IMMUNOFLUORESCENCE OF MUCOCUTANEOUS LESIONS

ImmunofluorescenceMicroscopy

• Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes.

• Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.

• Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.

FLUORESCENCE MICROSCOPY

There are two ways of doing IF staining◦ Direct immunofluorescence◦ Indirect immunofluorescence

1. Direct immunofluorescence Ag is fixed on the slide Fluorescein labeled Ab’s are layered over it Slide is washed to remove unattached Ab’s Examined under UV light in an fluorescent

microscope The site where the Ab attaches to its specific Ag

will show apple green fluorescence Use: Direct detection of Pathogens or their Ag’s in

tissues or in pathological samples

Direct immunofluorescence

2. Indirect immunofluorescence:

Indirect test is a double-layer technique The unlabelled antibody is applied directly to

the tissue substrate & Treated with a fluorochrome-conjugated anti-

immunoglobulin serum Advantage over direct IF

◦ Because several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test.

Principles of fluorescence ∎ Light is an electromagnetic wave, characterised by

frequency and wavelength ∎ The electromagentic spectrum: 300m - 3 x 10 -12 m ∎ UV and visible light: 300nm – 750nm

∎ Fluorescence is the result of a 3 stage processExcitation coefficient Excitation Emission

Extinction coefficient is the light capturing ability of thefluorophore molecule (high desired). A=Ecl.∎ Quantum yield is the efficiency of fluorescence ie. Ratio of emitted photons: absorbed photons (high desired)∎ Stoke’s Shift is the difference between energies of thethe absorbed photon and emitted photon.

∎ Implications-□ High Stoke’s shift means greater heat produced□ Low Quantum yield means other non-radiativeprocesses are more likely to occur eg. collisionalquenching, internal conversion, intersystem crossing□ Photobleaching results from reaction with oxygen in excited state. To avoid use low intensity excitation, anti-fade agents, sensitive detection system

Principle of Fluorescence

Fluorescence and phosphorescence are both types of luminescence.When molecules with luminescent properties absorb light, they emit light of a different wavelength. • With fluorescence the emission of light occurs extremely

rapidly after the absorption of excitation light, whereas with phosphorescence emission continues for milliseconds to minutes after the energy source has been removed.

• Fluorescent materials give off light because of their atomic structure.

• Electrons are arranged in discrete energy levels surrounding the atom’s nucleus with each level having a predetermined amount of energy.

• When an electron absorbs the energy from a photon of light (Figure 3) it becomes “excited” and jumps to a higher, less stable energy level.

• The excited state does not last long. The half-life of the excited state is generally less than 10 (8) seconds.

• The electron loses a small amount of energy as heat and the remainder of the extra energy is given off in the form of a photon.

• The emitted fluorescence has a lower energy than the absorbed light, so the wavelength of the emitted light is longer than that of the excitation light (except in the case of multiphoton excitation)

Figure 3

• A range of wavelengths of light can excite the electrons of a fluorochrome. For example, fluorescein will fluoresce when hit by light with any wavelength between 450 nm and 520 nm.

• However, the closer the excitation wavelength is to 495 nm, the more fluorescence will be produced.

• This optimal wavelength is called the excitation peak.

• Similarly, the light produced by fluorochromes has a range of wavelengths.

• The emission of light from fluorescein ranges from 490 nm to 630 nm, and the emission peak is approximately 515 nm.

Since the phenomenon of fluorescence was first explained by a British scientist, Sir George Stokes, in 1852, the shift in wavelength from short to long during fluorescence is called “Stokes shift” (Figure 4).

Figure 4. Excitation and emission spectrum of fluorescein. When fluorescein is excited at a wavelength other than its peak excitation (470 nm in this example),the shape of the emission curve (darker green) remains the same, but the relative intensity is reduced. The efficiency of the excitation at 470 nm is 45% of peak

Some fluorochromes have a small Stokes shift while other fluorescent compounds have large Stokes shifts. For example, the fluorochrome fluorescein can be excited by blue-green light, and its Stokes shift is only about 20 nm, which means that the light emitted is green. This contrasts with another fluorochrome, phycoerythrin, which also can be excited by blue-green light, but has a large Stokes shift. Thus, the light emitted is yellow-orange. In immunofluorescence, a single wavelength can be used to excite several fluorochromes with differentStokes shifts and thereby produce a variety of fluorescent colors asshown in Figure .ImmunofluorescenceExcitation spectrum: BlueEmission spectrum: GreenNucleus

The example in Figure shows a single wavelength at 488 nm(blue line) exciting three different fluorochromes identified bytheir absorption curves on the left of the figure (blue line). Eachfluorochrome is excited at a different efficiency and, therefore,the resulting emission will be at different intensities for equivalentfluorochrome concentrations. Knowing the excitation and emissionproperties of fluorescent compounds makes it possible to selectcombinations of fluorochromes that will work together. However, fora fluorochrome to be useful in a biological application it must attachto or be contained within a structure of biological significance.

Applications of Immunofluorescencein Pathology

Some practical applications of immunofluorescence in diagnosticpathology are: Analysis of antigens in fresh, frozen or fixed tissues; sub-cellularlocalization of antigens in tissue culture monolayers; observationof bacterial or parasitic specimens;

Detection and localization of the presence or absence of specificDNA sequences on chromosomes; and

Defining the spatial-temporal patterns of gene expression withincells/tissues.

• The most important application is in the field of autoimmune.

• It has wide application in demonstration of enzymes, hormones, plasma proteins, cells & its constituents.

• In identifying viral, protozoal, bacterial and parasitic antigen.

• IF methodes also have the potential to define antigen-antibody interactions at the subcellular level, such as detection of antibodies against mitochondrias, microsomes and smooth muscle fibres.

• As well as identifying small cell surface structures such as receptors of lymphocytes.

Immunofluorescence protocol

After the biopsy,

- The specimen can be placed on saline-saturated gauze & transported to the laboratory in a sealed plastic container with ice

- A pH of 7 to 7.2 should be maintain during transport to reduce variable staining

- According to Carson (1997) extensive time in medium can increase Autofluorescence

Freezing of fresh unfixed tissue:

- Tissue, that will not be frozen for several hours is best preserved using a transport medium such as ZEUS or MICHEL medium

- Tissue should be fresh & freezing should be done as rapid as possible using optimal cryostat temperature - Slow freezing can cause ice crystal formation which distorts tissue morphology & antigen binding sites

The frequently used freezing techniques are as follows;

Liquid nitrogen (-190)Isopentane cooled by nitrogen (-150)Carbon dioxide ‘cardice’ (-70)Carbon dioxide gas (-70)Aerosol spray (-50)

Preferably 4um thick frozen sections are produced using cryostat

Preparation of slides:

- The slides should be prepared carefully prior to procedure & can safely be stored for an indefinite period at room temp. in dust free packing

The slides are first thoroughly cleaned as,

Wash slides in detergent 30 min

Wash slides in running tap water 30 min Rinse slides in distilled water 2-5 min

Wash slides in 95% alcohol 3-5 minAir dry 10

min

‘Gelatin Formaldehyde’(1% Gelatin 5ml +2% Formaldehyde 5ml)

is used to avoid section floating off during staining

Coat slides with this adhesive & allow to dry at

37c for one hour before picking up sections

Staining & Incubation Procedures

Direct Immunofluorescence:

- Is used to detect Autoantibodies that are bound to the patient’s tissue

- First the human immunoglobulins are inoculated in a goat which creates the antibodies directed against these immunoglobulins

- The antibodies are produced in the goat in response to human immunoglobulins

- Then these are harvested from the goat & tagged with fluorescein

- A frozen section of patient’s tissue is placed on the slide & this is incubated with fluorescein - conjugated goat antihuman antibodies - These antibodies bind to the tissue at any site where human immunoglobulin is present

- The excess antibody suspension is washed off & the section is viewed with fluorescence microscope

Staining technique:

Slide preparation:

Unfixed cryostat section, air dried, 4 microns thick

Method:

1) Circle the location of the tissue on the back of the slide using a permanent pen; this will assist with identifying the tissue following staining

2) Place air dried slides in Tris buffer pH 7.6 for 5 minutes

3) Tap of and quickly remove excess buffer

4) Place FITC-labeled antibody or negative serum at its previously validated concentration on to the tissue section for 30 minutes 5) Rinse with Tris buffer to remove excess antibody

6) Rinse with deionizer water

7) Coverslip using an aqueous mounting medium

8) Seal the edge of the coverglass with clear fingernail polish or permanent mounting medium

9) Place slides in a slide tray

10) Store the slides in a cool dark place until review

Indirect Immunofluorescence:

- Is a semiquantative procedure in which a double immunolabeling is carried out to evaluate, the presence & titer of circulating antibodies or to specifically localize antigen in the skin - First a frozen section of tissue that is similar to human oral mucosa (monkey esophagus) is placed on the slide & incubated with the patient’s serum

- If there are Autoantibodies directed against epithelial attachment structures in the patient serum

- Then they will attached to homologous structure on the monkey esophagus

- The excess serum is washed off

- Fluorescein conjugate goat antihuman antibody is incubated with section

- The excess is washed off & the section is examined under fluorescent microscope to detect the presence of Autoantibodies that might have been in serum.

Staining technique: (Weller & coons 1954)

Slide preparation:

Unfixed cryostat section, air dried, 4 microns thick

Method:

1) Circle the location of the tissue on the back of the slide using a permanent pen; this will assist with identifying the tissue following staining

2) Place air dried slides in Tris buffer pH 7.6 for 5 minutes

3) Tap of and quickly remove excess buffer

4) Apply unlabeled antibody at its previously validated concentration on the tissue section for 30 Minutes

5) Rinse using Tris buffer pH 7.6 for 5 minutes

6) Tap off & quickly remove excess buffer

7) Apply avidin D for 15 minutes

8) Rinse using Tris buffer pH 7.6 for 5 minutes

9) Tap off & quickly remove excess buffer

10) Apply d-biotin to tissue for 15 minutes

11) Rinse using Tris buffer pH 7.6 for 5 minutes

12) Tap off & quickly remove excess buffer

13) Apply biotinylated horse anti-mouse antibody for25 minutes

14) Rinse using Tris buffer pH 7.6 for 5 minutes

15) Tap off & quickly remove excess buffer

16) Apply fluorescein-streptavidin for 15 minutes

17) Rinse using Tris buffer pH 7.6 for 5 minutes

18) Rinse using deionized water for 5 minutes

19) Coverslip using an aqueous mounting medium

20) Seal the edge of the coverglass with clear fingernail polish or permanent mounting medium

21) Place slides in a slide tray

22) Store the slides in a cool dark place until review

Common dyes: fluorescein, rhodamine Dyes chosen are excited by a certain light

wavelength, usually blue or green, and emit light of a different wavelength in the visible spectrum◦ Eg. Fluorescein emits green light ◦ Eg. Rhodamine emits orange/red light

By using selective filters in a fluorescence microscope only the light from the dye is detected

Available fluorescent labels now include red, blue, cyan or yellow fluorescent proteins

This can be used to detect the distribution of any protein

By attaching different dyes to different antibodies the distribution of two or more molecules can be determined in the same cell or tissue sample

Uses:

CLASSIFICATION:1. Subcorneal split- Bullous impitigo Pemphigus foliacious Pemphigus erythematosus Subcorneal pustular dermatoses Staphylococcal scalded skin syndrome

MUCOCUTANEOUS DISEASES

2. Spinous layer split- Friction blister Hailey hailey disease Ig A pemphigus Epidermolytic hyperkeratosis

3. Suprabasal split - Pemphigus vulgaris Dariers disease Grovers disease

4. Subepidermal basement membrane zone destruction-(a) Lamina dura Epidermolysis bullosa Bullous pemphigoid Cicatritial pemphigoid Dermatitis herpatiformis

(b)Sublamina densa Epidermolysis bullosa dystrophica & acquistica Lineal IgA bullous dermatitis Bullous SLE

5. Subepidermal split- Epidermolysis bullosa simplex Erythema multiforme Oral lichen planus.

Direct Immunofluorescence is used to demonstrate the presence of immunoglobulins ,predominantly IgG but sometimes in combination with C3, IgA & IgM, in the intercellular substance in either the oral epithelium of the lesions or of clinically normal epithelium adjacent to the lesions.

This test is carried out by incubating a biopsy specimen with a fluoroscein-conjugated antiglobulin.

IMMUNOFLUOROSCENT TESTING

Indirect Immunofluorescence is accomplished basically by incubating normal animal or human mucosa with serum from the patient suspected of having the disease and adding the fluorescein-conjugated human antiglobulin.

A positive reaction in the tissue indicates the presence of circulating immunoglobulin antibodies.

DIF testing is very reliable & sensitive diagnostic test for pemphigus vulgaris, in that it demonstrates lacelike IgG in the squamous intercellular/cell surface areas in upto 95% of cases, including early cases & those with very few lesions,& in upto100% cases with active disease.

IIF shows circulating IgG autoantibody in squamous intercellular substance in 80%-90%of cases.

Pemhigus vulgaris

DIF testing of perilesional skin is positive in vast majority of cases. Two pattern of pemphigus antibody deposition have been described. In most cases there is full thickness squamous intercellular substance deposition of IgG. Rarely IgG may be localised only to superficial portion of epidermis.

IIF testing of serum reveals squamous intercellular substance deposition of IgG in 80% to 90% of cases.

Pemphigus foliaceus

DIF testing of perilesional skin reveals squamous intercellular substance deposition of IgG in>70% of cases & granular deposition of IgM & IgG at dermal epidermal junction.

IIF study using monkey esophagus as substrate reveals squamous intercellular substance deposition of IgG in 80% of cases. Antinuclear antibodies are observed in 30%-80% of cases.

Pemphigus erythematosus

DIF testing reveals IgA deposition in squamous intercellular substance throughout the epidermis with increased intensity in the upper layers in some case of subcorneal pustular type. However some case may have both IgG & IgA present & thus may make a specific diagnosis difficult(i.e. pemphigus vulgaris vs. IgA pmphigus).

IIF results are positive in <50% of cases. In SPD(subcorneal pustular dermatosis) type IgA autoantibodies have been shown to recognize desmocollin 1. In IEN(intraepidermal neutrohilic)type dermatosis the autoantigens remain to be identified; the antibodies have been variously characterised as reacting to desmoglein 1 or desmoglein 3 in a subset of patients.

IgA pemphigus

Incubation of normal or patient skin in 1mol/L NaCl results in split of epidermis in the lamina lucida. Pemphigoid antibodies bind solely to the lower aspect of basal keratinocytes(the blister roof) in 80% of cases; in about 20% of cases antibodies bind to both lower basal keratinocytes(the roof)and the superior aspect of the dermis(the blister floor).

When direct salt split skin technique is used in pemphigoid IgG is present on the roof or on the roof & the floor.

Localization to only dermal base is characteristic of EBA (epidermolysis bullosa aquisita) & p-200 pemphigoid.

Salt-split skin IF studies

DIF testing of perilesional skin has shown linear C3 deposition at the dermal epidermal junction in virtually 100% of cases & IgG in 65% to 95%.

IIF studies reveal circulating anti-basement membrane zone IgG antibodies in 70% to 80%. Similarly deposited IgA & IgM are observed in about 25% of cases.

Bullous pemphigoid

Mucous membrane pemphigoid:

- Autoimmune disease with autoantibodies directed against antigens BP180 (BPAg2) & epilligrin (laminin-5) BM appears to detach with the epithelium from underlying connective tissue

- DIF shows Continuous linear band mainly of IgG & C3 at the BM zone, roof pattern of staining

- IDIF shows Positive in only 5% of cases

DIF studies reveal linear IgG & C3 in lesional & perilesional skin in approximately 80% of cases. Occassionaly IgA & IgM are also present.

IIF testing of serum shows circulating antibodies more readily demonstrated when salt-split human skin is used as substrate, in which IgG may be localised only to the roof or, as in antiepiligrin subgroup, to the base of the induced seperation.

Ciccatretial pemphigoid

Systemic lupus erythematosus:

- DIF shows Shaggy, particulate deposits of IgM, IgG or C3 at BM zone & Positive lupus band test - IDIF shows 95% - anti-nuclear antibodies ,double- stranded DNA antibodies, Sm protein antibodies which are very specific

Lichen planus: - DIF shows absence of immunoglobulin deposition in the basal lamina - The fluorescent pattern follows basement membrane & fibrin deposition probably accounts for the eosinophilic thickening of this region on routine staining

- The fluorescence projects downwards in to sub mucosal in to an ‘icicle’ or ‘stalactite’ appearance

- Using IDF a subpopulation of patients has been shown to possess circulating antibodies against cytoplasm of basal keratinocytes

- This phenomenon is more prevalent with lichenoid drug reactions than among

idiopathic drug reactions

DIF reveals IgA along the basement membrane zone in perilesional skin in 100% cases. In lamina lucida type of LAD, IgA antibodies bind to the epidermal side of salt-split skin, whereas in sublamina densa type, such as IgA-mediated EBA, IgA antibodies bind to the dermal side of salt- split skin.

Linear IgA Dermatosis

Examination of perilesional skin using DIF reveals linear deposition of complement at the basement membrane zone in vast majority of cases.

IgG is by far most common immunoglobulin found , but IgA & IgM may be present as well.

IIF reveals circulting anti-basement membrane zone antibodies in upto 50%

The use of salt- split skin technique lead to appropriate diagnosis in most cases. The antibodies in EBA have specificity for the globular carboxyl terminus of typeVII collagen & are deposited beneath the lamina densa. Therefore in salt-split skin studies IgG is on the floor & not on the roof of the split.

Epidermolysis Bullosa Aquisita

EPIDERMOLYSIS BULLOSA

In all reported cases, IgG & C3 are deposited at the epidermal basement membrane zone. The pattern was linear in more than 50% & was referred to as “granular bandlike” in approxymately 25%.IgM &IgA were fluorescent in approximately 50% to 60% of cases, respectively.

IIF study of serum rarely reveals circulating anti-squamous basement membrane zone antibodies that are detected against typeVII collagen.

A salt- split skin preparation using patient serum reveals localisation to the split floor, as in EBA

Bullous Systemic Lupus Erythematous

Presence of granular deposits of IgA within the dermal papillae in both lesional & nonlesional skin. Fibrillary IgA deposits may also be present.

Circulating IgA antibodies that react against reticulin, smooth muscle endomyceum, the dietary antigen gluten, bovine serum albumin, B-lactoglobin may be present.

Using monkey or pig gut as a substrate, IIF has been used to detect antiendomysial antibodies ,which are present in 52% to 100% patient.

Dermatiits Herpetiformis

In many patients with EM, deposits of IgM & C3 are found in the walls of the superficial dermal vessels. Granular deposits of C3, IgM & fibrinogen may also be present along the dermal-epidermal junction.

Nonspecific deposition of immune reactants in apoptic & necrotic keratinocytes may be observed in other disorders with apoptosis, lymphocyte satellite necrosis,& epidermal necrosis.

Erythema Multiforme

In general, IF results are often said to be negative. However, nonspecific granular basement membrane zone staining of C3 alone or in combination with IgM may be found.

Transient Acantholytic Dermatosis (Grover’s Disease)

Advantages

1) It has high sensitivity making possible the demonstration of substances in very low

concentrations or in particles below the resolution of transmitted-light microscope.

2) It can define the antigen-antibody interactions at sub cellular level, such as detection of antibodies against mitochondria, microsomes & smooth muscle fibers.

3) It is useful in identifying small cell surface structures such as receptors on lymphocytes.

4) It has the ability to identify the exact site of antigen-antibody reaction in tissue.

5) It can identify the multiple antigens present on a single cell at a time, hence it is very useful in Research.

Disadvantages

1) The stained section cant be reused, requires fresh section every time.

2) It is very technique sensitive, require a fluorescent microscope.

3) The ultraviolet lamp used in the fluorescent microscope has very short life span , so there is frequent need to change the lamp, also the lamp is too costly

4) Ultraviolet light used in immunofluorescence technique is harmful to eyes.

5) The observed immunofluorescence has to be stored in the form of photographs, as the fluorescence diminishes with time.

6) It is not useful in electron microscope.

7) False positive results can occur, as some substance shows auto fluorescence.

summary

- Immunofluorescence is the adjunctive method for diagnosis of some diseases &

premier method for diagnosis of autoimmune & mucocutaneous diseases.

- For using this technique, complete knowledge of immunology on the molecular

level must be understood

- Fluorescent microscope

-Types: direct & indirect

Class textbook (Human Molecular Genetics 3)

www.antibodystation.com/immunofluorescence-microscopy/

National University of Singapore website www.med.nus.edu.sg/path/services/immunofluor.htm

BD Biosciences website www.bdbiosciences.com

Eversole Skin Histopathology

References:

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