Introduction of

Imunohistochemistry (IHC)

Upik Andriani Miskad

Department of Pathology
Hasanuddin University

Central Dogma
Gene

Genome
Transcription

RNA

Transcriptome

Translation

Protein

Proteome

Methods

DNA
PCR
Southern Blotting

RNA
RT PCR
Real time PCR
In situ hybridization
Northern blot

PROTEIN
Immunohistochemistry
Immunofluorescence
Western Blotting
Elisa

IMMUNOHISTOCHEMISTRY
(IHC)
Immunohistochemistry is a method of
detecting the presence of specific
molecule or proteins in cells or tissues
based on antigen- antibody reaction.
Materials (on glass Slide)
Tissue
(Paraffin block/frozen section)
Cell (smear cell by FNA or Cell Culture)

Goal of IHC
To study cellular markers that define
specific phenotypes for:
1. Diagnostic
2. Prognostic
3. Therapeutic
4. get Any information related to disease status
and biology.

DIAGNOSIS
LCA

AE1/3

VIM

S100

-/+

KARSINOMA -

-/+

SARKOMA

-/+

-/+

MELANOMA

LIMFOMA

KPLST, 24-25/03/07, IAP KOL

History of IHC
Albert H. Coons and his colleagues (1941,
1955) were the first to label antibodies with a
fluorescent dye, and use it to identify
antigens in tissue sections.
1970s new detection systems using
peroxidase-anti-peroxidase system
1980 ABC (Avidin Biotin Complex) system
level.

Principle of IHC Method

1)primary antibody binds to
specific antigen;
2) antibody-antigen complex is
bound by a secondary, enzymeconjugated, antibody;
3) in the presence of substrate
and chromogen, the enzyme
forms a colored deposit at the
sites of antibody-antigen
binding.

TERM
Antigen : any molecule that has generated an antibody
response. Epitope is part of antigen which react with
antibody.
Antibody: Immunoglobulin (mainly IgG) or glycoprotein
that bind with high affinity and specificity to antigen.
Polyclonal ab : are produced by different cells
Monoclonal ab : the product of an individual clone of plasma
cell

Commercial antibody available.

 Antigen retrieval: any process that restores

immunoreactivity for fixed formalin tissues.
Protease digestion methode
Heat induced epitope retrieval methode
Microwave
Autoclave
Pressure cooker

Antibodies

LCA for define limfoid lineage

Actin : smooth muscle cells
VEGF : angiogenesis marker
Ki67/ PCNA : Proliferative marker
BCl 2 : apoptotic
Cytokeratin : Epithelial marker
P53, Her2, ER/PR, Endoglin, PRL-3 and
others

Polyclonal vs Monoclonal
Polyclonal:
Quicker and simpler to obtain by immunization of
animal
More sensitivity but less specificity than monoclonal,
cause heterogenitas nature of antibody.

Monoclonal:
Monospecifcity (to single epitope)
Consuming time to generate but immortality

Hybridoma: A cell produced by the fusion of a lymphocyte and a mutant neoplastic

plasma cell capable of growing only in special media.

Detection system on IHC

DIRECT
The primary antibody has the label

INDIRECT
Using labeled secondary antibody

Indirect detection
Peroxidase anti peroxidase (PAP method)
Localize primary antibody with bridgig ab (secondary
ab) to tertiary complex which contain the labels
Tertiary complex develope from the same animals in
primary ab for succesfull bridging.

Avidin Biotin Peroxidase (ABC method)

Using glycprotein avidin which has 4 high affinity biotin
binding sites as tertiary complex.
Most widely uses in lab.

Labeling
Enzymes
Horseradish Peroxidase (HRP)
Alkaline Phosphatase (ALK)
Glucose Oxidase

Substrates for enzymes

DAB (Diaminobenzidine)
Brown
AEC (3-Amino-9-ethylcarbazole) Red
Napthol
Blue

VEGF + in placenta

PROGESTERON RECEPTOR
Staining

Nuclear
staining
judge as
positive

IHC of ESTROGEN Receptor

ER,brown
staining in the
nucleus

Immunohistochemistry
Various factor contribute good result in IHC
Preanalytic phase
IHC needs proper tissue handling by the Surgeon
IHC needs proper tissue processing by pathologist
Fixation, processing

Analytic phase
Laboratory of pathology
Reagents, IHC technique and controls

Post analytic phase

Competency, Interpretation, quality assurance

Practical uses of IHC in breast pathology

Assesment of early carcinoma cell invasion
beyond the myoepithelial layer in lesion
predominantly CIS.
Assesment of micrometastasis in lymph nodes.
Assesment of hormone receptor (ER/PR)
Measure of tumor cell proliferation rate.
Assesment of oncogen expression (Her2).
Identification of metastatic breast cancer when
presenting as metastatic disease of unknown
origin.

Breast Specimen handling

Specimen should be oriented by the surgeon
Specimens should be place into at least 2x the volume of
fixative
Specimen incision could be perform if any delay time
between surgery and specimens receipt in laboratory, but
orientation must be mark for re excision in the laboratory.
Never allow large breast specimens to fix without slicing

IHC scoring: semi-quantitative

interpretation of HER2 expression

0 (negative)

1+ (negative)

2+ (equivocal)

3+ (positive)

Important Issues
Prognostic factors
Morphology based

molecular based

Molecular profile for target therapy

The most important MARKER???

 Breast cancer with the same histological

feature can show different molecular profile
Information of traditional prognostic factor
is insufficient to accurately access
individual risk.
There is a risk of treating patient with
chemotherapy without benefit.

Prognostic factor
College of American Pathologist 1999
I
Stage
Histological type
Histological grade
Mitosis
ER/PR
III.
EGFR, TGF, BCL2,
Cathepsin D

II
Her2
Proliferation index
Vessel invasion
P53

HER2 pada tahun2003

masuk kategori I

Conventional therapy

Molecular targeted
therapy

Nonselective
High morbidity
Poor quality of life

Selective
Low morbidity
Better quality of life

Prognosis and therapy

Based on molecular profile

Molecular profile can be determined by

IMMUNOHISTOCHEMISTRY (IHC)

The most important marker ??

Many molecular markers identified but
only 3 of proven value for breast cancer

ER, PR and Her2/Neu

Predictive and prognostic therapy
Targets for tailored treatment

The most important marker

ER+

ER/PR+

Favorable DFS/OS
Response to hormon
therapy 80%
Response to hormone
therapy 60%
Low grade histology

Cathepsin D +
Metastatic potential

The most important tumor marker

Her 2

P53

Aggressive course
Resistance to
hormonal th/
Resistance to CMF
Responsive to
antracyclin and anti
her2
High histological
grade

Unfavourable Survival
rate
Recurrence>>
Short survival
Resistance to
chemotherapy

Negative staining of ER

Estrogen Receptor staining

Progesteron receptor staining

Immunohistochemistry of
Her2/Neu

Cytoplasmic staining
of Her2 positive