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Introduction to and the History of
ImmunoHistoChemistry (IHC)
Histoteknikerforeningens seminar
20 March 2015
Helle Nielsen, Dako
Page 1
IHC – Introduction
Immunohistochemistry (IHC) combines histological, immunological
and biochemical techniques for the identification of specific tissue
components by means of a specific antigen/antibody reaction tagged
with a visible label.
IHC makes it possible to visualize the distribution and localization
of specific cellular components within a cell or tissue.
Detection af antigens
by antibodies and
visualized
in a microscope
Page 2
IHC – Parameters
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Page 3
Fixation & tissue preparation
Pre-treatment / epitope retrieval
Primary antibody
Detection system
Control material
Milestones in IHC
Coon
Taylor & Huang
Shi
“Us”
Human Genome
Label. Ab
Protease
HIER
Experience
IgK/L
Frozen
IgK/L
Paraffin
B&T cells
>100 CD
Diagnostic
Predictive
1940
1974
2003
2005
Page 4
1991
Spec. Ab
Historical overview – detection
1941
IF
labeled ab
1967
Enzyme
labeled ab
Poor sensitivity
Page 5
Historical overview – detection
1970
Secondary
Un labeled
ab
1978
APAAP
db staining
Page 6
Historical overview – detection
1995
ENVISION™
Polymer
Based
Detection
Dako
Avoiding endogenous biotin when high pH HIER is used
-can interfere with the interpretation !
Sensitivity increased
Page 7
Historical overview – detection
2008
FLEX+
Mouse/rabbit
Linker
Page 8
Historical overview – detection
1999
Bond™
Polymer Refine
Detection
(Leica)
2009
UltraView
Multimer
Detection
(Ventana/Roche)
Page 9
2012
OptiView
Multimer
Detection
(Ventana/Roche)
Historical
overview – enzymes
IHC
- Chromogen
Horse Radish Peroxidase (HRP)
+ high sensitivity
+ precise chromogenic reaction
+ can be amplified by metal (Cu/Ni)
Alkaline Phosphatase (AP)
+ suitable for cryostate sections and cytology (hematology)
+ double IHC staining
- granular chromogenic reaction
- relative low sensitivity
Page 10
Historical overview – chromogens
HRP:
Diaminobenzidin (DAB) - brown
Most commonly used chromogen!
Alcohol resistant
Amino-Ethyl-Carbazol (AEC) - red
Not alcohol resistant
NovaRed – red/brown
Alcohol resistant
AP:
Fast Red TR – bright red
Not alcohol resistant
More Vendors have now Red Chromogens for
AP which are alcohol resistant
Page 11
Newly released chromogens:
HRP:
AP:
Purple
Blue
Green
Green
Black
Blue
Double/Trippel/Quatro/ .......staining
Historical overview – antibodies
Quality of antibodies – not optimal in the beginning.....
Only polyclonal antibodies, batch to batch variation, unspecific reaction
Dr. Niels Harboe realized the need of standardization of the
antibodies to make reliable tests for diagnose !
He founded Dako in 1966 together with 110 rabbits!!
He began the developement of antibodies to make a
immunhistochemstry test reproducible.
Page 12
Historical overview – antibodies
1975: Invention of monoclonal antibodies (Kohler & Milstein)
Hybridoma technique was born and production of specific
antibodies gave a rapid growth in IHC both in research and in
diagnosis of cancer.
1991: Heat induced antigen retrieval (Shi et al)
Protease
1974
Introduction of HIER (Heat-Induced Epitope Retrieval) IHC could
be used in a much broader range of applications in FFPE tissue
sections, but a new factor got added to the protocol!
Fixation......Preanalytic step.....Standardization?...
Page 13
Historical overview
”Immunohistochemistry” articles listed in PubMed in 40 years
35000
30000
25000
20000
15000
10000
5000
0
1973
Page 14
1983
1993
2003
2013
Antibodies
Mouse monoclonals - one clone to one antigen determinant
High specificity - low to high sensitivity
Rabbit polyclonals - more clones to more antigen determinants
Low to high specificity - high sensitivity
Rabbit monoclonals High specificity – high sensitivity
Page 15
Antibodies
Monoclonal antibodies
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
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Mouse or rabbit hybridoma
Tends to be ‘cleaner’
Very consistent batch-to-batch
Supernatant / Ascites
Polyclonal antibodies
 Many different species
 Tends to have more non-specific reactivity
 Can have very different
avidity/affinity batch-to-batch
Page 16
IHC – Reaction Pattern
IHC staining is widely used in the diagnosis and treatment of cancer
IHC is identification of specific antigens (proteins) in cells / tissue
using antibodies against the antigen of interest.
Antigens (proteins) can be localized in the
 Cell membrane
 Cytoplasm
 Nucleus
Page 17
IHC – Reaction Pattern
Page 18
IHC – why ?
Cases for microscopy (FFPE sections)
70 – 90 % are diagnosed by H&E = standard staining
30 % additional special stains
IHC is needed in 10-25% of malignant tumours
 Narrowing of possibilities
 Specific diagnosis
 Unsuspected diagnosis
Page 19
IHC – why ?
Important to achieve a correct and specific diagnose !
The patient will get the right treatment/therapy !
Diagnose and treatment are linked together.
Breast cancer:
Ductal
Lobular
In-situ.......
Lung cancer:
Small cell
NSCLC
Adenocarcinoma
Planocell. Carcinoma
Metastasis ?
You often have to use antibodies in a panel!
Page 20
IHC – why ?
Classification
Invasive
Therapy
Micro organism
H&E staining
Page 21
IHC – why ?
Helps you to
find the cells
and structures
Page 22
IHC – why ?
Glioma ?
Metastatic carcinoma ?
Malignant lymphoma ?
Other ?
Page 23
IHC – Primary Panel
Page 24
Pan-CK
S-100
CD45
VIM
IHC – Primary Panel
Page 25
IHC – why ?
Genomic instability in cancer cells
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Page 26
Mutation
Deletion
Insertion
Translocation
Amplification
Lack of one or more DNA-repair systems
IHC – Mutation
Beta Catenin
Page 27
IHC – Amplification
Guidelines for Scoring HercepTest TM
Score:0
HER2 CISH
Score: 1+
HER2 FISH
Score: 2+
Score: 3+
Chromosome 17
HER2
Over expression of oncoprotein
Page 28
IHC – Translocation
Anaplastic large cell Lymphoma
Follicular Lymphoma
Mantle cell Lymphoma
MALT Lymphoma,
PNET/ES,
DSRCT,
Page 29
t(2;5)
t(14;18)
t(11;14)
t(1;14), t(11;18)
t(11;22)
t(11;22)
ALK-1
BCL-2
Cyclin-D1
BCL-10
FLI-1
WT-1
IHC – Translocation
Over expression of Bcl-2 caused by translocation (t14;18)
Immunoglobulin genes (heavy chain or light chain genes)
Over expression of Bcl-2 prevent apoptosis
Normal lymph node
Page 30
Bcl-2 positive follicular lymphoma
IHC - Translocation
Using IHC instead of ISH present a cost-effective
screening for ALK rearrangement in NSCLC
Page 31
IHC – Lack of DNA repair gene
Page 32
IHC – Lack of DNA repair gene
MSH2
Page 33
IHC – Dilution
The optimal dilution is a staining giving the maximum signal/noise
ratio
100
90
80
70
60
Spec.
50
Unspec.
40
30
20
10
0
FP
Signal is the
specific signal
FN
Noise is the unspecific –
back-ground signal
1:10.
Page 34
1:25.
1:50.
1:100.
IHC – Dilution
Case 1
Case 2
Pan-CK AE1/AE3 staining in two labs.
Serial sections from a multi tissue block with 3 cases of SCLC
Page 35
Case 3
IHC- RTU
Dakos concept of RTU:
RTU is not only an antibody
RTU
Antibodies
RTU is a system of parameters:
• antibody
• protocol
• software
• instrument
RTU
Protocols
EnVision Flex
Automation
“Dako Omnis”
“Autostainer”
Key opinion leaders
• low expressors
• high expressors
Page 36
IHC- RTU
Fra NordiQC run 42 / B18 2014:
Antibody
name
Antibody
clone
Antibody
format
Amount
of labs
% optimal +
good results
Melan A
A103
Conc ab*
100
60
Melan A
A103
Dako RTU
43
91
CD15
Carb-3
Conc ab*
55
85
CD15
Carb-3
Dako RTU
49
96
PGR
PgR636
Conc ab*
49
90
PGR
PgR636
Dako RTU
62
97
*all vendors
Page 37
IHC – “old days”
In the past IHC was a manual staining process....
Wash, apply, wash, apply.....
Every slide was handle separately......time consuming,
risk of errors...
Page 38
IHC – “old days”
Cadenza, Shandon
ES, Ventana
Page 39
TechMate Dako
IHC – Semi-automation
LabVision/Thermo Scientific Autostainer
Dako 48 link Autostainer
Nexes Ventana
Biocare IntelliPath
Dako Autostainer Plus
Page 40
IHC – Pre-treatment
Dako PT Link
MBO
LabVision/ThermoScientific
PT module
Pressure Cooker
Waterbath
Page 41
IHC – Total automation
Ventana Benchmark Ultra
Leica Bond-III
BioGenex
Xmatrx ULTRA
Page 42
Dako Omnis – Total automation IHC/ISH
True walk-away solution
• 15 minutes of daily set-up time
Same day patient case completion
• IHC 2:30 hours – run time
• ISH 3:40 hours – run time
Unmatched throughput
• Capability to load up to 165 IHC slides or
60 ISH slides in a typical workday
(8 hours/shortest protocols)
Including over night run
Protocols:
• IHC – single/double stain
• IF – direct/in direct
• ISH – HER2
• Coming: Probes (own and 3rd party)
Page 43
Questions
Page 44