IHC

Question 1

is used in histology to detect the presence of specific protein marker that can assist with accurate tumor classification and diagnosis

Answer 1

Immunshstochemistry (IHC)

Question 2

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.

Answer 2

IHC

Question 3

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

Answer 3

surgery operating room

interpretation of the stain

Question 4

Pre analytical steps

BAG TES

Answer 4

Biopsy
Acessioning
Grossing
Tissue processing and Embedding
Sectioning

Question 5

Immunshstochemistry (IHC) is used in histology to detect the presence of specific______ that can assist with accurate tumor classification and diagnosis,

Answer 5

protein marker (antigen)

Question 6

(surgically removed tissue specimen or needle biopsy) from the surgery room arrives in fixative at the pathology laboratory.

Answer 6

BIOPSY

Question 7

the sample details are entered into the_______. A barcoded label can ensure track and trace capabilities

Answer 7

ACCESSIONING

laboratory information system (LIS)

Question 8

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

Answer 8

Grossing

Question 9

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

Answer 9

TISSUE PROCESSING AND
EMBEDDING

Question 10

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.

Answer 10

SECTIONING

barcode

Question 11

ANALYTICAL STEPS

Answer 11

Antigen retrieval
Blocking
Primary antibody
Enzyme HRP

Question 12

It is performed to recover the antigens
that may have been altered by fixation,

Answer 12

Antigen Retrieval

Question 13

Endogenous enzymes are blocked (this step can also be performed after
primary antibody incubation)

Answer 13

Blocking

Question 14

A_______ is applied that specifically binds to the antigen of interest

Answer 14

primary antibody

Question 15

The______ antibody carries the label (enzyme) upon application it binds to the primary antibody

Answer 15

Enzyme-HRP

secondary

Question 16

ARS

Answer 16

Antigen Retrieval Solution

Question 17

Antigen retrieval is not necessary in

Answer 17

Frozen sections

Question 18

Performed to visualize nuclei and overall tissue architecture

Answer 18

Counterstaining

Question 19

DAB

Answer 19

diaminobenzidine

Question 20

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.

Answer 20

positive and negative tissue

bright field

oncologist

Question 21

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

Answer 21

charged slides or APES coated

Question 22

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.

Answer 22

INTERNAL TISSUE CONTROLS

Question 23

Chromogen

Answer 23

DAB

Brown

Question 24

IHC STAINING PROCEDURE
MATERIALS

Answer 24

• 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

Question 25

• Envision FLEX Target Retrieval Solution (high pH) • Envision FLEX Target Retrieval Solution (low pH)

Answer 25

Blue Pink/ Magenta

Question 26

Counterstaining

Answer 26

Hematoxylin

Question 27

For ________the sensitivity and specificity are the core elements.

Answer 27

primary antibodies

Question 28

Ideally, the primary antibody must provide both a______ and______ to produce an accurate and robust IHC

Answer 28

high sensitivity and a high specificity

Question 29

have become widely used because of their high specificity, consistency, purity and commercial availability.

Answer 29

Monocional antibodies

Question 30

One binding site

Answer 30

Monoclonal antibody

Question 31

Monoclonal antibodies, produced in______according to the ______method developed by_____.

Answer 31

mice In vitro hybridoma Kohler

Question 32

, 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.

Answer 32

Polycional antibodies Rabbits traditional immunization techniques sensitivity

Question 33

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

Answer 33

TARGET RETRIEVAL SOLUTION

Question 34

These improve antigen expression of your samples by breaking down formalin induced______, re-exposing epitopes on the antigen to antibody binding.

Answer 34

TRS antigen cross-linking

Question 35

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.

Answer 35

heat induced epitope retrieval (HIER) pH6 or pH9

Question 36

TRS

Answer 36

Target Retrieval Solution

Question 37

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. 6. Immediately soak slides in a staining jar with diluted_____

Answer 37

TRS 90-95°C. 25 20 Wash Buffer (WB).

Question 38

1. Tap and wipe off excess buffer and draw a circle around the Uissue section with_____. 2. Apply two drops (100 ul) or enough __________ Incubate for 5 mlnutes. 3. Put sildes in the_______. 4. 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. 6. Rinse gently as in Step 4. 7. Tap off excess buffer and apply two drops or enough______, Incubate for 30 minutes. 8. Rinse gently as in Step 4. 9. Tap off excess buffer and apply two drops or enough _______Incubate for 10 mlnutes. 10. 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

Answer 38

Dako pen Dual Endogenous Enzyme Block humidity chamber Dako Primary Antibody Labeled-polymer HRP DAB+ Substrate Working Solution hematoxylin