Immunohistochemistry

Practical issues
Dr Santosh Menon
Assistant Professor, Pathology
Tata Memorial Hosipital
What is IHC?
A method that uses antibodies to
identify, locate, and stain specific
protein molecules in tissue sections
(visualized using a microscope)
Used to diagnose the type of cancer
and to help determine the patient's
prognosis or as a predictive marker of
therapeutic response.
WHY IS IHC IMPORTANT in
surgical pathology practice?
cell lineage and tissue type
prognostic markers
Quantification; it is no longer enough that the
'stain' is there; rather it is a question of 'How
much is there?'
Predictive markers for targeted therapy
Patients are knowledgeablethanks to internet
CD20 and Rituximab
CD20
C-erbB2 and Herceptin in breast CA
C-kit and imatinib in GIST
Immunohistochemistry
Manual
Automated
Basis of Immunology

Simple Principle
Antigen + Antibody = Complex
Immunohistochemistry is a technique based on
the selective binding of specific antibodies to
specific antigenic sites on a cell, in a precise
lock and key mechanism
What is an Antigen?
Any substance that can trigger the
production of antibodies is called an antigen
What is an Antibody?
Antibodies are proteins called
immunoglobulins
Antigen
Antibody
Anti
gen
Primary Antibody
Labeled
Secondary
Antibody
Cell with antigens on surface
Anti
gen
B
B
PX
PX
PX
B
Avidin Biotin
Complex
DAB+H
2
O
2
IHC
Primary Antibody
Labeled
Secondary
Antibody
Cell with antigens on surface
Detection system
Chromogen
Detection system
Primary Antibody
Tissue section
with antigen
Primary Antibodies
Polyclonal antibodies: Heterogeneous
population of antibodies, produced against
several epitopes on a single antigen- multiple
clones produce the antibodies
More tolerant to retrieval techniques
Specificity is less
Monoclonal antibodies:
Homogenous population of antibodies for a
single epitope from a single clone of B-cells
More specific
Less cross reactivity
Less background non specific staining
Pure single Ab
After immunization, the mouse spleen contains B cells producing specific antibodies.
Each B cell produces only one kind of antibody, which binds to its specific antigen.
Conventional antiserum is the mixture of all antibodies produced by B cells from spleen.
If a single B cell was picked up and cultured, then it will produce only one kind of antibody.
But B cell can not survive well in the culture.
Myeloma cell can be cultured in the test tube, but can not produce useful antibody. Each hybridoma line can produce pure single antibody, called monoclonal antibody. If B cell is fused with myeloma, the fused cell might be cultured and produce antibody.
Adapted from Milstein (1980) Scientific American, Oct. p.58
1
2
3
4
m
m
m
m
1
2
3
4
1 2 3 4
Monoclonal
antibodies
Cell fusion
Spleen cells
+
Myeloma
x
Antiseum
Antigen
Immunization
A mixture of all Ab
1 2
3 4
B
A
L
B
/
c
1 2 3 4
1 2
3 4
B cell
Primary Antibody vial
How do we start with a new
antibody?
If you have acquired a new
antibody Titration is a must
Date Antibody
Used
Dilution Appropriate
dilution
Remarks
04-04-08 CD20 1:50,
1:100
1:200
1:100 GOOD
05-05-08 CD3 1:100
1:200
1:300
1:200 BEST
Positive controls
CYTOKERATIN
CD3
CD20
Negative control
Same steps but with omission of primary
antibody
Validation of new antibody
Antibody_CD138______ Clone M115 CodeNo._M7228
Vendor _ ___ Batch/Lot No._00037097__________
Comments:
Sign: Date:
S.N0 BLOCK MANUFACT.DILn. REMARKS
PATHNO. TISSUE 1:50
1 32320BX Satisfactory.
2 32210BX Satisfactory
3 18083BX Satisfactory
4 14597BZ Satisfactory
Workflow in IHC lab
Workflow in IHC: Making an
entry
Making the required entries
Blocks are cut in routine manner
Tissue sections on
poly-L-lysine
coated slides
Bind Primary antibody
Wash
Biotinylated 2
nd
antibody
Wash
Chromogen development
Counterstain dehydrate
coverslip examine
Block non-sp
binding
Deparaffinize
Xylene
Block
endogenous
peroxidase
Antigen Retrieval
Antigen retrieval -
standardization
Proteolytic enzyme methods
Heat induced epitope retrieval
Microwave
Pressure cooker
Achieving the desired temperature
pH of buffer- calibration
Holding times
Why is antigen/epitope retrieval
necessary? ..Formalin Fixation
Slides immersed in buffer solution
Scientific pressure cooker
Microwave
Primary Antibody
Primary Antibody vial -1ml
After opening make 20
aliquots of 50 microlitre each
For daily use make fresh
dilutions of primary
antibodies from aliquots
Micropippettes
Micropippettes
Have a daily dilution chart as a
spreadsheet
Antibody dilution chart Date 10/06/2010
Antibody Retrieval method Dilution
1 CD20 TE-Pascal 1:100
2 MPO SC-Microwave 1:300
3 CD23 TE-Microwave 1:100
4 ER SC-Pascal 1:50
Making the dilutions of antibodies with buffer/diluent
Washings and wiping
Primary antibody on slide
Incubation in humidity chambers
What are we looking for in IHC?
Coloured visual product demonstrating
an antigen antibody reaction:
Brown colour (DAB)
Red colour (PAP-APAAP)
Greenish yellow in
immunoflourescence
Issues related to interpretation
Where should we look for the colour?
Knowledge of Ag location is a must
Knowledge of pattern of staining is
essential
Cytoplasmic (diffuse,paranuclear,
perinuclear)
Nuclear (diffuse, nucleolar)
Membranous (Continuous, broken)
Interstitial
The cell of interest (larger tumor cell etc)
An Example of normal lymph node
CD20
CD3
CD23
Bcl2
Nuclear positivity
ER
Mib-1
p63 PR
Cytoplasmic positivity
Vimentin
Desmin
Membrane positivity
CD20
c-kit
Golgi zone positivity
CD15
CD15
Cell of interest
CD30
LCA
RS cells in Hodgkin lymphoma
CD30
LCA
Cell of interest
CD20
CD163
Cell of interest
C-kit
LCA
Is everything brown
positive?
No, beware
All that is brown is not
real
Background staining
False positivity including
artifacts
Problem of false brown stain
Hydrophobic and electrostatic
interactions
Endogenous peroxidase
Endogenous biotin
Antigen diffusion
Antigen retrieval
Polyclonal antibody
Necrotic tissue
Unexpected results.rules rather than
exceptionawareness is the key
p63 positivity in B-cell lymphomas
C-kit positivity in nasopharyngeal
carcinomas
Aberrant CD4 in myeloid leukemias
CD138 in myelomas and carcinomas
.and the list goes on and on..
Background staining
Polyclonal antibody Necrotic tissue
Antigen diffusion
k light chain
lambda light chain
False positivity
Cross reactivity due to epitope sharing
may result in false positivity
An example is CD79a, a B cell marker
cross reacts with smooth muscle
Error in data entry and
mislabelling(MyoD1 and Mib1)
Artifacts in IHC
Edge and trapping artifacts
Desquamation artifacts
Bubble artifacts
Drying artifacts
Artifacts of poor fixation
Precipitated DAB artifacts
Biotin artifacts
Edge and trapping artifacts
Crushed tissue artefact
Deposits artifact
When do you call an IHC as
negative?
Do not call an immunostain negative
without checking out the positive
controls
Remember the best control is positive
internal control
An example of breast cancer
Breast Cancer
Negative hormonal markers
Positive ER Positive PR
When to call tissue immunodead?
Vimentin immunostain used to decide the
immunoreactivity/preservation of
antigenicity of tissue.
Vimentin is virtually present in all tissues
and even smallest of biopsies usually
show some vimentin reactivity, if well
preserved
Quality issues and
validation
Antibody Clone Vendor Dilution 1 2 3-- 31
ER ID5 DAKO !:100 +6 +6 +6
LCA 2B11 DAKO 1:200 +3 +3 +2
Sign
Month: May2008
Daily positive control chart
Daily controls
Validation of New Control
Old Block New Block
Tissue Path No. Remark Tissue Path No. Remark
BREAST 6223CD DEPLET
ED
BREAST 5960CD VALIDA
TED
FOR ER
Antibody_ER____________
Vendor_DAKO___ Clone No.__ID5___ CODE No._M7047_________
Batch No.00000072_________ Dilution Factor__1:100____________
Comments:
Sign: Date:6/5/2008
How to overcome practical
difficulties of variation in IHC
staining related to human
errors?
AUTOMATION
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Acknowledgement
Without a technical hand
who has immense
dedication and interest in
IHC, it is impossible to
achieve required results.
Acknowledgements
Dr NA J ambhekar Prof & Head
Dr Sumeet Gujral & Dr Subramanian
Mrs Rekha Thorat Senior technical officer
Mr Mahendra Palkar
Mr Aamir Khan
Mr Pritam
Mr Dinkar
Mr Shinde