QUIZ Flashcards
T/F: The enzyme label for immunoperoxidase methods contains horseradish peroxidase
True
T/F: Monoclonal antibodies are often preferred over polyclonal antibodies because there is little bath-to-batch variability
True
T/F: DAB is an alcohol soluble chromagen
False
Synthetic resins may be used with DAB as it can be dehydrated and cleared.
What is the most common fixation/processing protocol for IHC?
Formalin fixed paraffin embedded (FFPE)
T/F: Harris hematoxylin cannot be used with AEC because of the alcohol present
True
Regulations regarding predictive marker staining of tissue places responsibility for the fixation time documentation on the lab
True
T/F: Precut control slides may be stored at room temperature indefinitely.
False
Must store in fridge
T/F: The procedure for staining cytology smears is the same as for routine slides.
False
Each slide must have its own protocol on the stainer
In the bloody areas of a tissue section stained with the immunoperoxidase technique, there is a marked reaction with RBCs. What happened?
Failure to use hydrogen peroxide
Paraffin sections stained with the immunoperoxidase technique show excessive background staining. What happened?
Nonimmune serum was not applied
If the first protein solution applied to the tissue is the primary antibody, nonspecific binding can occur
The skin control section for S100 was stained with the immunoalkaline phosphatase technique using fast red TR as the chromogen. It shows negative staining. What happened?
Sections were accidentally dehydrated and cleared causing the chromogen to breakdown
T/F: There is a negative correlation between extended cold ischemic time and loss of antigen ability.
False
The correlation is positive - longer times means worse staining
T/F: Prediluted antibody should always be used as provided by the manufacture
False
Always need to be validated before use
T/F: Negative tissue antigen controls may be run by substituting for the primary antibody the diluent in which the antibody is prepared
True
Negative controls omit the primary antibody
T/F: One standard protocol may be written to cover all specimens
False
Procedures need to be written for all non-standard tissue protcols
T/F: Zinc formalin preserves immunoreactivity very well
True
T/F: Multilink antibodies can only be used with monoclonal primary antibodies
False
Can be used with both mono and poly antibodies
T/F: Blocking reactions are used to block endogenous activity of the same enzyme used for the enzyme immune complex
True
Alpha, delta, gamma, and epsilon are all examples of:
Light polypeptide chains
Allergens
Epitopes
Heavy polypeptide chains
Heavy polypeptide chains
An antigen is a substance that triggers the production of:
immunogens
epitopes
hormones
immunoglobulins
immunoglobulins
Cells that engulf and destroy foreign substances and microorganisms are called:
Phagocytes
Myeloma Cells
Eosinophils
Natural Killer Cells
Phagocytes
A molecule which stimulates an immune response is called a(n):
immunoglobulin
epitope
immunogen
determinant
immunogen
Monoclonal antibodies are usually produced in which animal?
Cows
Goats
Mice
Cats
mice
Which of the following is a true statement about polyclonal antibodies?
Polyclonal antibodies are less sensitive than monoclonal antibodies.
Polyclonal antibodies produce less background staining than monoclonal antibodies.
Polyclonal antibodies can bind to multiple epitopes on one antigen.
Polyclonal antibodies are harvested from the spleen or lymph node of the host that they are produced in.
Polyclonal antibodies can bind to multiple epitopes on one antigen.
Antibodies are produced by:
B lymphocytes
Myeloma cells
Rabbit fusion partner cells
Phagocytes
B lymphocytes
Another term for background staining is:
enhanced staining
maximum staining
nonspecific staining
fluorescent staining
nonspecific staining
B cells are harvested with myeloma cells to produce:
hybirdomas
fusion cells
natural killer cells
rabbit fusion partner cells
hybirdomas
When using an IHC alkaline phosphatase staining method, what is the mechanism that produces the color?
An enzyme that hydrolyzes the substrate to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes.
An enzyme that catalyzes the reduction of hydrogen peroxide (H2O2) to water and oxygen.
An enzyme dissolves red food dye to tint the section
An enzyme that hydrolyzes the substrate to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes
A histotech is performing a CD-20 stain and receives a false-negative result on the specimen. What could be a cause for this happening?
Under fixation
T/F: Additives in alternative fixatives can cause either false-negative or false-positive immunohistochemical (IHC) staining.
True
Which of the following is an ADVANTAGE to using a polymer or micro polymer detection system?
Background free (typically) staining
Pretreatment of tisse for ISH can affect which two factors?
Signal strength
Background staining
T or F: The higher the temperature of the HIER solutions, the more effective the recovery of the epitope is
True
T/F: Most antigen retrieval methods apply temperatures near the freezing point of water.
False: boiling point
T/F: T or F: Certain non-epithelial tumors also stain positive for keratin
True
T/F: Vimentin is a sensitive marker for muscle differentiation and can be used to identify tumors derived from smooth, skeletal and cardiac muscle.
False: ACTIN
T/F: Melanomas and schwannomas always stain positive for vimentin
True
T/F: Vimentin has limited use as a stand-alone stain, but it can be very helpful when combined with other specific tumor markers.
True
T/F:T Neurofilament (NF) is most widely used to confirm the diagnosis of astrocytoma
False: Glial fibrillary acidic protein (GFAP)
Tumors that show neuronal or neuroendocrine differentiation will stain positive for what other marker?
neurofilament
A combination of keratin and chromogranin positivity is typical of _______
neuroendocrine carcinoma
Chromogranin positivity with a negative keratin stain is typical of ________
paraganglioma
T/F: PLAP is positive in majority of seminomas.
True
_______ are derived from neural crest and will be reactive for S100 protein
Melanocytes
The intensity of staining for S100 is usually ______________ proportional to the melanin content of the tumor.
Inversely
The best screening marker for lymphoma is _____.
LCA (leukocyte common antigen)
CD45
What are the immunophenotypic subclassification of lymphoma?
T cells (CD3, CD4, CD5)
B cells (CD19, CD20, CD23)
Reed-Sternberg cells (CD15, CD30)
immunoglobulin light and heavy chains
What is the most common immunohistochemical markers used to assess proliferation of tumor cells?
Ki-67 (MIB-1) and PCNA (proliferating cell nuclear antigen)
T/F: Chromogenic IHC staining can generate dense deposits that are easy to detect but difficult to quantitate.
True
T/F: Chromogenic detection is considered to be a less sensitive method than immunofluorescence.
False: more sensitive
The optimal incubation time for linking antibodies with peroxidase conjugates is ______________ at room temperature
30 to 60 minutes
T/F: Direct technique is less sensitive compared to indirect techniques.
True
T/F: The indirect technique of immunohistochemistry is a 2 or 3 step procedure that involves application of the unconjugated primary antibody, followed by a labeled antibody directed against the first antibody
True
In the indirect technique, cross-reactivity is eliminated by using _______.
preabsorbed secondary antiserum
What would be the purpose of the addition of a third layer of antibody?
To amplify the signal
T/F: Proteolytic enzyme digestion is necessary in renal and skin biopsies
True
Combining horseradish peroxidase with the most common chromogen, diaminobenzidine (DAB), results in a stable ________________ end product when antigen is present in the tissue.
insoluble dark brown reaction
In the PAP method, to block endogenous peroxidase activity, the sections are pre-incubated in ________ containing hydrogen peroxide
absolute methanol
Alkaline phosphatase antibodies raised in __________ , can also be used to form alkaline phosphatase-anti-alkaline phosphatase complexes (APAAP)
Mouse
T/F: The major advantage of APAAP procedure compared to the PAP technique is the lack of interference from exogenous peroxidase activity.
False: endogenous peroxidase activity
Endogenous alkaline phosphatase activity is usually blocked by adding ________ to the substrate solution
levamisole
T/F: Monoclonal antibodies, compared to polyclonal antibodies, depend more on environmental factors such as pH and solute for optimum performance.
True
What method uses avidin (derived from egg white) because of its marked affinity for biotin?
Avidin-Biotin Complex technique
Biotinylated label and avidin or streptavidin, and are added together _________ before use.
30 mins
T/F: Cryostat sections give much better antigen preservation than paraffin sections
True
T/F: Snap-frozen fresh tissue, without supporting media, can be stored at -10°C for long periods of time without appreciable antigen loss.
False: -70°C
Prolonged storage of OCT-embedded frozen tissue will cause gradual loss of cellular antigens
True
What fixative is generally used as fixative to preserve the antigen, to destroy harmful infective agents, and to allow a wide range of primary antibodies to be employed without destroying many of the epitopes.
Acetone
T/F: Formalin fixed paraffin-embedded material appears to be the best choice in a diagnostic setting of ISH.
True
T/F: In an aqueous environment, the thermodynamically stable conformation of a nucleic acid is that of a single-stranded helix.
False: double-stranded helix
The commonly used denaturants in the ISH method are:
Heat
Formamide
In a hybridization assay, the two sources are the ___________ and ____________.
target (sample) and the probe nucleic acids
The process of searching a sample for specific nucleic acid sequences is termed ________________.
hybridization reaction
T/F: RNA is much more unstable than DNA
True
SBB is positive in:
Acute Myeloid Leukemia (AML)
SBB staining is possibly a little more sensitive for the early myeloid cells
True
T/F: Metamyelocytes stains negative for SBB
False: metamyelocytes stain strongle positive
A piece of skin has been stained according to a standard melanoma protocol. You notice that after counterstaining the visible IHC product has been removed. What most likely occurred to cause this result?
Incorrect pre-treatment used
Dehydrated in alcohol
Incorrect antibody dilution
Chromogen was outdated
Dehydrated in alcohol
In immunohistochemical staining of formalin-fixed tissue, proteolytic enzyme pretreatment of a tissue section:
enhances background staining
unmasks antigen
epitopesis needed to demonstrate all antigens
has precise end-points
unmasks antigen epitopes
Which of the following is NOT a section in the MSDS?
-PPE
-physical hazards
-primary routes of entry
-staining procedures
staining procedures
How many mls of a 1:5 dilution of 100% alcohol would be water?
180 ml
190 ml
200ml
160 ml
160 ml
160 ml
After an alkaline phosphatase immunostain run was completed, the slides were manually counterstained in hematoxylin, dehydrated, cleared and coverslipped. The entire run was negative. What is the most likely source of error?
-the wrong type of hematoxylin was used as counterstain
-the primary antibodies were all omitted
-the counterstaining solution masked alkaline phosphatase activity
-the end-product for alkaline phosphatase activity was dissolved by alcohol
the end-product for alkaline phosphatase activity was dissolved by alcohol
Of the following, the preferred fixative for demonstration of intermediate filaments contains:
alcohol
formalin
mercury
dichromate
alcohol
According to OSHA, which of the following records can be kept for less than 30 years?
exposure records for specified carcinogens
fit testing results for personal chemical respirators
medical records for exposure to hazardous chemicals
personal protective equipment training
fit testing results for personal chemical respirators
A biotin block is often used as part of an IHC staining protocol for the reason of suppressing staining of endogenous biotin. All of the following are tissues that require biotin blocking except:
Kidney
Liver
Thyroid
Brain
Thyroid
When staining for neuroendocrine tumors the most beneficial markers of identification are:
s-100 and cytokeratin
chromogranin and synaptophysin
neurofilament and neuron specific enolase
neuron specific enolase
chromogranin and synaptophysin
The primary advantage for using non-formalin fixatives in immunochemistry it to:
reduce exposure to toxic chemicals
prevent loss of antigenicity
better preserve tissue morphology
reduce cost
prevent loss of antigenicity
Which statement is true concerning pH?
a liquid is more acidic if its pH is higher
adding sodium hydroxide will make the pH higher
indicator paper always turns blue if the pH is high
the pH increases by 2 units for a 10?C temperature rise
adding sodium hydroxide will make the pH higher
mmunologic stains and flow cytometry are performed on malignant cells found in pleural effusion specimen. The following results are obtained: B72.3 positive; carcinoembryonic antigen (CEA) positive; DNA content aneuploid. Based on this profile, the cells most likely represent:
reactive mesothelial cells
malignant mesothelioma
metastatic adenocarcinoma
Hodgkin’s lymphoma
metastatic adenocarcinoma
A section labeled with an antibody has no background but the antigen of interest stains very weakly. This is an example of:
low specificity, high sensitivity
high specificity, low sensitivity
high specificity, high sensitivity
low specificity, low sensitivity
high specificity, low sensitivity
As a chromogenic substrate, amino-ethylcarbazole (AEC) would be preferred to diaminobenzidine (DAB):
if permanent preparation is desired
when staining pigmented lesions
to reduce contact with a potential carcinogen
for subsequent metallic color enhancement
when staining pigmented lesions
A common fluorochrome used in immunohistochemistry is:
fluorinated isotryptophan
fluorine isothiocyanate
fluorescein isothiocyanate
fluoroisothiocyanate
fluorescein isothiocyanate
The chemical used to quench endogenous alkaline phosphatase in tissue sections is:
biotin
hydrogen peroxide
levamisole
EDTA
levamisole
The regulatory agency responsible for establishing and enforcing laws concerning the disposal of chemicals down the drain is:
EPA
GLP
NIOSH
NTP
EPA
The primary base units used in the metric system are:
liters, grams, meters
ounces, grams, meters
pounds, gallons, meters
meters, grams, pints
liters, grams, meters
A single plasma cell (activated B-cell) produces:
all the antibodies necessary to neutralize foreign antigens
polyclonal antibodies against a specific antigen
antibodies specific to a single antigenic determin
antantibodies specific to hypervariable determinants
antibodies specific to a single antigenic determinant
A negative control consisting of normal animal serum shows background staining. The primary slide, however, shows strong specific staining with no background. The likely cause of this discrepancy is that the normal serum:
was more dilute
has antibodies to something in the tissue, while the primary serum does not
was not an absorbed antiserum, while primary is an absorbed antiserum
had exceeded the expiration date, and probably contained a contaminant microorganism
has antibodies to something in the tissue, while the primary serum does not
Convert 3.5 gallons to liters
12.5 liters
14 liters
13.247 liters
13 liters
13.247 liters
Fluorochromes are available in several colors. In order to see these colors, a fluorescence microscope must be equipped with:
a color video camera
brightfield illumination
the correct wavelength filters
a quartz halogen bulb
the correct wavelength filters
In the indirect immunohistochemistry staining method, the:
test slide is first treated with unlabeled antiserum
test slide is first treated with enzyme-labeled to the primary antibody
second step is treatment with enzyme labeled antiserum to the primary antigen
second step is treatment with unlabeled antiserum to the primary antibody
second step is treatment with enzyme labeled antiserum to the primary antigen
When selecting reagents for peroxidase-anti-peroxidase (PAP) staining, the PAP complex should be prepared in the same (or closely related) animal species as the:
chromogen
secondary antibody
primary antibody
bridging antibody
primary antibody
The specific substrate of horseradish peroxidase is:
diaminobenzidine
ethylene diamine tetra acetic acid
hydrogen peroxide
polyethylene glycol
hydrogen peroxide
B-lymphocytes can produce specific antibodies without T-lymphocyte help only if the antigen is:
processed by macrophages
a cluster designation
greater than 10,000 kd
T-independent
T-independent
The primary reason that frozen sections destined for immunohistochemistry are fixed in some way after sectioning is to:
keep the tissue from falling off the slide
thaw the tissue in a controlled manner
prevent diffusion of the tissue antigens of interest
kill any infectious organisms that may present
prevent diffusion of the tissue antigens of interest
The best positive control tissue for IHC is one that is:
bought from a reputable vendor
as fresh as possible
processed identically to patient tissue
is available in large quantities
processed identically to patient tissue
When staining sections for examination by immunofluorescence microscopy:
tissue antigens are “stained” by fluorescent-labeled antibodies
tissue antibodies are “stained” by fluorescent-labeled antigens
fluorescent dyes are sandwiched between antigen and antibody
fluorescent dyes are bound to tissue with heavy metal mordants
tissue antigens are “stained” by fluorescent-labeled antibodies
The ability of an antibody to recognize and bind with a specific protein antigen is due to its:
molecular size
carbohydrate structure
the dimensional structure
animal species of origin
the dimensional structure
After fixation for 24 to 48 hours in formalin, tissue for IHC should be stored in which of the following fluids to preserve antigenicity?
10% buffered formalin
50% acetone
70% ethanol
Hanks buffered saline
70% ethanol
Which of the following are enzymes commonly used in immunohistochemistry to unmask antigenic determinants?
papain, propane, chymase, lactase
pepsin, tryptophan, kinase, proteinase K
pronase, trypsin, proteinase K, pepsin
lactase, pepsin, trypsin, pepsin
pronase, trypsin, proteinase K, pepsin
Which of the following is a chromogen used in peroxidase histochemistry that generates a product that is soluble in alcohol or xylene but not in water?
9-aminoethylcarbazole (AEC)
3,3’-diaminobenzidine (DAB)
alpha-naphthyl phosphate (ANP)
hexazonium pararosaniline (HPR)
9-aminoethylcarbazole (AEC)
During QC of an IHC stain it is noticed that a particular marker has not stained and needs to be repeated. The block, however, has been depleted. All of the following are acceptable ways to re-stain the specimen except:
Staining an unstained slide with a positive control
Staining over a negative control
Staining a previous HE of the same specimen
Staining over the IHC slide that did not work
Staining over the IHC slide that did not work
Formalin fixation causes cross-linkages, what affect might this have on IHC staining?
Masks epitopes
enhances staining
allows antigens to bind faster and easier
removes the need for pre-treatment
Masks epitopes
Staining of a blood or marrow smear for peroxidase :ctivity is sometimes helpful in the diagnosis of myeloid leukemia, but intensity of staining is sometimes low. To ensure that the incubation has been adequate:
incubate a control slide that has been smeared with a material known to have enzyme activity, such as a peroxidase-labeled antiserum
incubate until some staining is seen, because there is always some stainable peroxidase activity, even in normal leukocytes
treat the smear with 1% hydrogen peroxide in methanol for 10 minutes before incubating in the peroxidase reaction medium
incubate a control smear with added sodium azide to inhibit peroxidase
incubate until some staining is seen, because there is always some stainable peroxidase activity, even in normal leukocytes
he staining present in this specimen is the result of:
lymphocytes stained with leukocyte common antigen (LCA)
inadequately blocked biotin in red blood cells
inadequately blocked horseradish peroxidase in red blood cellsin
adequately blocked biotin in lymphcytes
inadequately blocked horseradish peroxidase in red blood cells
In immunoperoxidase staining, efficiency of the chromogen-substrate reaction step can be checked by:
doubling the methanol, hydrogen peroxide concentration
extending the time in the substrate-chromogen
increasing the concentration of chromogen
omitting the blockage of endogenous peroxidase
omitting the blockage of endogenous peroxidase
Antibodies must always be stored at:
2-8 C
2-6C
1-8 C
according to manufacturers specifications
according to manufacturers specifications
Commercial monoclonal antibodies are produced by all of the following except:
synthesized utilizing automated instrumentation in a factory
a single plasma cell and its clones
mice in a lab
cell culture
synthesized utilizing automated instrumentation in a factory
Heat induced epitope retrieval (HEIR) may be used for:
all tissue sections, for any antibody
all formalin-fixed tissue sections
those antibodies for which it has been determined to be optimal
frozen sections
those antibodies for which it has been determined to be optimal
With regard to the avidin-biotin- complex (ABC) immunohistochemistry method:
the primary antiserum is avidin-labeled
no conjugation steps are involved
the ABC complex binds to biotin-labeled secondary antibody
bridging antiserum is added in excess
the ABC complex binds to biotin-labeled secondary antibody
The specific substrate of horseradish peroxidase or HRP is:
diaminobenzidineethylene
diamine tetraacetic acid
hydrogen peroxide
polyethylene glycol
hydrogen peroxide
A chemical group which gives the property of color to a chromogen is a:
chromatophore
chromatography
chromophore
chromogranin
chromophore
Monoclonal antibodies are highly specific but may show low sensitivity because:
the dilution is too high
the specific binding site in the tissue may be altered
of the difficulty of preparing monoclonals
of poor technique by the technologist
the specific binding site in the tissue may be altered
Optimization can best defined as
The clinical process of confirming that the ‘reactivity’ that is observed
The technical process of adjusting various conditions in order to produce the greatest amount of specific staining (sometimes referred to as ‘signal’).
Developing an IHC staining protocol that definitively confirms that the results produced are ‘biologically ‘correct’
The process of documenting a protocol
The technical process of adjusting various conditions in order to produce the greatest amount of specific staining (sometimes referred to as ‘signal’).
Antibody diversity is due to different amino acid sequences of the:
variable regions of the heavy chain
variable regions of the light chain
variable regions of both heavy and light chains
Fc regions of the heavy chains
variable regions of both heavy and light chains
Which of the following is the substrate of the enzyme peroxidase?
HRP
DAB
H2O2
TRIS
H2O2
In peroxidase staining, the colored end product is formed following:
reduction of a chromogenic substrate and oxidation of horseradish peroxidase
reduction of hydrogen peroxide and oxidation of a chromogenic substrate
oxidation of hydrogen peroxide and reduction of a chromogenic substrate
oxidation of horseradish peroxidase and reduction of hydrogen peroxide
reduction of hydrogen peroxide and oxidation of a chromogenic substrate
Which of the following is true of a polyclonal antibody?
lot to lot consistency
sustained production
reacts with a specific epitope
reacts with multiple epitopes on a single molecule
reacts with multiple epitopes on a single molecule
Because of the harshness of IHC procedures on tissue slides, which of these factors in slide preparation are very important to a high quality IHC slide?
Charged slides
Drying time of tissue on slides
Well fixed tissue
All of the above
All of the above
Which one of the following is most correct regarding heat induced epitope retrieval (HIER) techniques?
Steamers, pressure cookers, microwaves and waterbaths can all be successfully used for HIER.
Lead thiocyanate formic acid and water have all been successfully used as HIER solutions.
You cannot “over retieve” tissue using HIER techniques.
Both “Steamers, pressure cookers, microwaves and waterbaths can all be successfully used for HIER.” and “Lead thiocyanate formic acid and water have all been successfully used as HIER solutions.” are correct.
Both “Steamers, pressure cookers, microwaves and waterbaths can all be successfully used for HIER.” and “Lead thiocyanate formic acid and water have all been successfully used as HIER solutions.” are correct.
In immunohistochemical procedures using horseradish peroxidase (HRP), a solution of hydrogen peroxide is used to:
block endogenous peroxidase
enhance background staining
affect reactivity of antibodies and antigens
intensify the coloring reaction
block endogenous peroxidase
An appropriate immunohistochemical reaction is dependent upon:
incubation time
incubation temperature
antibody dilution
All of the above
All of the above
A section of intestine is labeled with an anti-keratin antibody and visualized with alkaline phosphatase. The section shows strong staining along the entire brush border of the intestinal epithelium. What artifact must you be aware of while interpreting this stain?
nothing, the intestine shows background staining with all chromogens
anti-keratin antibody often stains collagen and smooth muscle
the intestinal brush border cells contain endogenous alkaline phosphatase
formalin pigment can mimic the pattern of keratin staining in the intestine
the intestinal brush border cells contain endogenous alkaline phosphatase
An anaplastic metastatic malignant tumor is submitted for diagnosis with the request to rule out malignant melanoma. The best antibody panel to study this problem should include:
vimentin, cytokeratin, S-100 and HMB45
cytokeratin, S-100, CEA, EMA
HMB-45, actin, desmin, CD99
S-100, chromogranin, vimentin, NSE
vimentin, cytokeratin, S-100 and HMB45
An immunohistochemistry labeling method in which a molecule of avidin is directly labeled with an enzyme is called:
avidin-biotin complex
labeled avidin-biotin
peroxidase-antiperoxidase
alkaline phosphatase-antialkaline phoshatase
labeled avidin-biotin
Formalin fixation causes cross-linkages, what affect might this have on IHC staining?
Masks epitopes
enhances staining
allows antigens to bind faster and easie
removes the need for pre-treatment
Masks epitopes
Which of the following panel results are improbable?
Keratin +, vimentin -, LCA -, S100-
Keratin +, Vimentin +, LCA +, S100 +
Keratin -, Vimentin +, LCA -, S100 +
Keratin -, Vimentin +, LCA - S100 -
Keratin +, Vimentin +, LCA +, S100 +
n IHC, a negative control is used to:
determine what steps contribute to any non-specific staining
show what the tissue would stain like if it was negative
test the dilution of the primary antibody
save primary antibody
determine what steps contribute to any non-specific staining
Following immunoperoxidase staining, it is observed that the positive control stains well, but there is no staining of a specimen expected to be positive. Of the following, the most likely cause is that:
the specimen was improperly counterstained
the specimen was not properly fixed
staining steps were not performed in the correct order
excess buffer or nonimmune serum diluted the primary antibody
the specimen was not properly fixed
Which statement about the pH of a solution is true?
the pH number is the concentration of hydrogen ions in gram-ions or moles per liter
a concentrated acid can have a negative pH, such as -1.5
the pH is a meaningful value for all liquids, even those that contain no water
a solid object, such as a crystal, a hair or a section of tissue has a pH value
a concentrated acid can have a negative pH, such as -1.5
In immunohistochemical staining of formalin-fixed tissue, proteolytic enzyme pretreatment of a tissue section:
enhances background staining
unmasks antigen epitopes
is needed to demonstrate all antigens
has precise end-points
unmasks antigen epitopes
A “checkerboard” titration of primary vs. secondary antibodies is used to determine the:
best primary antibody concentration only
best secondary primary antibody concentration
optimal combination of concentrations of each antibody
optimal chromogen concentration for each antibody
optimal combination of concentrations of each antibody
A single plasma cell (activated B-cell) produces:
all the antibodies necessary to neutralize foreign antigens
polyclonal antibodies against a specific antigen
antibodies specific to a single antigenic determinant
antibodies specific to hypervariable determinant
antibodies specific to a single antigenic determinant
Which of the following combinations would yield the best double-stain?
DAB as chromogen with ER and new fuchsin as the chromogen with PR
DAB as chromogen with kappa and new fuchsin as chromogen with lambda
Fast red as chromogen with high molecular weight keratin and new fuchsin as chromogen with pan keratin
Fast Blue as chromogen for CD3 and BCIP-NBT as chromogen for CD20
DAB as chromogen with kappa and new fuchsin as chromogen with lambda
Fixatives that tend to mask antigenic sites and hamper immunohistochemical localization of antigens contain:
mercury
phosphates
aldehydes
alcohol
aldehydes
The peroxidase staining procedure in which reagents are linked exclusively by antigen-antibody reactions without any conjugation steps is:
avidin-biotin-peroxidase complex method
peroxidase-anti-peroxidase
indirect immunoperoxidase method
direct immunoperoxidase method
peroxidase-anti-peroxidase
A brain biopsy reveals a poorly differentiated malignant tumor. The differential diagnosis is primary versus metastatic brain cancer. The most useful antibody panel to resolve this differential is:
cytokeratin, vimentin, muscle common actin
S-100, cytokeratin AE1/3, synaptophysin
glial fibrillary acid protein, cytokeratin AE1/3, synaptophysin
carcinoembryonic antigen, leukocyte common antigen, S-100
glial fibrillary acid protein, cytokeratin AE1/3, synaptophysin
If your antibody is FDA approved but your protocol is different how many tests do you need to run when validating?
10 previously known positive cases
20 positive and 20 negatives cases
10 negative cases
15 positive and 15 negative cases are run
20 positive and 20 negatives cases
The step in the IHC stainings process that allows the exposure of the sought after epitopes is called:
direct staining
detection
antigen retrieval
antibody matching
antigen retrieval
Identify the chromogen used in the image which is insoluble in alcohol and other organic substances.
diaminobenzidine
3amino - 9 - ethylcarbazole
4 chloro 11 napthol
diaminopropylene
diaminobenzidine
Paraffin sections for IHC should be cut at:
1 to 2 um
3 to 5 um
6 to 10 um
10 to 20 um
3 to 5 um
In order to assess the effects of over-fixation and the potential need for antigen retrieval on a given specimen, the inclusion of an antibody against what ubiquitous intermediate filament is suggested in all initial antibody panels?
ubiquitin
cytokeratin
vimentin
actin
vimentin
What marker is considered almost a universal control for over-fixation?
keratin
CD30
vimentin
kappa
vimentin
The size of a tissue sample is important for immunohistochemistry because:
smaller samples are easier to section
the fixative must penetrate the entire sample
larger samples are easier to handle
larger samples will stay on the slide better
the fixative must penetrate the entire sample
DAB may be enhanced by the use of:
Copper, silver, gold or iodine
Cobalt, nickel, silver or gold
Potassium, sodium, silver or gold
Cobalt, iodine, copper or nickel
Cobalt, nickel, silver or gold
In immuno-alkaline phosphatase staining, which control is useful to determine the level of endogenous alkaline phosphatase?
incubate in chromogen only
incubate overnight in levamisole
apply all steps except the enzyme
stain several slides at different time periods
incubate in chromogen only
Fluorochrome-conjugated primary antibodies are an example of:
direct labeling
indirect labeling
the ABC system
the PAP system
direct labeling
Monoclonal antibodies are highly specific but may show low sensitivity because:
the dilution is too high
the specific binding site in the tissue may be altered
of the difficulty of preparing monoclonals
of poor technique by the technologist
the specific binding site in the tissue may be altered
With regard to the avidin-biotin- complex (ABC) immunohistochemistry method:
the primary antiserum is avidin-labeled
no conjugation steps are involved
the ABC complex binds to biotin-labeled secondary antibody
bridging antiserum is added in excess
the ABC complex binds to biotin-labeled secondary antibody
mmunoperoxidase-stained tissues show reaction of red blood cells and granulocytes with the chromogenic substrate. The most likely explanation is that:
they contain antigen that reacted with the primary antiserum
the specimens were improperly counterstain
edendogenous peroxidase was not blocked
specimens were allowed to dry during the staining procedure
endogenous peroxidase was not blocked
The primary advantage for using non-formalin fixatives in immunochemistry it to:
reduce exposure to toxic chemicals
prevent loss of antigenicity
better preserve tissue
morphology
reduce cost
prevent loss of antigenicity
In immunoperoxidase staining, efficiency of the chromogen-substrate reaction step can be checked by:
doubling the methanol, hydrogen peroxide concentration
extending the time in the substrate-chromogen
increasing the concentration of chromogen
omitting the blockage of endogenous peroxidase
omitting the blockage of endogenous peroxidase
The three critical parameters of the heat-mediated antigen retrieval procedure are:
correct microwave wattage, pH, and volume
proper temperature, incubation time, and pH
volume of solution, temperature, incubation time
incubation time, pH, and style of steamer
proper temperature, incubation time, and pH
A metastatic carcinoma of unknown origin is presented for diagnosis. All of the following are useful to determine the primary site of the tumor except:
vimentin
cytokeratin 7 and 20
CEA
S-100
S-100
All enzymes:
change the rate of chemical reactions
catalyze oxidation-reduction reactions
act upon extracellular substrates
have enhanced activity at 60C
change the rate of chemical reactions
The two principle defense mechanisms of the body’s immune system are:
humoral and enzymatic
hydrolytic and cell-mediated
humoral and cell-mediated
hydrolytic and enzymatic
humoral and cell-mediated
Following avidin-biotin-peroxidase complex staining, the cytoplasm of scattered single cells in the stroma of both positive and negative control slides is stained. The most likely explanation is that the:
cells represent avidin-containing mast cells
primary antibody was applied to all slides
endogenous peroxidase was not blocked
staining steps were not performed in the correct order
cells represent avidin-containing mast cells
A negative control consisting of normal animal serum shows background staining. The primary slide, however, shows strong specific staining with no background. The likely cause of this discrepancy is that the normal serum:
was more dilute
has antibodies to something in the tissue, while the primary serum does not
was not an absorbed antiserum, while primary is an absorbed antiserum
had exceeded the expiration date, and probably contained a contaminant microorganism
has antibodies to something in the tissue, while the primary serum does not
As a chromogenic substrate, amino-ethylcarbazole (AEC) would be preferred to diaminobenzidine (DAB):
if permanent preparation is desired
when staining pigmented lesions
to reduce contact with a potential carcinogen
for subsequent metallic color enhancement
when staining pigmented lesions
The peroxidase staining procedure in which reagents are linked exclusively by antigen-antibody reactions without any conjugation steps is:
avidin-biotin-peroxidase complex method
peroxidase-anti-peroxidase
indirect immunoperoxidase method
direct immunoperoxidase method
peroxidase-anti-peroxidase
Commercial monoclonal antibodies are produced by all of the following except:
synthesized utilizing automated instrumentation in a factory
a single plasma cell and its clones
mice in a lab
cell culture
synthesized utilizing automated instrumentation in a factory
To eliminate the problem of endogenous staining a particular IHC method is optimal. What method would that be?
LSAB+
ABC
Polymer-Based
PAP
Polymer-Based
A cryostat may be defined as a/an:
device that maintains a controlled low temperature
instrument that cuts sections of frozen specimens
microtome contained in a freezing cabinet
section cut from a frozen specimen
microtome contained in a freezing cabinet
Light chain monoclonality is the hallmark of:
B-cell non-Hodgkin’s lymphoma
Hodgkin’s disease
Hairy cell leukemia
T-cell non-Hodgkin’s lymphoma
B-cell non-Hodgkin’s lymphoma
The red chromagen used in this image is particularly helpful for this type of specimen because it:
Aids in diagnosis of melanocytic lesions
Reduces background staining
Is better for quantitative image analysis
Is more sensitve
Aids in diagnosis of melanocytic lesions
The antibody class most frequently demonstrated in immunofluorescent and immunoenzyme staining is:
IgA
IgE
IgG
IgM
IgG
A cell antigen that can be demonstrated by immunofluorescent staining of fresh-frozen tissues is not visible by immunoperoxidase staining of formalin-fixed paraffin-processed tissue. The difference in the procedures that most likely accounts for the lack of staining is:
masking of antigen binding sites by fixation
difference in chromogen
the sequence of staining steps
interpretation under different microscopy systems
masking of antigen binding sites by fixation
Heat induced epitope retrieval (HEIR) may be used for:
all tissue sections, for any antibody
all formalin-fixed tissue sections
those antibodies for which it has been determined to be optimal
frozen sections
those antibodies for which it has been determined to be optimal
The specific substrate of horseradish peroxidase or HRP is:
diaminobenzidine
ethylene diamine tetraacetic acid
hydrogen peroxide
polyethylene glycol
hydrogen peroxide
In immunohistochemical staining, a limitation of polyclonal antisera as opposed to monoclonal antisera is:
that the production of polyclonal antibodies is technically more difficult
the greater cross-reactivity of polyclonals with similar antigens
the limited availability of polyclonal antisera
the extreme specificity of polyclonal antibodies
the greater cross-reactivity of polyclonals with similar antigens
Polymer based detection uses:
A structure made of polymer and biotin that contains secondary antibodies
A backbone of polymer with the addition of enzymes
A polymer backbone with secondary antibodies and hydrogen peroxide incorporated in the structure
A polymer backbone that includes secondary antibodies and enzymes
A polymer backbone that includes secondary antibodies and enzymes
Which of the following is a very useful positive control for lymphatic IHC stains?
stomach
tonsil
kidney
brain
tonsil
Sensitized T-cells respond to antigenic challenge by secreting a series of biological mediators termed:
prostaglandins
cytokines
kinins
c-reactive proteins
cytokines
In the indirect immunohistochemistry staining method, the:
test slide is first treated with unlabeled antiserum
test slide is first treated with enzyme-labeled to the primary antibody
second step is treatment with enzyme labeled antiserum to the primary antigen
second step is treatment with unlabeled antiserum to the primary antibody
test slide is first treated with unlabeled antiserum
S-100 protein shows reactivity with all of the following except:
Schwann cells and glial tissue
chrondrocytes and adipocytes
smooth muscle and skeletal muscle
Langerhans’ histiocytes and interdigitating reticulum cells
smooth muscle and skeletal muscle
Individuals who are hypersensitive to environmental allergens usually have above normal circulating levels of:
IgE
IgG
IgM
IgD
IgE
When using an alkaline phosphatase detection system:
It is always necessary to perform a hydrogen peroxidase quench
It is never necessary to perform a hydrogen peroxidase quench, but you must always perform an alkaline phosphatase quench.
It is recommended that an alkaline phosphatase quench be performed on frozen tissues
Using a quench of any type will result in very weak staining when using an alkaline phosphatase detection system
It is recommended that an alkaline phosphatase quench be performed on frozen tissues
All of the following are intermediate filaments except:
Vimentin
Keratin
Glial fibrillary protein
Actin
Actin
To eliminate the problem of endogenous staining a particular IHC method is optimal. What method would that be?
LSAB+
ABC
Polymer-Based
PAP
Polymer-Based
Which statement is true concerning pH?
a liquid is more acidic if its pH is higher
adding sodium hydroxide will make the pH higher
indicator paper always turns blue if the pH is high
the pH increases by 2 units for a 10?C temperature rise
adding sodium hydroxide will make the pH higher
Which of the following is a chromogen used in peroxidase histochemistry that generates a product that is soluble in alcohol or xylene but not in water?
9-aminoethylcarbazole (AEC)
3,3’-diaminobenzidine (DAB)
alpha-naphthyl phosphate (ANP)
hexazonium pararosaniline (HPR)
9-aminoethylcarbazole (AEC)
A cell antigen that can be demonstrated by immunofluorescent staining of fresh-frozen tissues is not visible by immunoperoxidase staining of formalin-fixed paraffin-processed tissue. The difference in the procedures that most likely accounts for the lack of staining is:
masking of antigen binding sites by fixation
difference in chromogen
the sequence of staining steps
interpretation under different microscopy systems
masking of antigen binding sites by fixation
Name two preservatives used in commercial preparation that can reduce antibody reactivity
Sodium chloride
Azides
What buffer should not be used as an antibody diligent unless specifically recommended by the manufacturer?
PBS
Archival unstained sections are stained for ER and PR because the original slides were lost; the newly stained slides were negative for both ER and PR, despite having been reported previously as positive. What is the likely cause of the discrepancy?
tissues were overfixed in formalin
improper antigen retrieval method was used
the sections are too old
the ER and PR antibodies have expired
the sections are too old
Polyclonal antibody serum often labels non-specific antigens in the tissue because polyclonal:
-antibodies can shift their hypervariable regions to conform to other epitopes
-antibodies are stickier than monoclonal antibodies
-antiserum is thicker than monoclonal serum
-antiserum contains many antibodies
antiserum contains many antibodies
The red chromagen used in this image is particularly helpful for this type of specimen because it:
-Aids in diagnosis of melanocytic lesions
-Reduces background staining
-Is better for quantitative image analysis
-Is more sensitve
Aids in diagnosis of melanocytic lesions
What is the staining pattern in the image?
-Nuclear
-Nuclear and cytoplasmic
-Membranous
-Cytoplasmic
Nuclear and cytoplasmic
This is an image of Melan-A staining using alkaline phosphatase detection with a red chromagen. The tissue is skin. Which of the following statements is true?
The tissue is stained appropriately. Melan-A has nuclear visualization.
The tissue is stained inappropriately. Melan-A has neither cytoplasmic visualization or nuclear visualization
The tissue is stained inappropriately. Melan-A has neither cytoplasmic visualization or nuclear visualization
The tissue is stained inappropriately. Melan-A has neither cytoplasmic visualization or nuclear visualization
This image is of prostate tissue stained with P63 antibody. Which of the following statements is true?
The tissue is stained inappropriately. P63 is a membranous marker, but there is also aberrant nuclear visualization.
The tissue is stained inappropriately. The basal cells are not staining with P63.
The tissue is stained inappropriately. P63 stains basal cells with nuclear visualization, but there is also aberrant membranous staining.
The tissue is stained appropriately.
The tissue is stained inappropriately. P63 stains basal cells with nuclear visualization, but there is also aberrant membranous staining.
Which of the following immunostains would be appropriate to request on this tissue section?
Cytokeratin 7, cytokeratin 20, and TTF-1
High molecular weight keratin and PSA
CD 3, CD5, CD20, and CD79a
Estrogen receptor, progesterone receptor and HER2/neu
Cytokeratin 7, cytokeratin 20, and TTF-1
The staining present in this specimen is the result of:
-lymphocytes stained with leukocyte common antigen (LCA)
- inadequately blocked biotin in red blood cells
-inadequately blocked horseradish peroxidase in red blood cells
-inadequately blocked biotin in lymphocytes
inadequately blocked horseradish peroxidase in red blood cells
Identify the chromogen used in the image which is insoluble in alcohol and other organic substances.
-diaminobenzidine
-3amino - 9 - ethylcarbazole
-4 chloro 11 napthol
-diaminopropylene
diaminobenzidine
The red chromagen used in this image is particularly helpful for this type of specimen because it:
-Aids in diagnosis of melanocytic lesions
-Reduces background staining
-Is better for quantitative image analysis
-Is more sensitive
Aids in diagnosis of melanocytic lesions
