it is a excellent presentation in the field of immunofluorescence microscopy and its utility in dermatology.

1. Immunofluorescence
Microscopy in Dermatology
Dr. Niraj Dhinoja

2. 1 Minute of Meditation

3. Introduction and History:
• Definition: It is a well-established technique used for the detection of a wide
variety of antigens in tissues or on cells in suspension.
• It acts as a valuable adjunct to clinical and histopathological diagnosis, especially in
vesiculobullous and connective tissue disorders.
• Coons developed IF in the 1940s with the blue fluorescing compound, β-anthracene.
• Diagnostic immunopathology in dermatology started in 1963 with the description of
the lupus band test (LBT), i.e. deposits of immunoglobulins and complement at the
dermo-epidermal junction.
• In 1964, Beutner and Jordon used the indirect IF technique to demonstrate antibodies
in the sera of pemphigus patients.

4. Principle:
• IF technique involves viewing of antigen–antibody complexes under
ultraviolet microscope using corresponding antibodies tagged to a
fluorochrome.
Fluorochrome:
• Fluorochromes are compounds containing electrons which when
irradiated with a light of a particular wavelength achieve an unstable
higher energetic state.
• On returning to the ground state as a spontaneous process, they emit
light of a longer wavelength.

5. Simple conjugation process, retention of the antibody activity in the
labeled protein, and stability of the fluorescent conjugate are
prerequisites of an ideal fluorochrome.
Fluorochromes, currently in use:
1. Fluorescein isothiocyanate (FITC) which produces apple-green color
2. Tetramethyl rhodamine isothiocyanate (TRITC) with a red color

6. IMMUNODERMATOLOGICAL METHODS:
1. Direct IF
2. Indirect IF
3. complement fixation
4. Immunoelectron microscopy.

7. Direct Immunofluorescence:
• Single-step procedure
• Demonstrates the antibodies bound in vivo to antigens in the
skin or mucosae.
• A 3–4 mm punch biopsy is optimum for DIF Study to get a
maximum yield
• It is important to take biopsy from an appropriate site.

8. Disease Site Remarks
Autoimmune blistering diseases
(AIBDs)
Perilesional skin May be negative if the
biopsy is taken from lesional skin as
the in vivo-bound
autoantibodies are consumed by
the inflammation.
Vasculitis Freshly erupted purpuric spot in the
most proximal part of the limb
IgA deposits may undergo
degradation in older lesions
Discoid lupus erythematosus (DLE),
amyloidosis, and lichen planus (LP)
Lesional biopsy Oldest, untreated lesion on non-sun-
exposed skin
Systemic lupus erythematosus
(SLE) and other connective tissue
diseases
Two or three biopsies are taken
(lesional/sun exposed and
nonlesional/sun protected skin)
Sun-exposed lesional skin should be
used to substantiate an initial diagnosis
of LE so as to avoid the problem of
false-negative results due to decreased
sensitivity in sun-protected areas.
Porphyria cutanea tarda (PCT) • Biopsy should be taken
preferably from the lesional skin;
a second biopsy from the
perilesional
• Normal skin may be considered,
especially if the patient has an
intact blister.

9. When two biopsies are planned for routine
histopathology and DIF
Which Should be taken first ?

10. Transport Media:
1. Phosphate-buffered saline (PBS)
2. Biopsy sample can be transported to the remote test center in Michel’s
medium (MM) or Zeus media.
MM medium contains:
• Ammonium sulfate
• N-ethyl-maleimide
• potassium citrate buffer, magnesium sulfate
• Distilled water.
• It probably preserves immunoantigenicity of the specimen by its ability
to precipitate macromolecules while inhibiting proteolytic enzymes.
• Immunoreactants may be demonstrable by DIF even at 6 months.

11. 3. Normal saline is also shown as a useful transport medium
if the samples can be shipped to the IF laboratory
within 24 h.
4. Liquid nitrogen

12. Biopsy specimen received in MM is washed in PBS, preferably at 4°C. It is
then oriented and embedded in optimal cutting temperature compound and
snap frozen.
Sections of 4–6 μm thickness are then cut using a cryostat
Two frozen sections are taken in each panel, and there are five such
panels each for anti-IgG, anti-IgM, anti-IgA, anti-C3, and anti-fibrinogen.
Treated with adequately diluted FITC-labeled conjugates (IgG, IgM, IgA,
C3, and fibrin) and incubated for 1 h in a moist chamber at room
temperature.
The sections are then washed in PBS (three washes of 10 min each) and
mounted in buffered glycerol and examined under fluorescent microscope.
PROCESS:

13. Direct Immunofluorescence of Hair
• Outer root sheath of anagen hair is structurally analogous to epidermal
keratinocytes; hence, pemphigus-specific fluorescence pattern can be
demonstrated in the plucked hair.
• Hair is plucked using rubber-tipped artery forceps and approximately five
anagen hairs are chosen.
• They are initially washed with PBS for 10 min following which they are
incubated with the fluorescent-labeled conjugates for 1 h.
• At the end of this process, they are once again washed in PBS before
examining under fluorescent microscope.

14. Interpretation of Direct Immunofluorescence:
Based on the following four parameters:
a. The primary site of immune deposits
b. The type of immune deposit
c. The number of immune deposits, if multiple to identify the most intense
deposits
d. Sites of deposition other than the primary.

16. • C3 much higher intensity than IgG favors pemphigoid group of
diseases
• IgG much higher intensity than C3 favors EBA and bullous SLE
• Multiple deposits at the BMZ favor bullous SLE and EBA over the
pemphigoid group
Serrated pattern analysis
• To distinguish BP from EBA
• EBA typically demonstrates the “u-serrated” pattern, whereas BP
shows “n-serrated” pattern.

18. Lupus Band Test:
• Deposition of immunoglobulins and complements in skin in patients
of LE, demonstratable as linear band at BMZ by DIF.
• Test is considered positive when one or more immuno-reactant (IgG,
IgA, IgM,C3) DEJ.
• 90-95% of patients with SLE/DLE have positive LBT.
• A positive LBT on sun protected non lesional skin provides useful ans
specific criteria for disgnosis.

19. Basement Membrane Zone and Blood Vessel Wall Staining:
DIF microscopy in porphyrias (PCT, pseudo–PCT, and erythropoietic
protoporphyria) is characterized by a homogeneous deposition of
IgG, IgA, and less frequently C3 along the BMZ as well as within
superficial blood vessel walls.
Vasculitis- Exclusively Blood vessel staining, negative DIF does not
exclude vasculitis
Psoriasis:
Psoriatic lesions may demonstrate bright continuous bands of
granular positivity along the DEJ with IgG, IgM, C3 and fibrinogen.

24. Indirect Immunofluorescence
• This test is carried out to detect the circulating autoantibodies in
patient’s serum.
• In addition to diagnosis, IIF titers may correlate with the disease severity
and hence predicts the prognosis and helps to monitor the response to
therapy.

25. • It is a two-step procedure.
• In the first step of IIF, serial dilution of patient’s sera is incubated
with frozen sections of a suitable substrate.
• The second step of this technique is similar to DIF and involves
staining of frozen sections with FITC conjugated IgG ± IgA.
• Monkey esophagus-PV
• Normal human skin (NHS)-PF
• Rat bladder-PNP
• For all AIBDs, salt-split skin is the ideal substrate.
• ANA testing- Hep-2
• Anti DNA- Crithidia luciliae (Protozoan)

26. Serum samples:
• About 3 ml of blood without anticoagulants are collected, and the
serum is separated from the clotted blood.
• For diagnosis of hereditary EB, about 2–5 ml of EDTA blood is
needed for mutation analysis.

29. Antigen Mapping/ Immune mapping
• A modified IIF technique using the patient’s own skin as a substrate
known as immunomapping (antigen mapping) is used to determine the
exact site of cleavage in various forms of hereditary epidermolysis
bullosa (EB).
• Biopsy for antigen mapping is ideally taken from an artificially induced
blister. This can be achieved by mechanically rubbing the skin with an
eraser till faint erythema develops.
• Shave biopsy if preferred over the punch biopsy as traction of punch
may dislodge the epidermis, especially in severe forms of EB.

30. • Frozen sections of patient’s skin are then stained with commercially
available monoclonal antibodies directed against different antigenic
components of BMZ/epidermis such as keratin 5/14 (K5/14),
laminin-332, type VII collagen, and type IV collagen.
• Staining with monoclonal antibodies with respect to the artificial
cleft in the frozen section enables one to subclassify EB.
• For example, staining with all the three antigens (laminin-332, type
VII collagen, and type IV collagen) is seen on the “floor” of the
artificially induced blister in EB simplex, whereas in dystrophic EB,
staining with laminin-332 and type IV collagen is seen on the “roof.”

31. • On IIF of sera, ANCA associated small vessel vasculitis (AASVV)
are characterized by the presence of circulating ANCA.
• Various ELISAs directed against various ANCA specificities
(including PR3, MPO, lactoferrin, etc.) are routinely available.

34. Salt split skin test:
The salt split skin test (SST) is further of two types:
Direct salt split skin test (D-SSST)
Indirect salt split skin test (I-SSST)
• D-SSST is performed on patient’s skin biopsy that is either freshly taken
(from clinically normal appearing ‘patient’ skin) or on the one that has
previously been investigated by routine DIF.
• This allows determining the deposition of in vivo bound autoantibodies
either on the blister roof or on the blister floor or on both sides of the
artificial split within the BMZ.
• A major drawback is the loss of the patient’s skin biopsy sample for
reinvestigation with routine DIF.

35. • For the I-SSST, a sample of NHS is used as a substrate. After
artificially inducing the junctional split, cryocut sections are
prepared and then IIF with
• Patient’s serum is carried out. This test is much more
sensitive than routine IIF and is very helpful in patients where
a biopsy is not available.

37. Use of BIOCHIP mosaic slides
• BIOCHIP mosaic slides has been found to be useful in screening for
autoantibodies in patients with autoimmune blistering diseases (AIBDs).
• These ready-to-use slides are available commercially and contain six
different substrates (monkey esophagus, primate salt-split skin,
recombinant BP180 NC16A, membrane-bound Dsg1 ectodomain, Dsg3
ectodomain, and the C-terminal globular domain of BP230) in a
miniature field.
• Technically, this is a modified IIF, wherein serum from patients with
suspected AIBD is added to these slides and examined under
fluorescence microscopy.

38. • The advantage of this technique is that it is a useful tool to screen
autoantibodies in AIBDs as well as to identify the target antigen. This
technique avoids the need to take frozen sections of a suitable
substrate.
• BIOCHIP mosaic is a simple, standardized, and readily available novel
tool which will further facilitate the diagnosis of AIBDs.
• Validation of the BIOCHIP showed high specificity and high sensitivity
for PV, PF, and BP.

39. Complement fixation:
• This is another type of IIF.
• After the patient’s serum is layered on the substrate, a source of
complement is added.
• Fluoresceinated anticomplement antibodies are then used to
detect the presence of complement in the tissue. This test can
detect small quantities of complement fixing antibodies.

40. Immunoelectron microscopy (IEM):
• It can be performed in an analogous fashion to detect DIF or IIF.
• Instead of fluoresceinated antibodies, the antibodies are labeled with an
enzyme, such as horseradish peroxidase or a heavy metal, such as colloidal gold.
• This test provides subcellular or ultrastructural localization of immunoreactants.
• This technique may be helpful in the differential diagnosis of subtypes of
hereditary EB, where antigen mapping is not significant. There are several
advantages of IF over immunoelectron microscopy.
• IF is a technically simpler and shorter procedure than IEM, and it is more
quantitative and reliable. It is also less costly in terms of technician time and
reagents; however, tests are less permanent than those made with peroxidase
staining. Another major difference is that the biopsy requires specialized cutting
of thin sections for immunoelectron microscopy.

41. Immunofluorescence in Infections :
• In infectious diseases caused by CT and HSV, the microbial agents can be easily
visualized in the smears with DIF and circulating antibodies against these
agents are found by IIF.
• By using the monoclonal antibody conjugates directed against specific antigens
of the corresponding microbial agents DIF highlights
• Cytoplasmic staining in
HSV type 1-infected cells
• Nucleolar staining in HSV
type 2-infected cells,
Elementary bodies in
CT-infected samples

42. Thank you