions

• Multiloculated subepidermal bullae develop

• Intense neutrophilic inflammatory infiltrate within the blister cavity

Dermatitis Herpetiformis

• Perilesional skin should be sampled

• Granular deposits of IgA seen in papillary dermis

• Granular-linear pattern may also be seen

Pemphigus

• Pemphigus vulgaris• Pemphigus vegetans• Pemphigus foliaceous• Paraneoplastic pemphigus• IgA pemphigus

Pemphigus Vulgaris

• Most common (80% cases)

• Middle age onset• Begins in mouth (50%)• Spreads to involve the

skin within weeks/months• Bullae and large and flacid

and rupture easily• Autoantibodies to

desmoglein 3

Pemphigus Vulgaris

• Suprabasal bullae• Acantholysis• Dermal papillae project

into cavity like villi• ‘Tombstone’ pattern –

layer of basal cells remain attached to dermis

Pemphigus Vulgaris

• Acantholytic cells round, eosinophilic & pyknotic nuclei

• Occasional eosinophils & neutrophils

• Dermal perivascular infiltrate composed of eoinophils and neutrophils

Pemphigus IMF (perilesional skin)

• Intercellular deposition of IgG and C3

• Individual keratinocytes outlined like chicken wire

• Serum antibodies can be demonstrated with indirect IMF using monkey oesophagus

Lichen Planus

• ‘Sawtooth’ epidermal hyperplasia

• Wedge shaped hypergranulosis

• Civatte and colloid bodies• Basal vacuolar

degeneration• Band-like lymphocytic

infiltrate in papillary dermis

Lichen planus IMF

• Helps exclude SLE and other bullous disease in difficult cases

• Direct IMF highlights colloid bodies in papillary dermis

• Colloid bodies can stain for IgM and C3

• Irregular band of fibrinogen along basal layer

Lichen planus pemphigoides

• LP associated with pemphgoid like blisters

• More in common in men, 4-5th decade

• Usually preceded by typical LP

• Blisters more common on extremities

Lichen planus pemphigoides

• Lichenoid lesions are typical

• Bullous lesions show subepidermal blister

• Cell rich or poor variants both occur

Lichen planus pemphigoides IMF

Direct IMF Linear deposition of IgG and C3 along dermoepidermal junction

Salt-split skin indirect IMF • Serum contains IgG basement membrane antibody in 50-60%

• IgG labels roof of blister

Leukocytoclastic Vasculitis

• Biopsy lesional skin• Early lesions < 6 hours

old more commonly positive

• Deposition of fibrinogen, C3 and IgM all seen in vessel walls

Henoch-Schonlien Purpura (leukocytoclastic Vasculitis)

• Represents 10% of all cutaneous vasculitis

• Purpuric rash on lower legs

• Histology indistinguishable from other LCV

• Deposition of IgA in vessel walls in involved and uninvolved skin