IHC TECHNIQUES AND ITS ROLE IN THE

FIELD OF IDENTIFICATION OF TUMORS

AND MICROBES

SPEAKER- DR S.BHATTACHARYYA
MODERATOR-
DR S.MOHANTY
 INTRODUCTION
IHC is a method for localizing specific antigens in tissues or
cells based on Ag-Ab recognition

The principle of IHC is a sharp visual localization of target

compounds in tissues , based on satisfactory signal to noise
ratio

Amplifying the signal while reducing non specific b/g

staining (noise) has been a major strategy
 HEADINGS
• 1.TERMINOLIGIES
• 2.METHODS AND TECHNIQUES
• 3.STEPS/PROTOCOL
• 4.CONTROL
• 5.ANTIGEN RETRIEVAL
• 6.LABELS
• 7.CHROMOGEN
• 8.GENERATION OF IHC RESULTS
• 9.IHC INTERPRETRATION ACC TO LOCATION IN
• CELL
• 10.MARKERS OF DIFFERENTIATION
 HISTORY OF IHC
A FLUOROSCENT Microscope is required to visualize the
fluorochrome but they have a tendency to fade.
So IHC was developed

Here enzymes are used as labels and visualized with

appropiate chromogen using light microscope

1974-IHC was first performed on routine formalin fixed

paraffin embedded sect

1991- Heat induced antigen retrieval technique in IHC was

done
1995-polymer technology introduced
 TERMINOLOGIES
ANTIGENS - Molecules that induces formation of
an Ab and is foreign to the animal into which it is
introduced

Sites on Ag that are capable of inducing Ab

formation are known as – EPITOPES/ ANTIGENIC
DETERMINANT – the exact site on the Ag with
which the Ab combines
EPITOPES CAN BE CLASSIFIED AS:-
Continuous – Consisting of
continuum of residues in
a polypeptide chain

Discontinuous – c/o residues from

different parts of a polypeptide
chain brought together by folding of
protein conformation
•ANTIBODIES – IgG is the most frequently used Ab for
IHC
• Paratope of Ab binds to the epitope of Ag

•IHC technique prove that Ig molecules can serve both

as Ab and Ag

•Monoclonal Ab is better than polyclonal Ab

(polyclonal Ab gives more non-specific staining)
IHC CAN BE PERFORMED ON –
Formalin fixed paraffin
embedded sections
Frozen sections
Smears
Imprints
Cytospins
 METHODS
• One step staining method
DIRECT conjugate • Labeled Ab reacts directly
labeled antibody
procedure with Ag in tissue

• Unlabeled primary Ab
INDIRECT/SANDWICH reacts with tissue Ag
PROCEDURE • Conjugated second Ab
reacts against primary Ab
 TYPES
1. Peroxidase-antiperoxidase method (PAP)
2. Biotin-avidin complex method (ABC)
3. Labeled streptavidin-biotin method (LSAB)
4. Alkaline phosphatase- anti alkaline phosphatase
methos (APAAP)
5. Polymer based labeling
 IHC PROTOCOL
Fixation and processing

Section cutting

Deparaffinisation and rehydration

 CONTD….
BLOCKING ENDOGENOUS PEROXIDE (BY H202)
(IF ENZYMES SIMILAR TO THOSE USED AS AB LEVEL ARE PRESENT IN TISSUE
THEY MAY REACT WITH SUBSTRATE,GIVE FALSE POSITIVE)

Blocking non-specific antibody binding

(TO UNMASK THE EPITOPES)

Antigen retrieval

Primary antibody
 CONTD… DILUTION REQD TO
ELIMINATE FALSE NEGATIVE
PARTICULARLY IN ANTIGEN
SECONDARY ANTIBODY RICH TISSUES

Chromogen

Chromogen enhancement Stringent washing

between reagents
Counterstain By PBS
TO PREVENT FORMATION OF
Ag -Ab COMPLEX THAT WILL
PRECIPITATE ON THE SECTIONS &
Mount B/G STAINING
TO REMOVE UNBOUND Ab
STEPS OF IHC IN SUM HOSPITAL
CONTD
 AUTOPROCESSER
SECTION CUTTING TISSUE EMBEDDING
SLIDE WARMING TABLE
TISSUE FLOATATION BATH
 CONTROL AND TYPES
CONTROLS ARE USED TO VALIDATE IHC RESULTS,TEST THE SPECIFICITY
OF ANTIBODY AND DISCARD EPITOPE SIMILARITIES
contain target molecule in its known location and can be
POSITIVE visualized by a stain eg normal reactive lymphocytes when
CONTROL Staining with an antibody to LCA to identify a suspected
lymphoma

Involves either omissiion of primary ab or replacement of specific

NEGATIVE primary ab by an immunoglobulin
CONTROL sssgoal is to demonstrate that the reaction visualized is due to the
interaction of the target molecule and paratope of the
antibody/affinity reagent
ABSORPTION inhibition of staining via absorption of primary
CONROL antigen/immunogen prior to use

ISOTYPE is used to ensure that the observed staining is due to antibody

CONTROL binding desired antigen & not to some general unspecific binding
of immunoglobulin to tissue
•MC used Buffer– 10% neutral buffered formalin
(NBF) (pH- 7-7.6)

•Length of fixation is imp – excessive fixation may

cause Ag denaturation and masking and too little
fixation can cause tissue deterioration
•Generally 6-72 hrs is acceptable

•IHC can be performed on decalcified tissue

•However some markers may not work well on such
tissues – eg – CD43, Ki67, ER, PR

•Paraffin embedding temperature s/b maintained

between 56-60◦C
 ANTIGEN RETREIVAL
UNMASKING OF ANTIGEN SITES
When formalin based fixatives are used

Intermolecular and intramolecular cross-bridges are

formed
with certain structural proteins

These are responsible for masking of tissue

antigens
The degree of masking of antigens depends on –
1. Length of time in fixative
2. Temperature
3. Concentration of fixative

• Many labs use automated IHC staining systems –

some of which have the ability to perform on-
board Ag retrieval- using enzymes or heat
MANUAL METHODS FOR ANTIGEN RETREIVAL
INCLUDE –
1. Proteolytic enzyme digestion
2. Microwave oven radiation
3. Combined enzyme and microwave
4. Pressure cooker heating
5. Decloaker heating
6. Pressure cooker inside a microwave oven
7. Autoclave heating
8. Water bath heating
9. Steamer heating
 PROTEOLYTIC ENZYME
DIGESTION
•MC used enzymes – trypsin and protease
•Others – chymotrypsin
• pronase
• proteinase K
• pepsin

•The digestion by these enzymes breaks down formalin cross-

linking and hence the epitopes are uncovered
• Digestion time needs to be tailored to individual Abs
• - underdigestion results in too little staining
• - overdigestion leads to false +ve staining and high b/g
staining
 HEAT INDUCED ANTIGEN RETREIVAL (HIER)

MICROWAVE ANTIGEN RETREIVAL

PRESSURE COOKER ANTIGEN RETREIVAL

STEAMER

WATER BATH

AUTOCLAVE
 MICROWAVE ANTIGEN
RETREIVAL
Most popular antigen retreival
solutions are –
1. 0.01M citrate buffer at pH 6
2. 0.1mM EDTA at pH 8
3. TRIS(Trisaminomethane)-EDTA at pH 9

Disadvantage – uneven heating and production of

hot spots
At no stage should the sections dry out during
antigen retreival
 PRODUCTION OF PRIMARY AB
1. POLYCLONAL ANTIBODIES
Produced by immunizing an animal with a purified specific
molecule (immunogen) bearing the Ag of interest

Animal mounts an immune response to the immunogen

Abs produced are harvested by bleeding the animal to

obtain
Ig- rich serum

Animal will produce numerous clones of polyclonal plasma

cells

Each clone will produce different Abs with different specificity

to the variety of epitope on a single antigen
2. MONOCLONAL ANTIBODIES –
•One pure Ab with high specificity is produced

•Background staining in such cases is minimal

3. LECTINS –
•Plant or animal proteins that can bind to tissue carbohydrates
with a high degree of specificity
•Carbs may be characteristic of a particular tissue, lectin
binding may have diagnostic significance
•They can be labeled in similar ways to Abs
 FALSE POSITIVE AND NEGATIVE

FALSE POSITIVE
1.CROSS REACTIVITY OF ANTIBODY WITH ANTIGEN
2.NON SPECIFIC BINDING OF ANTIBODY TO TISSUE
3.PRESENCE OF ENDOGENOUS PEROXIDASE
4.ENTRAPMENT OF NORMAL TISSUE BY TUMOR CELLS

FALSE NEGATIVE
1.ANTIBODY MAY BE INAPPROPIATE,DENATURED OR USED AT WRONG
CONCENTRATION
2.LOSS OF ANTIGEN THROUGH AUTOLYSIS OR DIFFUSION
3.PRESENCE OF ANTIGEN AT A DENSITY BELOW THE LEVEL OF DETECTION
 LABELS

ENZYME COLLOIDAL
LABELS METAL LABELS

FLUORESCEN RADIOLABELS
T LABELS
 ENZYME LABELS

•Mc used labels in IHC are enzymes

•Enzymes used are –

HORSERADISH PEROXIDASE (HRP)

ALKALINE PHOSPHATASE (CALF INTESTINAL)

GLUCOSE OXIDASE

β-D GALACTOSIDASE (BACTERIAL DERIVED)

CHROMOGENS
(Acted by reported enzymes
To form a colored precipitate)
 ALKALINE
PHOSPHATAS
CHROMOGENS E
 CHROMOGEN
ENHANCEMENT

•Place staining dish in 25◦C and incubate the slides in

0.5% copper sulfate solution for 1-5 mins

•Then wash under running tap water

 GENERATION OF IHC RESULTS

PRE-ANALYTICAL FACTORS
1. Time taken to remove the tissue at surgery
2. The ensuing ischemia
3. Interval between surgical resection and
fixation
4. Type of fixative
5. Length of fixative

ANALYTICAL FACTORS –
It pertains to lab procedures
 POST ANALYTICAL EVALUATION
–
•Very crucial
•Interpreting immunostains as merely +ve or –ve
without appreciating the following is inappropriate

STAINING PATTERN

% OF CELLS SHOWING POSITIVITY

INTENSITY OF STAINING
 QUALITY CONTROL
• FACTORS AFFECTING STAIN QUALITY
1.Fixation
2.Processing
3.Reversal of fixation(epitope reversal)

REAGENT FACTORS
1.Buffer & diluents
2.Temperature
3.Validation of antibodies
4.Positive & negative tissue control

PROCEDURAL FACTORS
1.Block & slide storage conditions
2.Monitoring stain quality
3.Deparaffinization
4.Complete antigen retrieval
 IHC INTERPRETATION IN DIFF LOCATION OF
THE CELL
Nuclear Cytoplasmic Membranous

OCT 4 Actin Her-2/neu

ER/PR Alpha fetoprotein CEA
Myogenin Chromogranin CD 99
Myo D1 Factor VIII related antigen CD20
P53 Desmin EMA
P63 GFAP
TTF 1 Hep-Par 1
MIB-1 HMB 45
Melan A/MART
CK, vimentin, NFP
 MARKERS OF
DIFFERENTIATION
EPITHELIAL DIFFERENTIATION

MUSCLE DIFFERENTIATION

NERVE SHEATH DIFFERENTIATION

NEUROENDOCRINE AND NEUROECTODER,AL DIFFERENTIATION

MELANOCYTIC DIFFERENTIATION

VASCULAR DIFFERENTIATION

GLIAL DIFFERENTIATION
 MARKERS OF
EPITHELIAL
DIFFERENTIATION
1. CYTOKERATINS –
• Currently used CK designation syatem is known as
MOLL’s catalogue
• 20 CKs known
• Divided into –
• Type I – acidic keratins – 12 types – CK9 to CK20
• Type II – basic keratins – 8 types – CK1 to CK 8
• Also divided into –
• High molecular weight CK (HMWCK)
• Low molecular weight CK (LMWCK)
•HMWCK – aa squamous keratins
•Exp. In squamous epithelium
•Ultrastructurally/EM – known as tonofilaments –
hallmark of squamous cell carcinoma

•LMWCK – aka simple/non squamous keratins

•Exp. In glandular epithelium and visceral parenchyma
(liver, kidney)

•Intermediate molecular weight CK – aka basal

keratins
•exp. In basal cells
ANTIBODY
COCKTAIL
AE1/AE2 •a pan-CK
•Can recognise both LMWCK and HMWCK
•Cannot identify HCC – as the combination do not contain CK18 which
is exp by HCC

CAM5.2 • can recognise LMWCK (including CK18)

•But may miss squamous cell carcinoma – as these exp HMWCK

35BE12 stains basal cells – used to distinguish well differentiated prostate adenocarcionm
from benign lesion of prostate

EMA EMA- neoplastic epithelia,mesotheliomas,some mesenchymal tissues,perineura

CEA fibroblasts,notochord,anaplastic large cell lymohoma,DLBCL,etc
CEA-ADENOCA-breast,colorectal,pancreas,HCC
p63 3. – HMWCK equivalent
Marker of squamous and urothelial epithelia

BerEp4 5. expressed in adenocarcinoma

•To differentiate lung adenocarcinoma (+ve) from mesothelioma (-ve)
•Favored marker in effusion cytology – selectively labels adenocarcinoma, where
b/g mesothelial cells are –ve (CK would label both)
•Some CK are aberrantly expressed by
mesenchymal cells and tumors – CK8, 18 and 19

•Mesenchymal neoplasms that are CK +ve are –

1. Synovial sarcoma
2. Epithelioid sarcoma
3. Chordoma
 MARKERS OF MUSCLE DIFFERENTIATION

SKELETAL MUSCLE as in rhabdomyoma and rhabdomyosarcoma

DIFFERENTIATION (RMS)

TRUE SMOOTH MUSCLE as in leiomyoma and LMS

DIFFERENTIATION

PARTIAL SMOOTH MUSCLE – as in myofibroblasts – these constitute a

DIFFERENTIATION significant population of cells in healing
wounds and stromal reaction to tumors . And
also in nodular fascitis and myofibroblastoma
1. DESMIN – ass. with both skeletal and smooth
muscle
Not exp by myofibroblast

NON muscle cells that exp desmin are –

A. fibroblast reticulum cells of lymph node
B. Endometrial stromal cells
C. Submesothelial fibroblast
2. ACTIN – divided into muscle and non-muscle
isoforms
• Interpretation is quantitative rather than
qualitative
• Muscle cells have far more actin than many other
cells
• Smooth muscle isoform – also expressed by
myofibroblast – show a characteristic tram track
pattern - (exp only in the periphery of their
cytoplasm)
• This distinguish them from smooth muscle cells
(uniform cytoplasmic +vity)
ACTIN– left – tram track pattern in myofibroblast
right – cytoplasmic +ve in true smooth
muscles
3. MYOGLOBIN – O2 binding heme protein
•Found in skeletal and cardiac muscle
•Not in smooth muscle

RECOMMENDATION FOR USE OF MUSCLE MARKERS –

•For identifying smooth muscle differentiation –
myogenin and myoD1

•For identifying skeletal muscle differentiation – desmin

and smooth muscle actin

•For identifying myofibroblasts desmin –ve and SMA –

tram track +vity
 MARKERS OF NERVE SHEATH
DIFFERENTIATION

1. S-100 protein – calcium binding protein

• 2 subunits –α and β – they combine to

form 3 isotypes
• α-α isotype – found in myocardium , skeletal muscle
and neurons
• α - β isotype – melanocytes, chondrocytes, glia
and skin adnexae
• β-β isotype – langerhan cells and schwann
cells
• Is of most value as a marker of benign and
malignant nerve sheath tumors and melanoma
• MPNST – show patchy and weak exp of S-100
• Benign nerve sheath tumors – strong and
uniform
+vity

• Perineural cells are -ve

2. CLAUDIN-1 –
•+ve in perineural cells
•Useful marker in perineuromas – granular membrane
+vity
•-ve in NF and schawannomas

2. GLUT-1,EMA – perineural cells +ve

3.CD57 – found in NK cells , T cells, oligodendroglial

cells and schwann cells
 MARKERS OF MELANOCYTIC
DIFFERENTIATION

1. HMB-45 (human melanoma black) – monoclonal

Ab HMB-45 identifies Pmel17 gene product gp100
(present in premelanosomes)
+ve in immature melanocytes and –ve in mature
melanocytes
+ve in - -ve in -
a. melanoma a. Nevi
b.PEComa b. Resting melanocyte

Desmoplastic and spindle cell melanomas – usually

negative
•Less sensitive than melan-A and S-100
•But more specific

2. Melan-A
•Product of MART-1 gene(Melanoma Ag recogniton
gene)
•Also +ve in nevi and resting melanocytes
•+ve in 50% cases of desmoplastic melanoma
•Also +ve in – adrenal cortical tumors and other steroid
producing tumors
3. MiTF – Micropthalmia transcription factor
Product of micropthalmia (mi) genenuclear
+vity

4. TYROSINASE – enzyme involved in synthesis of

melanin

5. S-100 –
Highly sensitive for melanoma
-ve for S-100 makes melanoma highly unlikely
Only 2-3% melanomas are –ve for S-100
6. SOX10
7. PNL2 RECENT MARERS
8. MUM1
 NEUROECTODER
M
1. CD99 – AL AND NEURO
• Product of MIC2 gene
• Transmembrane gp ENDOCRINE
• MARKER
Most imp use is in diagnosis of ES/PNET –
membrane +vity
Other SRCBT that are CD99 +ve–
• lymphoblastic lymphomas
• PD synovial sarcoma
• Mesenchymal chondrosarcoma
• Small cell OS
• DSRCT
never seen in neuroblastoma (NB)
2. CD56 – mediates calcium independent cell-cell
binding
•Exp in Normal cells like –
•Neurons, astrocytes, glia, NK cells
•+ve in high grade NE neoplasms – esp small cell
carcinoma(which may be –ve for all other NE
markers)
•+ve in NB

3. NB-84
Highly sensitive for neuroblastoma
4. SYNAPTOPHYSIN (SYN) and CHROMOGRANIN A
(CHR)-
•1st line markers for NE differentiation
•Mark neurosecretory granules – show granular
cytoplasmic positivity
•SYN is more sensitive
•CHR is more specific
•NON NE neoplasms which are SYN +ve but CHR –ve
are-
•Adrenocortical neoplasms and pancreatic solid
pseudopapillary tumors, Parathyroid adenoma
5. NSE (neuron specific enolase)

6. CK in NE neoplasms –
NE neoplasms fall in 2 categories-
a. Epithelial –carcinoid, pancreatic NE tumor, small cell
carcinoma
b. Non epithelial/neural – pheochromocytoma,
paraganglioma , NB

Epithelial NE neoplasms – CK+ve

Non epithelial NE neoplasms – CK-ve
• Distinctive feature of high grade NE carcinoma (small
cell carcinoma and merkel cell carcinoma ) – CK
shows dot like (punctate) perinuclear pattern
• CK20 – esp +ve in merkel cell Ca
 MARKERS OF VASCULAR
DIFFERENTIATION
1. CD31 – more sensitive and specific
2. CD34
3. Factor –VIII (actual Ag is vWF)
4. Ulex europaeus I
5. CD141 (thrombomodulin)
6. Fli-1
7. ERG – a new promising vascular marker which is
also positive in prostate adenocarcinoma
8. D2-40 (podoplanin) – novel marker for lymphatic
endothelial cells
KEY APPLICATIONS OF VASCULAR MARKERS
a. To identify vascular nature of Poorly differentiated
neoplasms – angiosarcoma, epithelioid
hemangioendothelioma, hemangiopericytoma, kaposi
sarcoma
b. To highlight vessels to help identify LV invasion in
tumors

CD34+ve non vascular tumors –

a. Solitary fibrous tumor
b. DFSP
c. GIST
d. Epithelioid sarcoma
e. Nerve sheath tumors
f. Granulocytic sarcoma
 ORGAN SPECIFIC
GI stains- arginase 1,CD117,CDX2,DOG 1,chromogranin,ki67,
PAS, Synaptophysin

Adrenal- Calretinin, chromogranin,GAT 3,Inhibin, S 100

Kidney –AE1/AE3, AMACR,CAIX,CAM 5.2,CD10,CK7,CKIT,EMA

PAX 8,RCC, Vimentin,
Endometrium-AMACR,ARID 1A, bhcG, cyclin E, E cadherin,ER,
GATA 3,HNF 1B,inhibin,Melcam,napsin A,6, p53,p57,PAX2,PAX8

Fallopian tube-Bcl2, ki67, p53

Ovary-AE1/3,ARID1A,B CATENIN,CDX2,CK20,CK7,EMA,ER,
FOXL2,CALRETININ,Glycipan 3, inhibin,MUC2,MUC5A,MUC6
P16,p53,pax8,PTENWT1,SALL4,SATB2
CERVIX-BCL2,CDX2,CEA,CK 7,ER, GATA3,HIK1086,KI67,MUC6,p16, p40, p53,p63
PAX8, PR,VIMENTIN

UTERUS-ALCIAN BLUE,ALK,CALDESMON,CD10,Cyclin D1,DESMIN,EMA,HMB45

Melan A,myogenin,myoglobin,p16,p53

VAGINA- CD34,CK17,desmin,p16,p53

Testis- AFP,CD30,CD117,Glypican 3,hcG,OCT3/4,PLAP

PROSTATE-AR,AMACR,NKX3.1,Prostein,PSA,PSAP,PSMA

THYROID-CALCITONIN, CK19,GALECTIN 3,HBME,THYROGLOBULIN,TTF1

BREAST-AR,CK7,E-CADHERIN,ESTROGEN,GATA3,GCDFP 15,HER2,Ki67
Mammaglobilin

LUNGS-TTF,CK 20,CK7,NAPSIN

LIVER-HepPAR1,GLYPICAN 3,AFP,PCEA
 APPLICATION OF IHC IN ROUTINE
SETTINGS
DIAGNOSIS OF TUMORS

PROGNOSTIC MARKER

PREDICTIVE OR THERANOSTIC MARKERS

IDENTIFICATION OF INFECTIOUS ORGANISMS

 DIAGNOSIS OF
TUMORS

1. Maximum utility of IHC is in distinguishing

carcinoma from lymphoma, sarcoma and melanoma
2. Workup of hematolymphoid neoplasms
3. Metastatic carcinoma of unknown primary (CUP)
4. Soft tissue neoplasms – 4 common diagnostic
setting
a. Small round cell tumors
b. Monomorphic spindle cell tumors
c. Epithelioid soft tissue tumors
d. Pleomorphic spindle cell tumors
 CONTD…

5. In bone – to differentiate primary from metastatic

non –osseous tumors
6. CNS tumors
7. Germ cell tumors
 PROGNOSTIC
MARKERS

1. Loss of myoepithelial or basal cells or

basement membrane/collagen type IV – these
allow assessment of microinvasion
2. Endothelial markers – assist in identification of
lymphovascular spaces to ascertain tumor
embolism
3. ER, PR and her2/neu
4. Ki-67 /MIB-1 – proliferation markers
 PREDICTIVE OR
THERANOSTIC
MARKERS
1. ER/PR – tamoxifen in Ca. breast
2. Her 2 – herceptin in breast cancer
3. C-kit – gleevac/imatinib in GIST, CML
4. CD20 – rituximab in B-cell NHL
5. EGFR – erlotinib in lung cancer
DIAGNOSIS OF TUMORS
 TITLES
DIFFERENCE BETWEEN TE OMMON NEOPLASMS
METASTATIC CARCINOMA OF UNKNOWN PRIMARY
SOFT TISSUE TUMORS
GERM CELL TUMORS
PLASMA CELL NEOPLASMS
T/NK CELL NEOPLASMS
T CELL NEOPLASMS
B CELL NEOPLASMS
APPROACH TO LUNG TUMORS
APPROACH TO SALIVARY GLAND TUMORS
PROGNOSTIC MARKERS
OVARIAN TUMORS
APPROACH FOR BREAST AND PROSTATE
LARGE CELL TUMORS
PEDIATRIC SMALL CELL TUMORS
HISTIOCYTIC & DENDRITIC CELL TUMORS
DISTINGUISHING TUMORS WHICH ARE OF SIMILAR TYPES
CNS TUMORS
RECENT ADVANCES
GENOGENIC IHC
 In distinguishing carcinoma from
lymphoma, sarcoma and melanoma
METASTATIC CARCINOMA OF
UNKNOWN PRIMARY (CUP)
CK7/CK20 EXPRESSION
 SOFT TISSUE NEOPLASM
a. Small round cell tumors
b. Spindle cell tumors
c. Epithelioid soft tissue tumors
d. Pleomorphic tumors
e. Myxoid tumors
SMALL ROUND CELL TUMORS
 NEOPLASMS CK & S100 SMA DESMIN CD34 OTHERS
DIFFERENTIATI EMA
ON
MUSCLE LM - - + + - S
LMS + - + + - CD99
RMS - - - + - Myogenin/myod1 P
NERVE NF - +focal - - + I
SHEATH SCHWANNOMA - +diff - - +
MPNST +focal +focal - - + N
VASCULAR ANGIOSARCOMA - - - - + Factor viii, CD31 D
KAPOSI
L
MYOFIBRO FIBROMATOSIS
NODULAR FASCITIS
-
-
- + -/+ - B CATENIN E
BLASTIC - + - - CD8
IMT -
-/+
- +/- -/+ - ALK
C
FIBROHISTIOCY DF - - - - - Factorxiiia+
TIC DFSP - - - - + factorxiiia- E
L
ADIPOSE DEDIFFERENTIATED - + - - -/+ CDK4,MDM2
LIPOSARCOMA L
OTHERS GIST - - +30 - + C KIT,DOG1,BCL2
SFT & HFC
SYNOVIAL SARCOMA
-
+focal
-
+30
-
-
-
-
+
-
BCL2
Calponin,CD56,
T
SPINDLE CELL CA
SPINDLE CELL
-
+diff
-
-
-
-
-
-
CD99,BCL2
For sqCC,CK90
U
MELANOMA
P63 M
O
R
EPITHELIOID SOFT TISSUE
TUMORS
 PLEOMORPHIC TUMORS

PLEOMORPHIC MPNST-S100
PLEOMORPHIC LEIOMYOSARCOMA-SMA
PLEOMORPHIC LIPOSARCOMA-CDK4, MDM2
PLEOMORPHIC RHABDOMYOSARCOMA-MYOD1
PLEOMORPHIC UNDIFFERENTIATED SARCOMA-MYOGENIN

MYOGLOBIN
GERM CELL TUMORS
PLASMA CELL NEOPLASMS
T/NK CELL NEOPLASMS
T CELL NEOPLASMS
 IMMUNOPHENOTYPIC
ALGORITHM FOR CLASSIFICATION
OF THE MAJOR MATURE B-CELL
LYMPHOID NEOPLASM
 HODGKIN LYMPHOMA
– BASIC PANEL
Classic HL NLPHL

LCA -ve +ve

CD30 +ve -ve

CD15 +ve -ve

CD20 -ve +ve

EMA -ve -/+

 ROLE OF BCL2
• In normal tumour BCL2 stains mantle zone of
Lymph node
• But in Follicular Ca it stains germinal centre

• BCL2 +ve DLBCL seen in 50% cases

• BURKITTS-bcl2-ve
• Bcl2 and c-myc +ve- Double expressive
lymphoma
SALIVARY GLAND TUMOR
APPROACH TO LUNG CARCINOMA
 CONTD……
• LUNG ADENOCARCINOMA
• CK7+20-,TTF1(also thyroid),Napsin 80%(also some RCC),CD56,CK 5/6,p63
• LUNG SQUAMOUS CA,BOTH BASALOID AND USUAL TYPES
• CK 7-20-70%, CK7+20-25%
• TTF/NAPSIN neg,CD56 - 0-10%
• P63,34BE12 and CK5/6+100%
• LUNG SMALL CELL (OAT CELL)CA
• CK7-20-90%,CK20 rare
• TTF1 90%,CD56 -95%
• P63 neg,CK5/6 –ve,34BE12 scattered pos cells 12%
• Synaptophysin 50%, chromogranin 40%
• Keratin usually absent to patchy or dot like
HISTIOCYTIC & DENDRICTIC CELL
TUMOURS
 P53
–VE- ENDOMETROID CA OVARIAN TUMORS
STRONGLY +VE -SEROUS CA
WEAKLY FOCAL +VE -CLEAR CELL CA

CK7 CK 20 WT1 CA125 CEA CDX2 VIM

serous + - + +

TCC + - + +

Endometroid + - - + +

Mucinous intestinal + + - + +
type

Clear cell + - -

Metastatic colorectal - + - + +

TCC is +ve for UROTHELIN

 LARGE CELL TUMORS
TUMORS CK Mel S100 CD45 CD15 CD30 ALK CD20 OCT4 Mp0
an A
DLBCL - - - + - -/+ - + - -

Carcinoma + - -/+ - -/+ -/+ - - - -

Melanoma - + + - - - - - - -

Seminoma - - - - - - - - + -

CHL - - - -/+ +/- + - +/- - -

ALCL - - - +/- - + +/- - - -

Myeloid - - - +/- +/- - - - - +

sarcoma
 PEDIATRIC SMALL CELL
TUMORS
TUMORS CK CD99 NB84 WT1 CD56 DESMN MYOD1
Lymphobl 100%
lymphoma

neuroblastoma 40% 100% 100%

nephroblastoma 75 70% 70%

%

Ewings 100% focal focal

/PNET

RMS 100% 100% 100% 97%

DSCRT 90 35-80 50% 100% 50-100%
%
Synovial 62 62%
sarcoma %

Malignant 99
Rhabdoid %
tumour
•
COMPARISON OF THE TUMORS WHICH ARE
OF VERY SIMILAR TYPES AND CAN BE EASILY
DISTINGUISHED BY HISTOPATHOLOGICAL
EXAMINATION ALONE
CD34+VE IN NON VASCULAR TUMORS SARCOMAS THAT ARE +VE IN EMA
1.SOLITARY FIBROUS UMOR 1.EPITHELIAL ANGIOSARCOMA
2.DFSP 2.EPITHELIAL SARCOMA
3.GIST
4.EPITHELOID SARCOMA
5.NERVE SHEATH TUMORS
6,GRANULOCYTIC SARCOMA

MESENCHYMAL NEOPLASMS SHOWING CK+VE

1.SYNOVIAL SARCOMA
2.EPITHELOID SARCOMA
3.CHORDOMA
CA BREAST MALIGNANT SWEAT GLAND
TUMOR
P63-VE P63+VE

COMPLETE MOLE PARTIAL MOLE

P57-VE
PROSTATIC ADENOCARCINOMA ADENOCARCINOMA BLADDER
CK7- +
P63 - +
AMACR+ +
PSA+ -
PSAP+ ,PROSTEIN+ +
 RENAL MARKERS
VIMENTIN CD117 CK7
CLEAR CELL 85% NEG/FOCAL
PAPILLARY 90% 20-80%
CHROMOPHOBE 80% 70%
ONCOCYTOMA 90% NEG/SCATERRED

RENAL CELL ONCOCYTOMA ONCOCYTIC VARIANT OF RCC

CD117+VE CD117-VE
 FOLLICULAR & PAPILLARY TTF1,THYROGLOBULIN
• THYROID & THYMUS
MEDULLARY
TTF1,CALCITONIN,CHROMOGR
ANIN

PAPILLARY-HBME 1,GALACTIN 3,CK17 +VE

THYMOMA PAX8,
P63+VE
CD5+VE

THYMIC CARCINOMA PAX8, P63,CD5-VE

NEOPLASTIC BILIARY EPITHELIUM BENIGN BILIARY EPITHELIUM
P53+
KI67 HIGHER IN P53 patient

PANCREATIC ADENOCA CHRONIC PANCREATITIS

LOSS OF SMAD4
 ENDOMETRIAL STROMAL LEIOMYOMA
TUMOUR
CD10 + -
SMA - +
DERMAL NEVUS MELANOMA
HMB45+, KI67 LESS THAN 5% HMB45+, KI67 MORE THAN 10%

DESMOPLASTIC MELANOMA-HMB45-VE,MELAN A –VE

BUT S100+VE
 endometrial endocervical
ER and vimentin Diffuse or +ve -ve
p16 and CEA -ve +ve

ENDOMETRIAL ENDOMETRIAL
MUSCLE STROMAL
CALDESMON 70% 5%
CD10 35% 95%
DESMIN 90%
B CATENIN -VE 50-100%
 HCC BENIGN NON NEOPLASTIC
LESIONS
GLYPICAN 3 + -
HEP PAR1 + -

CD34 is used to identify thickened plates of hepatocytes

Polyclonal CEA gives a canalicular pattern in HCC

HCC CHOLANGIOCA

CK7
- +
 Inhibin Melan A SYNAP CHRG 5NO CALRETIN
IN

Adrenocortical + + + _ _ +
carcinoma

Pheochromocyto _ _ + + + _
ma
 PROGNOSTIC MARKERS
MARKERS ON ASSESSMENT OF
INVASION
1. Collagen type IV – component of basement
membrane
2. Basal cells and myoepithelial cells – in prostate
and breast carcinoma respectively
• These are absent in invasive carcinomas
3. Racemase (AMACR) – for prostate carcinoma
Exp in malignant acinar cells but is –ve in benign
acinar cells
PROSTATE AND BREAST COMPARISON
METASTATIC PROSTATE CARCINOMA
NKX3
ANDROGEN RECEPTOR
PSA FOCAL
 BREAST MARKERS
• BRST2
• ER PR
• MAMMAGLOBULIN
• EGFR, GATA3,CA125
• GROSS CYSTIC DISEASE FLUID PROTEIN
INVASIVE DUCT INSITU
CA
P63 L0ss
 ER PR Her 2 neu
*IHC results
Interpretation (% of tumor cells with nuclear **Control
staining)

1% or more

JCO, 2010: ASCO/CAP guidelines for ER PR Testing

Allred Scoring for ER and PR –
Guidelines

>0 to 1% >1 to 10% >10 to 33% >33 to 67% >67 to 100%

Modified from: Allred, Mod Pathology, 1998

Recent ASCO/CAP HER2 Testing Guideline
 ER PR POSITIVE TUMORS

• breast, ovary,endometrium,papillary thyroid,

• Sarcomas,skin adnexal tumors,
• meningioma(PR)
• Solid pseudopapillary neoplasm of
pancreas(PR)
• NON NEUROENDOCRINE TUMORS SHOWING
NE MARKER+VITY
• ADRENOCOTICAL CARCINOMA
• SOLID PAPILLARY NEOPLASM PANCREAS
• PARATHYROID ADENOMA
 PLEURAL LUNG CARCINOMA
MESOTHELIOMA

CALRETININ 90% 15%

CK5/6 90% 10%
D2-40(PODOPLANIN) 90% 0-7%
WT1 90% 25%
TTF1 NEG 70-80%
CD15 Very rare 95%
CEA Very rare 90%

BerEp4 15% 95%

MOC31 10% 100%

B72.3 10% 100%

OVARIAN SEROUS VS PERITONEAL MESOTHELIOMA
OVARIAN SEROUS PERITONEAL
MESOTHELIOMA
CALRETININ 7-34% 100%
CK5/6 17% 100%
THROMBOMODULIN 30% 74%
PAX8 75-90% 9%
BerEp4 100% 9%
CD15 60% NEG
S100 27% NEG

B72.3 80% NEG

MOC31 97% WEAK

WT1
MESOTHELIOMA LUNG ADENO CA
CALRETININ TTF1

REACTIVE MESOTHELIOMA
MESOTHELIAL
CELLS
GLUT 1 - +

DESMIN + -

EMA - +
 CNS MARKERS
• GLIAL TUMOURS
• ASTROCYTOMA,GLIOBLASTOMA,OLGODENDR
OGLIOMA- GFAP,IDH,P53
• NEURONAL TUMOURS
• -SYNAPTOPHYSIN
• CHROMOGRANIN
• NSE
• PRIMITIVE TUMOURS-
RECENT ADVANCES IN FUTURE
DIRECTIONS
1. Genogenic IHC for diagnosis
2. Sequential double staining method
3. Develop better monoclonal Abs with recombinant
technology
4. Technician free automation of IHC procedures
5. “Pathologist-free” microscope image analysis
technology for interpretation of IHC
 GENOGENIC IHC

Identification of underlying molecular changes by IHC

–
1. Markers to monitor drug resistance –
P-glycoprotein which is the product of mdr (multidrug
resistance) gene
2. BRCA-1 gene
3. DNA repair genes (microsatellite instability)
4. Loss of E-cadherin
5. ALK overexpression to recognise t(2;5) in ALCL
6. FLI-1 overexpression for t(11;22) in ES
7. WT-1 overexpression for t(11;22) in DSRCT
 SEQUENTIAL DOUBLE
STAINING
TECHNIQUE
•The procedure involves sequential application
of 2 staining systems
•To demonstrate more than 1 Ag in a single
section
•Must produce contrasting colors to be
effective
•Eg -In 1st staining system – secondary Ab may be
conjugated with HRP and AEC as substrate
•In 2nd system – secondary Ab may be conjugated with
Alkaline phosphatase and fast blue as substrate
APPLICATION OF IHC IN MICROBES

VIRUS
• Normally diagnosis of viral infection has relied on
cytopathic changes and intracellular inclusions.
• But in some cases these changes are subtle
• And sparse.
• In those cases IHC is more sensitive and specific
HERPES SIMPLEX VIRUS- Monoclonal antibodies against To distinguish between HSV 1
VIRUS and HSV2

KAPOSI
DETECTION OF VIRUS
2. To demonstrate association of HHV8 with Kaposis sarcoma,Primary
effusion lymphoma, Castleman disease

3.Immunostaining of Kaposi sarcoma latent associated nuclear

antigen 1 is used to confirm diagnosis of kaposis sarcoma

CMV
4.CMV-Ihc is useful in detection of CMV infections in steroid
refractory ulcerative colitis,occult cases

EBV virus-Latent membrane protein 1 in case of Hodgkin lymphoma and

post transplant lymphoproliferative disorder
EBV causes BURKITTS LYMPHOMA
NASOPHARYNGEAL ADENOCA
Various tumors are EB+DLBCL,HODGKINS,
 CONTD…
HEP B & HEP C

RABIES WHERE NEGRI BODIES ARE INCONSPICOUS

HIV

PARVOVIRUS19

EBOLA VIRUS
 CONTD…
ADENOVIRUS

RSV

INFLUENZA
VIRUS

BK VIRUS renal transplant specimen associated with mononuclear interstitial

infiltrates and tubular atrophy and helps it to distinguish from acute
cellular rejection

YELLOW FEVER, DENGUE HEMORRHAGIC FEVER

VIRUS WITH IHC EXAMPLES
CONTD…
CONTD…
 BACTERIA
H.PYLORI To detect Helicobacter pylori infection because by conventional
histopathology diagnostic accuracy is 66%

BARTONELLA Bartonella infection-

To identify
B.henslea
B,quintana
WHIPPLES

Q FEVER COXIELLA BURNETTI

LEPTOSPIROSIS
 CONTD…
SPIROCHETES IN A PATIENT OF SYPHILIS

SOFT TISSUE INFECTION A/W GRP A STREPTOCOCCUS

STAPHYLOCOCCUS
AUREUS

CLOSTRIDIUM SP
CONTD…
CONTD…
CONTD…
 FUNGUS
ASPERGILLUS Aspergillus- monoclonal abs against aspergillus ag (WF AF1,
164G,611F) is highly sensitive in identifying A.fumigatus, A.niger,
A.flavus

CANDIDA
FUSARIUM
Pseudoallerscheria boydii
Scedosporium sp

Cysts and trophozoites of P.jirovecci

distinguish Penicillium marneffi from histoplasma capsulatum,

cryptococcus neoformans and candida albicans by EBA1 monoclonal
ab
 PROTOZOAL INFECTION
• Diagnosis of Leishmania when morphology of
the parasite is distorted by tissue necrosis or
autolysis in case of unusual presentation of
the disease
REFERENCES –

1.Diagnostic immunohistochemistry – Dabbs

2.Enzinger and Weiss
3.Rosai and Ackerman 10th editionsurgical pathology
4.Handbook of practical histochemistry
5.Immunohistochemistry in surgical pathology practice
: nirmala jambhekar
6.Walts jc surgical pathology in infectious disease
7.Guarner et al diagnosis of infectious disease
8.Harrisons General medicine
9.Bancrofts theory and practise of histological