Dr Claire Murray

Normal Skin
Perilesional skin
Lesion
Ellipse incisional biopsy helps preserve an intact blister. Punch biopsies are more likely to disrupt the roof
For histology
For IMF

• 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

• 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

• 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

• Most common subepidermal autoimmune bullous disorder
• Typically affects elderly
• Common sites are lower abdomen, groins, legs and arms

• Unilocular, subepidermal blister
• Roof attenuated or normal in early lesions.
May become necrotic in large or older lesions
• Blister contents: fibrin, inflammatory cells

• 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

• Festooning of dermal papillae = preservation of outline dermal papillae
• Severe dermal oedema
• Perivascular eosinophils and histiocytes
• Eosinophilic spongiosis in adjacent epidermis

Homogenous, linear deposition of IgG and/or C3 along the dermoepidermal junction

Direct IMF
Bullous
Pemphigoid
Epidermolysis
Bullosa
Bullous SLE
Linear IgG and C3 Linear IgG and C3 Linear IgG and C3
Indirect IML
Salt-split skin
IgG antibodies
75 – 80%
Roof
IgG antibodies
25 – 50%
Floor
IgG antibodies
60%
Floor

• 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

• 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

• Non-inherited variant
• Onset in mid-life
• Development of noninflammatory bullae after minor trauma
• Extensor surfaces of limbs most affected

• 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

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

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

Lupus Band Test for Lupus erythematosus
• Direct IMF performed on lesional and nonlesional 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 sunexposed skin biopsies from unaffected patients
• Negative IMF can occur in early lesions, treated lesions, lesions from the trunk, when in remission.

• 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

• 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

• 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 sunexposed sites

• 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

• IgG, IgM and C3 outline vessels in the papillary dermis ‘doughnut’ distribution
• Linear deposition of
IgG, IgM and C3 at the dermoepidermal junction.

• Associated with coeliac disease
• Affect all ages
• Lesions on posterior scalp, back, buttocks, backs of arms and legs
• Intensely pruritis, widespread, papulovesicular erruption

• Neutrophilic abscesses within the dermal papillae in early lesions
• Multiloculated subepidermal bullae develop
• Intense neutrophilic inflammatory infiltrate within the blister cavity

• Perilesional skin should be sampled
• Granular deposits of IgA seen in papillary dermis
• Granular-linear pattern may also be seen

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

• 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

• Suprabasal bullae
• Acantholysis
• Dermal papillae project into cavity like villi
• ‘Tombstone’ pattern – layer of basal cells remain attached to dermis

• Acantholytic cells round, eosinophilic & pyknotic nuclei
• Occasional eosinophils
& neutrophils
• Dermal perivascular infiltrate composed of eoinophils and neutrophils

• Intercellular deposition of IgG and C3
• Individual keratinocytes outlined like chicken wire
• Serum antibodies can be demonstrated with indirect IMF using monkey oesophagus

• ‘Sawtooth’ epidermal hyperplasia
• Wedge shaped hypergranulosis
• Civatte and colloid bodies
• Basal vacuolar degeneration
• Band-like lymphocytic infiltrate in papillary dermis

• 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

• LP associated with pemphgoid like blisters
• More in common in men, 4-5 th decade
• Usually preceded by typical LP
• Blisters more common on extremities

• Lichenoid lesions are typical
• Bullous lesions show subepidermal blister
• Cell rich or poor variants both occur

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

• 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