1. Welcome Introduction to IHC
Ventana
3/31/2017
Welcome
Introduction to IHC
A basic overview of theory and techniques relating to immunohistochemistry
History
Theory
Techniques
Questions and Answers
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2. Ventana
3/31/2017
IHC History
The principle of immunohistochemistry has existed since the 1930s
Started in 1941 with Coons identifying pneumococci using a direct fluorescent method.
Single antibody labeled with FITC molecule for visualization
Next came the indirect method, the addition of horseradish peroxidase, the peroxidase anti-peroxidase technique of and the use of the Avidin and Biotin complex in the early 1980s.
Methods utilizing enzyme conjugated antibodies were developed independently by Nakane and Pierce and Avrameas and Uriel
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3. IHC Theory Fixation Cutting Eptitope Retrieval Primary Antibody
Ventana
3/31/2017
IHC Theory
Fixation
Cutting
Eptitope Retrieval
Primary Antibody
Secondary Labeling
Enzyme Reaction
Substrate Labeling
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4. Ventana
3/31/2017
IHC Theory
Fixation
Stops the autolysis of proteins and maintains the properties of the tissue
Cross-Links Proteins with the cell
Essentially stops the meat from rotting
Should be done as soon as possible preferably in the Operating Room with an adequate amount of fixative
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5. IHC Theory Cutting Performed on a microtome Only well-fixed specimen
Ventana
3/31/2017
IHC Theory
Cutting
Performed on a microtome
Only well-fixed specimen
Charged slides recommended (VMSI instruments requires this for optimal results)
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6. IHC Theory Eptitope Retrieval Heat Induced (HIER)
Ventana
3/31/2017
IHC Theory
Eptitope Retrieval
Heat Induced (HIER)
Uses physical heat to uncover cross-linked antigens
In theory, water will achieve this but there are better more specific buffers available
Protoelytic Induced (PIER)
Uses chemical enzyme to cut proteins
Pronase, Trypsin, Protinase K are all examples of enzymes that will achieve specific results
General protease works well most of the time
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7. IHC Theory Primary Antibody
Ventana
3/31/2017
IHC Theory
Primary Antibody
2 animals predominantly used in antibody production
Rabbit and Mouse
until recently rabbit antibodies were polyclonal and mouse antibodies were monoclonal
Rabbit as polyclonal antibody production
Cells of interest injected into rabbit (e.g. skin for keratin antibody)
Immune response takes place
Rabbit blood serum is harvested and purified to obtain a “clean” antibody
Very sensitive antibody is created
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8. IHC Theory Primary Antibody Mouse as monoclonal antibody production
Ventana
3/31/2017
IHC Theory
Primary Antibody
Mouse as monoclonal antibody production
A monoclonal antibody is a preparation of antibody that all have the same exact specificity, and in fact, is all the identical protein
one immunizes an animal, removes their spleen and purifies the B-cells that make antibodies.
These cells are then separated from one another, and each are fused to another cell that confers the ability to replicate and survive outside of the animal. (hybridoma)
The culture supernatants of these hybrid cells are tested for the antibody that you want. Once you find a hybrid cell making the antibody you want, you can grow as much of the now monoclonal antibody as you need.
This creates an antibody with great specificity
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9. IHC Theory Secondary Labeling Typically an antibody derived from goat
Ventana
3/31/2017
IHC Theory
Secondary Labeling
Typically an antibody derived from goat
Directed against the type of antibody used for your primary reaction i.e. anti-mouse goat or anti-rabbit goat
May be have many different labels
The labels include biotin, fluorescein, rhodamine, horseradish peroxidase and calf intestinal alkaline phosphatase.
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10. IHC Theory Enzyme Reaction – Avidin Binding Complex Ventana 3/31/2017
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11. IHC Theory Enzyme Reaction – Labeled Avidin Ventana 3/31/2017
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12. IHC Theory Substrate Labeling
Ventana
3/31/2017
IHC Theory
Substrate Labeling
Immunoenzymatic staining of tissues results from the reaction of a soluble substrate with an enzyme to produce an insoluble, colored product.
The intensity of the color produced when the substrate is added should correlate to the concentration of the primary antibody and the respective tissue antigen
The most common selections are horseradish peroxidase and calf intestinal alkaline phosphatase.
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13. Questions?

14. IHC Techniques Fixation Cutting Eptitope Retrieval Primary Antibody
Ventana
3/31/2017
IHC Techniques
Fixation
Cutting
Eptitope Retrieval
Primary Antibody
Secondary Labeling
Enzyme Reaction
Substrate Labeling
PowerPoint Presentation

15. IHC Techniques Fixation
Ventana
3/31/2017
IHC Techniques
Fixation
As described before optimal fixation occurs if the specimen is placed into fixative immediately
Industry standard is 10% Neutral Buffered Formalin
Specimens should be fixed on processor a minimum of six hours to allow for adequate fixation
In order to maintain quality this practice should be as standardized as possible
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16. IHC Techniques Cutting Well fixed specimens cut the best
Ventana
3/31/2017
IHC Techniques
Cutting
Well fixed specimens cut the best
For immunohistochemistry to work optimally cutting should be done between three and six microns
A sharp cutting blade helps eliminate knife chatter on the specimen.
Slides stain best when applied to charged slides
Slides have either a chemically or physically applied charge to the surface. This helps the slightly negatively charged specimen adhere to the glass.
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17. IHC Techniques Eptitope Retrieval - HIER
Ventana
3/31/2017
IHC Techniques
Eptitope Retrieval - HIER
A method in which the cross linkage of proteins that occurs during fixation is undone
May be performed in a decloaker (pressure cooker), steamer, or in the microwave
Buffers of varying pH are available to optimize different antibodies
Cooking of the tissue varies from 10 to 60 minutes depending on the method used
The temperature achieved by these methods appears to be the critical variable.
Standardization at this step is the key to successful staining
Shi S-R, Cote C, Kalra KL, Taylor CR, Tandon AK (1992a) A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry. J Histochem Cytochem 40: [Abstract]
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18. IHC Techniques Eptitope Retrieval – PIER
Ventana
3/31/2017
IHC Techniques
Eptitope Retrieval – PIER
A method in which the cross linkage of proteins that occurs during fixation are cut
Protein specific proteases are available for this as well as general proteases
The difference between the two is the proteins they target
Excessive time in protease will start to break down proteins destroying the morphology of the cell
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19. IHC Techniques Primary Antibody Applied directly to the specimen
Ventana
3/31/2017
IHC Techniques
Primary Antibody
Applied directly to the specimen
Applied for 4 minutes to as long as overnight
Should be performed in a humidity chamber to avoid evaporation of antibody
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20. IHC Techniques Secondary Labeling
Ventana
3/31/2017
IHC Techniques
Secondary Labeling
In actuality this is simply an antibody directed against a mouse or rabbit epitope
Applied directly to the specimen
Typical incubation is 10 to 30 minutes
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21. IHC Techniques Enzyme Reaction – ABC Method Ventana 3/31/2017
Staining intensity is a function of the enzyme activity and improved sensitivity can be achieved by increasing the number of enzyme molecules bound to the tissue. The multiple binding sites between the tetravalent avidin and biotinylated antibodies (bound to the antigen) are ideal for achieving this amplification. The avidin-biotin complex (ABC) method was developed by Hsu and colleagues. A biotinylated enzyme (HRP or AP) is pre-incubated with avidin, forming large complexes to be incubated with the biotinylated antibody. Typically, the avidin and biotinylated enzyme are mixed together in a specified ratio for about 15 minutes at room temperature to form the complex. An aliquot of this solution is then added to the tissue, and any remaining biotin-binding sites on the avidin bind to the biotinylated antibody that is already bound to the tissue. The result is a greater concentration of enzyme (3 enzyme molecules to one avidin molecule) at the antigenic site and therefore an increase in signal intensity and sensitivity
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22. IHC Techniques Enzyme Reaction – LAB Method Ventana 3/31/2017
If the avidin-biotin complex becomes too large to penetrate the tissue, a second method developed by the Guesdon and colleagues can be used. The labeled avidin-biotin (LAB) method employs an avidin-enzyme conjugate (or streptavidin-enzyme conjugate) to detect the bound biotinylated primary antibody on the tissue section. This smaller complex allow better tissue penetration. This is labeled with horseradish peroxidase or calf intestinal alkaline phosphatase.
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23. IHC Techniques Substrate Labeling
Ventana
3/31/2017
IHC Techniques
Substrate Labeling
An insoluble colored end product that allows visualization of the chemical reaction that was performed on the tissue.
DAB (diaminobenzamidine) and a red or blue substrate for calf intestinal alkaline phosphatase
Other reagents may be added to enhance the color e.g. copper or nickel for DAB
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24. Questions?

25. Current Trends in IHC Routine surgicals Imaging New Molecular Tests
Ventana
3/31/2017
Current Trends in IHC
Routine surgicals
Imaging
New Molecular Tests
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26. Current Trends in IHC Routine surgicals
Ventana
3/31/2017
Current Trends in IHC
Routine surgicals
Routine surgical biopsies are being done more and more as an outpatient procedure removed from the hospital completely
These specimens are then transported to an off-site location for processing and interpretation
Typically done on prostate and skin samples
It appears that specialized testing will soon be the heart of the hospital laboratories and routines will be handled strictly on an outpatient basis
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27. Current Trends in IHC Imaging
Ventana
3/31/2017
Current Trends in IHC
Imaging
Imaging of specimens is increasing at most large institutions
This allows for digital images of the specimen to be analyzed by an algorithm and assist the pathologist in interpretation
This does not take the place of the physician but gives them another tool to make a diagnosis
Also in the future may allow for collaboration around the world or around the corner
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28. Current Trends in IHC New Molecular Tests
Ventana
3/31/2017
Current Trends in IHC
New Molecular Tests
With few exceptions most of the testing done in the IHC lab is based upon proteins and their expression
As drugs are developed that target specific genes rather than proteins new molecular test will need to be developed
The best example of this currently is FISH for Her2neu
This tests enables the pathologist to diagnosis an equivocal to either a positive or negative interpretation
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29. Goodbye!