Exam 4 Flashcards
cause of background staining on a negative reagent control
Reagent contamination
A preferred method for the storage of control slides to prevent loss of antigenicity would be
Dipped in paraffin
This method of amplification involves the repeating or reapplying of amplification reagents
Cycled TSA
Loss of antigenicity due to slide oxidation more readily affects which type of antigen?
Nuclear
There is no staining on both the specimen and control on the same slide. The protocol was performed on an automated IHC stainer. The slide was recut and the same protocol repeated on the stainer using the same control. The results were optimal. What could be a possible explanation for the first slide
Instrument malfunction
In tyramide signal amplification, the tyramide conjugate is applied
After the HRP detection enzyme
Which of the following could be a cause of background staining on the patient specimen, control (both on the same slide) and on the slide surrounding the tissue
Waterbath adhesive or additive was used
Macrophages have ingested the target antigen and are staining positive. This would be referred to as
Antigen diffusion
Which would be an appropriate counterstain for HRP-AEC detection
Mayer hematoxylin
The purpose of this type of control is to determine if the IHC reagents in general are contributing to non-specific staining
Negative reagent control
The introduction of polymer-based detection systems has had what effect in negative reagent controls
Reduced the need
Disadvantage of TMA is
Assembly time
Amplification is used to
Increase the staining affinity
A specimen that is underfixed will show what type of staining pattern?
Optimal staining on the edge, no staining to weak staining in the center
Of the following, which would be the best choice for a single tissue type control
A single tissue type known to contain both positive and negative components
Once activated the tyramide conjugate is covalently attracted to
Proteins
Which may be considered an advantage of TMAs
Allows for sampling of many different tissue types
For a positive tissue control, it is acceptable, but not optimal, to run
A batch control
Slide oxidation can be prevented by
Storing in an airtight container
Which of the following is not a type of control used in immunohistochemistry
Positive reagent control
An IHC stained slide shows optimal intensity in the specimen, but the control is weak (both on the same slide). What could be the cause
The control tissue is oxidized
The control tissue fell off the slide during IHC staining, but the patient specimen remains (they were both placed on the same slide). How might the slide still be usable for diagnosis
If the specimen contains areas that do and do not contain the antigen
After IHC staining, the control stains adequately, but the specimen shows unexpected weak intensity, especially in the center. What is the most likely cause
The specimen was not properly fixed
There is non-specific staining in only the red blood cells of the negative reagent control, tissue control and the specimen. The protocol uses HRP-DAB detection. What is the most likely explanation
Hydrogen peroxide blocking was omitted from the protocol
The degradation of protein, DNA and RNA in paraffin sections is called
Slide oxidation
There is weak staining intensity with a concentrated antibody. How could the protocol be modified to correct this
Decrease the primary antibody dilution
The expected localization pattern of an antigen
Can be found in the Antibody Specification Sheet
Which would be the best choice for the negative reagent control
Non-immune serum of the same isotype
Which of the following counterstains would be used with fluorescent detection
DAPI
In tyramide signal amplification, the tyramide conjugate is activated by
Horseradish peroxidase
The optimal method for utilizing a negative reagent control would be to
Run a separate patient tissue section for each block
Which of the following would be a disadvantage of TMAs
Human error
Negative reagent controls should be used for avidin-biotin based detection systems to detect
Endogenous reactivity
Upon microscopic review of an IHC stained slide using LSAB detection, there are areas of inconsistent background staining in the specimen and control tissue (both are on the same slide). What is a possible cause
Biotin blocking was omitted
A negative tissue control is used to
Detect primary antibody cross reactivity
Which of the following is used for Ig amplification
Intermediary antibodies targeting the primary antibody
Which of the following would be an advantage to TMA
Donor tissue is conserved
When using Ig amplification with a mouse primary antibody, you would use
Rabbit anti-mouse IgG
Which of the following factors would not influence IHC results
Antigen localization
An optimal positive tissue control is one that is
Fixed and processed identically as the patient
Which antibody is typically used as a tissue processing control to determine adequate fixation
Vimemtin
This type of control would confirm that the IHC protocol has been performed correctly
Positive tissue control
There is usually a high chance of false positive or negative results with cytology specimens because
Controls treated identically to cytology specimens are not readily available
Which of the following is considered the best practice for positive and negative tissue controls
Multi-tissue block
Non-specific or background staining on the negative reagent control may indicate
Reagent contamination
On an IHC stained slide, the patient specimen has weak positivity. The control stains adequately. Both are on the same slide. What could cause this
A fixative other than 10% NBF was used to fix the patient tissue
Both the control and patient tissue unexpectedly show non-specific or background staining (both are on the same slide). The most likely reason is
Endogenous activity was not blocked
There is optimal intensity with non-specific staining. What protocol parameter would you suggest changing
Decrease the antigen retrieval aggressiveness
Which type of tissue control is commercially prepared to contain negative, weak and strongly positive cells to improve standardization
Cell line control
This type of tissue control can improve standardization across many different laboratories
Cell line control
A pathologist orders two IHC tests on the same specimen block. The antigen retrieval for each protocol is different. If you were asked to run only one negative reagent control, which would be true
Choose the most aggressive antigen retrieval of the two
There is incomplete cell membrane staining in cells known to contain the antigen meaning all of the antigen is not being detected. This would be referred to as
Weak specificity/sensitivity
A spectrum of staining in the positive tissue control is important to
Detect subtle changes in antibody sensitivity
When selecting positive tissue control, it is optimal to select tissue with
Low to high intensity staining
The purpose of amplification is to
Increase intensity
Specific staining in an unexpected area of localization on the positive tissue control may indicate
Antigen diffusion
A pathologist returns an IHC slide to the histology lab stating that there is non-specific staining in the specimen, however the control is optimal (both are on the same slide). The patient tissue is liver. What could be an explanation for this
Reactivity with hepatitis antigen
When creating a TMA, the tissue core is removed from the
Donor block
An IHC result shows weak staining in the correct area of localization in both the patient and control tissue on the same slide. This is an example of
Optimal specificity, low intensity
Autopsy tissue is a poor choice for positive tissue controls because
The tissue is often subject to autolysis and putrefaction
Positive tissue controls are necessary to
Assess the performance of the primary antibody
Upon microscopic review the control tissue demonstrates optimal staining, and the patient specimen is weak (both are on the same slide). What could be a cause
The IHC reagents did not adequately cover the entire slide
An isotype control is also referred to as a/n
Negative reagent control
An IHC stained slide shows weak intensity, but good specificity on the control and specimen (both on the same slide). A possible cause is
The primary antibody dilution is too high
The most common type of hematoxylin used as a counterstain for IHC is
Mayer
Non-specific binding of serum (Ig) proteins will not be detected with this type of negative reagent control
Diluent
A positive tissue control is also used to
Verify that the protocol was performed correctly
Intensity that is too dark in the patient specimen, but optimal in the control (both on the same slide) can be indicative of
Over incubation of DAB chromogen
Control tissues with low to high levels of antigen expression are necessary to
Reduce the risk of false positive or negative results
After completing an IHC protocol, positive staining is absent in the control and specimen (both on the same slide). What could be a possible cause
Inadequate deparaffinization
Which of the following would be omitted for the negative reagent control
Primary antibody
The staining intensity is too dark and the specificity is too weak. What are two potential protocol modifications that could improve results
Increase the antigen retrieval time, decrease the antibody incubation time
Which of the following is not a purpose for controls in immunohistochemistry
Determine the appropriate section thickness
There is excessive background on an IHC stained patient specimen, however the control is adequate (both on the same slide). What could be a cause
The specimen is necrotic
When the patient tissue contains tissue elements that express both positive and negative staining, it is called a(n)
Intrinsic control
