IMMUNOFLUORESCENCE

AARYA.H.NAIR
PG Resident
 CONTENTS
• Introduction
• Definition
• Principle
• Types
• Materials required for carrying out IF tests
• Method of IF testing
• Interpretation
• Quality control
• Applications
• Advantages & disadvantages
• Variants of IF technique
 INTRODUCTION
• Very common laboratory techniques -
diagnostics and research
• Detect specific proteins in cells
• Anciliary diagnostic aid
• Early diagnosis , treatment & subsequent
monitoring of disease activity
• Immunofluorecsence means
– Location of antigen or antibody
– Tissue section or smear
– Pattern of fluorescence
– Exposed to the specific antibody or antigen
– Labeled with a fluorochrome
 DEFINITION
• Various techniques
• For detecting an Ag or Ab in a sample
• Coupling its specifically interactive Ab or Ag
to a fluorescent dye / compound
• Mixing with the sample
• Observing the reaction under an UV – light
fluorescent microscope
• No visible change
• Readily identifiable label must be irreversibly
bound to the antibody so that its localization
can be recognized
 HISTORY
• Gold standard for the diagnosis of autoimmune
blistering diseases

1942: Dr. Albert Coons et al

• 1964: Beutner and Jordon: Indirect

immunofluorescence.
• 1971: Jordon et al. : DIF on lesional and
perilesional skin

• Newer substrates and modified substrate

PRINCIPLE OF IMMUNOFLUORESCENCE
Photoluminescence
•Fluorescence
•Phosphorescence

FLUOROCHROMES
• Substance used by Coons - Beta-anthracene :
blue fluorescence
• Currently :
– Fluosrecein isothiocyanate (FITC) : apple-green
color
– Tetramethylrhodamine isothiocyanate (TRITC) :
red colour
– Phycoerythrin : red fluorescence
FLUORESCENCE MICROSCOPE
– Mercury-vapor or xenon light
source, and appropriate exciter
and barrier filters
• Exciter filter :
– Shed light of necessary
wavelength on the examined
slide
• Barrier filter :
– Stops the exciting photons,
letting through only the
fluorescent light
 Types of fluorescence
• Specific fluorescence
– Reaction between the substrate and the protein labeled
with fluorochrome (antigen-antibody reaction).
• Nonspecific fluorescence
– Coloration of tissues by free fluorescent dye or
fluorescent proteins or both.
• Autofluorescence
– Natural fluorescence of tissues (yellow, blue) when
exposed to ultraviolet light
FACTORS TO BE CONSIDERED
WHILE TAKING BIOPSY
CLINICAL DIAGNOSIS & DENTAL
/ MEDICAL HISTORY
• Communication with the pathologist
• Definitive diagnosis depends on correlation of
clinical findings & medical history with H&E
& DIF

• Eg: Lichen Planus v/s Licenoid Reaction

• Comprehensive history of systemic diseases

• Stop topical steroids at least a month before
the biopsy procedure
 SITE SELECTION
• 2 biopsies:
(1)Lesional / perilesional site with intact epithelium
(2) Adjacent normal tissue

–Avoid areas of blister formation and

ulceration
• Epithelial covering required : diagnosis
based on epithelial connective tissue
junction
• Ideal lesions
– Fresh (less than 24–48 hold), intact, and
nonexcoriated vesiculobullae, with normal or
erythematous perilesional skin included in the
biopsy field.
• Biopsy from Multiple sites - better diagnostic
sensitivity
• H & E biopsy from lesional site adjacent to
DIF biopsy site
PATHOLOGY SITE OF BIOPSY

Autoimmune vesico-bullous Inflamed unblistered perilesional

dermatosis area

Collagenosis Active lesion in evolution (avoid

recent lesions, with less than 60
days)

Vasculitis Recent lesions with up to 24 hours

of evolution

Lupus erythematisus Lesional areas

Dermatomyositis
Vasculitides
Lichen planus
Cutaneous porphyria
Pseudoporphyria
 BIOPSY TECHNIQUE
• Punch biopsy samples
greater detection rate than
scalpel biopsy

• PUNCH BIOPSY : PROBLEMS ASSOCIATED:

– Lack of experience
– Ease with which the
epithelium slides off
– Difficulty on unattached
mucosa & all sites of the oral
cavity
• Wedge – shaped incisional biopsy from
perilesional site bisected
– Large sample
• Biopsy procedure similar for all the mucosal
tissues except for gingiva

• GINGIVA:
– Extending to the bone & must be dissected from
the underlying periosteum
• SITE OF BIOPSY :
– Incisional / punch?

– Palatal & gingival sites : Incisional

biopsy: technique of choice

– Incisional biopsy : elliptical – length

approx. Three times the width
• Length - 4-5 mm length
• Depth – 3-4 mm (include basement
membrane & underlying Connective tissue)
• Orientation markers :
– Excisional biopsy
– To establish if a lesion has been adequately excised
– Placed using sutures
– Not required in Punch / incisional biopsy
 METHOD OF SUBMISSION &
SPECIMEN HANDLING
• H & E BIOPSY
– 10% formalin
– Dehydration in gradations of alcohols
– Cleared in xylene
– Embedded in paraffin
– 5 micron tissue sections cut using microtome
– Mounted on glass slides
– Stained using H&E & examined under light
microscope
• DIF BIOPSY
– Tissue snap frozen in dry ice / liquid nitrogen
– Transported in frozen state

– MEDIUM FOR TRANSPORT:

– MICHEL et al
• Received
• Washed in a buffer solution
• Removed from wash buffer & mounted on a
block by snap freezing on dry ice
• Four- to sixmicron tissue sections
• Two to three sections are placed on each five-
well adhesive coated slide
• Dry at ambient temperature for 20 minutes
• Wash in phosphate buffered saline containing
sodium azide (PBS-A)
• Cover-slipped using a drop of buffered glycerin (90%
glycerin in PBS) and examined under the
fluorescence microscope
• Stained for bound IgG, IgA, IgM, fibrin, and
complement C3 using fluorescein-labeled goat
antihuman conjugates
TYPES & TECHNIQUE
• Direct
• Indirect
• Complement indirect immunofluorescence.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
 DIRECT IMMUNOFLUORESCENCE
• One-step procedure
• Skin biopsy specimen
– Perilesional skin or mucosa
– Uninvolved site should be taken to maximize the
chances of a positive finding.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• Following antisera are recommended:
– Anti-IgG, anti-IgA, anti-IgM, anti-C3 and anti-
fibrinogen

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
INDICATIONS FOR DIF BIOPSY
• Persistent vesiculobullous or erosive or
ulcerative oral lesions : immune-mediated
disease
• By request of the oral pathologist after H & E
biopsy – gold standard for diagnosis of
immune – mediated blistering diseases
• Definitive diagnosis
 INDIRECT
IMMUNOFLUORESCENCE
• 2 step procedure
• Circulating Abs in a patient’s serum
• 10 times more sensitive than DIF
• Patient's blood (5-10 ml) is taken, centrifuged
to extract serum after centrifugation. Serial
dilutions (1:10, 1:20, 1:40…) are made.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• First Step
– Contact between the serum and sections of
appropriate substrate, which contains the
corresponding antigen.
– Tissue substrates : monkey esophagus, guinea-pig
lip/esophagus, etc
– Sections of substrate on glass slides are incubated
with (primary unlabelled) patient's serum for 30
minutes
Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of
Pakistan Association of Dermatologists 2003; 13: 76-88.
• Second Step
– Identical to the staining procedure of the DIF
technique
– Sections are treated with FITC conjugated anti-
IgG, IgA, IgM at 37°C for another 30 minutes.
– After the final three washes (10 minutes each in
PBS), the sections are air-dried, mounted with
buffered glycerol and examined with a
fluorescence microscope.
Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of
Pakistan Association of Dermatologists 2003; 13: 76-88.
 Complement Indirect Immunofluorescence
(Patient’s serum)
• Very sensitive
• 3-step serologic technique
• Circulating antibodies are detected by means
of their high affinity to fix complement.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• (1) Normal tissue substrate is overlaid with
plasma, serum – heated to 56 deg. For 30 mins &
then washed
• To destroy the complement fixing activity of Ag-Ab complexes
• (2) Tissue sections incubated with a source of
complement & washed again
– Activates the complement and generates C3
• (3) Incubated with fluorescien labeled antihuman
Abs , washed again, examined under microscope
– Binds to C3
• Pemphigoid gestationis :
– HG factor is a complement-fixing anti-BMZ IgG
that is present in the sera of patients with PG in
minimal amounts, and is not detectable by the
standard IIF test.
– With complement IIF, HG factor can be
demonstrated in half of the cases.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
VARIANTS OF TECHNIQUE
 SALT SPLIT TECHNIQUE
• Distinguish between subepidermal blistering
conditions with similar DIF
• Normal human serum is incubated in 1 M
NaCl for 48-72 hrs to split it at the level of
lamina lucida
• Bullous pemphigoid : roof & floor of the
blister
• Epidermolysis bullosa : bind solely to the floor
of the split skin
 ANTIGENIC MAPPING
• Adjunct to electron microscopy
• Differentiate between major forms of
epidermolysis bullosa
• Blister induced by rubbing the skin with a
pencil eraser for 1 minute – biopsy – snap
frozen – slides prepared
• IIF – polyclonal / monoclonal Abs used –
dermal-epidermal junction : Bullous
pemphigoid
 SANDWITCH TECHNIQUE
• Appropriate fixed tissue sections are reacted with a
solution of the antigen for which specific antibody is
to be identified in the section
• Incubation & washing
• FITC-labeled antiserum with the same specificity as
that to be identified in the section applied
• The labeled antiserum again identifies the location of
the tissue component
• Term ‘sandwitch’ - antigen is sandwitched
between two layers of the same specific
antibody ,one labeled and one not
MULTIPLE LAYER TECHNIQUE
• Extension of sandwich technique
• Antigen(antibody) made more sensitive by successive layering
of antibodies
• Eg: if mouse antibody (globulin) is to be detected, the
specimen is treated with unlabeled rabbit – anti – mouse
gamma globulin
• This will combine with any mouse globulin that may be
present
• Uncombined antisera are washed off
• Tissue is treated with unlabeled goat anti-rabbit sera which
will combine with the rabbit globulin
 APPLICATIONS OF IF
• DIRECT IMMUNOFLUORESCENCE
– SKIN/MUCOSA
• Bullous pemphigoid
• Pemphigus vulgaris
• Lupus
– RENAL
– PULMONARY TRANSPLANTATION
• INDIRECT IMMUNOFLUORESCENCE
– Pemphigus vulgaris
– Paraneoplastic pemphigus
– Bullous and / or cicatrical pemphigoid
• OTHER FIELDS OF APPLICATIONS
– Amyloid , mucin , lipid
– Bone marrow
– Cell surface markers
– Infective disorders
• Viral (influenza , ebv , adeno virus , herpes virus ,
varicella zoster , cmv and hiv)
• Bacterial ( m. Pneumoniae , m. Tuberculosis ,
t.Pallidum)
• Parasitic (entamoeba,giardia,cyclospora)
INTERPRETATION
• Using fluorescence microscope XCITE 120 bulb
• 4 main features of fluorescence are particularly
important
a) Main site of deposition
b) The class of immunoglobulin or type of
immunoreactant
c) The number of immunoreactants
d) Any deposition in other sites besides the main site
Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of
Pakistan Association of Dermatologists 2003; 13: 76-88.
• i) Deposition of IgG in the ICS only
– Hallmark of pemphigus group of diseases.
– Pemphigus vulgaris, pemphigus foliaceus and their
variants :
• Appears as typical linear deposition on the surfaces of
keratinocytes
• It resembles a "Chicken wire or net-work pattern".
• fluorescence : in suprabasal area in PV and the upper
epidermal area in PF

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
Intercellular space deposition of IgG in Pemphigus
ii) IgA deposition in the ICS
• IgA pemphigus
– Subcorneal pustular dermatosis (SPD)-type :
upper epidermis
– Intraepidermal neutrophilic (IEN)-type : entire
epidermis

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
IgA pemphigus intercellular,intraepithelial
IgA
 Deposition in the ICS and BMZ
• Pemphigus erythematosus and paraneoplastic
pemphigus
• Pemphigus erythematosus:
– BMZ - granular or as a fibrillar band
– ICS: a net-work pattern
• PNP : IgG and C3 are deposited in the ICS
and BMZ

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
Paraneoplastic pemphigus. Linear, intercellular,
intraepidermal and homogeneous, focal IgG in the BMZ
 Basement Membrane Zone
Deposition
• 4 features have to be observed:
• a) the type of immunoreactants
• b) their number
• c) morphologic pattern
• d) deposition present at any other site

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
i) IgG or C3 deposition at the BMZ
• BP, CP, PG, EBA, and bullous LE
• They are present in a continuous, fine and
linear pattern

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
Linear and continuous band of C3 deposit along the
basement membrane zone (BMZ) (DIF) pemphigoid
• Bullous pemphigoid
– Linear deposits of IgG and C3
– If the perilesional skin is not available for biopsy
then anterior aspect of thigh or flexor aspect of
forearm is a suitable alternative site.
• Lichen planus pemphigoides shows C3
deposits at BMZ

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
Lichen Planus: Fibrin deposition along the BMZ extending as
irregular
strands into the superficial lamina propria (DIF)
• Pemphigoid gestationis
– Homegeneous linear deposits of C3 at the BMZ

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• Cicatricial pemphigoid
– Linear deposits of IgG (IgG4 isotype) along with
C3 are seen.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• EBA and bullous LE
– IgG (IgG1 and IgG4 subclasses) is almost always
present, followed by C3 and then IgA
– BMZ deposition : thick, broad and homogeneous
band
EPIDERMOLYSIS BULLOSA ACQUISITA
ii) IgA deposition at the BMZ only
• Dermatitis hepetiformis
– Granular deposition of IgA in the papillary dermis
of uninvolved skin

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• Linear IgA disease
– Linear band of IgA deposition at the BMZ
– IgA belongs to IgA1 isotype and lacks J-chains and
secretory component

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
 Anti-ICS antibodies
i) IgG class
• Pemphigus:
• Circulating IgG anti-ICS antibodies in a chicken
wire pattern are characteristic of PV , PF , PE,
some cases of drug-induced pemphigus and PNP.
– Monkey esophagus is the sensitive and specific
substrate for PV
– Guinea pig tissue (esophagus or lip) is the preferred
substrate for PF.
• Titer used to follow progress and response to
therapy
Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of
Pakistan Association of Dermatologists 2003; 13: 76-88.
ii) IgA Class
• IgA pemphigus:
– IgA anti-ICS antibodies are characteristic of IgA
pemphigus.
– Deposition occurs in the upper epidermis in SPD-
type and entire epidermis in IEN-type.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
 B. Anti-BMZ antibodies
i) IgG class
• BP, CP, PG, EBA and bullous LE

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• Bullous pemphigoid
– Positive result - 70-80%
– Not predictive of disease severity
– Using monoclonal antibodies:
• Serum level of anti-BP180 correlates with the disease
activity and can be used as a guide for therapy.

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• Pemphigoid gestationis
– IgG (IgG1 class) serum anti-BMZ antibodies are
detected in only 20% of patients

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
• Epidermolysis bullosa acquisita
– About 50% of patients with EBA have serum
autoantibodies.
– IIF on salt-split skin shows the dermal pattern of
deposition.
– This is helpful in differentiating between EBA and
BP

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
ii) IgA class
• Linear IgA disease
– Circulating autoantibodies are found in 80% of
children and 30% of adults with LAD.
– IIF on salt-split substrate demonstrates epidermal,
dermal and combined patterns, with epidermal
being the most common

Zahida Rani :Immunofluorescence in immunobullous diseases :Journal of

Pakistan Association of Dermatologists 2003; 13: 76-88.
QUALITY CONTROL
• Potentially infectious
• Universal precautions (gloves)
• Use soap for routine hand washing
• Use biohazard waste disposal guidelines
LIMITATIONS
 PHOTOBLEACHING
• Exposure of fluorescence stained sample to
light causes photochemical destruction of the
fluorescence dye (FITC or some other
fluorophore) and this phenomenon is called
photobleaching.
• In diagnostics photobleaching can result in
erroneous/ wrong result.
 AUTOFLUORESCENCE
• Certain biological structures such as
mitochondria, riboflavin, melanin, elastin and
collagen, etc. fluoresce after absorbing light,
emit light with fluorescence without the
addition of fluorophores like FITC

• Interferes with the reading of test result

ADVANTAGES & DISADVANTAGES
 Advantages of Immunofluorescence
• Relatively simple and reproducible technique
• Short procedure time
• Sensitivity
– Sensitivity of the indirect or complement method
is estimated to be between five and ten times
higher than that of the direct one
– IIF : diagnostic importance + prognostic value,
particularly for pemphigus cases
Disadvantages of immunofluorescence
• Delicate immune reaction
• Careful examination
• Selection of materials
• Skillful observation and judgement
• Specific fluorescence must be distinguished
from nonspecific fluorescence.
 CONCLUSION
• In most cases of vesiculobullous diseases the
definitive diagnosis is based on a combination
of clinical, histologic, immunopathologic, and
sometimes serologic findings.
• In some of the cases the diagnosis may be very
descriptive and inconclusive
• IF serves as a cheap and effective tool in
diagnosis – use of newer substrates may
enhance the sensitivity & specificity.
Thank you...