Immunohistochemistry

Contents
PART 1

Basics -history -principle -procedure -quality control -selective use of monoclonal antibodies

Immunohistochemistry is a molecular technique that combines principles from both immunology and biochemistry,to the study of histology and pathology by revealing molecules and patterns within cells and tissues.

It is a process of localizing proteins in a tissue section exploiting the principle of application of labeled or enzyme bound antibody to identify specific antigen which is visualized under light microscopy by means of a color indicator.

TERMINOLOGIES

Immunohistochemistry Immunocytochemistry Immunophenotyping Cytogenetics Flourescent InSitu Hybridization

why IHC ??

H&E Vs IHC

H&E Principle : affinity for acidic and basic groups. Procedure : basic dye hematoxylin, colors basophilic structures with blue-purple hue, and alcohol-based acidic eosin Y, colors eosinophilic structures bright pink.

IHC Principle : formation of Ag-Ab complexes Procedure : Many antigens hidden. Retrieves Ag exposes via a color reaction.

Approach : diagnostic

Approach :diagnostic, therapeutic response, prognostic

MILE STONES

The first to describe immunohistochemistry was Dr.Coons.

Albert Hewett Coons June 28, 1912 September 30, 1978 By Hugh O. McDevitt

ALBERT COONS, PROFESSOR IN the Department of Bacteriology and Immunology at Harvard Medical School and a member of the National Academy of Sciences since 1962, died September 30, 1978, at the age of sixty-six. He was born in Gloversville, New York, on June 28, 1912, the son of Albert S. and Marion (Hewett) Coons. He was educated in Gloversville public schools, graduated from Williams College in 1933, and received the M.D. degree from Harvard Medical School in 1937.

The original immunohistochemistry method antibody tagged with a fluorescent probe which was developed in rabbits, which was mixed with tissue sections and searched for, under a fluorescent microscope following a period of incubation.

Avrameas et.al E labeling to demonstrate ag-ab complexes. Taylor & Burns IHC to formalin fixed ,paraffin embeded tissues. Sternberger unlabelled Ab(PAP)method Heitzman & richards ABC method Kohler & milstein one step direct conjugate,multistep indirect method.

Samples
Tissue sections -formalin fixed paraffin blocks -frozen section -cell block Cell smear FNAC smear

PROCEDURE

1.Tissue processing -fixing -embedding -section cutting 2.Antigen retrieval 3.Blocking 4.IHC staining method -incubation with primary antibody -incubation with secondary antibody-E complex -incubation with substrate 5.Mount and observe.

1.TISSUE PROCESSING

Relavent points-no particular optimal fixative -staining results fixatives Ph osmolarity temperature length of treatment tissue types 10%NBF,10%zinc formalin,10%formal saline good Ag preservation 10%formal acetic acid,Bouins fixative poor Ag preservation

2.ANTIGEN RETREIVAL
Most crucial step Process of breaking down the protein cross link. Unmask the hidden Ag sites.

Enzyme proteolytic enzyme retrieval trypsin (glucagon in rectal tumors) pepsin protease neuraminidase (myelin)

Heat heat induced epitope retrieval (most popular) microwave pressure cooker water bath steamer autoclave Different temp for different antigens Eg- ER-PR :95deg

Heat
Deparaffinised slides placed in plastic coplin jars containing AR solution Heated in a microwave for 10min(95-120 deg) Washed in phosphate buffer solution for 5min Stained.

AR solution???

AR solution
Distilled water Various buffer solution

Major influencing factors: time temp Ph

3.BLOCKING
To block/inhibit the endogenous tissue components from leaking out background staining false positive results. Endogenous peroxidase Endogenous biotin Endogenous avidin

Procedure
Must be done before incubation with primary antibody. Incubate sections in avidin (egg white)solution /biotin(skimmed milk) 15min Followed by brief rinse in phosphate buffer solution Peroxidase is blocked by the pretreatment with H.peroxide.

4.IHC STAINING

Direct
Indirect

Direct method
Application of monospecific Ab directly onto the tissue sections.

Adv: short and quick Disadv: not enough for visualization less sensitive To enhance/amplify: Ab+biotin+labelled avidin/streptavidin.

Streptavidin
Derrived from Streptococcus avidini. Uncharged does not bind to other elctrostatic tissues. No carbohydrate groups- does not bind to tissue lectins.

Signal amplification in IHC

C.A.R.D (catalyzed reporter deposition) biotinylated tyramine

horseradish peroxidase
free radical formation radicalised biotinylated tyramine Covalently binds electron rich prt mol (tryp,his,ph.al)-near the antibody site. More deposition

signal amplification

Horseradish (Armoracia rusticana, syn. Cochlearia armoracia) is a perennial plant of the Brassicaceae family, which also includes mustard, wasabi, and cabbages. The plant is probably native to southeastern Europe and western Asia, but is popular around the world today. It grows up to 1.5 metres (five feet) tall and is mainly cultivated for its large white, tapered root.

The enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.

Alone, the HRP enzyme, or conjugates thereof, is of little value; its presence must be made visible using a substrate that when oxidized by HRP using hydrogen peroxide as the oxidizing agent, yields a characteristic change that is detectable by spectrophotometric methods.

Numerous substrates for the horseradish peroxidase enzyme have been described and commercialized to exploit the desirable features of HRP. These substrates fall into several distinct categories. HRP catalyzes the conversion of chromogenic substrates (e.g. TMB, DAB) colored molecules while the HRP products of chemiluminescent substrates (e.g. SuperSignal, ECL) generate light.

Indirect methods (Sandwich method)

-labelled

indirect Ab

technique -unlabelled indirect antibody technique.

Labelled IAT
Application of unlabelled primary antibody to the target Ag and the labeled secondary ab reacts with primary Ab.

Unlabelled IAT
Uses three layers of antibody. Third antibody is anti- enzyme Ab against, -peroxidase -alkaline phosphatase -glucose oxidase Add-chromogen Enzymes covert the substrate(chromgen) specific colour.

Markers
Biotin
Flurochromes-

flourescein isothiocyanate(FITC) Colloidal gold Enzymes

enzyme
Substrate
unlabelled ter.Anti-E Ab

Coloured substance

unlabelled sec anti Ig Ab

Antibody against the same animal sps Recognises the epitopes of both prim & sec.

Primary unlabelled Ab

Raised in animal sps

Ag

Ag

Tissue section

Advantages
Unlabelled IAT(3step) more sensitive Polyvalent secondary Ab can detect multiple sites on the Fc and Fab portions of the prim and teritiary Ab Primary Ab is more accessible to the sec Ab then to the Ag (modified by fixation & embedding)

Recently used techniques

Peroxidase-anti peroxidase(PAP) Avidin-biotin-complex(ABC) Biotin-streptavidin complex Polymer technique.(newer technique) Transcription factors

ImmunoEnzyme systems
Immunoenzymatic staining of tissue results from a reaction of a soluble substrate producing an insoluble colored product. Intensity of the color = concentration of the primary Ab+Ag complex.

Peroxidase
Diaminobenzidine(DAB) DAB with enhancement Brown Black

3amino9ethylcarbazole(AEC)
4chloro1naphthol(CN) Hanker-Yates reagent

Red
Blue-black Blue

Alpha naphthol pyronin

3,3,5,5 TMB

Red
Blue

Alkaline phosphatase
Fast blue BB
Fast red TR New fuschin BCIP-nitroblue tetrazolium(NBT)

Blue
Red Red Blue

Glucose oxidase
Tetrazolium Blue

Tetranitroblue tetrazolium

Black

Immunogold
Colloidal gold Red

With silver enhancement Black

Double IE technique
Double staining Immunoperoxidase Immuno alk.ph Eg: Kappa & lambda positive cells and their ratio for clonality of plasma cells One E with 2substrates Three E, different substrates.

Polymer technique
Dextran Permits binding of large number of Enzyme molecules to a sec.Ab/teritary.Ab via dextran back bone. Increase in sensitivity Decrease in non specific background. Decrease in total number of steps.

Newer aspects
Area of great significance Transcription factors Nuclear proteins required for certain genes tissue specific Adv over traditional markers : Nuclear stains can be combined High specificity No diffusion Eg:myogenin

Quick labelling method

Less than 7min Used to diagnose intra-operative material Like sentinel lymphnode

Sausage tissue block

Simultaneous evaluation of more than 100 different tissue samlples on a single slide.

One drop of Ab
Multi block / TISSUE MICROARRAY Modern 100-1000 T.blocks HIGH THROUGHPUT TISSUE MICROARRAY

DISADV..
If tissue cylinders are below certain diameter risk of no representative area EDGE ARTEFACT. Preferred diameter 3mm.

Advantages of IHC
S/S Applicability to routinely processed material(even if stored for long periods) Feasibility of an accurate co-relation with traditional morphological parameters. Compatible with most tissue fixatives. Even in decal. material,previously stained sections,totally nectrotic material. Used simultaneously with other stains.

Uses of ihc

Analysis of tumors of uncertain origin.. Detect unknown primaries.. Microinvasin.. Pseudoinfiltration.. Occult micromets.. Lymphnode mets.. Tumor cell proliferation.. Oncogenes.. Receptors growth factors.. Response to therapy.. Prognosis..

Pit falls
Due to -technique -Ab activity -proper use of positive & negative controls.

Quality control
Positive control section that contains Ag of interest & is stained the same way as the patients specimen. Negative control is the same specimen stained with the same procedure as the +ve control without the prim.Ab prim.Ab is replaced by the Ab diluent/non immune Tg from the same sps of the prim.Ab.

False negatives
Ab inappropriate denatured used in wrong conc. Loss of Ag diffusion / autolysis ( even after fixation prefer paraffin blocks instead of tissues fixed in formalin for long periods )

False positives

More dangerous.. Cross reactivity with different antigen Nonspecific binding of Ab to the tissue. Endogenous peroxidase Avidity for ABC Entrapment of normal tissues by tumor cellseg:skeletal muscle in a soft tissue tumor Release of proteins from cytoplasm of normal cells absorption by tum.cellseg: thyroglobulin.

Selective use of monoclonal antibodies for staining.

2 approaches -algorithmic approach -panel approach

Algorithmic
Multi step screening Decision depends on the results of Ab reactions in the previous step. Interpret according to the algorithmic sequence

Panel
On the basis of preliminary (clinical &/ morphological) For targeted diagnosis & D/D.

For eg.
Poorly differentiated tumor

CD 45 (hem.neo)
CD3 T cell

Pancytokeratin (epi.tumors)
CD20 B cell Ig(for clonality) Bcl2 CD43 CD10,CD5 CD23,bcl6 (for sub classification)

References.
Anderson Ackerman Biogenics (hyd..) Internet

To be continued.