Download - Basic IHC Final
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Immunohistochemistry Practical issues
Dr Santosh MenonAssistant Professor, Pathology
Tata Memorial Hosipital
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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.
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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 knowledgeable…thanks to internet
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CD20 and Rituximab
CD20
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C-erbB2 and Herceptin in breast CA
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C-kit and imatinib in GIST
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Immunohistochemistry
• Manual
• Automated
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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
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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
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Antigen
Primary Antibody
Labeled Secondary Antibody
Cell with antigens on surface
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Antigen
B
B
PX
PX
PX
B
Avidin Biotin Complex
DAB+H2O2
IHC
Primary Antibody
Labeled Secondary Antibody
Cell with antigens on surface
Detection system
Chromogen
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Detection system
Primary Antibody
Tissue section with antigen
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Primary Antibodies
• Polyclonal antibodies: Heterogeneous population of antibodies, produced against several epitopes on a single antigen- multiple clones produce the antibodiesMore tolerant to retrieval techniquesSpecificity is less
• Monoclonal antibodies: Homogenous population of antibodies for a single epitope – from a single clone of B-cellsMore specific Less cross reactivityLess background non specific staining
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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
12
34
mmm
m
12 3
4
1 2 3 4
Monoclonalantibodies
Cell fusion
Spleen cells
+Myeloma
x
Antiseum
Antigen
Immunization
A mixture of all Ab
1 23 4
BALB/c
1 2 3 4
1 23 4
B cell
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Primary Antibody vial
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How do we start with a new antibody?
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If you have acquired a new antibody…… Titration is a must
Date Antibody Used
Dilution Appropriate dilution
Remarks
04-04-08 CD20 1:50, 1:1001:200
1:100 GOOD
05-05-08 CD3 1:1001:2001:300
1:200 BEST
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Positive controls
CYTOKERATIN
CD3
CD20
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Negative control
Same steps but with omission of primary antibody
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Validation of new antibodyAntibody_CD138______ Clone M115 CodeNo._M7228
Vendor _ ___ Batch/Lot No._00037097__________
Comments:
Sign: Date:
S.N0 BLOCK MANUFACT.DILn. REMARKSPATHNO. TISSUE 1:50
1 32320BX “ Satisfactory.
2 32210BX “ Satisfactory
3 18083BX “ Satisfactory
4 14597BZ “ Satisfactory
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Workflow in IHC lab
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Workflow in IHC: Making an entry
Making the required entries
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Blocks are cut in routine manner
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Tissue sections on poly-L-lysine coated slides
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Bind Primary antibody
Wash
Biotinylated 2nd
antibody
Wash
Chromogen development
Counterstain dehydrate coverslip examine
Block non-sp binding
Deparaffinize
XyleneBlock
endogenous peroxidase
Antigen Retrieval
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Antigen retrieval -standardization
• Proteolytic enzyme methods• Heat induced epitope retrieval
MicrowavePressure cooker
Achieving the desired temperaturepH of buffer- calibration
Holding times
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Why is antigen/epitope retrieval necessary? ……..Formalin Fixation
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Slides immersed in buffer solution
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Scientific pressure cooker
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Microwave
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Primary Antibody
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Primary Antibody vial -1ml
After opening make 20 aliquots of 50 microlitre each
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For daily use make fresh dilutions of primary
antibodies from aliquots
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Micropippettes
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Micropippettes
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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
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Making the dilutions of antibodies with buffer/diluent
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Washings and wiping
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Primary antibody on slide
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Incubation in humidity chambers
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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
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Issues related to interpretation
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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)
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An Example of normal lymph node
CD20 CD3
CD23Bcl2
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Nuclear positivity
ER Mib-1
p63 PR
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Cytoplasmic positivity
VimentinDesmin
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Membrane positivity
CD20 c-kit
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Golgi zone positivity
CD15 CD15
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Cell of interest
CD30
LCA
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RS cells in Hodgkin lymphoma
CD30 LCA
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Cell of interest
CD20
CD163
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Cell of interest
C-kit LCA
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Is everything brown positive?No, beware
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All that is ‘brown’ is not real
• Background staining
• False positivity including artifacts
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Problem of false brown stain
• Hydrophobic and electrostatic interactions
• Endogenous peroxidase• Endogenous biotin • Antigen diffusion• Antigen retrieval• Polyclonal antibody• Necrotic tissue
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Unexpected results….rules rather than exception…awareness 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……..
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Background staining
Polyclonal antibody Necrotic tissue
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Antigen diffusion
k light chain lambda light chain
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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)
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Artifacts in IHC
• Edge and trapping artifacts• Desquamation artifacts• Bubble artifacts• Drying artifacts• Artifacts of poor fixation• Precipitated DAB artifacts• Biotin artifacts
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Edge and trapping artifacts
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Crushed tissue artefact
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Deposits artifact
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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
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An example of breast cancer
Breast Cancer Negative hormonal markers
Positive ER Positive PR
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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
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Quality issues and validation
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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
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Daily controls
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Validation of New Control
Old Block New Block
Tissue Path No. Remark Tissue Path No. Remark
BREAST 6223CD DEPLETED
BREAST 5960CD VALIDATED 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
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How to overcome practical difficulties of variation in IHC
staining related to human errors?
AUTOMATION
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wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww
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Acknowledgement• Without a technical hand
who has immense dedication and interest in IHC, it is impossible to achieve required results.
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Acknowledgements• Dr NA Jambhekar 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
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