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
1St 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:
1. Specific fluorescence
2. Non-specific fluorescence
3. 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:
1. Ammonium sulfate (55 g)
2. Buffer solution (100 ml) with pH 7.2
• Buffer solution consists of:
1. 1M Na/K citrate buffer (2.5 ml)
2. 0.1 M MgSO4 (5 ml)
3. 0.1 M N-ethylmaleimide (5 ml)
4. 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 follows:

11. • 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

12. PATTERNS
EPITHELIUM BMZ
Intercellular
Fluorescence
Nuclear (ANF)
Fluorescence
Pemphigus
group
Connective
Tissue d/o
1. Linear
2. Granular
3. Shaggy
Dermal
1. DH
2. Vasculitis
3. Porphyria
4. HSP
Pemphigoid
group, etc.
1. Granular
2. Vascular

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

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

16. 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

17. BULLOUS PEMPHIGOID

18. BP EBA

19. MMP

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

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

22. 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%

23. 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

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

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

26. 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

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

29. 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

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

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

32. 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.

33. 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.