An
advanced diagnostic method
in
surgical pathology

Rosai : “The H & E technique”
Old mistress apologue

Mainstay of surgical pathology
The H & E technique ,1998
pathologica
“Hemotoxylum campechianum”
Bloody red bark tree
Mexico
Campeche
H&E Advantages
Relatively quick
Inexpensive
Suitable for most situations
Easy to master
Allows accurate Dx of the large majority of specimens
Nevertheless, Cannot answer all the questions
SPECIAL TECHNIQUES
Special stains
Enzyme histochemistry
Tissue culture
Electron microscopy
Immunohistochemistry vs Immunocytochemistry !
Flow cytometry
Cytogenetics
Molecular pathology
Histometry
Chromogene
Tag
2nd Ab
1st Ab
Ag
To detect target antigens
cytoplasmic
cytokeratin
vimentin
chromogranin A
nuclear
estrogen receptor
progesterone receptor
Ki-67
P-53
memebranous
Her 2-neu
E-cadherin
EGFR
Basic structure:
Polypeptide
Glycoprotein
Lipoprotein
Antigen
• The small site on an antigen
to which a complementary antibody
may specifically bind is called
an epitope.
• This is usually one to six monosaccharides
or 5–8 amino acid residues on the surface
of the antigen.
Antigen
• Because antigen molecules exist in space,
•specific three-dimensional antigenic conformation
(e.g., a unique site formed by the interaction
of two native protein loops or subunits),
or the epitope may correspond to a simple primary
sequence region.
• Such epitopes are described as conformational and linear.
Antigen
•The range of possible binding sites is enormous, with each
potential binding site having its own structural properties
derived from:
covalent bonds,
ionic bonds and
hydrophilic and
hydrophobic interactions.
Ag-Ab reaction
For efficient interaction to occur between
the antigen and the antibody:
the epitope must be readily available for binding.
If the target molecule is denatured, e.g.
through fixation,
reduction,
osmolalaity changes,
pH changes,
temperature,
chemical agents,
the epitope may be altered
and this may affect its ability to interact with an antibody.
Characteristics of a Good Antigen Include:
1.Areas of structural stability and chemical complexity within
the molecule.
2.Lacking extensive repeating units.
3.A minimal molecular weight of 8,000–10,000 Daltons,
although haptens with molecular weights as low as 200 Da
have been used in the presence of a carrier protein.
4.The ability to be processed by the immune system.
Characteristics of a Good Antigen Include:
5.Immunogenic regions which are accessible to the antibodyforming mechanism.
6.Structural elements that are sufficiently different from the
host.
7.For peptide antigens, regions containing at least 30% of
immunogenic amino acids: K, R, E, D, Q, N.
8.For peptide antigens, significant hydrophilic or charged
residues.
Class/
Subclass
Heavy
Chain
Light Chain
Molecular Weight
(kDa)
Structure
Function
lgA1
lgA2
a1
a2
l or κ
150 to 600
Monomer to tetramer
Most produced lg; protects mucosal surfaces; resistant to
digestion; secreted in milk.
lgD
d
l or κ
150
Monomer
Function unclear; Works with lgM in B-cell development.
mostly B cell bound
lgE
e
l or κ
190
Monomer
Defends against parasites; causes allergic reactions
lgG
lgG2a
lgG2b
lgG3
lgG4
g1
g2
g2
g3
g4
l or κ
150
Monomer
Major lg in serum; good opsonizer; moderate complement
fixer (lgG3); can cross placenta
lgM
µ
l or κ
900
Pentamer
First response antibody; Strong complement fixer; Good
opsonizer
Monoclonal vs. Polyclonal
2nd Ab : Mouse / goat / rabbit anti- 1st Ab
1st Ab : Mouse / goat / rabbit anti-human
Antigen-Antibody Interaction
The specific association of antigens and antibodies is dependent
on hydrogen bonds, hydrophobic interactions, electrostatic
forces, and van der Waals forces.
These are all bonds of a weak, non-covalent nature-Like
antibodies.
Antigen-Antibody Interaction
Antigens can be multivalent, either through multiple copies of the
same epitope, or through the presence of multiple epitopes that
are recognized by multiple antibodies.
Interactions involving multivalency can produce more stabilized
complexes, however multivalency can also result in steric
difficulties, thus reducing the possibility for binding.
Antigen-Antibody Interaction
All antigen-antibody binding is reversible, however, and
follows the basic thermodynamic principles of any
reversible bimolecular interaction:
where KA is the affinity constant, Ab and Ag are the molar
concentrations of unoccupied binding sites on the antibody
or antigen respectively, and Ab–Ag is the molar
concentration of the antibody-antigen complex.
Antigen-Antibody Interaction
Affinity describes the strength of interaction between
antibody and antigen at single antigenic sites. Within each
antigenic site, the variable region of the antibody “arm”
interacts through weak non-covalent forces with antigen at
numerous sites; the more interactions, the stronger the
affinity.
Antigen-Antibody Interaction
Avidity is perhaps a more informative measure of the overall
stability or strength of the antibody-antigen complex. It
is controlled by three major factors:
1.antibody-epitope affinity;
2. the valence of both the antigen and antibody;
3.and the structural arrangement of the interacting parts.
Polyclonal vs monoclonal
1.Polyclonal antibodies often recognize multiple epitopes,
making them more tolerant of small changes in the nature of the
antigen.
Polyclonal vs monoclonal
2.Polyclonal antibodies are often the preferred choice for detection
of denatured proteins
Polyclonal vs monoclonal
3.Polyclonal antibodies may be
generated in a variety of species,
including rabbit, goat, sheep,
donkey, chicken and others,
giving the users many options
in experimental design.
Polyclonal vs monoclonal
4.Polyclonal antibodies are sometimes used when the nature of the
antigen in an untested species is not known.
Polyclonal vs monoclonal
5.Polyclonal antibodies target multiple epitopes and so they
generally provide more robust detection.
Monoclonal vs polyclonal
6.Because of their specificity,
monoclonal antibodies are excellent
as the primary antibody in an assay,
or for detecting antigens in tissue,
and will often give significantly
less background staining than
polyclonal antibodies.
Monoclonal vs polyclonal
7.When compared to that of
polyclonal antibodies, homogeneity
of monoclonal antibodies is very high.
If experimental conditions are kept
constant, results from monoclonal
antibodies will be highly reproducible
between experiments.
Monoclonal vs polyclonal
8.Specificity of monoclonal antibodies
makes them extremely efficient for
binding of antigen within a mixture
of related molecules, such as in the
case of affinity purification.
Slide 28
© 2003 By Default!
Basic concepts
&
Historical aspects
A Free sample background from www.powerpointbackgrounds.com
Slide 29
© 2003 By Default!
Basic Concept
Immunohistochemistry is
1.The localization of antigens in tissue sections by the use of
2.labeled antibody as specific reagents through
3.antigen-antibody interactions
4.that are visualized by a marker such as
fluorescent dye,
enzyme,
radioactive element or
colloidal gold.
A Free sample background from www.powerpointbackgrounds.com
Slide 30
© 2003 By Default!
There are numerous immunohistochemistry
methods that may be used to localize antigens.
The selection of a suitable method should be
based on parameters such as :
the type of specimen under
investigation and
the degree of sensitivity required.
A Free sample background from www.powerpointbackgrounds.com
Slide 31
© 2003 By Default!
Immunohistochemistry
Historical aspect
1.Albert H. Coons 1944- 1955; fluorescent dye
2.Nakane and Pierce 1966 ; enzyme labels / peroxidase;
Mason and Sammons 1978 alkaline phosphatase
3.Faulk and Taylor 1971; Colloidal gold by both light and
electron microscopy level
4.Radioactive elements, and visualization by autoradiography.
A Free sample background from www.powerpointbackgrounds.com
Slide 32
© 2003 By Default!
With the expansion and development of immunohistochemistry
technique,
enzyme labels
have been introduced such as
peroxidase (Nakane and Pierce 1966; Avrameas and Uriel 1966)
and
alkaline phosphatase (Mason and Sammons 1978).
A Free sample background from www.powerpointbackgrounds.com
TO APPLY IHC OR ICC FOR …?

1.Diagnostic purpose.

2.Prognostic purpose

3.Therapeutic purpose

4.Preventive purpose?
Applications of IHC
Diagnostic purpose:
Applications of IHC
Diagnostic purpose:
IHC profile for metastatic carcinoma of unknown origin
Female
Male
ER/PR
PSA
CK 7
CK 7
CK 8
CK 8
CK 20
CK 20
CD X2
CD X2
CEA
CEA
TTF 1
TTF 1
Diagnostic purpose of IHC
CK
7
+
-
CK 8 CK 20
Dx
+
-
Follicular adenocarcinoma ,Thyroid
Adenocarcinoma ,Pancreas
+
+
-
+
-
Hepatocellualr carcinoma
Squamous cell carcinoma CK 5/6
Thymoma
CK 5/6
Diagnostic purpose of IHC
CK
8
+
CK 18
Dx
+
Follicular adenocarcinoma ,Thyroid
Papillary adenocarcinoma ,Thyroid
Adenocarcinoma,Pancreas
Hepatocellualr carcinoma
+
-
+
Trichoepithelioma
-
-
Meningioma
Collectind duct carcinoma
Thymoma
Adenocarcinoma , Ampullary
Applications of IHC
Diagnostic purpose:
an example
IHC profile for prostatic carcinoma
Slide 40
© 2003 By Default!
IHC in prostate Cancer

Indications:
1- Distinction of Benign from Malignant
• High molecular weight cytokeratin (34E12, CK5/6)
– Negative cytoplasmic marker (in basal cells)
• P63
– Negative nuclear stain (in basal cells)
• AMACR (P504S)
– Positive cytoplasmic marker (in tumor cells)
– Also positive in HGPIN, 31% of Bladder Ca. &
70% of Colorectal Ca.
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Slide 41
HMW-CK (34E12)
© 2003 By Default!
Normal Glands
Negative in Carcinoma
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Slide 42
© 2003 By Default!
AMACR
(P504S) stain
in Carcinoma
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Slide 43
© 2003 By Default!
Increasing IHC
resolution:
P63 / AMACR
cocktail
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Slide 44
© 2003 By Default!
Increasing IHC
resolution:
34E12 / P63 / AMACR
2-chromogen cocktail
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Slide 45
© 2003 By Default!
IHC in prostate Cancer (Cont.)

Indications:
2- Differential Dx from urothelial carcinoma:
PSA
PSAP 34E12 Leu7 CK5/6
Prostate Ca
+
+
–
+
-
Urothelial Ca
–
–
+
–
+
A Free sample background from www.powerpointbackgrounds.com
Slide 46
© 2003 By Default!
IHC in prostate Cancer (Cont.)

Indications:
3- Differential Dx in metastatic carcinoma:
Bone Tumor:
PSA stain
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Applications of IHC
Prognostic purpose
in breast carcinoma
Poor prognostic markers
Good prognostic markers
ER(-)
ER(+)
PR(-)
PR(+)
pS2(-)
pS2(+)
Her-2 (3+)
Her-2 (0/1+)
Cathepsin D(+)
Cathepsin D(-)
P 53 >10%
P 53 <10%
EFGR (+)
EGFR( - )
Ki-67>23%
Ki-67<11%
Prognostic purpose of IHC

ER/PR/pS2:

pS2 : Cystein-rich peptide induced by estrogen secreted from
breast cells.

pS2 expression has been found to be associated with longer
overall & disease free survival.

ER +/ PR +/ pS2 + : 85% to 97% have good prognosis
ER +/ PR +/ pS2 - : only 50% to 54% have good prognosis.

Clinical data
inconsistency
How to Assess an IHC
immunostain?
reproducibility
Inter-observer variation
An example
Assessment of Her2 status
in breast cancer
Published guidelines (ASCO)
Her2 over-expression should be evaluated on every
primary breast cancer either at the time of diagnosis or
at the time of recurrence.
J Clin Oncol 19:1865-1878, 2001
Importance of getting it right
• Poorer prognosis if HER2 positive
• Role in the selection of the most appropriate
adjuvant therapy
• Herceptin improves survival if HER2 positive,
but not HER2 negative
®
– false negatives
• deny life-extending treatment
– false positives
• false hope
• complications & cost of the treatment
HER2 technical approaches
• Gene amplification
– Southern or dot (slot) blotting
– quantitative PCR
– FISH/CISH
• mRNA over-expression
– Northern blotting
– quantitative RT-PCR
• Protein over-expression
– immunohistochemistry
– Western blotting
– Elisa
POINT MUTATIONS
m-RNA
C-proto
-onc
C-onc protein over
production with
Increased activity
C-onc
Point Mutation
M-RNA
overexpression
IHC
vs
FISH
•
•
•
•
•
•
•
Fast
Cheap
Easy
False-negative rate
False-positive rate
Subjective interpretation
Difficult to standardise
•
•
•
•
Long
Expensive
Difficult
Accurate (#signals)
•
Theoretically does not identify pts with
overexpression without gene amplific
Standardised
•
IHC/FISH concordance
0
1+
2+
3+
FISH -
207
28
67
21
FISH +
7
2
21
176
3%
7%
24%
89%
Overall concordance: 82%
Mass R et al.: Proc ASCO 2000
Her2 testing algorithm
Patient tumour
sample
IHC
–
FISH
2+
3+
+
Retest with
FISH
Herceptin®
therapy
Herceptin®
therapy
+
Herceptin®
therapy
–
Slide 58
© 2003 By Default!
How to interpret?

1.Intimacy with pattern of staining:
Nuclear
ER
PR
P 53
Ki 67
Membranous
Her 2
EGFR
CD
20
CD
45
Cytoplasmic
CK
Vime
ntin
Desm
in
NSE
Cytonuclear
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SMA
HMB45
S100
Calret
inin
How to interpret?
2.Intimacy with scoring methods
Score
Pattern
Assessment
0
Staining of 0-10% cells
Negative
1+
Faint and partial
membrane staining
Weak to moderate
complete membrane
staining
Moderate to strong
complete membrane
staining
Negative
2+
3+
Positive/
Negative
Positive
Proficiency surveys of HER2 testing
2001 College of American Pathology IHC survey
• A breast tumor sample unreactive by IHC and
FISH was distributed to 415 laboratories
– 72% reported negative results
– 28% reported immunoreactivity
– 9% reported 2+ or 3+ scores
Conclusion
 Any HER2 assay performed at a non-reference laboratory
(<100 cases/month) will require validation at a reference
laboratory
Potential for misdiagnosis
(IHC)
• Antibodies (>28 commercially available)
• Technical performance
• Interpretation
– scoring
– artifacts
Common problems in HER2 IHC
• Underestimation of expression:
– (over)fixation in NBF
– poor antigen retrieval (unmasking)
– choice of antibodies
• Overestimation
– alcoholic (post)fixation (check normal
cells!)
• Cytoplasmic staining
Who can interpret an IHC?
Normal lobule
3+ breast carcinoma
Lobular cancerization
2+ breast carcinoma
2+ breast carcinoma?
Normal lobule
Cytoplasmic staining
What to do?
• Central Reference or large local laboratories
– high number of cases (>250 cases/year* or >100
cases/months [NSABP])
– quality assurance controls (internal and external)
– automated IHC
– high level of training (technique/interpretation)
• Small laboratories (<250 cases/yr or <100 cases/mos)
– do not do it
– send to larger laboratories
*Ellis I, et al: J Clin Pathol 2004
… but
• There are labs capable of performing
quality testing with lower volumes
• A high test volume does not ensure an
accurate test result
• If an individual lab can properly validate
an assay and perform acceptably in an
external validation, then it should be
permitted to offer the test
Hsi ED & Tubbs RR: JCP 2004
Applications of IHC
therapeutic purpose
Chemotherapy in breast carcinoma
ER (+)
PR (+)
Tamoxifen
/ Femara
ER (-)
PR (+)
Tamoxifen
/ Femara
ER (+)
PR (-)
Tamoxifen
/ Femara
Her-2
psoitive
Her-2
negative
Trastazumab
(Herceptin)
-
Cost :300 000 000 Rials
-
Applications Of IHC



Therapeutic purpose: PharmaDx Abs
Chemotherapy in breast carcinoma
ER - / PR - / Her-2 Triple negative breast carcinoma
Treatment is completely different:
Cisplatin
Technical points
Handling of Antibodies

RTU Abs have a shorter shelf life

Upon receipt reagents should be stored promptly according to
manufacturer’s recommendation.

Record: lot No, expiration date, date of receipt, invoice number.
Storage Containers

Containers:





Negligible protein absorptivity
Polypropylene, polycarbonate, borosilicate glass
Clear & colorless containers
Labels should allow access for inspection
Solutions containing very low concentrations of protein
(<10-100 microg/mL) should receive inert protein such
as 0.1 % to 1.0 % albumin to reduce polymerization &
adsorption onto the container.
Storage Temperature







Accurate and consistent temperature
Temperature alarm & emergency backup system
Store RTU Abs & kits at 2-8 C
Store concentrated Abs at -20 C in aliquots
Prevent from contamination, heat, excessive light exposure.
Sterile , clean pipette tips
Prompt return to storage temperature.
Antibody Titer
Antibody Dilution

How to determine optimal dilution? Titration
First select a fixed incubation time.
Make small volumes of experimental dilutions.
100 to 400 microL per section

What is the optimal dilution?






Peak in intensity
Minimal background
Maximal signal to noise ratio
Effecte of pH & ion strength on Ab dilution

All monoclonal Abs could be diluted higher &
stained more intensly at pH 6.0 .

IgG3 is an exception at pH 9.0
PBS suppress the reactivity of all monoclonal
Abs

Commercial Ab diluents are preferred.

PBS is usually used for dilution nevertheless it
leads to less reaction and decrease in affinity,
thus not recommended.
Antibody Dilution Buffers
Antibody dilution buffer is used for diluting primary and secondary
antibodies as well as some detecting reagents.
Primary Antibody Dilution Buffer
1% BSA (stabilizer and blocking)
0.1% cold fish skin gelatin (blocking)
0.05% sodium azide (preservative)
0.01M PBS, pH 7.2
Antibody Dilution Buffers
TBS as Ab diluent
1)TBS pH 7.6 used in primary antibody dilution buffer produces
weaker staining;
2)Antibodies diluted using this buffer can be stored at 4 ºC for 6
months without reducing binding activity;
3) This buffer can not be used for diluting HRP conjugated
antibodies since sodium azide is an inhibitor of HRP.
How to dilute?
1:10 dilution = one part of stock solution + nine
parts of diluents.
Then , two-fold serial dilutions
How to dilute

To prepare 1.0 mL of a 1:1000 dilution:
Step 1: 10 micro + 90 micro ------- > 1:10
 Step 2: 10 micro + 990 micro ----- > 1:100
 Final dilution is 1:1000

Checkerboard titration
Checkerboard titrations are used to determine the optimal dilution
of more than one reagent simultaneously.
1.The optimal dilutions of the primary Ab & the streptavidin-HRP reagent are found.
2.While the dilution of the biotinylated link Ab is held constant.
3.Nine tissue sections are required for testing three dilutions.
4.If results achieved by use of several dilutions are identical or similar, reagent costs
may become an additional factor in selecting optimal dilutions.
Incubation Time
Inverse relationship between incubation time & antibody titer.
The higher the Ab titer , the shorter the incubation time required
for optimal result.
In practice:
First set a suitable incubation time then determine the optimal
dilution.
Incubation Time
The most widely used incubation time:10-30 min
High concentration , high affinity , optimal pH,
Optimal ion strength ------ > shorter incubation
24 hr incubation for economy purpose ----- > allow higher dilutions
Incubation Time
Low titer / low affinity Abs must be incubated for
long periods in order to reach equilibrium.
But nothing can be gained by prolonging primary Ab incubation
beyond the time at which the tissue is saturated with antibody.
Incubation Time
Equilibrium is usually not reached before 20 min.
Consistent timing is important.
Inconsistent timing leads to variations in overall
stain quality & intensity.
Incubation temperature
Equilibrium may achieved more quickly at 37C
compare to RT.
Incubation at 37C allows higher dilution +
shorter incubation time ------ > consistency in
incubation time becomes even becomes more
crucial.
Incubation at 37C ----- > increase background
Incubation temperature
4C is used in combination with overnight or longer incubations.
Slides incubated for extended periods , or at 37C should be placed
in humidity chamber to prevent evaporation and drying of tissue
sections.
Tissue incubated at RT in a very dry or drafty environment will
require the use of a humidity chamber.
Specimen requirements

Paraffin-embedded blocks vs. fresh frozen vs.
cytospin prep. cytological slides.









Comprising the representative lesion. Not too small.
Devoid of necrosis.
Devoid of extensive hemorrhage.
Not too old Paraffin blocks < 3 years old.
Free from over fixation.
Don’t treat with overheated paraffin during embedding &
processing.
Proper labeling.
Clear and precise IHC request.
Corresponding conventional pathology report.
Fixation
Fixation


Each tissue has finite amount of Ag
Most steps in IHC process destroy some of the Ags
particularly : fixation
Fixation

The purpose of fixation:
1. To change protein structure in order to preserve them from
elution,
degradation,
and other modifications
2.To preserve the position of the Ag ;
 Nuclear
 Cytoplasmic
 Membrane-bounded
3.Preserve secondary and tertiary structue
4.To provide a target for Abs
Fixation
Poor or inadequate
fixation



Incorrect
interpretation
Elution of ER protein from nucleus to
cytoplasm
Elution of Cerb-b2 from membrane to
cytoplasm
Therefore diagnostically the stain is useless.
Fixation
Tissue :A
Tissue :A
Neutral buffered formalin
Neutral buffered formalin
ER as target Ag
ER as target Ag
Monoclonal Ab clone E15
Monoclonal Ab clone E45RT
Result: negative
Result: positive
Fixation may destroy specific epitopes thus may lead to improve
two different reactions with two different monoclonal Abs
What is the solution?

Standardization of the fixative and fixation
protocols would be an start.
Fixation & Ag retrieval
AB
Ag
Aldehyde cross link after formalin
fixation


There is no one universal fixative that is ideal for the
demonstration of all antigens.
However, in general, many antigens can be successfully
demonstrated in formalin-fixed paraffin-embedded
tissue sections.
The discover and development of antigen retrieval
techniques further enhanced the use of formalin as
routine fixative for immunohistochemistry in many
research laboratories.




Some antigens will not survive even moderate
amounts of aldehyde fixation.
Under this condition, tissues should be rapidly fresh
frozen in liquid nitrogen and cut with a cryostat without
infiltrating with sucrose.
The sections should be kept frozen at -20 C or lower
until fixation with cold acetone or alcohol.
After fixation, the sections can be processed using
standard immunohistochemical staining protocols
Ten percent neutral buffered formalin , pH 7
(10% NBF)

Fresh
&
Buffered to pH of 7.0-7.6
Formalin 40%
Dibasic sodium phosphate,
anhydrous, Na2HPo4
Monobasic sodium phosphate,
monohydrate, KH2Po4
DW
100 mL
6.5 g
4.0 g
900 mL
Most common fixatives
a) 4% paraformaldehyde in 0.1M phosphate buffer
b) 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate
buffer
c) PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2%
lysine in 0.1M phosphate buffer
d) 4% paraformaldehyde with 0.05% glutaraldehyde (TEM
immunohistochemistry)
Other aldehyde-based fixative

Glutaraldehyde 2%

Act similarly to 10% NBF and are used much less
frequently
Mercuric chloride fixatives

Used in the past

Mechanism: react with amino acid residues such as thiols, amino
groups, imidazole, phosphate, and hydroxyl groups.

Fixation time : is short , as a positive point

Highly toxic with special disposal procedures, as a negative point
Mercuric chloride fixatives

1.B5 fixative
Mercuric chloride fixatives

2.Zenker’s fixative
Alcoholic Fixatives

Carnoy’s

In looking at lymphocytes using CD-specific markers

In looking for immunoglubins such as IgG, IgA, IgM
Tissue pretreatment
1. Tissue not dry out : onto moist absorbent paper ,in a covered
container
2. Rapid delivery to the path. Lab.
3. Trimming and cut for fixation
4. Into blocks no more than 2 cm square by four mm thick
Tissue pretreatment
5. Thickness is important:
1. Penetration: ideally fast
2. Fixation : usually slow
6. Optimum fixation time: 6-12 hr
7. Over fixation can pose problems: increased cross linking
8. How to repair this damage?
Heating the fixed tissue in boiling water to 95 degrees for 15-20 min
Tissue & slide processing

Once the tissue is well-fixed, subsequent steps seem to have little
effect on antigen detection.

Variation in xylol processing, alcohol rehydration, wax
temperature, time or formulation, instrumentation used etc ,
provide satisfactory results.
Tissue & slide processing

No process should raise temperature to higher
than 60C,
as this will cause severe loss of antigenicity that
may not be recoverable.
Tissue & slide processing

Tissue fixation medium must be replaced by wax,
generally done through a series of incubations in
increasing alcohol concentrations to 100 percent,
followed by xylene and then hot wax.

This is to provide stability of the tissue (wax) in order
to make cutting the sections easier.
Tissue & slide processing


Appropriate thickness: 3-4 microns
No more than 5 microns
Tissue & slide processing





Commercially available slides with positive
charge.
Albumin coated slides
Silane coated slides
Poly-L-Lysine coated slides
Sections that are not flat and that have nonadherent ridges likely will be digested or torn
off the slide during immunostain.
Tissue & slide processing :
De-waxing protocol
A. Circle & label the specimen with a diamond pencil
B. Place in 60C oven for 30 minutes
C. Transfer immediately to a fresh xylene bath for three minutes.
D. Repeat step C above with a second xylene bath.
E. Place in a fresh bath of absolute alcohol for three minutes.
F. Repeat step E above with a second bath of absolute alcohol.
G. Place in a bath with 95 percent ethanol for three minutes.
H. Repeat step G with the second 95 percent ethanol bath.
I. Rinse under gently running water.
J. Do not let dry, store in buffer; begin required Ag treatment
or immunostaining
Note: 50 slides per 250 mL of xylene is the limit before the xylene
Is no longer effective and residual wax begins causing artifacts in
the final stained tissue.
Procedure
1.3 micron slide sections
2.Xylol
3.Xylol
4.Absolute alcohol
5.96 alcohol
6.70 alcohol
7.DW
8.3%H2O2/methanol v/v
9.DW
10.Citrate buffer pH=6
11.Cooling
37c
RT
RT
48hr
15min
15min
RT
RT
RT
RT
RT
RT
microwave
RT
15min
15min
15min
rinse/*2/5min
30min
rinse/*2/5min
14min
gradually
Procedure
12.PBS
13.Protein block
14.1st Ab
15.PBS
16.2nd Ab
17.PBS
18.Detection system
19.PBS
20.Chromogen DAB
21.DW
22.Hematoxyline
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
rinse/*2/5 min
10min
10-60min
rinse/*2/5 min
30min
rinse/*2/5 min
30min
rinse/*2/5 min
10min
rinse
3 dips (10 sec)
Citrate Buffer pH=6.0
2.1 gr
acid citric monohydarte
900mL
DW
13mL
NaOH 2 normal
Up to 1000mL
PBS 10x
Na2HPo4
11.5 gr
NaCl
80 gr
KCL
2
KH2Po4
3.4 gr
Up to
1000mL
gr
Antigen Retrieval Protocols
Break the protein cross-links formed by formalin fixation
and thereby uncover hidden antigenic sites.
Heat Induced Epitope Retrieval (HIER)
Hydrochloric Acid Method (pH 1)
Formic Acid Method (pH 2)
Citrate Buffer Method (pH 6)
Citrate-EDTA Buffer Method (pH 6.2)
EDTA Method (pH 8)
Tris-EDTA Method (pH 9)
TBS Method (pH 9)
Tris Buffer Method (pH 10)
Heat Induced Epitope Retrieval (HIER)




Pressure cooker
Autoclave
Microwave
Ag retrieval solutions:
0.01 M citrate pH=6
10x
 1 mM EDTA
10x
 20 mM Tris/0.65 mM EDTA/0.0005% Tween 20
pH=9
10x

Proteolytic Induced Epitope Retrieval (PIER)
Proteinase K Method
Trypsin Method
Pepsin Method
Pronase Method
Protease Method
Frozen Section Epitope Retrieval
SDS Method
Heating En Bloc Method
Blocking:

The main cause of non-specific background staining
is non-immunological binding of the specific
immune sera by hydrophobic and electrostatic forces
to certain sites within tissue sections.

This form of background staining is usually
uniform and can be reduced by blocking those
sites with normal serum.
Blocking:

Background staining may be specific or non-specific.

Inadequate or delayed fixation may give rise to false positive results
due to the passive uptake of serum protein and diffusion of the
antigen.

Such false positives are common in the center of large tissue blocks or
throughout tissues in which fixation was delayed.
Non-immunological binding
Inadequate or delayed fixation
common in the center of large tissue blocks
binding of the specific immune sera by hydrophobic and electrostatic
non-specific background staining is non-immunological binding
usually uniform and can be reduced by
blocking those sites with normal serum.
Blocking:
Antibodies, specially polycolonal antibodies, are
sometimes contaminated with other antibodies
due to impure antigen used to immunize the
host animal.
Immunological binding
impure antigen used to immunize the host animal
contaminated polyclonal antibodies with other antibodies
Non-specific Immunological binding
Non-immunological background
Peroxidase Block
Endogenous peroxidase activity is found in many tissues and can be
detected by reacting fixed tissue sections with DAB substrate.
The solution for eliminating endogenous peroxidase activity is by
the pretreatment of the tissue section with hydrogen peroxide prior
to incubation of primary antibody.
Peroxidase Blocking Solution (3% H2O2 in Methanol)
30% H2O2 ------------------------- 2 ml
Methanol --------------------------- 18 ml
Mix well and store at 4 ºC.
Block sections for 20-30 minutes after primary
antibody incubation.
Note: The solution must be fresh.
Endogenous alkaline phosphatase (AP) activity
Many tissues also contain endogenous alkaline
phosphatase (AP) activity and should be blocked by
the pretreatment of the tissue section with
levamisole if using AP as a label.
Blocking:

Some tissues such as liver and kidney have
endogenous biotin.

To avoid unwanted avidin binding to
endogenous biotin if using biotin-avidin
detection system, a step is necessary for these
tissues by the pretreatment of unconjugated avidin
which is then saturated with biotin.
Avidin/Biotin Block
Avidin 0.001% in PBS
Biotin 0.001% in PBS
Store these blocking solution at 4 ºC.
Incubate sections for 10-15 min each and rinse with
PBS between steps. Recommended to block before
primary antibody incubation.
Controls:
Positive control
To use the tissue of known positive as a control.
If the positive control tissue showed negative staining, the protocol
or procedure needs to be checked until a good positive staining
is obtained.
Controls:
Negative control is to test for the specificity of an antibody
involved.
First, no staining must be shown when omitting primary antibody or
replacing an specific primary antibody with normal serum (must
be the same species as primary antibody).
This control is easy to achieve and can be used routinely in
immunohistochemical staining.
Controls:
Second, the staining must be inhibited by adsorption of a primary
antibody with the purified antigen prior to its use, but not by
adsorption with other related or unrelated antigens.
This type of negative control is ideal and necessary in the
characterization and evaluation of new antibodies but it is
sometimes difficult to obtain the purified antigen, therefore it is
rarely used routinely in immunohistochemical staining.
Direct Method:
Direct method is one step staining method, and involves a labeled
antibody (i.e. FITC conjugated antiserum) reacting directly with
the antigen in tissue sections:DFA
This technique utilizes only one antibody and the procedure is short
and quick. However, it is insensitive due to little signal
amplification and rarely used since the introduction of indirect
method.
Indirect Method:
Indirect method involves an unlabeled primary antibody (first layer)
which react with tissue antigen,
and a labeled secondary antibody (second layer) react with primary
antibody (Note: The secondary antibody must be against the IgG
of the animal species in which the primary antibody has been
raised).
Indirect Method:
This method is more sensitive due to signal amplification through
several secondary antibody reactions with different antigenic
sites on the primary antibody.
In addition, it is also economy since one labeled second layer
antibody can be used with many first layer antibodies (raised
from the same animal species) to different antigens.
PAP Method (peroxidase anti-peroxidase method)
Three layer method
Rabbit antibody to peroxidase,
coupled with peroxidase
Unconjugated goat anti-rabbit
gaba-globulin
PAP Method (peroxidase anti-peroxidase method)
The sensitivity is about 100 to 1000 times higher since the
peroxidase molecule is not chemically conjugated to the anti IgG
but immunologically bound, and loses none of its enzyme
activity.
It also allows for much higher dilution of the primary antibody,
thus eliminating many of the unwanted antibodies and reducing
non-specific background staining.
Avidin-Biotin Complex (ABC) Method
Is standard IHC method.
Avidin, a large glycoprotein, can be labeled with peroxidase or
fluorescein and has a very high affinity for biotin.
Biotin, a low molecular weight vitamin, can be conjugated to a
variety of biological molecules such as antibodies.
Avidin-Biotin Complex (ABC) Method
Three layers method.
1.The first layer is unlabeled primary antibody.
2.The second layer is biotinylated secondary antibody.
3.The third layer is a complex of avidin-biotin peroxidase.
4.The peroxidase is then developed by the DAB or other substrate
to produce different colorimetric end products.
Labeled StreptAvidin Biotin (LSAB) Method
Streptavidin, derived from streptococcus avidini, is a recent innovation for
substitution of avidin.
The streptavidin molecule is uncharged relative to animal tissue, unlike
avidin which has an isoelectric point of 10, and therefore electrostatic
binding to tissue is eliminated.
In addition, streptavidin does not contain carbohydrate groups which might
bind to tissue lectins, resulting in some background staining.
Labeled StreptAvidin Biotin (LSAB) Method
1.The first layer is unlabeled primary antibody.
2.The second layer is biotinylated secondary antibody.
3.The third layer is Enzyme-Streptavidin conjugates (HRPStreptavidin or AP-Streptavidin)
A recent report suggests that LSAB method is about 5 to 10 times
more sensitive than standard ABC method.
Polymeric Methods:
Polymeric Methods:
Dextran polymer technology.
Binding of a large number of enzyme molecules (horseradish
peroxidase or alkaline phosphatase) to a secondary antibody via
the dextran backbone.
The benefits are many, including
increased sensitivity,
minimized non-specific background staining
reduction in the total number of assay steps
Polymeric Methods:
Procedure:
i) Application of primary antibody;
ii) Application of enzyme labeled polymer;
iii) Application of the substrate chromogen.
EnVision+ was developed after EnVision to provide increased
sensitivity.
Chromogen Substrate Solutions
DAB-Peroxidase Substrate Solution (Brown)
DAB-Peroxidase Substrate Soluiton (Gray)
DAB-Peroxidase Substrate Solution (Black)
DAB-Peroxidase Substrate Solution (Blue)
AEC-Peroxidase Substrate Solution (Red)
BDHC-Peroxidase Substrate Solution (Blue)
TMB-Peroxidase Substrate Solution (Blue)
New Fuchsin Alkaline Phosphatase Substrate Sulution (Red)
BCIP/NBT Alkaline Phosphatase Substrate Solution (Blue)
Protocol for DAB Peroxidase Substrate Solution
DAB Peroxidase Substrate Solution – Brown
Final Dilution:
0.05% DAB - 0.015% H2O2 in 0.01M PBS, pH 7.2
Stock Solutions:
1% DAB (20x) in Distilled Water:
Add 0.1g of DAB (3,3’-diaminobenzidine tetrahydrochloride, Sigma) in 10
ml distilled water. Add 10N HCl 3-5 drops and solution turns light brown
color. Shake for 10 minutes and DAB should dissolve completely. Aliquot and
store at –20 C.
0.3% H2O2 (20x) in distilled water:
Add 100ul of 30% H2O2 in 10 ml distilled water and mix well. Store at 4 C
or aliquot and store at –20 C.
Counterstain Solutions
Gill's Hematoxylin Solution (Blue)
Mayer's Hematoxylin Solution (Blue)
Nuclear Fast Red Solution (Red)
Methyl Green Solution (Green)
PI Counterstain Solution (Fluorescent Red)
DAPI Counterstain Solution (Fluorescent Blue)
Slide 155
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© 2003 By Default!
TO APPLY IHC OR ICC FOR …?

1.Diagnostic purpose.

2.Prognostic purpose

3.Therapeutic purpose

4.Preventive purpose?
Applications of IHC
Diagnostic purpose:
Applications of IHC
Diagnostic purpose:
IHC profile for metastatic carcinoma of unknown origin
Female
Male
ER/PR
PSA
CK 7
CK 7
CK 8
CK 8
CK 20
CK 20
CD X2
CD X2
CEA
CEA
TTF 1
TTF 1
Diagnostic purpose of IHC
CK
7
+
-
CK 8 CK 20
Dx
+
-
Follicular adenocarcinoma ,Thyroid
Adenocarcinoma ,Pancreas
+
+
-
+
-
Hepatocellualr carcinoma
Squamous cell carcinoma CK 5/6
Thymoma
CK 5/6
Diagnostic purpose of IHC
CK
8
+
CK 18
Dx
+
Follicular adenocarcinoma ,Thyroid
Papillary adenocarcinoma ,Thyroid
Adenocarcinoma,Pancreas
Hepatocellualr carcinoma
+
-
+
Trichoepithelioma
-
-
Meningioma
Collectind duct carcinoma
Thymoma
Adenocarcinoma , Ampullary
Applications of IHC
Diagnostic purpose:
an example
IHC profile for prostatic carcinoma
Slide 162
© 2003 By Default!
IHC in prostate Cancer

Indications:
1- Distinction of Benign from Malignant
• High molecular weight cytokeratin (34E12, CK5/6)
– Negative cytoplasmic marker (in basal cells)
• P63
– Negative nuclear stain (in basal cells)
• AMACR (P504S)
– Positive cytoplasmic marker (in tumor cells)
– Also positive in HGPIN, 31% of Bladder Ca. &
70% of Colorectal Ca.
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Slide 163
HMW-CK (34E12)
© 2003 By Default!
Normal Glands
Negative in Carcinoma
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Slide 164
© 2003 By Default!
AMACR
(P504S) stain
in Carcinoma
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Slide 165
© 2003 By Default!
Increasing IHC
resolution:
P63 / AMACR
cocktail
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Slide 166
© 2003 By Default!
Increasing IHC
resolution:
34E12 / P63 / AMACR
2-chromogen cocktail
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Slide 167
© 2003 By Default!
IHC in prostate Cancer (Cont.)

Indications:
2- Differential Dx from urothelial carcinoma:
PSA
PSAP 34E12 Leu7 CK5/6
Prostate Ca
+
+
–
+
-
Urothelial Ca
–
–
+
–
+
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Slide 168
© 2003 By Default!
IHC in prostate Cancer (Cont.)

Indications:
3- Differential Dx in metastatic carcinoma:
Bone Tumor:
PSA stain
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Applications of IHC
Prognostic purpose
in breast carcinoma
Poor prognostic markers
Good prognostic markers
ER(-)
ER(+)
PR(-)
PR(+)
pS2(-)
pS2(+)
Her-2 (3+)
Her-2 (0/1+)
Cathepsin D(+)
Cathepsin D(-)
P 53 >10%
P 53 <10%
EFGR (+)
EGFR( - )
Ki-67>23%
Ki-67<11%
Prognostic purpose of IHC

ER/PR/pS2:

pS2 : Cystein-rich peptide induced by estrogen secreted from
breast cells.

pS2 expression has been found to be associated with longer
overall & disease free survival.

ER +/ PR +/ pS2 + : 85% to 97% have good prognosis
ER +/ PR +/ pS2 - : only 50% to 54% have good prognosis.

Clinical data
inconsistency
How to Assess an IHC
immunostain?
reproducibility
Inter-observer variation
An example
Assessment of Her2 status
in breast cancer
Published guidelines (ASCO)
Her2 over-expression should be evaluated on every
primary breast cancer either at the time of diagnosis or
at the time of recurrence.
J Clin Oncol 19:1865-1878, 2001
Importance of getting it right
• Poorer prognosis if HER2 positive
• Role in the selection of the most appropriate
adjuvant therapy
• Herceptin improves survival if HER2 positive,
but not HER2 negative
®
– false negatives
• deny life-extending treatment
– false positives
• false hope
• complications & cost of the treatment
HER2 technical approaches
• Gene amplification
– Southern or dot (slot) blotting
– quantitative PCR
– FISH/CISH
• mRNA over-expression
– Northern blotting
– quantitative RT-PCR
• Protein over-expression
– immunohistochemistry
– Western blotting
– Elisa
POINT MUTATIONS
m-RNA
C-proto
-onc
C-onc protein over
production with
Increased activity
C-onc
Point Mutation
M-RNA
overexpression
IHC
vs
FISH
•
•
•
•
•
•
•
Fast
Cheap
Easy
False-negative rate
False-positive rate
Subjective interpretation
Difficult to standardise
•
•
•
•
Long
Expensive
Difficult
Accurate (#signals)
•
Theoretically does not identify pts with
overexpression without gene amplific
Standardised
•
IHC/FISH concordance
0
1+
2+
3+
FISH -
207
28
67
21
FISH +
7
2
21
176
3%
7%
24%
89%
Overall concordance: 82%
Mass R et al.: Proc ASCO 2000
Her2 testing algorithm
Patient tumour
sample
IHC
–
FISH
2+
3+
+
Retest with
FISH
Herceptin®
therapy
Herceptin®
therapy
+
Herceptin®
therapy
–
Slide 180
© 2003 By Default!
How to interpret?

1.Intimacy with pattern of staining:
Nuclear
ER
PR
P 53
Ki 67
Membranous
Her 2
EGFR
CD
20
CD
45
Cytoplasmic
CK
Vime
ntin
Desm
in
NSE
Cytonuclear
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SMA
HMB45
S100
Calret
inin
How to interpret?
2.Intimacy with scoring methods
Score
Pattern
Assessment
0
Staining of 0-10% cells
Negative
1+
Faint and partial
membrane staining
Weak to moderate
complete membrane
staining
Moderate to strong
complete membrane
staining
Negative
2+
3+
Positive/
Negative
Positive
Proficiency surveys of HER2 testing
2001 College of American Pathology IHC survey
• A breast tumor sample unreactive by IHC and
FISH was distributed to 415 laboratories
– 72% reported negative results
– 28% reported immunoreactivity
– 9% reported 2+ or 3+ scores
Conclusion
 Any HER2 assay performed at a non-reference laboratory
(<100 cases/month) will require validation at a reference
laboratory
Potential for misdiagnosis
(IHC)
• Antibodies (>28 commercially available)
• Technical performance
• Interpretation
– scoring
– artifacts
Common problems in HER2 IHC
• Underestimation of expression:
– (over)fixation in NBF
– poor antigen retrieval (unmasking)
– choice of antibodies
• Overestimation
– alcoholic (post)fixation (check normal
cells!)
• Cytoplasmic staining
Who can interpret an IHC?
Normal lobule
3+ breast carcinoma
Lobular cancerization
2+ breast carcinoma
2+ breast carcinoma?
Normal lobule
Cytoplasmic staining
What to do?
• Central Reference or large local laboratories
– high number of cases (>250 cases/year* or >100
cases/months [NSABP])
– quality assurance controls (internal and external)
– automated IHC
– high level of training (technique/interpretation)
• Small laboratories (<250 cases/yr or <100 cases/mos)
– do not do it
– send to larger laboratories
*Ellis I, et al: J Clin Pathol 2004
… but
• There are labs capable of performing
quality testing with lower volumes
• A high test volume does not ensure an
accurate test result
• If an individual lab can properly validate
an assay and perform acceptably in an
external validation, then it should be
permitted to offer the test
Hsi ED & Tubbs RR: JCP 2004
Applications of IHC
therapeutic purpose
Chemotherapy in breast carcinoma
ER (+)
PR (+)
Tamoxifen
/ Femara
ER (-)
PR (+)
Tamoxifen
/ Femara
ER (+)
PR (-)
Tamoxifen
/ Femara
Her-2
psoitive
Her-2
negative
Trastazumab
(Herceptin)
-
Cost :300 000 000 Rials
-
Applications Of IHC



Therapeutic purpose: PharmaDx Abs
Chemotherapy in breast carcinoma
ER - / PR - / Her-2 Triple negative breast carcinoma
Treatment is completely different:
Cisplatin