IMMUNOHISTOCHEMISTRY Prt 1 Flashcards
Pre-analytical steps
A. Biopsy
B. Accessioning
C. Grossing
D. Tissue Processing and Embedding
E. Sectioning
Analytical Steps
A. Antigen Retrieval
B. Blocking
C. Primary Antibody
D. Enzyme-HRP
E. Counterstaining
F. Dehydration, Mounting, and Coverslipped
• Used in histology to detect the presence of specific protein marker that can assist with accurate tumor classification and diagnosis.
IMMUNOHISTOCHEMISTRY (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.
IMMUNOHISTOCHEMISTRY (IHC)
The factors that influence the_____ result start in the surgery operating room and end at the interpretation of the stain by the pathologist, which ultimately leads to treatment decision by the oncologist.
immunohistochemical staining
In the Accessioning room, the sample details are entered into the…
Laboratory Information System (LIS)
• A _____can ensure track and trace capabilities.
barcoded label
• The specimen is visually examined for suspicious areas that require further examination.
GROSSING / GROSS EXAMINATION
Samples from the specimen that require further microscopic testing are excised as tissue blocks and placed in..
barcoded cassettes
processed into a form and condition suitable for making ultrathin microscopic sections.
• Typically, the tissue is fixed in formalin and then dehydrated before it is embedded in paraffin.
TISSUE PROCESSING AND EMBEDDING
• The steps where the tissue block is
The fine art of cutting the paraffin-embedded tissue blocks into ultrathin (_____) sections and placing them onto glass slides.
-4 um
A_____ on the slide can ensure traceability and may also contain protocol information for the requested test for that particular section.
barcode
• After sectioning, the sample will be fished-out from the flotation bath and will undergo ______to know if the sample is positive or negative.
If positive =_______
H&E staining
request for IHC
END PRODUCTS OF PRE-ANALYTICAL STEPS:
If we already have these four, then we can request an IHC.
- Histopath result/report
- Blocks
- Slides -› read by the pathologist for confirmation.
- Histopath Worksheet (sometimes only)
• The analytical part of the IHC process.
• It encompasses antigen retrieval, application of the primary antibody and visualization system, and ending with counterstaining.
STAINING
STAINING
It encompasses…
antigen retrieval
application of the primary antibody and visualization system
ending with counterstaining.
ANALYTICAL STEPS
- Antigen Retrieval
- Blocking
- Primary Antibody
- Enzyme-HRP
- Counterstaining
- Dehydration, Mounting, and Coverslipped
• 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
Can be done before or after primary antibody.
Block nonspecific antibodies.
BLOCKING
• It is applied to specifically binds to the antigen of interest.
PRIMARY ANTIBODY
(HRP)
ENZYME-HORSERADISH PEROXIDASE
• The secondary antibody carries the label (enzyme)
ENZYME-HORSERADISH PEROXIDASE (HRP)
• Upon application, it binds to the primary antibody.
ENZYME-HORSERADISH PEROXIDASE (HRP)
• It is performed to visualize nuclei and overall tissue architecture.
COUNTERSTAINING
But before counterstaining.____ is performed:
DAB+
Diaminobenzidine (DAB)
• A chromogen -> gives color to the dyes.
Will bind to the secondarv
antibody to give color.
• If there will be a color, everything is identified as positive.
Diaminobenzidine (DAB)
There is also an H&E staining that does not utilize secondary
antibody.
Antigen + Antibody + Color (DAB)
• Counterstain used for IHC.
HEMATOXYLIN
In H&E staining, it is a primary stain while in IHC it is a counterstain.
Hematoxylin
In H&E staining procedure, dehydration is…
95, 100, 100, Xylene, Xylene, then mount.
Since we used xylene, we have a high refractive index of the sample.
The mounting media to be used should be a…
_____mounting media is not allowed because of xylene. The sample will have a white/milky appearance.
resinous mounting media/mountant.
Aqueous
IHC slides should be coverslipped because:
• It is expensive to perform
• It uses expensive reagents
• It should be long-lasting
POST-ANALYTICAL PROCESS
• The_____ interprets the stains in context with positive and negative tissue controls, using bright field microscopy.
• The results are reported to the____ for treatment decision.
pathologist
oncologist
POSITIVE AND NEGATIVE TISSUE CONTROLS
• When fishing out, the____ is placed only on half of the slide (bottom part). While the_____ is placed on the other half (upper part).
• Every slide has a_____.
sample
control
•
IV. SECTIONING
Use high-quality slides to provide High-Quality sections.
Take particular care to use thin, flat sections that have been thoroughly dried onto the slide.
Preferably use_______ or ______ for IHC
charged slides
APES-coated slides
o Much more expensive and durable than frosted-end slides.
Charged slides
During IHC, the temperature used in the water bath is around…
Withstand high temperatures…
95C
Charged slides
• These controls must be processed identically to the specimen but contain the target protein.
INTERNAL TISSUE CONTROLS
In some cases, it will be advantageous to have this control tissue stain only _______as to monitor not only for the presence of the antigen but also for any possible loss of sensitivity.
marginally positive
IHC STAINING PROCEDURE MATERIALS
• Drying Oven
• Pipettes and Tips
• Control Tissue (+)
• Tissue Paper
• Humid Chamber
• DAKO FLEX
• Staining Jar
• Dako Concentrated
• Timers
• Coverslips
• Distilled Water
• Dako Antibody Diluent
• Charged Slides
• Envision FLEX Target Retrieval Solution (TRS) (high pH)
• Reagents For Deparaffinization
• Envision FLEX Target Retrieval Solution (TRS) (low pH)
• Water Bath › 95-99 Degrees Celcius
• Temperature-controlled water bath should
• Wash buffer
• Hematoxylin
• Dako pen
• the sensitivity and specificity are the core elements
PRIMARY ANTIBODY
• must provide both a both high sensitivity and a high specificity to produce accurate and robust
IHC assay.
PRIMARY ANTIBODY
- they are able to detect or react immediately to the presence of antigen
- the antibody must react to a
specific antigen only and nothing else
Sensitive
Specific
2 categories of primary antibody
(1) Monoclonal (2) Polyclonal
• they have become widely used because of their high specificity, consistency, purity and commercial availability.
MONOCLONAL ANTIBODIES
MONOCLONAL ANTIBODIES
• produced in____ according to the in______ method developed by_____
mice
vitro hybridoma
Köhler
one: reacts to one antigen (Ag)
thus it has high specificity since it only reacts to a specific type of antigen
Monoclonal
POLYCLONAL ANTIBODIES
• produced in____ by ______techniques, with booster immunizations to maximize the reactivity against the target antigen
rabbits
traditional immunization
• frequently give a higher sensitivity (avidity) compared to monoclonal antibodies, as the many antibody ‘species’ present react with more antigen sites
Polyclonal antibody
many: reacts to many antigen (Ag)
thus it has high sensitivity since it is sensitive to many types of antigen
Polyclonal antibody
• the solution used in antigen retrieval, where it unmasks the antigens, for it to be visible and be binded by the primary antibodies
TARGET RETRIEVAL SOLUTION (TRS)
• basically, used to reveal antigenic sites/epitopes
TARGET RETRIEVAL SOLUTION (TRS)
the major drawback of
Formalin-Fixed
Paraffin-Embedded (FFPE) tissue
MASKING
when formalin-induced molecular modification of proteins (antigens) result in loss of the ability of the antibody to react with the antigen
MASKING
a loss that can only be corrected by the restoration (retrieval) of the ‘formalin-modified’ antigen molecular structure
Masking
TARGET RETRIEVAL SOLUTION (TRS)
These improve antigen expression of samples by:
(1) breaking down formalin induced antigen cross-linking
(2) re-exposing epitopes on the antigen to antibody binding
TRS
Heat and enzyme retrieval are both employed, with_____, which is now being the most commonly used.
Heat-Induced Epitope Retrieval (HIER)
Heat-Induced Epitope Retrieval (HIER)
Temperature:
Buffer:
> 95
pH6 or pH9
TRS
• 2 categories:
• HIGH pH (pH 9)
o blue color
• LOW pH (pH 6)
o pink color
• ER and PR can be processed together in TRS since both of them utilizes _____ pH
• HER2/neu will be processed separately since it utilizes____ pH
high
low
Machine used in trs
• Microwave
• Pressure cooker
• Water bath
HYDROPHOBIC PEN
• Ex.
Dako Pen, Pap Pen
its purpose is to prevent the water from escaping within the encircled area
HYDROPHOBIC PEN
- read by MTs
It refers to the percentage of the positive results in the sample
Proportion Score
- read by Pathologists
Intensity Score
Allred score =
positive =
PS + IS
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