Direct Immunofluorescence in Dermatology

This is a PowerPoint presentation of DIF in Dermatology and its clinical importance. This PPT is made by Dr. Jerriton Brewin, 1st year PG in DVL at SVMCH, Pondy.

1. DIRECT IMMUNOFLOURESCENCE IN DERMATOLOGY Dr. Jerriton Brewin 1 St Year PG DVL, SVMCH, Pondy .

2. INTRODUCTION The beginnings of direct immunofluorescence date back to 1942, when Albert Coons et al. showed the labeling of anti-pneumococcal antibodies with fluorescein in the pulmonary tissue. Immunofluorescence was introduced into Dermatology in the 1960s, when Beutner and Jordon revealed through this technique tissue and circulating antibodies in autoimmune vesicobullous dermatoses .

3. DIF DIF is a one‐step procedure used to detect and localize immunoreactants deposited in vivo in the patient’s skin or mucosa. The immunoreactants include antibodies, complement components and fibrinogen.

4. Fluorochromes are dyes that absorb radiation (ultraviolet light), are excited by it and emit visible light. One of the most used fluorochromes is fluorescein isothiocyanate (FITC), of green color, and Rhodamine , of red color. Three distinct forms of fluorescence should be listed in the readings of IF assays: Specific fluorescence Non-specific fluorescence Autofluorescence PRINCIPLE

5. Specific fluorescence is due to a reaction between the substrate and the protein labeled with fluorochrome (antigen-antibody reaction). Nonspecific fluorescence occurs with coloration of tissues by free fluorescent dye or fluorescent proteins or both. Autofluorescence happens due to the natural fluorescence of tissues (yellow, blue) when exposed to ultraviolet light. 

6. SITE OF BIOPSY CONDITION SITE Vesiculobullous Perilesional SLE Lesional skin Non- lesional sun-exposed & non-sun-exposed skin Vasculitis , DLE, LP, Amyloid, PCT Lesional skin

7. SITE OF BIOPSY In autoimmune vesicobullous dermatosis , the best site is the perilesional region; In collagenosis , the biospy should be done in the active lesion in evolution (avoid recent lesions, with less than 60 days); In vasculitis , preference should be given to recent lesions with up to 24 hours of evolution.

8. TECHNIQUE After biopsy, if DIF facilities are not available, it can be transported in Michel’s medium and should reach the lab within 2 weeks. Composition of Michel’s medium: Ammonium sulfate (55 g) Buffer solution (100 ml) with pH 7.2 Buffer solution consists of: 1M Na/K citrate buffer (2.5 ml) 0.1 M MgSO 4 (5 ml) 0.1 M N- ethylmaleimide (5 ml) Distilled water (87.5 ml)

9. TECHNIQUE In the lab, tissue specimens received in Michel’s medium are washed extensively in phosphate buffered saline (PBS) to remove ammonium salts and any residual blood proteins. Then snap-freezing is done as f ollows:

10. After storing in liquid nitrogen, frozen sections 4–5 μm in thickness are cut with the cryotome and placed on slides. These are dried with an electric fan. The slides are then washed in PBS at a pH of 7.4 to remove surrounding OCT compound. The sections are fan dried once more and incubated with FITC‐ labelled antihuman IgG , IgA, IgM , fibrinogen and the C3 component of complement at 37°C. The sides are again washed in PBS to remove unbound antibodies, fan dried and mounted in buffered glycerol. They are then viewed with the fluorescence microscope. TECHNIQUE

11. PATTERNS EPITHELIUM BMZ I ntercellular Fluorescence Nuclear (ANF) Fluorescence Pemphigus group Connective T issue d/o Linear Granular Shaggy Dermal DH Vasculitis Porphyria HSP Pemphigoid g roup, etc. Granular Vascular

12. INTERCELLULAR STAINING PATTERN 1. Pemphigus Vulgaris 2. Pemphigus Vegetans 3. Pemphigus Foliaceus 4. Paraneoplastic Pemphigus 5. Pemphigus Erythematosus 6. IgA Pemphigus

13. INTERCELLULAR STAINING PATTERN 1. Pemphigus Vulgaris 2. Pemphigus Vegetans 3. Pemphigus Foliaceus 4. Paraneoplastic Pemphigus 5. Pemphigus Erythematosus 6. IgA Pemphigus

14. LINEAR BMZ PATTERN IgG + C3 BP EBA MMP LP PEMPHIGO-IDES IF PATTERN IgG + C3 in 90% cases IgG (100%) and C3; occasionally IgA (66%) or IgM (50%) IgG , C3, IgA (60%) IgG and C3 with changes of lichen planus ADDITIONAL FINDING SSS: Epidermal SSS: Dermal SSS: Epidermal/ Dermal Cytoid bodies & shaggy BMZ with fibrinogen

15. BULLOUS PEMPHIGOID

16. BP EBA

17. MMP

18. LINEAR BMZ PATTERN C3 PEMPHIGOID ( HERPES) GESTATIONIS C3 (100% of cases, diagnostic) IgG (approximately 25% of cases)

19. LINEAR BMZ PATTERN IGs, C3, FIBRIN BULLOUS LUPUS ERYTHEMATOSUS IgG and C3; IgM and IgA also (if perilesional biopsy of bullae) Seen in 40%

20. GRANULAR BMZ PATTERN SYSTEMIC LUPUS ERYTHEMATOSUS Granular BMZ pattern for IgG , IgM , IgA, C3 (sun-exposed involved skin >90%; sun-exposed nonlesional skin 50%; non–sun-exposed nonlesional skin 30%) Speckled epidermal nuclei pattern for IgG in 10% to 15%

21. GRANULAR BMZ PATTERN SUBACUTE CUTANEOUS LUPUS ERYTHEMATOSUS Granular BMZ pattern for IgG , IgM , C3 Epidermal/keratinocyte intracytoplasmic particulate deposition with IgG Cytoid bodies for IgM and IgA

22. GRANULAR BMZ PATTERN SCLERODERMA Granular BMZ pattern for IgM (sun-exposed 60%) Speckled epidermal nuclei pattern in 20% Shaggy BMZ with fibrinogen

23. GRANULAR BMZ PATTERN DERMATOMYOSITIS Granular BMZ pattern for IgM , IgG , and C3 (low intensity) Cytoid bodies for IgM and IgA, shaggy BMZ with fibrinogen

24. SHAGGY BMZ PATTERN Seen in: 1 . Lichen Planus 2. Lupus Erythematosus (DLE) and Other Connective Tissue Disorders (Scleroderma) 3. Drug Reactions 4. Photodermatoses Shaggy BMZ pattern for fibrinogen Cytoid bodies for IgM and IgA, occasionally IgG , C3 and fibrinogen

25. DERMAL VASCULAR STAINING PATTERN Seen in: 1 . Porphyria 2. Henoch-Schönlein Purpura 3. Vasculitis

26. DERMAL VASCULAR STAINING PATTERN Dermal vessels: homogeneous thick staining for IgG , ± IgA, C3, fibrin Granular BMZ for C3, IgM Weak, thick linear BMZ for IgG , IgA PORPHYRIA

27. DERMAL VASCULAR STAINING PATTERN Strong dermal vessels with IgA (± other conjugates) HENOCH-SCHONLEIN PURPURA

28. DERMAL VASCULAR STAINING PATTERN Strong dermal vessels with IgM , IgG , C3, fibrinogen VASCULITIS

29. CONCLUSION It is important to understand that immunofluorescence is not a complete substitute for histopathology but is in fact complementary to it. The values of positive or negative immunofluorescence findings are dependent on the experience and skill of the laboratory staff and also on the knowledge of the observer who reports them. A close cooperation with the clinician is essential, who in turn should select representative and fresh lesions for biopsy. Any break in these simple rules may result in the immunofluorescence findings being unhelpful or misleading.

30. REFERENCES Rook’s Textbook of Dermatology Bologina’s Dermatology Fitzpatrick’s Dermatology IADVL Textbook of Dermatology Amer N. Kalaaji , Atlas of Immunofluorescence in Dermatology: Patterns and Target Antigens, Mayo Clinic Scientific Press, 2006. Aoki V, Sousa JX Jr, Fukumori LM, Perigo AM, Freitas EL, Oliveira ZNP. Direct and indirect immunofluorescence . An Bras Dermatol . 2010;85(4):490-9.