Immunohistochemistry Practical issues

Dr Santosh MenonAssistant Professor, Pathology

Tata Memorial Hosipital

What is IHC?

• A method that uses antibodies to identify, locate, and stain specific protein molecules in tissue sections (visualized using a microscope)

• Used to diagnose the type of cancer and to help determine the patient's prognosis or as a predictive marker of therapeutic response.

WHY IS IHC IMPORTANT in surgical pathology practice?

• cell lineage and tissue type

• prognostic markers

• Quantification; it is no longer enough that the 'stain' is there; rather it is a question of 'How much is there?'

• Predictive markers for targeted therapy

• Patients are knowledgeable…thanks to internet

CD20 and Rituximab

CD20

C-erbB2 and Herceptin in breast CA

C-kit and imatinib in GIST

Immunohistochemistry

• Manual

• Automated

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

What is an Antibody?Antibodies are proteins called

immunoglobulins

Antigen

Antibody

Antigen

Primary Antibody

Labeled Secondary Antibody

Cell with antigens on surface

Antigen

B

B

PX

PX

PX

B

Avidin Biotin Complex

DAB+H2O2

IHC

Primary Antibody

Labeled Secondary Antibody

Cell with antigens on surface

Detection system

Chromogen

Detection system

Primary Antibody

Tissue section with antigen

Primary Antibodies

• Polyclonal antibodies: Heterogeneous population of antibodies, produced against several epitopes on a single antigen- multiple clones produce the antibodiesMore tolerant to retrieval techniquesSpecificity is less

• Monoclonal antibodies: Homogenous population of antibodies for a single epitope – from a single clone of B-cellsMore specific Less cross reactivityLess background non specific staining

Pure single Ab

After immunization, the mouse spleen contains B cells producing specific antibodies.Each B cell produces only one kind of antibody, which binds to its specific antigen. Conventional antiserum is the mixture of all antibodies produced by B cells from spleen.If a single B cell was picked up and cultured, then it will produce only one kind of antibody. But B cell can not survive well in the culture.Myeloma cell can be cultured in the test tube, but can not produce useful antibody.Each hybridoma line can produce pure single antibody, called monoclonal antibody.If B cell is fused with myeloma, the fused cell might be cultured and produce antibody.

Adapted from Milstein (1980) Scientific American, Oct. p.58

12

34

mmm

m

12 3

4

1 2 3 4

Monoclonalantibodies

Cell fusion

Spleen cells

+Myeloma

x

Antiseum

Antigen

Immunization

A mixture of all Ab

1 23 4

BALB/c

1 2 3 4

1 23 4

B cell

Primary Antibody vial

How do we start with a new antibody?

If you have acquired a new antibody…… Titration is a must

Date Antibody Used

Dilution Appropriate dilution

Remarks

04-04-08 CD20 1:50, 1:1001:200

1:100 GOOD

05-05-08 CD3 1:1001:2001:300

1:200 BEST

Positive controls

CYTOKERATIN

CD3

CD20

Negative control

Same steps but with omission of primary antibody

Validation of new antibodyAntibody_CD138______ Clone M115 CodeNo._M7228

Vendor _ ___ Batch/Lot No._00037097__________

Comments:

Sign: Date:

S.N0 BLOCK MANUFACT.DILn. REMARKSPATHNO. TISSUE 1:50

1 32320BX “ Satisfactory.

2 32210BX “ Satisfactory

3 18083BX “ Satisfactory

4 14597BZ “ Satisfactory

Workflow in IHC lab

Workflow in IHC: Making an entry

Making the required entries

Blocks are cut in routine manner

Tissue sections on poly-L-lysine coated slides

Bind Primary antibody

Wash

Biotinylated 2nd

antibody

Wash

Chromogen development

Counterstain dehydrate coverslip examine

Block non-sp binding

Deparaffinize

XyleneBlock

endogenous peroxidase

Antigen Retrieval

Antigen retrieval -standardization

• Proteolytic enzyme methods• Heat induced epitope retrieval

MicrowavePressure cooker

Achieving the desired temperaturepH of buffer- calibration

Holding times

Why is antigen/epitope retrieval necessary? ……..Formalin Fixation

Slides immersed in buffer solution

Scientific pressure cooker

Microwave

Primary Antibody

Primary Antibody vial -1ml

After opening make 20 aliquots of 50 microlitre each

For daily use make fresh dilutions of primary

antibodies from aliquots

Micropippettes

Micropippettes

Have a daily dilution chart as a spreadsheet

Antibody dilution chart Date 10/06/2010

Antibody Retrieval method Dilution

1 CD20 TE-Pascal 1:100

2 MPO SC-Microwave 1:300

3 CD23 TE-Microwave 1:100

4 ER SC-Pascal 1:50

Making the dilutions of antibodies with buffer/diluent

Washings and wiping

Primary antibody on slide

Incubation in humidity chambers

What are we looking for in IHC?

Coloured visual product demonstrating an antigen antibody reaction:Brown colour (DAB)Red colour (PAP-APAAP)Greenish yellow in

immunoflourescence

Issues related to interpretation

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)• Nuclear (diffuse, nucleolar)• Membranous (Continuous, broken)• Interstitial• The cell of interest (larger tumor cell etc)

An Example of normal lymph node

CD20 CD3

CD23Bcl2

Nuclear positivity

ER Mib-1

p63 PR

Cytoplasmic positivity

VimentinDesmin

Membrane positivity

CD20 c-kit

Golgi zone positivity

CD15 CD15

Cell of interest

CD30

LCA

RS cells in Hodgkin lymphoma

CD30 LCA

Cell of interest

CD20

CD163

Cell of interest

C-kit LCA

Is everything brown positive?No, beware

All that is ‘brown’ is not real

• Background staining

• False positivity including artifacts

Problem of false brown stain

• Hydrophobic and electrostatic interactions

• Endogenous peroxidase• Endogenous biotin • Antigen diffusion• Antigen retrieval• Polyclonal antibody• Necrotic tissue

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……..

Background staining

Polyclonal antibody Necrotic tissue

Antigen diffusion

k light chain lambda light chain

False positivity• Cross reactivity due to epitope sharing

may result in false positivity• An example is CD79a, a B cell marker

cross reacts with smooth muscle• Error in data entry and

mislabelling(MyoD1 and Mib1)

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

When do you call an IHC as negative?

• Do not call an immunostain negative without checking out the positive controls

• Remember the best control is positive internal control

An example of breast cancer

Breast Cancer Negative hormonal markers

Positive ER Positive PR

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

Quality issues and validation

Antibody Clone Vendor Dilution 1 2 3-- 31

ER ID5 DAKO !:100 +6 +6 +6

LCA 2B11 DAKO 1:200 +3 +3 +2

Sign

Month: May2008

Daily positive control chart

Daily controls

Validation of New Control

Old Block New Block

Tissue Path No. Remark Tissue Path No. Remark

BREAST 6223CD DEPLETED

BREAST 5960CD VALIDATED FOR ER

Antibody_ER____________

Vendor_DAKO___ Clone No.__ID5___ CODE No._M7047_________

Batch No.00000072_________ Dilution Factor__1:100____________

Comments:

Sign: Date:6/5/2008

How to overcome practical difficulties of variation in IHC

staining related to human errors?

AUTOMATION

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Acknowledgement• Without a technical hand

who has immense dedication and interest in IHC, it is impossible to achieve required results.

Acknowledgements• Dr NA Jambhekar Prof & Head• Dr Sumeet Gujral & Dr Subramanian• Mrs Rekha Thorat Senior technical officer• Mr Mahendra Palkar• Mr Aamir Khan• Mr Pritam• Mr Dinkar• Mr Shinde