IHC Flashcards
is used in histology to detect the presence of specific protein marker that can assist with accurate tumor classification and diagnosis
Immunshstochemistry (IHC)
It is used in the pathology laboratory as an aid in the differential diagnosis and classification of cancer, and for certain other diseases, including infections.
IHC
The factors that influence the immunohistochemical staining result start in the _____and end at the____ by the pathologist, which ultimately leads to treatment decision by the oncologist
surgery operating room
interpretation of the stain
Pre analytical steps
BAG TES
Biopsy
Acessioning
Grossing
Tissue processing and Embedding
Sectioning
Immunshstochemistry (IHC) is used in histology to detect the presence of specific______ that can assist with accurate tumor classification and diagnosis,
protein marker (antigen)
(surgically removed tissue specimen or needle biopsy) from the surgery room arrives in fixative at the pathology laboratory.
BIOPSY
the sample details are entered into the_______. A barcoded label can ensure track and trace capabilities
ACCESSIONING
laboratory information system (LIS)
the specimen is visually examined for suspicious areas that require further examination.
Samples from the specimen that require further microscopic testing are excised as tissus blocks and placed barcoded cassetes
Grossing
The steps where the tissue block is processed into a form and condition suitable for making ultrathin microscopic sections.
Typically, the tissue is fixed in formalin then dehydrated before it is embedded in paraffin
TISSUE PROCESSING AND
EMBEDDING
The fine art of cutting the parafin-embedded tissue blocks into ultrathin (-4 um) sections and placing them onto glass sides.
A_____ on the slide can ensure traceability and may also contain protocol information for the requested test for that particular section.
SECTIONING
barcode
ANALYTICAL STEPS
Antigen retrieval
Blocking
Primary antibody
Enzyme HRP
It is performed to recover the antigens
that may have been altered by fixation,
Antigen Retrieval
Endogenous enzymes are blocked (this step can also be performed after
primary antibody incubation)
Blocking
A_______ is applied that specifically binds to the antigen of interest
primary antibody
The______ antibody carries the label (enzyme) upon application it binds to the primary antibody
Enzyme-HRP
secondary
ARS
Antigen Retrieval Solution
Antigen retrieval is not necessary in
Frozen sections
Performed to visualize nuclei and overall tissue architecture
Counterstaining
DAB
diaminobenzidine
Post-Analytical Steps
• In the post-analytical process, the pathologist interprets the stains in context with________ controls, using ______microscopy.
• The results are reported to the_____ for treatment decision.
positive and negative tissue
bright field
oncologist
SECTIONING
Use High Quality Sections
Take particular care to use thin, flat sections that have been thoroughly dried onto the slide. Preferably use __________ or _______slides for IHC.
Uneven, poorly-adhering sections stain unevenly with variable background staining
charged slides or APES coated
Again, these controls must be processed identically to the specimen but contain the target protein.
In some cases, it will be advantageous to have this control tissue stain only marginally positive as to monitor not only for the presence of the antigen, but also for any possible loss of sensitivity.
This loss might not be apparent if only intensely staining controls are used.
Controls for loss of sensitivity would be particularly important, for example, when staining tumors.
INTERNAL TISSUE CONTROLS
Chromogen
DAB
Brown
IHC STAINING PROCEDURE
MATERIALS
• DRYING OVEN
• CONTROL TISSUE (+)
• HUMID CHAMBER
• STAINING JAR
• TIMERS
• COVERSLIPS
• DISTILLED WATER
• CHARGED SLIDES
• REAGENTS FOR DEPARAFFINIZATION
• WATER BATH 95-99 DEGREES CELCIUS
• PIPETTES AND TIPS
• DAKO FLEX Ready-to-use Primary Antibodies or
• Dako Concentrated Primary Rabbit or mouse
Antibodies
• Dako Antibody Diluent
• Envision FLEX Target Retrieval Solution (high pH)
• Envision FLEX Target Retrieval Solution (low pH)
• Wash buffer
• Hematoxylin
• Envision FLEX Target Retrieval Solution (high pH)
• Envision FLEX Target Retrieval Solution (low pH)
Blue
Pink/ Magenta
Counterstaining
Hematoxylin
For ________the sensitivity and specificity are the core elements.
primary antibodies
Ideally, the primary antibody must provide both a______ and______ to produce an accurate and robust IHC
high sensitivity and a high specificity
have become widely used because of their high specificity, consistency, purity and commercial availability.
Monocional antibodies
One binding site
Monoclonal antibody
Monoclonal antibodies, produced in______according to the ______method developed by_____.
mice
In vitro hybridoma
Kohler
, typically produced in_____ by ________techniques, with booster immunizations to maximize the reactivity against the target antigen, frequently give a higher______ (avidity) compared to monoclonal antibodios, as the many antibody “species’ present react with more antigen sites.
Polycional antibodies
Rabbits
traditional immunization techniques
sensitivity
The major drawback of Formalin-Fixed Paraffin-Embedded (FFPE) tissue is that formalin-induced molecular modification of proteins (antigens) may result in loss of the ability of the antibody to react with the antigen, a loss that can only be corrected by the restoration (retrieval) of the
“formalin-modified’ antigen molecular structure
TARGET RETRIEVAL SOLUTION
These improve antigen expression of your samples by breaking down formalin induced______, re-exposing epitopes on the antigen to antibody binding.
TRS
antigen cross-linking
Heat and enzyme retrieval are both employed, with______ now being the most commonly used.
In simple terms HIER involves heating your slides in buffer at___ or ____(depending on your antibody) using a microwave or a pressure cooker.
heat induced epitope retrieval (HIER)
pH6 or pH9
TRS
Target Retrieval Solution
HIER PROCEDURE
1.Place a staining jar containing diluted____ in awater bath. Cover the staining jar with lid to stabilize the temperature and prevent evaporation then cover the water bath.
2.Heat the water bath and the staining jar filled with TRS to_____
3.Measure temperature inside the staining jar with a calibrated thermometer to ensure correct temperature. Immerse the rack slides in the Preheat bed PRS in the staining jar. Cover the staining Jar and the
water bath
4.Bring the temperature of the water bath and the TRS back to 95-99°C. Incubate for____ minutes at 95-99°C.
5.Remove the entire staining jar with slides from the water bath. Allow the slides to cool in the solution for___ minutes at room temperature.
- Immediately soak slides in a staining jar with diluted_____
TRS
90-95°C.
25
20
Wash Buffer (WB).
- Tap and wipe off excess buffer and draw a circle around the Uissue section with_____.
- Apply two drops (100 ul) or enough __________ Incubate for 5 mlnutes.
- Put sildes in the_______.
- Rinse gently with wash buffer solution and place in a fresh wash buffer bath for five minutes.
5.Tap off excess buffer and apply two drops or enough______, Incubate for 30 minutes. - Rinse gently as in Step 4.
- Tap off excess buffer and apply two drops or enough______, Incubate for 30 minutes.
- Rinse gently as in Step 4.
- Tap off excess buffer and apply two drops or enough _______Incubate for 10 mlnutes.
- Rinse gently with distilled water and place in a fresh distilled water bath for 5 minutes.
11.Tap off excess water and apply two drops or enough______. Incubate then wash with distilled water and soak in wash buffer for 1 minute.
uh distilled-water-twise.
STAINING FROCEDURE - one rhanel and Xylene twice each for 1 min.
nountina medlum
Dako pen
Dual Endogenous Enzyme Block
humidity chamber
Dako Primary Antibody
Labeled-polymer HRP
DAB+ Substrate Working Solution
hematoxylin
