Sunteți pe pagina 1din 52

Advances in Diagnostic

Pathology
COURSE

COURSE TEACHER:

DR.M.L.SATYANARAYANA,PhD.,

PRESENTED BY :M.VETRIVEL

Immunohistochemistry is a technique which is used


to demonstrate the antigens in cells and tissues in their
exact localization by using a specific antigenantibody reaction.

Immunoperoxidaseis
refer

to

sub-class

a type of immunostain and it


of

immunohistochemical

or

immunocytochemical procedures in which the antibodies are


visualized via a peroxidase-catalyzed reaction.
Used in molecular biology medical research, and
clinical diagnostics

History
1941: Coons et al., have demonsrated antigens on
tissue sections by using an antibody which was linked
to an fuorescent label
1966: Nakane and Pierce reported the use of
secondary antibody which was conjugated with
peroxidase enzyme.
1979:
Sternberger
described
the
peroxidaseantiperoxidase method.
1981: Hsu et al., have defined the method of avidinbiotin-peroxidase complex which is abbreviated as ABC
method.

Principle
Antigen-antibody reaction taking place
For visualization/detection, an enzyme known as
a peroxidase is used to catalyze a chemical
reaction convert colorless chromogens into
visible colored end-products
Reaction can be detected by light microscope

Types of IPT
Direct IPT
Indirect IPT
PAP complex IPT
ABC IPT

Immunolabelling methods:
1. Direct method: The antigen directly binds to its

specific labelled antibody (primary antibody)

Fast to get the results


Labeling intensity is low
Used for kidney or skin biopsies

Direct method
A substrate-chromogen solution is added producing
a colored end-product

substratechromogen
Labeled
Antibody

Indirect method:
Two step indirect method
Three step indirect method

Immunolabelling methods:
Indirect method:
i) Two step indirect method
Primary antibody is unlabelled. A secondary antibody (which is labeled)
is used.
Secondary antibody must be raised against the immunoglobulin
of the species which the primary antibody is made in.
Example If the primary antibody (which is now the antigen) is made in
mouse, the secondary antibody must be against mouse immunoglobulin.

More sensitive than the Direct Method because several


secondary antibodies are likely to bind with a number of
various epitopes on the primary antibody increasing the
enzyme labels involved

Indirect method Two step indirect method

substratechromogen

Three-Step Indirect Method


Uses another layer of enzyme-labeled (tertiary)

antibody that is added to the previously


described Two-Step Indirect Method.
The added antibody layer increases signal and

staining intensity that is helpful when staining


antigens with few or limited available epitopes.
Both secondary and tertiary antibodies must

be conjugated to the same enzyme.

Soluble enzyme immune complex


techniques
PAP method
APAAP method

Methods include
peroxidase-antiperoxidase

(PAP)
alkaline phosphatase-antialkaline
phosphatase (APAAP)
The secondary antibody must be directed

against both the primary and the antibody of


the enzyme-antienzyme immune complex.

peroxidase-antiperoxidase (PAP)

chromogen

Mouse

Goat anti Mouse

Mouse monoclonal

Peroxidase
antiperoxidase
(PAP)

Alkaline phosphatase anti-Alkaline


phosphatase (APAAP)

Avidin biotin method


Uses the strong and high affinity of avidin

(egg white glycoprotein) for biotin (watersoluble vitamin).

Avidin has four binding sites for biotin


but fewer than four molecules of biotin
will actually bind to avidin.

The enzyme complex is prepared by mixing

biotinylated enzyme (HRP or AP) and avidin. This


preformed avidin-biotin-enzyme complex then
reacts with the biotinylated secondary antibody.

ABC - Procedure

Immunoperoxidase Steps
Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Blocking background staining
Primary antibody
Secondary antibody
Chromogen Substrate
Counterstain
Mounting

Microscopy
Slide 3 Observation
of 23

The First Step: Collection of Samp

Before doing IPT, tissues have to be collected an


preserved.
The time elapse between the death of the anima
the collection of tissues for IPT is usually critica
transfer into fixative is desired.
Specific criteria for tissues collected for IPT:
type of tissue (enzyme-rich tissues (intesti
pancreas) autolyze very fast
type of antigen (protozoa and fungi are pro
more resistant to autolysis than viruses)
distribution of antigen (antigens are usuall
evenly distributed through a lesion or organ
severity of lesions does not correlate with t
localization of infectious agent).

The Second Step: Specimen Fixa

Preserve cells and tissues in a reproducible a


life-like manner as possible.
Prevent autolysis and putrification.
Preserve antigenicity.
Make cellular elements more resistant to the
rigorous effects of the subsequent processing
and staining.
Samples to be fixed should not be thicker than
0.5 cm and ideally the ratio of fixative to sam
should be equal or higher than 10 to 1.
Options:
fixative type
fixative pH
fixation time
fixation temperature

Type of Fixation

Physical or Chemical Methods


Physical: heating, micro-waving,
freeze-drying
rarely used in routine laborato
Chemical: coagulant, cross-linking,
combination
of
used in most laboratories,
carried out by liquid fixatives

Physical Methods of Fixat

Heating: - precipitate proteins, dehydrate tissue


- used to accelerate other fixatives and
tissue processing, e.g. backing slides
Micro-waving: - speed fixation (reduce time)
- dangerous: vapors from
formalin-fixed tissues!
Freeze-drying: - immersed in nitrogen cooled
isopentane, kept at -40C
- used for highly soluble materials
- combination with coagulation fixati
immersion in acetone or alcohol at
remove water by dissolving ice crys
gradually raising temperature to +4
denature proteins (complete fixatio

Coagulant Fixatives

Organic and non-organic solutions coagulate.


proteins and render them insoluble (loss of functio

Types:

- dehydrant (alcohols and aceton)


- strong acid (picric acid, trichloracetic acid

Alcohol denatures proteins differently depending o


choice and concentration of alcohol, presence of o
and non-organic substances, pH, temperature.

Coagulant Fixatives of Dehydrant T

Proteins are normally soluble in aqueous solution


Fixation: removal and replacement of free water.
Destabilization of hydrophobic and hydrophilic
bonding (hydrophobic areas are released from t
repulsion of water and occupy a greater area).
Unfolding of tertiary structure
Disruption of
tertiary protein
structure.
Water
Result: partially Removal
reversed protein
hydrophobic
Denaturation
structure
hydrophilic
methanol: >80%
ethanol: >50%
Alcohol
Addition Water soluble protein
Eltoum et al., 2001

denatured protein
in alcohol

Non-Coagulant Fixatives of
Cross-Linking Type

Form cross-links in and between proteins, in and


between nucleic acids and between nucleic acids
and proteins.
Aldehydes: formaldehyde, glutaraldehyde, chlora
hydrate, glyoxal.
In aqueous solutions, formaldehyde forms methyl
hydrate, a methylene glycol:

H 2C = O +

H 2O

HOCH2OH

Methylene hydrate reacts with several side chain


proteins to form reactive hydroxymethyl side grou

Special Fixatives

Dichromate and Chromic Acid Fixation:


- used to identify chromaffin granules in endo
tissues, unreliable, > IPT
Metallic ions as fixative supplements:
- Hg2+, Pb2+, Co2+, Cu2+, Cd2+, Zn2+
- mercury, lead and zinc are used most comm
- pH is important (zinc formaldehyde better fi
Fixatives for electronmicroscopy:
- preservation cell organelles and membranes
fixatives that do not solubilize lipids
(e.g. glutaraldehyde)

Factors affecting the Qua


of Fixation
Buffers and pH
Duration of fixation and size of tissue
Temperature of fixation
Concentration of fixation
Osmolality of fixative
Ionic composition

Commonly Used Fixative

Formaldehyde-based fixatives
Neutral buffered formalin
Bouins solution (picric acid, glacial acetic
Mercuric chloride based fixatives
B5 (mercuric chloride, formalin, sodium ace
Zenckers Solution (mercuric chloride, glac
acetic acid, potassium dichromate, sodium
sulfate)
Acetic acid-zinc chloride (formalin, glacial acetic
Periodate-lysine-paraformaldehyde
Ethanol
Acetone

Mechanism of Antigen Retrie

Protein denaturation (some antigens are lost afte


Multiple pathways: breaking of cross-linkage, extr
of diffusible blocking proteins, precipitation of pr
rehydration of tissue (better penetration of antib
increased accessibility to antigen)

Mobilization of last traces of paraffin by microwav


energy, allowing antibody to better penetrate tis

Heat induced reversal of various chemical modific


of protein structure caused by formalin fixation

Protein +Formalin
AR: Heat
in Water

Chemical Modification

of Protein Confirmation
(Crosslinking)
Hydrolysis
Re-Modification
(Restoration of Ag)
Strong Intensity of IPT
AR-IPT
Ab

Negative with IPT

Ag

Ab

Ag

Heat Induced Epitope Retrieval Method

Influence of Temperature on
Antigen Retrieval

Cytokeratin AE1/AE3 staining in the lu

no heat

20 min heat40 min hea

Influence of pH on Ag
retrieval
The retrieval solution pH is important.

low pH buffer appear especially useful for nuclea


antigens
0. 1 M Citrate buffer solution pH: 6.0
0. 1 M EDTA buffer solution pH: 8.0
0. 5M Tris base buffer pH: 10.0

Influence of pH on Antigen Retr

Cytokeratin AE1/AE3 staining in the l

pH4

pH7

pH9

Proteolytic Enzyme Antige


Retrieval

Digest the tissue to some degree allowi


antibodies to recognize antigenic sites.
Types: trypsin, proteinase K, pepsin,
pronase
E, ficin

Combinations of heat and protein digest

Heat Retrieval and


Protein Digestion

Lysozyme staining in the lymph nod

no retrieval

proteinase Ktrypsin

Monoclonal Antibodie
Produced mostly in mice
Hybridoma technology = ascites vs. cell
supernatant

Specific for one epitope


Low cross-reactivity
Very sensitive to antigen changes during
fixation = antigen retrieval necessary

Polyclonal Antibodies
Classic antibodies

Produced in: rabbit, donkey, chicken, goat, etc


Recognition of multiple epitopes of the Ag
molecule

Heterogeneous composition = high affinity and


affinity
antibodies

Likely to recognize antigens even after formal


fixation

A Third Player

Rabbit Monoclonal Antibodi


vs. Mouse Monoclonals

Higher affinity
Recognize a greater variety of epitopes that are
immunogenic in mice
Many small compounds or peptides elicit good
immune response only in rabbits
Less need for antigen retrieval

Blocking of endogenous enzymes


Peroxidases are normally present in tissues
Endogenous peroxidase inactivated by 3 %

H2O2 in deionized water or methanol.

This reduces the non specific reaction

Enzyme label
Commonly used enzyme labels for IPT

procedures include Horseradish peroxidase


(HRP)

HRP, from horseradish plant, is an enzyme that

catalyze the reduction of hydrogen peroxide


(H2O2) to water and oxygen.

Substrate Chromogen
In order to visualize the enzymes labelling the
antibodies with light microscope, enzyme
substrate reactions, which convert colorless
chromogens into visible colored endproducts, is used.:
Peroxidase- hydrogen peroxide- diaminobenzidine
(DAB): BROWN
3-amino-9-ethylcarbazole (AEC): RED
4-chloro-1-naphthol (CN): KOYU
DARK MAV
BLUE

Summarize Step by Step


Antigen Retrieval
Rinse

Aq
M ueo
ou u
nt s

Deparaffinization and Rehydration

Block Endogenous Peroxidase


Rinse
Protein Block
Primary Antibody
Rinse

HRP
Rinse
Chromogen
Rinse
Counterstain
Rinse
Mounting

Colon
carcinoma
MS-1375 CEA

Rinse

SPECIME
N

Secondary Antibody

Advantages
Even under light microscope we can read the

results

It gives permanent preparation

Antigen inside the cell can be identified


Counter stain can be used

Disadvantages
Risk of nonspecific staining
Necessary to block endogenous peroxidase

enzyme
DAB is carcinogenic

Applications of Immunoperoxidase
staining
Clinical diagnostics, can be used to localized antigens and to

identify antibodies in serum of patients with various viral,


bacterial and parasitic diseases.

Cancer, to determine the origin of tumors and in sub-

classifying tumors

IPT can also be used to help diagnose skin diseases

conditions, and to sub classify amyloid deposits

Related techniques are also useful in sub-typing lymphocytes

which all look quite similar on light microscopy

Localize hormones

S-ar putea să vă placă și