Introduction of

Imunohistochemistry (IHC)

Upik Andriani Miskad

Department of Pathology

Hasanuddin University
 • DNA
Methods – 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

LIMFOMA + - -/+ -

KARSINOMA - + -/+ -

SARKOMA - -/+ + -/+

MELANOMA - - + +

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.

• 1970’s 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,
enzyme-conjugated, 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 INDIRECT
The primary antibody has the label 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 • II
Stage Her2
Histological type Proliferation index
Histological grade Vessel invasion
Mitosis P53
ER/PR

III. HER2 pada tahun2003

EGFR, TGF, BCL2, masuk kategori I
Cathepsin D
Conventional therapy Molecular targeted
therapy

• Nonselective • Selective
• High morbidity • Low morbidity
• Poor quality of life • 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
• Response to hormone therapy 80%
therapy 60%
• Low grade histology

Cathepsin D +
•Metastatic potential
 The most important tumor marker

Her 2 P53
• Aggressive course • Unfavourable Survival
• Resistance to rate
hormonal th/ • Recurrence>>
• Resistance to CMF • Short survival
• Responsive to • Resistance to
antracyclin and anti chemotherapy
her2
• High histological
grade
Negative staining of ER
Estrogen Receptor staining
Progesteron receptor staining
Immunohistochemistry of Her2/Neu

Cytoplasmic staining
of Her2 positive