04_ihc practical issues
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TMH proceedings 2010-2011,pdfTRANSCRIPT
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Immunohistochemistry Practical issues
Dr Santosh MenonAssistant Professor, PathologyTata Memorial Hospital
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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
B 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 antibodies
More 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
I
Adapted from Milstein (1980) Scientific American, Oct.
12
34
mmm
m
12 34
1 2 3 4
Monoclonalantibodies
Cell fusion
Spleen cells
+Myeloma
x
Antiseum
Antigen
Immunization
A mixture of all Ab
1 23 4
B A L B / 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
WashAvidin Peroxidase
complexes
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 Microwave Pressure cooker
Achieving the desired temperaturepH of buffer- calibration
Holding times
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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:Ø Brown colour (DAB)Ø Red colour (PAP-APAAP)Ø 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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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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What do we mean by quality?
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Looks sameTastes sameSmells same
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Quality in IHC means……
• Standardised procedures• Validated antibodies• Sensitivities and specificities of antibodies• Accurate, reproducible and comparable results• Checkpoints for corrective actions• Continuous monitoring and improvement
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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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Thankfully!• Automation cannot interpret Surgical
path and IHC slides…….• Otherwise I would have no job……
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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• Dr Saral Desai• Mrs Rekha Thorat Senior technical officer• Mr Mahendra Palkar• Mr Aamir Khan• Mr Pritam• Mr Dinkar• Mr Shinde
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THANK YOU