Basics of Immunohistochemistry

Dr. Smita Pathak

Professor
Department of Pathology,
MIMER Medical College, Talegaon, Pune

Dr. S.R. Joshi.

Professor & Head
Department of Pathology,
MIMER Medical College, Talegaon, Pune

Definition: Immunohistochemistry is a technique for identifying cellular or

tissue constituents (Antigens) by means of antigen-antibody interactions, the
site of antibody binding being identified either by direct labeling antibody or by
use of a secondary labeling method1.

Introduction: Immunohistochemistry is as the name implies, a marriage of two

disciplines immunology and histology. The Immunohistochemistry technique
is used not only to determine if a tissue express or does not express a particular
antigen but also to determine the antigenic status of particular cells within that
tissue and microantomic location of the antigen2.
Immunohistochemistry uses antibodies to distinguish antigenic difference
between the cells2.

History: Immunohistochemistry methods for diagnostic use were first described

by Coons and Jones using immuno-fluorescent techniques for the detection of
bacteria. However, it was in the late 1970s that the discovery of monoclonal
antibodies gave the much needed impetus for ushering objectivity in surgical
pathology diagnosis3.
Principle of Immunohistochemistry: Immunohistochemistry is a method for
localizing specific antigen in tissues or cells based on antigen antibody reaction.
The site of antibody binding 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 the more commonly used
enzymatic marker for light microscopy. Ideally maximal visual signal strength
on a contrast of least background or nonspecific staining is ideal to highlight the
antigen reactivity4.

Methods: Depending upon the type of antigen-antibody detection system used

these methods are dividing in two types:

A] Direct method It is one step staining method and involves a labelled

antibody reacting directly with the antigen in tissue sections. The technique
utilizes only one antibody and the procedure is short and quick. However it is
insensitive due to little signal amplification and has been rarely used ever since
the introduction of the indirect method. This involves identification of antigen
by a conjugated antibody directed specifically against it.

B] Indirect method In this method the primary antibody is not conjugated. A

Second antibody raised to the gammaglobulin of the species which produced the
primary antibody, is conjugated. This method is more versatile as different
primary antibodies raised in one species can be tagged with the single
conjugated labelled secondary antibody.

Indirect method involves an unlabeled primary antibody (first layer) which

reacts with tissue antigen and labelled secondary antibody (second layer) which
reacts with primary antibody. Indirect method is more sensitive due to signal
amplification through several secondary antibody reactions with different
antigenic sites on the primary antibody. In addition it is also economical since a
single labeled second layer antibody can be used with various first layer
antibodies against antigens4.

Technique: Immunohistochemistry technique includes following steps:

1. Deparaffinization of tissue sections taken on polylysine coated slides

2. Quenching of endogenous enzymes (which otherwise over react with
Immunohistochemical reagents giving false positive result). This is
usually done by 3% H2O2 or with free avidin.
3. Antigen retrivals.
4. Blocking of non-specific binding sites.

5. Binding primary antibody.

6. Binding with biotinylated secondary antibody
7. Detection methods using peroxidase anti peroxidase methods, avidinbiotin conjugates or the more recently widely used polymer labeling two
step methods.
8. Addition of chromogen substrate.
9. Counter staining, dehydrating and coverslipping the slide4.

Current applications of Immunohistochemistry:

1. Analysis of tumors of uncertain origin A common problem in
diagnostic surgical pathology is the classification of neoplasm
according to the type of cellular differentiation that they display.
Proper classification provides important information regarding the
likely clinical course and prognosis for particular disease conditions
and has significant impact on treatment decision by clinicians1.
2. A tumor cannot be staged and appropriate therapy cannot be
determined without specific classification. Generally tumors are
classified histologically (e.g. Epithelial, mesenchymal, neural etc.) or
by their tissue of origin (breast, colon, Lungs etc.) Most tumors can
be categorized in such fashion. At times, however, a tumor may defy
classification as when
i)
The tumor I first found as a metastatic deposit and the
primary site cannot be determined.
ii)
The tumor is so poorly differentiated that it exhibits no
specific morphological features.
iii) The tumor has morphologic appearance that is compatible
with more than one distinct tissue.
Immunohistochemistry methods have revolutionized the approach
to tumors of uncertain origin.

Commonly used Antigens for analysis of anaplastic tumors:

2. For predicting response to therapy Some neoplasms including

carcinoma breast, prostate, ovary are often responsive to hormones, a
property that has been exploited by the use of drug therapy to influence
hormone level or inhibit the effects of hormones on tumor cells. e.g. ER
PR receptors, androgen receptors.
3. Immunohistochemistry for infectious diseases Immunohistochemistry
may also be used for diagnosis of infectious processes by the
identification of infectious agents in the tissue sections.
Immunohistochemistry is superior to culture in that rapid results can be
obtained for agent that may be difficult to grow or require long
incubation. E.g. Bacteria such as H. pylori, T. pallidum, some
mycobacteria, fungi such as H.capsulatum, viruses such as Herpes
simplex virus, cytomegalo virus and parasites such as Toxoplasma gondii
and Pneumocystis carinii.

4. Immunohistochemistry allows greater specificity in diagnosis of

degenerative brain disease and muscular dystrophies.
5. Immunohistochemistry can also be used on cytologic preparations5.

Limitations of Immunohistochemistry:
1. Experience: Experience is critical in standardizing the procedure
including the selection and proper dilutions of necessary reagents and
regular performance of all the appropriate controls. Interpretation too
has its foundation in experience.
2. Availability of antibodies: The paucity of antibody with high degree of
specificity for cellular and tissue antigens was serious limitation until
recently. This has been remedied in part by using hybridoma
technique for monoclonal antibodies.

3. Antigen loss: The specificity of an antibody for particular antigen and

its ability to react with that antigen require the preservation of antigen
configuration2.
To conclude, Immunohistochemical analysis is far more objective than
routine morphologic examination. Indeed, Immunohistochemistry is
elevating surgical pathology to an entirely new level of specificity and is
allowing the pathologist to provide the information that is not available
by morphologic evaluation2.

A poorly differentiated Carcinoma stained with H&E(a) and with an

antibody to low molecular weight cytokeratin(b)

Immunohistochemical staining for infectious agents. (a) Spirochetes in

cutaneous infection
(b) Cytomegalovirus in fetal lung

References:
1. Peter Jackson and David Blythe. Immunohistochemical techniques
Chapter no 21. In, John D Bancroft, Marilyn Gamble (ed). Theory and

2.

3.

4.

5.

practice of Histological techniques, 6th edition. Churchill Livingstone,

Elsevier, 2008;433-72
Richard J Cole, Clive R. Taylor. Immunohistochemistry and related
marking techniques Chapter no.8. In, Ivan Damjanov James
Linder(ed). Andersons Pathology Volume I, 10th edition. St. Louis
Mosby, Elsevier,2009;136-75
Nirmala A Jambhekar, Anshuman C Chaturvedi, Bhulaxmi Prakash
Madur. Immunohistochemistry in Surgical pathology Practice: A
Current perspective of a simple, powerful yet complex tool. IJPM
2008;51(1):2-11
Nirmala Jambhekar, Santosh Menon. Introduction to
Immunohistochemistry. In, Tanuja Shet (ed) . An approach to
Immunohistochemical evaluation of common tumors. Tata Memorial
Hospital Mumbai, 2010;1-4.
Charles L. White, III. Immunohistochemistry Applications in
Pathology Chapter no 23. . In, John D Bancroft, Marilyn Gamble (ed).
Theory and practice of Histological techniques, 6th edition. Churchill
Livingstone, Elsevier, 2008; 493-515.