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Immunohistochemisty (IHC)

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Shaikha Al Kaabi

on 12 January 2014

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Transcript of Immunohistochemisty (IHC)

IHC is used for disease diagnosis, drug development and biological research.
Using specific Tumor Markers, physicians use IHC to:
Immunohistochemisty (IHC)

Presented by: Shaikha Al Kaabi
Student ID #: S990008283
Course: MLAB N450

The principle of IHC has existed since the 1930s, but it was not until 1941 that the first IHC study was reported.
Started in 1941 when Coons identified pneumococci using a Direct fluorescent method & the Indirect method. Addition of horseradish peroxidase
Peroxidase anti-peroxidase technique in 1979
Use of Avidin & Biotin Complex (ABC) in early 1980’s
1. History
4. Sample Preparation
2. Definition
3. Application
Detection Methods

Immunohistochemistry (IHC) is a technique that uses antibodies (matching molecules) which combines histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label that are either fluorescent dyes or enzymes (such as horseradish peroxidase)
1. Fixation
2. Tissue Processing
3. Embedding
4. Sectioning
5. Tissue Treatment (Antigen Retrieval)
6. Controls (Positive & Negative)
IHC Protocol (Blocking)
5. IHC Protocol (Blocking)

Sample Preparation:
Antigen (Ag) Retrieval:
is to unmask the cross reaction between the formalin and the antigen.
There are several steps that involves the Ag retrieval:


Heating Induced Epitope Retrieval (HIER) 98°C

pH ( using a Specific Buffer ) such as:
Citrate Buffer for pH 9 Alkaline e.g. P63 Antibodies
EDTA Buffer for pH 6 Acidic e.g. P53 Antibodies

Enzymatic Retrieval and the most common one is proteinase k

No Treatment e.g Factor XIII
1 . Peroxidase Block
This product is intended for use in peroxidase-based immunohistochemical (IHC) staining procedures on cell preparations, frozen tissue sections, and paraffin-embedded tissue sections

It will Minimize the amount of Background Staining and it is divided to Specific and Non-Specific
2 . Incubation with Primary Antibody
The aim is to obtain the maximum contrast between specific signal and background staining
3 . Incubation with Secondary Antibody Conjugated to a detection of Antibody
This step is required if primary antibody is unconjugated.
Wash it in Tris-Buffer to remove the access of Antibody and to retrieve the pH
4 . Adding Enzyme Substrate if detection conjugation is a enzyme
Enzymes requires a substrate and Chromogen

5. Coverslip and Observe under the Microscope
Examining the Slides are performed by the Histopathologist
8. IHC Tests
9. IHC Results
10. Reference

All specimens which are received in the Grossing Area for routine Histopathology should be fixed at 10% of Neutral Buffered Formalin NBF for 24 - 72hrs. This step is vital and necessary because of the following:
a) Kills any Bacteria or Fungus so the 10% NBF acts as anti-septic agent.
b) Prevent Purification
c) Endogenous enzyme that will regulate the tissue, so once it’s it is fixed that will prevent autolysis of the specimen.
d) Prevents denaturation of the internal structure of the protein i.e. stabilize the protein in the tissue.
e) It will change the reflected index inside the tissue to be able to see it under the microscope.
f) Formalin will hold and firm the tissue in order for the Histopatologist to gross a thin section or called Representative Section (RS) or Entirely Submitted (ES) and place it in the Cassette/s. In addition, all cassettes will placed in further fixative ( 24 or 48 or 72hrs depending on the sample size ) to preserve the sample as close to the natural state as possible.

Is the casting or blocking of tissue section, which involves the enclosure of the tissue in the infiltration medium used for processing in order for the medium to solidify. The Embedding Center is divided into:
a. Tissue Tank
b. Wax Tank
c. Hot Plate
d. Cold Plate
e. Cold Spot
f. Forceps
g. Waste
h. Molds that comes in different sizes to fit different sizes of tissue
Tissue Embedding Center
Different Sizes of Molds
The instrument utilized in order to produce these sections is known as the microtome, which is just a sharp knife with a mechanism that allow the advancing of a paraffin block standard distances across it.

The process of microtomy will allow us to produce a slide that will continue through a IHC staining technique with a final result of a diagnostical tissue section.
6. Detection

7. IHC Staining Method
There are two methods used:
Antibody Types
The antibodies used for specific detection can be either Polyclonal or Monoclonal.

: a heterogeneous mixture of antibodies directed against various epitopes of the same antigen.

: are a homogeneous population of immunoglobulin directed against a single epitope.

Note: Epitopes or called Antigenic Determinant are markers on the antigen by which the antibody binds on the same antigen. It is used to describe the part of an antigen that is directly bound by the antibody.
Direct Method

This technique is short and quick because it is a one-step staining method and only one antibody layer involves. An antibody reacting directly with the antigen in tissue section. The primary antibody must be labeled. The common labels includes fluorescent dye, biotin or enzymes.

The antibody can be applied directly to the section and will bind to its specific antigen, which can then be localized by the fluorescence.

This method is less commonly used.
Involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody.

In the indirect technique, the primary antibody is not labeled fluorescently. A second antibody capable of binding to the primary antibody is needed and this needs to be labeled with fluorescent dye.

Indirect Method
Avidin-Biotin Complex (ABC) Method: it is standard IHC method and one of the widely used technique for immunhistochemical staining. Avidin, a large glycoprotein, can be labeled with peroxidase or fluorescein and has a very high affinity for biotin. Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies.

The technique involves three layers:

1. The first layer is unlabeled primary antibody.
2. The second layer is biotinylated secondary antibody.
3. The third layer is a complex of avidin-biotin peroxidase.

The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products.
Detection Complex ( ABC )
1. Direct Methods
2. Indirect Methods
Detection Complex ( PAP )
Peroxidase Anti-Peroxidase (PAP) Method : is a further development of the indirect technique and it involves a third layer which the antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase anti-peroxidase complex.

The sensitivity is about 100 to 1000 times higher since the peroxidase molecule is not chemically conjugated to the anti-IgG but immunologically bound and loses none of its enzyme activity.

It also allows for much higher dilution of the primary antibody, thus eliminating many of the unwanted antibodies and reducing non-specific background staining.

Detection Complex ( LSAB )
Labeled StreptAvidin Biotin (LSAB) Method: Streptavidin, derived from Streptococcus avidini, is a recent innovation for substitution of avidin.

LSAB is technically similar to standard ABC method:
1. The first layer is unlabeled primary antibody.
2. The second layer is biotinylated secondary antibody.
3. The third layer is Enzyme-Streptavidin conjugates (HRP-Streptavidin or AP-Streptavidin) to replace the complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solutions to produce different colorimetric end products. The third layer can also be Fluorescent dye-Streptavidin such as FITC-Streptavidin if fluorescence labeling is preferred.

A recent report suggests that LSAB method is about 5 to 10 times more sensitive than standard ABC method.

There are further detection methods used in the Indirect Method called the Detection Complex such as PAP, ABC, LSAB & Polymer or Micro-polymer
IHC Staining Methods
1. Lymphoma & Leukemia
Below are some of the tests listed in this panel:
2. Immunoglobulins
Below are some of the tests listed in this panel:
3. Epithelial Panel
Below are some of the tests listed in this panel:
4. Tumor Marker
Below are some of the tests listed in this panel:
5. Breast & Prostate
Below are some of the tests listed in this panel:
Common Testes Requested for IHC
CK 7
CK 8
CK 14
CK 18
CK 19
CK 20
CA 15-3
CA 19.9
CA 125
Breast Panel:
Prostate Panel:
AMACR (P504)
IHC Staining one of the Tumor Markers e.g ( CA 125 )
IHC Staining Different Epithelial Panel
IHC Staining one of the Breast Panel e.g ( ER )
IHC Staining one of the Prostate Panel e.g ( PSA )
PSA: Prostate Specific Antigen is a protein produced by cells of the prostate gland. The higher a man’s PSA level, the more likely he has Prostate Cancer
CA-125: is used as a tumor marker, because it is 79% sensitive for Ovarian Cancer
ER: is Estrogen Receptor Antibody which is an essential component of the evaluation of Breast Cancers
Lets Start Our Tour with . .
Benchmark (IHC) Staining Instruments
Diagnose a cancer as benign or malignant, determine the stage and grade of a tumor.
Identify the cell type and origin of a metastasis to find the site of the primary tumor.
Direct Method, Indirect Method, ABC Method & LSAB Method ( Advantages & Limitations )
Tissue Processing
Summary of the Tissue Processing Steps
Tissue processing is concerned with the diffusion of various substances into and out of stabilize porous tissues i.e. impregnation or infiltration of the solid tissue with various substance. Below are the following steps using the Tissue Processor Instrument:

1. All the Blocks will be placed in the Tissue Processor Chamber
2. Fixation ( 10% Neutral Buffered Formalin NBF ) to ensure that all the tissue are fixed well
3. Dehydration ( removal of water and that will be performed gradually to avoid shrinking the tissue ) with Alcohol e.g. ( Ethanol is the common one in use or Methanol or Isopropanol )
4. Absolute Xylene. This step is essential to Clear and remove all the Alcohol from the tissue and to make it ready for the next step
5. Parraffin Wax ( infiltration of the tissue )
6. The total duration of time required for the above process is approximately 11 hours & 14 minutes
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