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.
2. INTRODUCTION
ā¢ 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.
ā¢ 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
ā¢ DIF is a oneāstep procedure used to detect and localize
immunoreactants deposited in vivo in the patientās skin or
mucosa.
ā¢ The immunoreactants include antibodies, complement
components and fibrinogen.
4. ā¢ Fluorochromes are dyes that absorb radiation (ultraviolet
light), are excited by it and emit visible light.
ā¢ One of the most used fluorochromes is fluorescein
isothiocyanate (FITC), of green color, and Rhodamine, of
red color.
ā¢ 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. ā¢ Specific fluorescence is due to a reaction between the
substrate and the protein labeled with fluorochrome
(antigen-antibody reaction).
ā¢ Nonspecific fluorescence occurs with coloration of
tissues by free fluorescent dye or fluorescent proteins or
both.
ā¢ 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
ā¢ In autoimmune vesicobullous dermatosis, the best site is
the perilesional region;
ā¢ In collagenosis, the biospy should be done in the active
lesion in evolution (avoid recent lesions, with less than 60
days);
ā¢ In vasculitis, preference should be given to recent lesions
with up to 24 hours of evolution.
8. TECHNIQUE
ā¢ After biopsy, if DIF facilities are not available, it can be
transported in Michelās medium and should reach the lab
within 2 weeks.
ā¢ Composition of Michelās medium:
1. Ammonium sulfate (55 g)
2. Buffer solution (100 ml) with pH 7.2
ā¢ 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
ā¢ In the lab, tissue specimens received in Michelās medium
are washed extensively in phosphate buffered saline
(PBS) to remove ammonium salts and any residual blood
proteins.
ā¢ Then snap-freezing is done as follows:
10.
11. ā¢ After storing in liquid nitrogen, frozen sections 4ā5 Ī¼m in
thickness are cut with the cryotome and placed on slides.
ā¢ These are dried with an electric fan.
ā¢ The slides are then washed in PBS at a pH of 7.4 to
remove surrounding OCT compound.
ā¢ The sections are fan dried once more and incubated with
FITCālabelled antihuman IgG, IgA, IgM, fibrinogen and the
C3 component of complement at 37Ā°C.
ā¢ The sides are again washed in PBS to remove unbound
antibodies, fan dried and mounted in buffered glycerol.
ā¢ They are then viewed with the fluorescence microscope.
TECHNIQUE
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
32. CONCLUSION
ā¢ It is important to understand that immunofluorescence is not a
complete substitute for histopathology but is in fact complementary to
it.
ā¢ 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.
ā¢ A close cooperation with the clinician is essential, who in turn should
select representative and fresh lesions for biopsy.
ā¢ Any break in these simple rules may result in the immunofluorescence
findings being unhelpful or misleading.
33. REFERENCES
ā¢ Rookās Textbook of Dermatology
ā¢ Bologinaās Dermatology
ā¢ Fitzpatrickās Dermatology
ā¢ IADVL Textbook of Dermatology
ā¢ Amer N. Kalaaji, Atlas of Immunofluorescence in Dermatology:
Patterns and Target Antigens, Mayo Clinic Scientific Press, 2006.
ā¢ 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.
Editor's Notes
OCT Compound is a water-soluble blend of glycols and resins that provides a convenient specimen matrix for cryostat sectioning at temperatures of -10ĖC and below.
S.S = Systemic Scleroderma
Changes of LP: cytoid bodies with IgM, IgA, C3, and shaggy BMZ with fibrinogen