1. Immunofluorescence and skin biopsies
Dr Claire Murray

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

3. 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
Monoantibodies specifc for IgA, IgG, IgM, C1, C3 and fibrinogen are applied directly to the tissue sections from lesional/perilesion skin. Very similar to immunohistochemistry but the here the antibodies are labelled with a fluorphore

4. 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

5. 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
Indirect immunofluorscence is more sensitive

6. 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

7. 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)

8. 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

9. 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

10. Bullous pemphigoid
Most common subepidermal autoimmune bullous disorder
Typically affects elderly
Common sites are lower abdomen, groins, legs and arms

11. 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

12. 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

13. Bullous pemphigoid
Festooning of dermal papillae = preservation of outline dermal papillae
Severe dermal oedema
Perivascular eosinophils and histiocytes
Eosinophilic spongiosis in adjacent epidermis

14. Bullous pemphigoid
Homogenous, linear deposition of IgG and/or C3 along the dermo-epidermal junction

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

16. 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
Pathogenesis of BP -
IgG localised to roof in bullous pemphigoid

17. 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

18. Epidermolysis Bullosa Acquisita
Non-inherited variant
Onset in mid-life
Development of non-inflammatory bullae after minor trauma
Extensor surfaces of limbs most affected

19. 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

20. Epidermolysis Bullosa Acquisita
Direct IMF: intense deposition of IgG and faint C3 along dermoepidermal junction

21. Epidermolysis Bullosa Acquisita
Salt-split skin IMF: antibodies bind to the floor of the blister

22. 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

23. 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.

24. 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

25. 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

26. 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

27. 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

28. 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.

29. 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

30. Dermatitis Herpetiformis
Neutrophilic abscesses within the dermal papillae in early lesions
Multiloculated subepidermal bullae develop
Intense neutrophilic inflammatory infiltrate within the blister cavity

31. Dermatitis Herpetiformis
Perilesional skin should be sampled
Granular deposits of IgA seen in papillary dermis
Granular-linear pattern may also be seen

32. Pemphigus Pemphigus vulgaris Pemphigus vegetans Pemphigus foliaceous
Paraneoplastic pemphigus
IgA pemphigus

33. 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

34. Pemphigus Vulgaris Suprabasal bullae Acantholysis
Dermal papillae project into cavity like villi
‘Tombstone’ pattern – layer of basal cells remain attached to dermis
Pemphigus blisters rupture easily so it is important to biopsy an early lesion. Acantholysis occurs due to damage to the intercellular bridges

35. Pemphigus Vulgaris
Acantholytic cells round, eosinophilic & pyknotic nuclei
Occasional eosinophils & neutrophils
Dermal perivascular infiltrate composed of eoinophils and neutrophils

36. 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

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

38. 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

39. 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

40. Lichen planus pemphigoides
Lichenoid lesions are typical
Bullous lesions show subepidermal blister
Cell rich or poor variants both occur

41. 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

42. Leukocytoclastic Vasculitis
Biopsy lesional skin
Early lesions < 6 hours old more commonly positive
Deposition of fibrinogen, C3 and IgM all seen in vessel walls

43. 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