Basic principles of immunohistochemistry in tissue diagnosis

Speaker: Dr Divya Moderator: Dr A L Hemalatha

INTRODUCTION

IHC is a technique for identifying cellular or tissue constituents by means of antigen antibody interactions.

It involves localisation of the antigens in the tissue sections by the use of labeled antibody through ag-ab interactions that are visualized by a marker such as flourescent dye, enzyme,radioactive element or colloid gold.

In ambiguous areas of histopathological diagnosis, IHC provides a way of identifying substances in tissues. It has emerged as powerful tool which can provide supplemental information to the routine morphological assessment of the tissues.

HISTORY OF IHC
1941-ALLERT H COONS developed immunofluroscence technique ,described new way of visualizing tissue constituents using a ab labeled with flouroscent dye.The first dye to be used was Fluorescein isocyanate [ apple green fluoroscence] 1966-Nakane & Pierce developed enzyme labeling

PRINCIPLE

Specific antigens in the tissues are localized based on the ag and ab recognition . The site of ab is identified either by tagging the antibody directly or indirectly with a visible label.The visual marker may be a fluorescent dye,colloidal metal,hapten,radioactive marker or enzyme.

PREREQUISITES FOR IMMUNOSTAINING

1.The substance to be localized and the tissue itself must be preserved in such a way that the antigenic groups are available for reaction with the applied antibody. 2.The antibodies must be of high affinity and react only with the substances being investigated. 3.They must be fully labeled in order to achieve the maximum impact . 4. The detection system for the label must be efficient.

ANTIGEN-a molecule that induces the formation of an antibody. Bears one or more ab binding sites. These sites are highly specific topographic regions known as epitopes ANTIBODIES- they are immunoglobulins found in blood ,tissue fluids and many secretions. 5 types-IgA,IgD,IgE,IgG,IgM. IgG is the commonest and used in IHC .

-Each ab composed of 2 heavy and 2 light chains. -They are formed in the humoral immune system by the B-lymphocytes after recognition of a foreign antigen.Terminal region of each arm varies in a sequence and is known as variable domain. This variability provides specificity for a particular epitope and enables the ab to bind specifically to ag against which it was raised.

ANTIBODY-ANTIGEN BINDING The amino acid side chain of the variable domain of of an antibody form a cavity which is geometrically and chemically complementary to a single type of antigen epitope. The ag and ab are held together by a combination of hydrogen bonds , electrostatic and vander waals forces.

 Antibody

types:

Poyclonal antibodies: Heterogeneous population of antibodies produced against several epitopes of a single antigen. More sensitive but less specific Monoclonal antibodies: Homogenous population of antibodies for a single epitope. More specific but less sensitive

Polyclonal antibodies are produced by immunizing an animal with a purified specific molecule bearing the antigen of interest . Humoral response to the immunogen numerous clones of plasma cellseach clone produces antibodies to variety of epitope present on the immunogen Antibodies harvested by bleeding the animal to obtain immunoglobulin rich serum

Monoclonal antibodies are produced as follows: .Mouse is injected with a purified antigen produces antibody against it. .When large amounts of antibodies are produced the mouse is sacrificed and spleen containing large amounts of B lymphocyte is removed .B cell suspension is mixed with myeloma cells in a medium that will cause the cell to fuse

.The resultant is a hybridoma.(hybridomafusion of myeloma cells and B cells of the same species) .Hybridoma is tested for the clone antibody against the desired epitope .Required hybridoma is grown in tissue culture, the supernatant fluid contains the antibody produced by the hybridoma

IHC STAINING TECNIQUES

Antibodies cannot be seen with a light microscope/ electron microscope unless they are labeled by some method. Numerous IHC techniques are used to localize and demonstrate tissue antigens. Selection of technique depends on the type of tissue ,type of preparation (paraffin/frozen), degree of sensitivity required.

Antibody labelling methods

1. Immunoenzyme method: Enzyme is used as the label. Chromogen (staining chemical) must be used Peroxidase, alkaline phospatase, glucose oxidase 2. Immunofluorescence method: Fluorochrome AMCA, Fluorescein, FITC, Rodomine, Texas Red. Fluorescence microscope with appropriate filter confocal laser scanning microscope
3. Immunogold method: Colloidal gold particles Usually used in electron microscopy.

IHC METHODS

Direct technique
One step method Labeled antibody reacts with antigen in tissue sections (Label conjugated may be a flurochrome or an enzyme) Advantages: rapid, short and easy Disadvantages: poor signal amplification, hence less sensitive Need to manufacture labeled ab against every ag to be tested

Uses: Demonstration of Ig & complement in frozen sections of skin and renal biopsies.

Direct Method

Labeled Antibody

Tissue Antigen

Indirect / sandwich method

Primary antibody(which reacts with the tissue ag)is not conjugated Uses a labeled secondary antibody directed against the immunoglobulin of species which produced the primary antibody. Horseradish peroxidase labeling is most commonly used with an appropriate chromogen.

Advantages
. More sensitive than the direct method . More versatile method, as a number of primary abs can be used with same conjugated secondary ab.

Two-Step Indirect Method

Secondary Antibody

Primary Antibody

Tissue Antigen

Unlabeled antibody method (enzyme-antienzyme method)

A secondary antibody links primary antibody to an antiperoxidase antibody bound to peroxidase. Peroxidase antiperoxidase method(PAP) Alkaline phosphatase antialkaline phosphatase method (APAAP) Avidin(streptavidin)-Biotin method(ABC)

PEROXIDASE ANTIPEROXIDASE METHOD .Peroxidase antiperoxidase complex(rabbit)-3 peroxidase molecule and 2 antiperoxidase antibodies. .They are bound to the unconjugated primary antibody(rabbit anti human antibody) by a second layer of bridging antibody(swine antirabbit ab).

.Secondary antibody is applied in excess so that one of its 2 identical binding sites binds to the primary ab and other to rabbit PAP complex

Alkaline phosphatase anti alkaline phoshphatase method(APAAP) .Principle is same as PAP method except that PAP complex is replaced by APAAP complex.

Advantages:
Increase in sensitivity,100 to 1000 fold increased signal amplification .

Peroxidase antiperoxidase (PAP)

Alkaline phosphatase anti-Alkaline phosphatase (APAAP)

Avidin-biotin conjugate method(ABC) .Relies on the strong affinity of avidin or streptavidin to vitamin biotin (nonimmunological) .3 step technique1st layer- unconjugated primary antibody 2ndlayer- biotinylated secondary antibody (against species of primary animal) 3rd layer- complex of peroxidase conjugated biotin and avidin

. Biotin cojugate with biological molecule such as antibodies and enzymes. . Avidin has 4 binding sites for biotin. . Free sites of avidin molecule allows binding to biotin molecule on the secondary antibody.

ABC Method
(avidin-biotin complex method )

Disadvantages .Avidin (glycoprotein) has tendency to bind to lectins in the tissue- non specific staining. .Avidin has isoelectric point =10, at neutral pH it is +vely charged hence binds non specifically to vely charged structures. .Tissues rich in endogenous biotin (liver, kidney)will require blocking of endogenous biotin before applying primary antibody by preincubation with avidin.

Labeled Streptavidin biotin method

.Streptavidin is isolated from streptomyces avidinii. .Has higher affinity for biotin. .Has isoelectric pH=7,and not a glycoprotein hence reduced non specific staining.

SP Method
(streptavidin peroxidase conjugated method)

Advantages
Increased sensitivity Allows higher dilution of the primary antibody

Polymeric labeling two step method

Uses unconjugated primary antibody. Followed by secondary antibody conjugated to an enzyme labeled polymer chain. Polymer chain contains a dextran backbone with 70 molecules of the enzyme and 10 molecules of the secondary antibody attached.

Conjugation of both anti-mouse and anti rabbit secondary antibodies enables the same reagent to be used for both monoclonal and polyclonal primary antibodies Advantages .Increased sensitivity .Minimized non-specific background staining .Reduction in the total number of assay steps

Other methods
Immunogold silver staining technique Hapten labeling technique Mirror image compliment antibody labeling technique(MICA) Biotinylated tyramide signal amplification

Tissue preparation in IHC

Good tissue preparation is the corner stone of IHC Steps are same as the routine tissue preparation ,with extra care taken at certain steps. Fixation: Good fixation is a challenge as on one hand the protein structure is intentionally changed to preserve it from degradation and on the other hand it should preserve the position of the ags & their antigenicity.

Neutral buffered formalin is the fixative most commonly used. Cross linkage of proteins formed by the formalin can mask the antigenic site if over fixed. Fixation time, pH, temperature can affect IHC reactions. Formalin should be fresh and buffered to pH of 7 to 7.6

Table 1. Comparison of Tissue Fixatives

Fixative Description Suggested Fixation Times* >8 hrs., 24-48 hrs. often necessary for complete fixation Reported Benefits Reported Disadvantages fixation is slow, may not be complete with shorter times; may not be suited to long- term storage of tissue for ICC; harder specimens; Antigen (Ag) crosslinking often results with need for Ag retrieval; partial or irretrievable Ag disappearance possible; special handling and disposal requirements possible quenching of primary fluorescence; special handling and disposal requirements

Formalin

10% neutral buffered formalin

most common fixative; readily available, penetrates tissue quickly

Zinc Formalin

mixture of zinc sulfate and formalin

4-48 hrs., as little as 46 hrs. for complete fixation

shorter fixation times; minimal need for antigen unmasking or retrieval; preserves better tissue Ag morphology shorter fixation times; better cryostat sections (cold EtOH/acetone); good preservation of cytoplasmic intermediate filaments

Alcohol/Acetone

70% or 95% EtOH; 90% EtOH/10% Acetone

variable; often occurs in tissue processing secondary to formalin fixation; 10-15 minutes for cryostat sections/cytology smears 1-12 hrs.

decreased quality/integrity of ICC staining; shrinking and/or hardening of the tissue

Bouin

mixture of formalin and picric acid

fixes tissues rapidly; useful particularly for endocrine tissues and tumors

decreased preservation of many types of Ags, particularly lipidcontaining Ags; longer fixation may result in brittle tissues; poor penetration may result in under-fixation; special handling and disposal requirements tissue hardening; surface Igs not well demonstrated; special handling and disposal requirements

B-5

mixture of mercuric chloride, sodium acetate, and formalin

1-6 hrs.

primary use in lymph node tissue; enhanced cytologic detail and immunoreactivity of cytologic immunoglobulin (Ig), intracytoplasmic Ags.

Paraffin embedding temperature should be maintained between 56 and 60.c Sections should be mounted on tissueadhesive coated slides sections which are not flat with non adherent ridges are likely to be digested or torn off during antigen retrieval or washing. .Poly-l-lysine .3-aminopropyltriethoxysilane .Electrostatically coated slides

Staining

Antigen retrieval:

. Most antigens are partially or completely masked after formalin fixation due to cross linking of the amino acid group. Prior to immunostaining, sections are pretreated to unmask the antigens. . Methods: -Enzyme epitope retrieval -Heat induced antigen retrieval (HIER) -Combination of HIER & enzyme retrieval

Enzyme epitope retrieval: .Trypsin / chymotrypsin method .Protease method .Pepsin method .Pronase or proteinase method -Period of 5 to 15 min @ 37.c -Enzyme treatment is believed to cleave the protein cross link .

Heat induced antigen retrieval: .Relies on application of heat to tissue sections in an aqueous medium (retrieval solution). .Commonly used retrieval solutions. Citrate buffer at pH 6 EDTA at pH 8 TRIS-EDTA at pH-9 .Degree of immunoreactivity restored depends on the duration of incubation(10 min to 1hr) and attained temperature.

Various methods of heating .Microwave processing .Pressure cooker .Water bath / steamer .Autoclave
Combination of HIER and enzyme retrieval method : .Alternate approach if other methods do not work .Useful when double or triple labeling for 2 or 3 antigens

MCA-497 staining of Liver tissue fixed in NBF for 28 days, no pre-treatment

MCA-497 staining of Liver tissue fixed in NBF for 28 days, with citrate buffer & heat-mediated antigen retrieval

BLOCKING ENDOGENOUS ENZYMES

Endogenous enzymes blocked prior to staining -to eliminate false positive reactions RBCs WBCs and muscle contain abundant peroxidase activity. Blocked by preincubation of section with absolute methanol containing 3% H2O2 . Other methods0.3% H2O2 .with 0.1%Na azide in PBS /TBS Glucose oxidase Acid hematin

BLOCKING ENDOGENOUS ALKALINE PHOSPHATASE ACTIVITY In paraffin sections lost in processing In frozen sections blocked with 1mM concentration levamisole

Section of liver illustrating endogenous biotin resulting in non-specific background staining.

Blocking non specific background staining

Non specific binding results from binding of antibodies(highly charged) to tissue components with reciprocal charge(eg:collagen) Unwanted binding sites blocked before incubating with the primary antibody. Blocking serum consists of dilute serum from the same species used for the production of secondary antibody

Proteins in the serum occupy charged sites on tissue sections. This serum neither interferes nor participates in immunological reactions during staining procedure.

(a) Pan-cytokeratin, mouse thymus. Left: Perithymic mediastinal tissues exhibit profound nonspecific background staining (brown). Right: GSH abolishes nonspecific background retaining thymic epithelial cell labeling . (b) c-myc, ffpe mouse liver. Left: Staining diffusely present in hepatocytes, including in tumor nodule (arrow). Right: GSH coincubation reveals that c-myc from the albumin-promoted transgene is concentrated in

(c) Pan-cytokeratin, ethanol-fixed mouse colon. Left: Nonspecific fluorescent signal (green) in colon wall and mesenteric adipocytes (arrows). Right: GSH coincubation enhances specific fluorescent visualization of colonic epithelium

Antibody standardization
For optimal staining it is necessary to use antibodies at correct dilutions Not the darkest staining but greatest contrast between the specific +ve staining and unwanted background non specific staining

Diluent for antibody - Freshly prepared Tris-buffered saline(TBS) or autoclaved TBS at 4.c -Bovine serum albumin(BSA) 0.1% .Titrations performed using manufacturers antibody titers by using different antigen retrieval methods and buffers .Once ideal method of ag retrieval is identified antibody titer optimized using different dilutions. .Dilution of ab has to be worked with each new batch

Antibody storage and maintenance

.If antibody is concentrated, smaller aliquots made and stored at -20.c after standardizing appropriate dilution .Once aliquot vial is thawed it should not be frozen again, as freeze and thaw cycle results in loss of potency of ab .Use autoclaved tips for making dilutions.

Washing buffers

To prevent formation of ag- ab complexes that will precipitate onto sections -interpretation problems and background staining. It is necessary to remove the unbound ab before incubating the next layer Achieved by washing the sections with TBS between the antibody incubations TBS with surfactant tween 20 is most often used

Chromogens
To visualize the enzymes labelling the antibodies Enzyme substrate reactions - convert colorless chromogens into visible colored end-products 3,3-diaminobenzidine hydrocloride(DAB) Brown end product Insoluble in alcohol or other organic solvents,hence can be subjected to routine dehydration and permanent mounting

3 amino-9ethyl carbazole(AEC) .on oxidation produces rose red end product ,soluble in alcohol .Non alcoholic counter stain like mayers haematoxylin is used. .Aqueous mouting done with glycerol. .AEC is susceptible to oxidation,exposure to light hence dark storage

Other chromogens used .4-chloro 11 naphthol .Hanker-yates reagent .Naphthol AS MX phosphate .Fast red TR .Fast blue BB .New fuschin .Nitro-blue tetrazolium

Control
Positive control: is known to contain ag under question and should be handled in the same fashion as the test tissue Negative control: treated in same fashion as that for the test except for the primary ab which is omitted and replaced with non immune serum or wash buffer. .Used to assess specificity of the method and presence of background staining

Internal control

The ag being tested is present in the adjacent normal tissue. eg: normal breast in case of ca breast for ER/ PR.

Double staining Two or more different antigens can be demonstrated on cells. Involves incubation of section with one set of reagents to stain one ag followed by incubation with second set

erad

Double staining for kappa & lambda of reactive lymph node using sequential horseradish technique & alk phosphatase method

SUMMARY OF STEPS

Deparaffinize /rehydration Heat slides in oven at 65.c for 1 hr Deparaffinize/hydrate:2 xylene washes followed by 100%ethanol rinses ,followed by 95%ethanol, 70%ethanol, 50%ethanol, 30%ethanol, followed by water and TBS for 5 min Antigen retrieval Staining Wash slides with TBST for 5 min on a shaker Inactivate endogenous peroxidase by covering tissue with 3% H2O2 for 10 min

Wash slides 3 times with TBS Block slide with blocking solution for 1 hr Dilute primary antibody Apply primary antibody to each section and incubate overnight in humidified chamber(4.c) Wash slides 3 times with TBST Apply to each sections the diluted secondary antibody for 1 hr at room temperature Wash slides 3 times with TBST

Add freshly prepared DAB substrate to the sections Incubate sections with substrate at room temperature until suitable staining develops(2 -5 min) Rinse sections with water Counter stain with hematoxylin Rinse section with water Dehydrate samples using two rinses with 100%ethanol followed by 2 rinses with xylene Mount coverslip on slide

Fresh Tissue
Fixative

Dehydrated through grades of alcohol (75%-100%) Clear with xylene

Impregnate and then embed in paraffin blocks

The First Steps Are Routine Histopathology

Block cutting

Paraffin ribbon

Mounting on Poly l lysin coated glass slides

Deparaffinize

Destroy endogenous peroxidase Antigen Retrieval Block non-sp binding

Bind Primary antibody

Xylene

PBS

Wash PBS Counterstain dehydrate coverslip examine

Biotinylated 2nd antibody

Wash

Chromogen development

IMMUNOHISTOCHEMISTRY (IHC-FR) - FROZEN SECTIONS

PROTOCOL Frozen sections: Once mounted on APES coated slides, frozen sections are best kept at -80C until needed. When required, warm at room temperature for 5 min. Pre-cool the fixative (acetone, methanol or ethanol) (Abcam recommends starting with acetone) at -20C for 30 min.

Fix with the pre-cooled fixative for 5 -10 min, at room temperature Rinse 3-4 X in PBS. Continue with the immunohistochemical staining protocol. The absence of formalin eliminates the need for an antigen retrieval step. However, if frozen tissue or cytological specimens have been fixed in formalin, antigen retrieval can be attempted although the friable nature of the specimens may compromise the success.

Trouble shooting : Week / No staining

Sources
Inadequate depaffinization

Solution
Longer deparaffinization/ fresh xylene

Inactive antibodies or inactive ABC Replace with a new batch, check reagents for proper storage Ab concentration too low Inadequate ab incubation time Increase the concentration / check for optimal dilution of abs Increase the incubation time

Improper tissue fixation

Tissue overfixed

Increase the duration / try different fixative

Reduce duration/ perform appropriate ag retrieval

Incompatible primary or secondary ab

Inadequate substrate incubation time Incorrect mounting media (AEC) Reagents applied in wrong order

Use secondary ab that interact with primary ab

Increase the time Use correct media Check procedure used

Over staining
High concentration of Proper dilution primary or secondary ab Incubation time too long High incubation temperature Section dried out Reduce the time

Reduce the temperature

Use humid chamber for incubation

High background
Inadequate washing of sections Wash 2 -3 times between steps

Endogenous enzymes Blocking using 3%H2O2 peroxidase, alk in methanol/ levamisole phosphatase in tissue prior to primary ab incubation Endogenous biotin Block agent prior to activity in tissue primary ab incubation

Non specific binding of primary antibody Non specific binding of secondary antibody Diffusion of tissue ags due to inadequate fixation Sections dried out

Use high dilutions of primary antibody Treat with normal serum from same species as secondary antibodies Increase duration of postfixation Use humid chamber

T H A N K Y O U