Immunohistochemistry

Practical issues


Dr Santosh Menon
Assistant Professor, Pathology
Tata Memorial Hospital
What is IHC?

A method that uses antiodies to

identify, locate, and stain specific
protein molecules in tissue sections
!"isuali#ed using a microscope$

%sed to diagnose the type of cancer

and to help determine the patient&s
prognosis or as a predicti"e mar'er of
therapeutic response(

WH) IS IHC IMP*+TA,T in
surgical pathology practice?

cell lineage and tissue type

prognostic mar'ers

-uantification. it is no longer enough that the

&stain& is there. rather it is a /uestion of &Ho0
much is there?&

Predicti"e mar'ers for targeted therapy

Patients are 'no0ledgeale1than's to internet

CD20 and Rituximab
CD20
C-erbB2 and Herceptin in breast CA
C-kit and imatinib in GIST
Immunohistochemistry

Manual

Autmated
2asis of Immunology
!
"
Simple Principle
Antigen 3 Antiody 4 Comple5
Immunohistochemistry is a techni/ue ased on
the selecti"e inding of specific antiodies to
specific antigenic sites on a cell, in a precise
loc' and 'ey mechanism
What is an Antigen?
Any sustance that can trigger the
production of antiodies is called an antigen
What is an Antibody?
Antiodies are proteins called
immunogloulins
Antigen
Antiody
Anti
gen
Primary Antiody
6aeled
Secondary
Antiody
Cell 0ith antigens on surface
Anti
#en
B
B
B
2
B
P
X
P
X
P
X
B
A$idin Bitin
Cmplex
DAB+H2O2
IHC
Primary Antiody
6aeled
Secondary
Antiody
Cell 0ith antigens on surface
Detection system
Chromogen
Detection system
Primary Antiody
Tissue section
0ith antigen
Primary Antiodies

Polyclonal antiodies% Heter#eneus

ppulatin & antibdies' prduced a#ainst
se$eral epitpes n a sin#le anti#en- multiple
clnes prduce t(e antibdies
Mre tlerant t retrie$al tec(ni)ues
Speci&icit* is less

Monoclonal antiodies%
Hm#enus ppulatin & antibdies &r a
sin#le epitpe + &rm a sin#le clne & B-cells
Mre speci&ic
,ess crss reacti$it*
,ess back#rund nn speci&ic stainin#

Pure single Ab
I
Adapted &rm Milstein -./001 Scientific American' 2ct3
1
2
3
4
m
m
m
m
1
2
3
4
1 2 3 4
Monoclonal
antibodies
Cell fusion
Spleen cells
+
M*elma
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 Antiody "ial
H4 d 4e start 4it( a ne4
antibd*5
If you ha"e ac/uired a ne0
antiody11 Titration is a must
Date Antiody
%sed
Dilution Appropriate
dilution
+emar's
7897897: CD;7 <=>7,
<=<77
<=;77
<=<77 G22D
06-06-00 CD7 .%.00
.%200
.%700
.%200 B8ST
Positi"e controls
C9T2:8RATI;
CD7
CD20
;e#ati$e cntrl
Same steps but 4it( missin & primar*
antibd*
?alidation of ne0 antiody
Antiody@CD<A:@@@@@@ Clone M<<> Code,o(@MB;;:
?endor @ @@@ 2atchC6ot ,o(@777AB7DB@@@@@@@@@@
Comments=
Sign= Date=
S3;0 B,2C: MA;<=ACT3DI,n3 R8MAR:S
>ATH;23 TISS<8

.%60
. 72720B? @ Satis&actr*3
2 722.0B? @ Satis&actr*
7 .0007B? @ Satis&actr*
A .A6/BBC @ Satis&actr*
Wor'flo0 in IHC la
Drk&l4 in IHC% Makin# an
entr*
Makin# t(e re)uired entries
2loc's are cut in routine manner
Tissue sections on
poly969lysine
coated slides
2ind Primary antiody
Wash
2iotinylated ;nd
antiody
Wash
A
"
id
in
P
e
ro
5
id
a
s
e

c
o
m
p
le
5
e
s
Chromogen de"elopment
Counterstain dehydrate
co"erslip e5amine
2loc' non9sp
inding
Deparaffini#e
Eylene
2loc'
endogenous
pero5idase
Antigen +etrie"al
Antigen retrie"al
9standardi#ation

Proteolytic en#yme methods

Heat induced epitope retrie"al

Micro0a"e
Pressure coo'er
Achie"ing the desired temperature
pH of uffer9 caliration
Holding times
Slides immersed in uffer solution
Scientific pressure coo'er
Micro0a"e
Primary Antiody
Primary Antiody "ial 9<ml
A&ter penin# make 20
ali)uts & 60 micrlitre eac(
For daily use ma'e fresh
dilutions of primary
antiodies from ali/uots
Micrpippettes
Micropippettes
Ha$e a dail* dilutin c(art as a
spreads(eet
Antibd* dilutin c(art Date .0E0FE20.0
Antibd* Retrie$al met(d Dilutin
. CD20 T8->ascal .%.00
2 M>2 SC-Micr4a$e .%700
7 CD27 T8-Micr4a$e .%.00
A 8R SC->ascal .%60
Ma'ing the dilutions of antiodies 0ith ufferCdiluent
Washings and 0iping
Primary antiody on slide
Incuation in humidity chamers
What are 0e loo'ing for in IHC?
Coloured "isual product demonstrating
an antigen antiody reaction=

2ro0n colour !DA2$

+ed colour !PAP9APAAP$

Greenish yello0 in
immunoflourescence
Issues related t interpretatin
Where should 0e loo' for the colour?

Hno0ledge of Ag location is a must

Hno0ledge of pattern of staining is

essential

Cytoplasmic !diffuse,paranuclear,
perinuclear$

,uclear !diffuse, nucleolar$

Memranous !Continuous, ro'en$

Interstitial

The cell of interest !larger tumor cell etc$

An I5ample of normal lymph node
CD20
CD7
CD27
Bcl2
,uclear positi"ity
8R
Mib-.
pF7 >R
Cytoplasmic positi"ity
Gimentin
Desmin
Memrane positi"ity
CD20
c-kit
Golgi #one positi"ity
CD.6
CD.6
Cell of interest
CD70
,CA
Cell of interest
CD20
CD.F7
Cell of interest
C-kit
,CA
Is e"erything ro0n
positi"e?
,o, e0are
All that is Jro0nK is not
real

Back#rund stainin#

=alse psiti$it* includin#

arti&acts
Prolem of false ro0n stain

Hydrophoic and electrostatic

interactions

Indogenous pero5idase

Indogenous iotin

Antigen diffusion

Antigen retrie"al

Polyclonal antiody

,ecrotic tissue
%ne5pected results1(rules rather than
e5ception1a0areness is the 'ey

pF7 psiti$it* in B-cell l*mp(mas

C-kit psiti$it* in nasp(ar*n#eal

carcinmas

Aberrant CDA in m*elid leukemias

CD.70 in m*elmas and carcinmas

HHH3and t(e list #es n and nHH33

2ac'ground staining
>l*clnal antibd*
;ecrtic tissue
Antigen diffusion
k li#(t c(ain
lambda li#(t c(ain
False positi"ity

Cross reacti"ity due to epitope sharing

may result in false positi"ity

An e5ample is CDBDa, a 2 cell mar'er

cross reacts 0ith smooth muscle

Irror in data entry and

mislaelling!MyoD< and Mi<$
Artifacts in IHC

Idge and trapping artifacts

Des/uamation artifacts

2ule artifacts

Drying artifacts

Artifacts of poor fi5ation

Precipitated DA2 artifacts

2iotin artifacts
8d#e and trappin# arti&acts
Crus(ed tissue arte&act
Depsits arti&act
When do you call an IHC as
negati"e?

D nt call an immunstain ne#ati$e

4it(ut c(eckin# ut t(e psiti$e
cntrls

Remember t(e best cntrl is psiti$e

internal cntrl
An example & breast cancer
Breast Cancer
;e#ati$e (rmnal markers
>siti$e 8R >siti$e >R
When to call tissue immunodead?

Gimentin immunstain used t decide t(e

immunreacti$it*Epreser$atin &
anti#enicit* & tissue3

Gimentin is $irtuall* present in all tissues

and e$en smallest & bipsies usuall*
s(4 sme $imentin reacti$it*' i& 4ell
preser$ed
-uality issues and
"alidation
What do 0e mean y
/uality?
6oo's same
Tastes same
Smells same
-uality in IHC
means11

Standardised procedures

?alidated antiodies

Sensiti"ities and specificities of antiodies

Accurate, reproducile and comparale results

Chec'points for correcti"e actions

Continuous monitoring and impro"ement

Antiody Clone ?endor Dilution < ; A99 A<
I+ ID> DAH* L=<77 3M 3M 3M
,CA 2B.. DA:2 .%200 I7 I7 I2
Sign
Month= May;77:
Daily positi"e control chart
Daily controls
?alidation of ,e0 Control
*ld 2loc' ,e0 2loc'
Tissue Path ,o( +emar' Tissue Path ,o( +emar'
2+IAST M;;ACD DIP6IT
ID
2+IAST >DM7CD ?A6IDA
TID
F*+ I+
Antiody@I+@@@@@@@@@@@@
?endor@DAH*@@@ Clone ,o(@@ID>@@@ C*DI ,o(@MB78B@@@@@@@@@
2atch ,o(777777B;@@@@@@@@@ Dilution Factor@@<=<77@@@@@@@@@@@@
Comments=
Sign= Date=MC>C;77:
H4 t $ercme practical
di&&iculties & $ariatin in IHC
stainin# related t (uman
errrs5
A%T*MATI*,
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Than'fullyL

Automation cannot interpret Surgical

path and IHC slides11(

*ther0ise I 0ould ha"e no No11

Ac'no0ledgement

Without a technical hand

0ho has immense
dedication and interest in
IHC, it is impossile to
achie"e re/uired results(
Ac'no0ledgements

Dr ;A Jamb(ekar >r& K Head

Dr Sumeet GuLral K Dr Subramanian

Dr Saral Desai

Mrs Rek(a T(rat Senir tec(nical &&icer

Mr Ma(endra >alkar

Mr Aamir :(an

Mr >ritam

Mr Dinkar

Mr S(inde
THANK YOU