Immunohistochemistry(IHC):

Basics & Applications

Dr Rajiv Kumar
Asst Professor,
Department of Histopathology, TMH
 What is IHC?

Immunohistochemistry (IHC) =

Immunology +histology + chemistry

• IHC is an application of antibodies to tissue

preparation
– for the localization of target antigens
– visualized using a microscope
 IHC: Indispensable ancillary tool
• “IHC testing is an essential component of the
pathologist’s evaluation of a tissue sample.

• It serves a critical need in patient care as the

results from IHC testing often directly guide
treatment”
-Patrick L. Fitzgibbons,
 Why is IHC important in surgical
pathology practice?
• Categorization of undifferentiated malignant tumors
– Carcinoma/ lymphoma/sarcoma

• Sub-categorisation of tumors
– Lymphomas

• Determination of site of origin of metastatic tumors

– Lung/GIT/ Prostate

• Detection of molecules that have prognostic or therapeutic

significance
– ER/PR/cerb-2 in breast carcinoma
– Alk-1 in Lung adenocarcinomas
CD20 and Rituximab

CD20
C-erbB2 and Herceptin in breast CA
C-kit and imatinib in GIST
IgG IgG4+/IgG+ IgG4
Ratio>90%
 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
 What is an Antigen?
Any substance that can trigger the production of
antibodies is called an antigen

Antibody

Antigen

What is an Antibody?
Antibodies are proteins called
immunoglobulins
 What cellular antigens
can we target?
• Cytoplasmic
• Nuclear
• Cell membrane
• Protein
 IHC

DAB+H2O2
Chromogen

PX
B
Labeled PX
Secondary B
Antibody
B
Avidin Biotin
Complex
Detection system
PX

Primary Antibody

Anti
gen
Cell with antigens on surface
Fresh Tissue Dehydrated through
Fixative grades of alcohol (75%-
100%)
Clear with xylene

Impregnate and then

embed in paraffin
blocks

The First Steps Are Routine

Histopathology
Block cutting

Paraffin tissue
ribbon

Mounting on Poly l
lysin/APES coated
IHC glass slides
 Immunohistochemistry

• Manual

• Automated
 Block non-sp
binding
Wash Bind Primary antibody
Deparaffinze Wash
Destroy Wash
endogenous
peroxidase Antigen Retrieval

Wash

Counterstain dehydrate
coverslip examine

Secondary antibody

Wash

Chromogen development
Wash
Heat induced epitope retrieval
HIER
 Primary Antibodies
• Polyclonal antibodies

• Monoclonal antibodies
Primary Antibody vial -1ml

After opening make 20 aliquots of

50 microlitre each
For daily use make fresh dilutions of primary
antibodies from aliquots with
buffers/diluents
Primary antibody on slide
Incubation in humidity chambers
 How to overcome practical
difficulties of variation in IHC
staining related to human errors?

AUTOMATION
5 milestones in the development and
advancement of the IHC field
1. The discovery of monoclonal antibodies

2. Heat induced antigen retrieval (AR) method

3. Highly sensitive secondary detection system,

4. An automated staining system

5. Digital pathology with imaging analysis

 Standardization & Validation of IHC
• Why we need to validate IHC markers ……

– Reagents/ antibodies may be of different

• origin,
• composition or conc.
• or sensitivity or specificity
• Can’t rely on the manufacturer’s

• We need to test them on in-house tissues

before start to use them in diagnostic use
 Validation of new antibody
Antibody_CD138______ Clone M115 CodeNo._M7228
Vendor _DAKO____ Batch/Lot No._00037097__________

S.N0 BLOCK DILUTION REMARKS

PATHNO. TISSUE 1:50

1 32320BX “ Not Satisfac.

2 32210BX “ Not Satisfac.

3 18083BX “ Not Satisfac.

4 14597BZ “ Satisfactory

Comments:
Sign: Date:
Further titration of selected block

Block Titration Used Remarks

Path No. 1 : 40 Not satisfactory

14597 BZ
1 : 50 Good
Tissue

1 : 100 Satisfactory

Comments: 1 : 100 dilution recommended.

Signature: Date:
 Controls in IHC

WHY WE NEED THEM??

 To ensure proper technique and specificity of

the staining method used

Essential for correct interpretation of

IHC result.
 Daily control
• Include positive and negative controls

• Negative control for each case and positive

control for each marker

• Test slides and controls are treated identically

CONTROLS in IHC

C-erB 2
COMPOSITE CONTROL FOR ON EACH
SLIDE
 Composite controls on each slide for
biomarkers
Composite block of

Negative,
Control Low positive and
high positive

Test
 POSITIVE EXTERNAL CONTROL
CD 3

CK

ck

ck

CD20
 POSITIVE INTERNAL CONTROL

• Best control as it has undergone same change as

target tissue so if positive you know that tissue
antigenicity is preserved
 Avoiding misleading IHC:
Pre analytic
• Ensure prompt adequate fixation and processing

• Use carefully selected limited antibody panel

– Choose panel based on morphological and/or clinical
differential diagnosis
– Panel should include positive and negative markers for
each differential diagnosis

“Shotgun” approach with large antibody panels rarely

helpful and may be misleading
 Avoiding misleading IHC:
Analytic

• Use optimal methodology

– Antibody, incubation time, retrieval etc.

• Quality Control
– Use of appropriate controls
 Avoiding misleading IHC:
Post-analytic- Interpretation
• Careful assessment of immunostaining
– Which cells positive?
– Proportion of cells positive?
– Pattern of immunoreactivity

• Morphological correlation

• Clinical/radiological correlation
 Interpretation of
Immunohistochemistry
• To recognize the brown colored cells in right
context

• “Everything that looks brown is not positive ”

 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)
• Cytokeratins, PSA
– Nuclear (diffuse, nucleolar)
• p63, ER
– Membranous (Continuous, broken)
• CD20, Her-2
• The cell of interest (larger tumor cell etc)
 An Example of normal lymph node

CD20 CD3

Bcl2
CD23
 Nuclear positivity

ER Mib-1

p63 PR
 Cytoplasmic positivity

Desmin
Vimentin
 Membrane positivity

CD20 c-kit
 Golgi zone positivity

CD30
Cell of interest

CD30

LCA
 Cell of interest

C-kit LCA
 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……..

 All that is ‘brown’ is not real
• Background staining

• False positivity including artifacts

 Problem of false brown( background)
stain
• Hydrophobic and electrostatic interactions
• Endogenous peroxidase
• Endogenous biotin
• Antigen diffusion
• Antigen retrieval
• Polyclonal antibody
• Necrotic tissue
 Background staining

Polyclonal antibody Necrotic tissue

 Antigen diffusion

k light chain lambda light chain

 Artifacts in IHC
• Edge and trapping artifacts
• Desquamation artifacts
• Bubble artifacts
• Drying artifacts
• Artifacts of poor fixation
• Precipitated DAB artifacts
• Biotin artifacts
Edge and trapping artifacts
Crushed tissue artefact
Deposits artifact
Drying artefact
 Evaluation of immunoreactivity
• Not just positive or negative

• Quantification of expression is important

– % of cells positive; patchy; focal; diffuse

– Focal positivity must be interpreted with caution

– Nuclear staining is more reliable than cytoplasmic staining

– Beware of interpreting immunoreports

– Review immuno slides

 IHC: What can go wrong?

• Interpretation of immunostaining:
(+) or (-)

• Interpretation of immmunoprofile
What does the positivity/negativity actually means
Do not call an IHC negative/positive without checking out the controls

Breast Cancer Negative hormonal markers

Positive internal
control for ER
 Cell of interest-Negative IHC

MLH1 & PMS2 –Neg …..Means tumor is MSI - Pos

 Interpreting an IHC test result:
Take with Pinch of salt
• Be cautious before reporting :
– If the known internal and external positive controls are
only weakly positive

– If the target tissue is only weakly positive in the presence

of background staining

– Focal staining in target tissue

• IHC need to be repeated if

– If the known internal and/or external positive controls
are negative
 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
 Ordering an IHC
Two questions should be asked before ordering any IHC
assay:

• ‘‘Why should I order this marker?’’

‘‘Does it change my diagnosis and patient care?’’

• “I don’t know,’’

Don’t order that Marker

 Ordering an IHC
• Check the Diagnostic Sensitivity and Specificity of Each IHC
Marker

• An entirely sensitive and specific IHC marker rarely exists

– A small panel of IHC markers rather than a single marker is strongly

recommended to avoid a potential diagnostic pitfall

• Begin With a Limited Panel of IHC Markers (First IHC Panel)

• Continue With Second IHC Panels if first panel inconclusive

 Immunohistochemistry
• Epithelial origin
– cytokeratins, AE1-AE3 cytokeratin • Lymphoma
cocktail, CK7, CK20, CEA and EMA – CD45
• Melanoma – CD20
– S100 – CD10
– HMB45 – CD3
• Germ Cell Tumour • Thyroid
– AFP – thyroglobulin
– bHCG – TTF1
– PLAP • Prostate
• Neuroendocrine – PSA
– chromogranin • Sarcoma
– Synaptophysin – AML
– CD56 – CD31
– CD34
Approach to an unknown primary
Case No 1
 CK7

P40 TTF-1

Adenocarcinoma of primary pulmonary origin

Case No 2
• 30 year old female
presented with
anterior thigh swelling
for 8 months.
S 100 P S 100 P
HMB 45 HMB 45

Melan A Bcl 2
 Diagnosis

Amelanotic spindle cell Malignant melanoma

(Clark level V)
Case No 3
26 yr/ M, c/o
– B/L nasal stuffiness since 2 months
– difficulty in swallowing 3 months

MRI
– Large lobulated mass lesion in the nasopharynx
(left > right) measuring 3.0 x 8.1 x 5.5 cm
– B/L level Ia, Ib, II, III and IV nodes
P 63
• Undifferentiated
carcinoma
nasopharyngeal type
AE1/AE3
P 63
LCA
CD20
CD3
MIB-1
 Diagnosis
• Diffuse large B- cell lymphoma with aberrant
p63 expression
Case No 4
• A 26 year old young male
– Fever, weight loss and right sided cervical swelling

• Lymph node biopsy was done

CD30
CD30
LCA
CD20
EBV
CK
Negative for
 OCT 2
 Pax5
 Alk -1
Positive for
 P63 (focal)
 Final Diagnosis
• Hodgkin lymphoma like undifferentiated
nasopharyngeal carcinoma

• CT SCAN revealed nasopharyngeal mass

– Patient treated with CT/RT
 To conclude
 IHC = immunology +histology + chemistry

 Has strengths and weaknesses

 IHC must always be interpreted in the context of

morphology

 Think about your planned panel of IHC before ordering

 IHC will just mislead and waste money in poorly

processed tissue