4.immunohistochemistry INTRO

Download as pptx, pdf, or txt
Download as pptx, pdf, or txt
You are on page 1of 25

Immunohistochemistry/

Immunocytochemistry
Presenter: Ms. Nabaasa Saphurah
Modulator: Mr. Ssedyabane Frank
OBJECTIVES OF THIS LECTURE
By the end of this lecture we should be able
to discuss;
 Introduction/history of IHC,
 Definition of terms antigen, antibody,
affinity, avidity, specificity, sensitivity,
 Mechanisms of antigen-antibody binding
 Applications of IHC.

10/22/2024 2
IHC introduction
Immunohistochemistry (IHC) 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.
. A brief summary of this basic step is to react with
"antibody" that specifically binds to an "antigen",
and "visualize" the site where the antigen-
antibody reaction has taken place with a visible
lable and observe it microscopically to confirm the
presence
10/22/2024 or absence of the disease. 3
IHC introduction..
Immunohistochemistry (IHC) uses antibodies to
detect cell and tissue proteins and provide semi-
quantitative data about target protein expression,
distribution, and localization.

Tissues are sectioned from fixed embedded (e.g.


IHC-Paraffin or plastic) or frozen blocks (e. g. IHC-
Frozen), and the sections are then probed with
primary antibodies against the antigens of
interest.
10/22/2024 4
IHC introduction..
Target expression can be evaluated with the
corresponding labeled primary antibody (direct
detection) or, more commonly, with
the addition of labeled secondary antibodies
(indirect detection).

The label, either fluorescent or enzymatic, is used


to visualize the antigen-antibody complex.

10/22/2024 5
IHC history
• Immunohistochemistry began over 120 years ago when Von Behring
discovered serum antibodies in 1890 and used them to cure diphtheria
and tetanus.
• Dr. John Marrack visualized the reaction by attaching dyes to antibodies.
Dr. Albert H. Coons developed the first fluorescent antibody labels,
officially launching light microscopic (LM) immunohistochemistry in 1941.
• Immuno-electron microscopy (EM) was officially launched in 1959 when
Dr. S.J. Singer attached ferritin to antibodies to detect viral surface
antigens.
• Drs. Ludwig Sternberger and Sam Spicer modified the immunoenzyme
protocol in 1969 with the use of antibody–antibody bridging, thus
avoiding chemical conjugation.
• Doctors Faulk and Taylor attached colloidal gold labels to antibodies in
1971.
• In 1982, Dr. S.-M. Hsu added avidin–biotin linkages, which increased
sensitivity and allowed the reliable detection of antigenic sites that were
difficult to preserve.
10/22/2024 6
Limitations of IHC
• IHC stains are not standardized
worldwide.
• While the cost of the procedure is
relatively inexpensive, the equipment
needed to perform IHC is costly.
• Quantifying results is difficult.
• IHC is subject to human error. Well-trained
personnel are paramount.

10/22/2024 7
Definition of terms
• Immunohistochemistry (IHC) is a technique
for identifying cellular or tissue antigens by
means of antigen-antibody interactions. IHC
takes its name from "immuno” for antigen-
antibodies reactions, and "histo” for tissue
or “cyto” for cell.
• IHC uses antibodies conjugated to labels that
catalyze reactions to form detectable
compounds to visualize and localize specific
antigens in a tissue sections. 8
Definition of terms..
• Antigen: is a substance foreign to the host
which stimulates formation of a specific
antibody and which will react with the
antibody produced.
Antigens have two main properties,
i.e.
1. Immunogenicity, which is the
ability to provoke an immune
response.
2. specific reactivity, which means
that the antigen can react with the
antibody specific to it in all
aspects.
10/22/2024 9
Definition of terms..
• An antibody is a serum protein that is formed
in response to exposure to an antigen. and
reacts specifically with that antigen to form
immune complexes.
• Epitope: This is the part of an antigen that a
specific antibody binds. It is also called
antigenic determinant.
• Paratope: This is the part of an antibody that a
specifically binds to an epitope of an antigen.

10/22/2024 10
Definition of terms..
• Avidity: the overall strength of binding between
an antibody and an antigen.
• Antibody specificity: is the characteristic of an
antibody to bind selectively to a single epitope or an
antigen.
• Sensitivity: is the relative amount of an antigen that
a technique is able to detect.
• Affinity: It is a measure of the binding strength
between antigen and antibody.

10/22/2024 11
10/22/2024 12
Definition of terms..

• Monoclonal antibodies: made using identical


immune cells (B cell) that are all clones
(daughters) of a specific parent cell. They thus
have affinity for the same antigen and same
epitope
• Polyclonal antibodies: made using several
different immune cells (B cells) they will have
affinity for the same antigen but different
epitopes on the same antigen.

10/22/2024 13
Definition of terms..
• Labels: A label is a specific tag attached to an
antibody to aid in detection of the complex
after antigen-antibody reaction.
Common labels used include:
1. Fluorochromes (e.g., fluorescein, rhodamine).
2. Enzymes (e.g., peroxidase,
alkaline phosphatase).
3. Electron-scattering compounds (used in electron
microscopy (e.g., ferritin, colloidal gold).

10/22/2024 14
Definition of terms..
Chromogen: Chemical that changes color in
presence of oxygen and used to visualize the
final colored reaction product in the enzyme
labels.
Antigen retrieval/ Epitope recovery: is the
process of uncovering the epitopes on the
antigens that were covered by the formaldehyde
fixative such that the antibodies can bind to the
epitopes on the antigen.
10/22/2024 15
Definition of terms..
Antigen retrieval methods;
None, Heat-induced Epitope Retrieval (HIER), and Protease-
induced Epitope Retrieval (PIER)
• Blocking
Blocking is essential for preventing non-specific
binding of antibodies or other reagents to the
tissue.
Normal Serum, BSA, Casein, Various Detergents,
and Salt Concentrations

10/22/2024 16
Principle of IHC
• Antigen in tissue sections are identified by the
use of labeled antibodies through antigen-
antibody interactions, which leads to a colored
reaction product, that are then visualized
using different methods such as fluorescent
dye, enzyme, radioactive element, colloidal
gold, etc.

17
18
Importance/significance of IHC
1. Diagnosis of tumors
2. Assessment of markers reflecting prognosis
(“prognostic” markers”)
3. Assessment of markers reflecting a therapeutic
response (“predictive” or “theranostic”
markers)
4. Detection of micrometastases
5. Identification of infectious organisms

19
1.Diagnosis of tumors:
a. Classification of poorly differentiated neoplasms:
 carcinoma (cytokeratin +) vs.
 lymphoma (CD45+) vs.
 melanoma (S100+, Melan-A+, HMB45+)
b. Diagnosis of carcinoma of unknown primary:
 colon (CDX2+) vs.
 lung (TTF-1+) vs.
 prostate (PSA+)
c. Diagnosis of invasion:
 loss of myoepithelial cells (breast cancer)
 loss of basal cells (prostate cancer)
 loss of basement membrane/collagen type IV (various
carcinomas, rarely used)
20
2.Assessment of markers reflecting prognosis
(“prognostic” markers”):

• Ki67/MIB1 (general proliferation marker)


• p53 (general marker of apoptosis)
• HER2 (adverse prognosis in breast cancer)
• CD38 (adverse prognosis in chronic
lymphocytic leukemia)

21
3.Assessment of markers reflecting a therapeutic
response (“predictive” or “theranostic”
markers):
• ER/PR +ve (Tamoxifen for breast cancer)
• HER2 +ve (Herceptin for breast cancer)
• C-kit +ve (Gleevec for GIST (gastrointestinal
stromal tumor) CML, other; mutations
more predictive than IHC)

22
4.Used to detect pathogens in tissue
• Viruses (HSV, CMV,EBV, HPV)
• Bacteria (H.Pylori)
• other organisms (Toxoplasma, Pneumocystis

23
Read and make notes
1. Mechanisms of antigen-antibody binding
2. Why do you need an IHC test after a biopsy?

10/22/2024 24
References
1. Quick Reference Handbook for Surgical
Pathologists by Natasha Rekhtman Justin A.
Bishop, Springer-Verlag Berlin Heidelberg
2011
2. Immunohistochemistry (IHC) Handbook by
novus biologicals.

25

You might also like