Immuno Histoche mistry

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Dr.Prathana samuel
MDS 2nd yr

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Definitio n It is a technique for identifying

cellular or tissue constituents (antigens) by means of antigen-antibody binding being identified either by direct labelling of the antibody or by use of a secondary labelling method.

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 Principle Localization of antigens in tissue sections by the use of labeled antibodies

through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold.

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Introdu ction
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Where does IHC come to play

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So what is it ???

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WHO AM I ????

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T CEL L

T CE LL

T CEL L

T L CE L

T L CE L

T CEL L
CE T L L

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Indications
To find the type of cell and tissue (in diagnosis aided by clinical data and histopathology )

to demonstrate histogenesis in undifferentiated tumours by looking for antigens related to differentiation

To quantify the Prognostic markers To Classify and Grade tumours In renal diseases to

demonstrate different types of immune complexes in glomeruli, important in the diagnosis of glomerulonephritis

In suspected infective disease - to detect specific 25-07-2011 1313 organisms

For a more specific IHC u need

least damage on the cell or tissue Usage of least amount of antibody in shortest time with least background staining.

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acids, lipids

IHC can Proteins, carbohydrates, nucleic identify.

Types of the secreting cells Membrane antigens Structural antigens within the Antigen localised in the nucleus
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cytoplasm

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P roto co l. . .
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P r o t o c ol
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Antigen Retrieva l

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Antigen Retrieval of It is a process

NORM ALTISS UE ANTIG EN recovering the antigenicity of tissue sections that had been masked by formalin fixation and paraffin imbedding.
AN RE TI TR GE IE N VA L

T PR ISS O UE CE SS IN G

NORM ALTISS UE ANTIG EN

DAMAGE D ANTIGEN
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Heat Induced Antigen Procedure: Retrieval

sections are soaked in an antigen retrieval solution and heated at 90C to 110C. Source: Pressure cooker Hot water bath Microwave oven Autoclave Steamer

ANTIGEN RETRIEVAL SOLUTIONS:

citrate buffer, EDTA, Tris base buffer (0.5 M, pH 10), glycine-HCl buffer, 1% periodic acid, various concentrations of urea, lead thiocyanate solutions, even distilled water or liquid hand soap.
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Proteolytic Antigen Retrieval Procedure.

Sections are incubated in enzyme (0.05 to 0.1%.) for 5-30 min at 37'C.

Enzymes Used

proteinase k Pronase pepsin

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Buffers For Wash ing

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Buffers for washing... Only freshly prepared

should be used.

buffers

Phosphate buffered saline (PBS) or in Tris-buffered saline (TBS) are commonly used. The pH of both solutions should be 7.4 (for optimal immunoreactivity of antibodies )

Do not use PBS for alkaline phosphatase-conjugated antibodies, since phosphate is an inhibitor of alkaline phosphatase. 25-07-2011 2323

Blocki ng Steps

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Blocking Frequent causes of background staining are steps activity, Fc receptors, endogenous enzyme

Blocking step 1: to block endogenous enzyme activity or endogenous biotin Wash sections in PBS or TBS Blocking Step 2: to block receptor,.
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endogenous biotin and autoflourescence of normal tissue components. These are first blocked before incubating with primary antibody.

endogenous
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Fc

Blocking Fc receptor incubate sections for 20 minutes with PBS steps containing 5% normal serum of or TBS

species in which the secondary antibodies were raised

Peroxidase Alkaline phosphatase Biotin

pre-treatment of cells and tissues sections with hydrogen peroxide (30%). 1 mM levamisole/0.03 0.5 N HCl or 1 M citric to the chromogen/substrate Incubate sections with Avidin block solution (avidin 0.01% in PBS) and biotin block (0.05% PBS) for 10 15 min each and rinse twice with PBS between steps.

Autoflouresce nce

spectral unmixing algorithms subtracting the background fluorescence.

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Antib ody
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Polyclonal Antibody

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Monoclonal Antibody

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Antibody for IHC

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Rabbit vs mouse
polyclonal antibodies produced by rabbits

are known to have a higher affinity than

antibodies raised in mice, which means to put it simply a better antigen recognition.

Additionally, the rabbit immuno-response

recognizes antigens / epitopes that are not immunogenic in mice.

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So the best choice is..

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Immunolabelli ng

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Immunolabelling methods
1.

Direct method: The antigen directly binds to its specific labelled antibody (primary antibody)

Direct method is one step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections Fast to get the results Labeling intensity is low Used for kidney or skin biopsies.

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Immunolabelling methods
2.

Indirect method: Primary antibody is unlabelled. A secondary antibody (which is labeled) is used.

Indirect method involves an unlabeled primary antibody which react with tissue antigen, and a labeled secondary antibody react with primary antibody. Getting results takes longer More sensitive More economic

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Primary antibodies: blot excess blocking solution from sections, and incubate for 60 min at room temperature or over night at 4C with a correspondingly diluted primary antibody. Wash sections in PBS or TBS for 2 -3 min. Secondary antibodies: incubate sections for 30 60 min at room temperature with a HRP- or AP-labelled secondary antibody raised against the corresponding IgG of the primary antibody. Wash sections in a buffer recommended for the corresponding enzyme substrate development 25-07-2011 3636 for 2 -3 min.

Antibody Labels. . .
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1.

Immunoenzyme method:

An enzyme is used as the label. Peroxidase, alkalin phosfatase, glucose oxidase Chromogen (staining chemical) must be used

2.

Immunofluorescence method:
A fluorochrome is used as the label. AMCA, Fluorescein, FITC, Rodomine, TRITC, Texas Red. Fluorescence microscope with appropriate filter or confocal laser scanning microscope must be used to visualize.

3.

Immunogold method:

colloidal gold particles are used as the label. Usually used in electron microscopy.

Substrate: incubate sections with an appropriate enzyme substrate until optimal colour develops. Wash sections first in the same buffer and thereafter in distilled water. 25-07-2011 3838

Chromogen s. . .
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CHROMOGEN PEROXIDASE

COLOUR

Diaminobenzidine (DAB) Brown DAB with enhancement Black 3-Amino-9-ethyl carbazole (AEC) Red 4-Chloro-1-naphthol (4-CN) Blue-black Hanker-Yates reagent Blue Alpha-naphthol pyronin Red 3,35,5-tetramethylbenzidine Blue ALKALINE PHOSPHATASE Fast blue BB Blue Fast red TR Red New fuchsin Red CIP-NBT Blue GLUCOSE OXIDASE Tetrazolium Blue Tetranitroblue tetrazolium Black IMMUNOGOLD With silver enhancement black With silver enhancement black 25-07-2011 4040

Nuclear Counterstaining
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NUCLEAR COUNTERSTAINING Recommended for

better interpretation both of immunostaining And tissue morphology. common nuclear stain - Mayers Hematoxylin. Advantages Of Mayers Hematoxylin This staining provides blue violet nuclear staining without obscuring antigen-specific chromogen deposition, giving excellent colour contrast with most commonly-used peroxidase and alkaline phosphatase substrates in bright field microscopy Points To Be In Mind While Staining - No acid differentiation - Short duration (5-10min) OthersMethyl Green and Nuclear Fast Red 25-07-2011 4242

Cont rols
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SPECIFIC CONTROLS The specificity of the results depends on two

independent criteria: -The specificity of the antibody -the method used. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG , and a positive control (testing the primary antibody with tissues known to contain the antigen).

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Trouble Shooting
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The recognition of inappropriate staining can be divided into 3 main categories based on the pattern of staining results on the test tissue as well as the control: Weak / Absence of staining Over staining Background staining
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TROUBLE SHOOTING

Weak or No Staining
Sources Inactive primary antibodies Solutions Replace with a new batch of antibodies Inadequate deparaffinization Deparaffinize sections longer or change fresh xylene Antibodies do not work due to store antibodies into smaller volumes and store in improper storage freezer (-20 to -70) and avoid repeated freeze and thaw cycles. Antibody concentration was too low Inadequate antibody incubation time Increase the concentration of antibodies. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio Increase antibody incubation time

Inadequate or improper tissue Increase duration of postfixation or try different fixation fixatives 25-07-2011 4747

drprathanasamuel@gm

Weak or No Staining
Sources Tissue overfixation Incompatible secondary and primary antibodies Solutions Reduce the duration of postfixation or perform an appropriate antigen retrieval procedure Use secondary antibody that will interact with primary antibody.

Inactive secondary antibody or Replace with a new batch of reagents other reagents Inadequate substrate incubation time Incorrect mounting medium Reagents applied in wrong order or steps omitted Increase the substrate incubation time Choose a correct mounting medium Check notes or procedure used

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Over-staining
Sources Solutions The concentration of antibodies was Reduce antibody concentration or perform a too high titration to determine the optimal dilution for primary and secondary antibodies Incubation time was too long Reduce incubation time Incubation temperature was too high Reduce incubation temperature Substrate incubation time was too long Sections dried out Reduce substrate incubation time Avoid sections being dried out

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High Background
Sources Inadequate washing of sections Tissue contains endogenous enzyme Tissue contains endogenous biotin activity Non-specific binding of primary antibodies to tissue or high antibody concentration Non-specific binding of secondary antibodies to tissue Solutions Wash at least 3 times between steps Block endogenous enzyme activities Block endogenous biotin activity using the avidin/biotin blocking reagent prior to incubation of primary antibodies. Non-specific binding may be reduced by using higher dilution of primary antibodies Treat tissue with normal serum block from the same species as secondary antibodies

Diffusion of tissue antigen due to Increase duration of postfixation inadequate fixation 25-07-2011 5050

INTERPRETA TION
It is based on the presence, pattern, and intensity of coloured chromogen products, which are deposited on the tissue as the result of specific antibody-antigen reactions in the cells. The resultant pattern of staining can be -focal or diffuse, -nuclear /cytoplasmic / membranous.

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Immunom arkers
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The antigen in/on a particular cell helps to identify the cell type and hence helps in diagnosis of a tumour. These immunomarkers can be broadly classified as the following:

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Intermediate filaments Epitheliail-related markers Germ cell tumour markers Neuroendocrine markers Mesenchymal tissue markers Basement membrane components Harmone receptors Oncoproteins Cell proliferation markers Leukocyte antigens
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INTERMEDI ATE FILAMENTS Cytokeratin s

Vimentin

Desmin

Neurofilam ent proteins Glial fibrillary acidprotein

EPITHELIA L RELATED MARKERS Epithelial Membrane Antigen (EMA) Carcinoem bryonic antigen Prostrate specific Antigen & acid Phosphata se Thyroglob ulin Other tissue Specific markers

GERMCELL TUMOUR MARKERS Placental Alkaline phosphata se Alpha fetoprotei n Human choreo gonodotro pin

NEUROEND OCRINE MARKERS Neuron specific enolase Chromogra nin Synaptophy sin

MESENCHYMA L TISSUE MARKERS Smooth muscle Actin S100 protein

Melanoma specific Antigens (HMB45)

Human placental lactogen

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BASEMENT MEMBRANE COMPONENT S Collagen IV

HARMONE RECEPTOR S Estrogen receptor Progestero ne receptor

ONCO PROTEIN S c-erbB2

CELL PROLIFERATION MARKERS Ki-67

Laminin

Bcl-2

Proliferating cell nuclear antigen / cyclin (PCNA)

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LEUKOCYTE ANTIGENS General lymphoid markers B cell markers T cell markers Hodgkins cell markers Ig light & heavy chain Bcl-2 oncoprotein Macrophage/Histioc yte markers Myeloid Cell markers Myeloid enzymes Erythroid cell Markers Megakaryocyte 25-07-2011 markers CD 45R, CD45RO,CD45RA CD45RB CD20, CD10, CD3, CD5, CD3 CD15, CD30, Kappa, lamda light chains

CD 68, Mac 387

CD 66 (Carcinoembryonic antigen, nonspecific cross reacting Antigen) Neutrophil elastase, myeloperoxidase, chloracetate esterase Erythrocyte-specific glycophorin CD 61 (platelet glycoprotein 3A) 5656

Limita tions
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The main uses

a good example is the MIC2 antigen which was initially thought to be specific for EWS-PNET but now is recorded in many tumors like osteosarcoma, mesenchymal chondrosarcoma with widely different treatment or chemotherapy options. This is where sound histology diagnosis, molecular diagnosis, clinical and X-ray picture will help.

Infidelity of markers

Aberrant expression of markers

Sometimes tumors express unusual markers e.g. RMS expresses LCA, desmin and cytokeratins are seen in EWSPNET group of tumors.

Undifferentiated tumors Need for strict quality control

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These tumors often have no markers and even lack histological clues to the diagnosis.

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histopathology is an important prerequisite so that a suitable IHC panel can be designed Hence IHC can never and requested.

So Forming a differential diagnosis on

be

considered superior Than histopathology

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Thank You
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Referen Bancroft JD, Stevens ces A. Theory and Practice of Histological Techniques. 4th Edition. Pierson
Professional Ltd. Hongkong, 1996:435-512 Buchwalow IB, Bocker W. Immunohistochemistry - basics and methods. Springer. Berlin, Germany. 2010 4 Dabbs DJ. Diagnostic Immunohistochemistry. 3rd Edition. Saunders, an imprint of Elsevier Inc. Philadelphia, 2010:1-41
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http://tmc.gov.in/clinicalguidelines/EBM/Vol5/pathology_