Laboratory Operations

Question 1

How should an optimal positive control be produced?

Answer 1

In the exact same conditions as the patient tissue (fixation, processing, staining) and cut onto the same slide.

Question 2

How can you validate a prediluted Ab?

Answer 2

By performing a serial dilution (1:2, 1:4, 1:8 and 1:16) and assessing for reactivity on known positive tissue.

Question 3

What is the one prediluted Ab that can be run without additional lot validation?

Answer 3

FDA approved pre diluted Ab.

Question 4

What is a concentrated Ab?

Answer 4

A non-diluted Ab in which several serial dilutions must be performed in order to find the optimal dilution. Called “prep kits” in Vancouver.

Question 5

Are diluted or concentrated Ab more practical?

Answer 5

Concentrated. Even though the dilution process takes longer, they are much more shelf stable and are much cheaper than diluted Ab. Concentrated Ab also give the user much more control over Ab expression in patient tissue.

Question 6

What does a 1:20 diluted Ab entail?

Answer 6

1 part Ab, 19 parts diluent totaling up to 20 parts.

Question 7

What is a lambda in reference to measurement?

Answer 7

An old fashioned way to say microliter. Same as micron=-micrometer.

Question 8

Why is Ab validation important.

Answer 8

To establish a standardized protocol also to assist with lab reproducibility and consistency.

Question 9

An appropriate immunohistochemical reaction is dependent upon:
A. incubation time
B. incubation temperature
C. antibody dilution
D. All of the above

Answer 9

D. All of the above.

Question 10

What is the antibody class most frequently demonstrated in immunofluorescent and immunoenzyme staining?

Answer 10

IgG. Of the immunoglobulin classes, IgG is found most abundantly in serum and consequently most antibodies produced belong to this class.

Question 11

Polyclonal antibodies are produced by injection of antigen into:

Answer 11

Animals. Specialized cell cultures can be used to produce monoclonal antibodies. Bacterial cultures will not produce antibodies.

Question 12

What is the chemical used to quench endogenous alkaline phosphatase in tissue sections?

Answer 12

Levamisole. The anti-helminthic drug, levamisole, is effective in quenching non-intestinal alkaline phosphatase.

Question 13

What is a “checkerboard” titration of primary vs. secondary antibodies is used to determine?

Answer 13

The optimal combination of concentrations of each antibody. It is done by varying the secondary antibody concentration for each of several primary antibody concentrations. When charted out, the results form a grid, or “checkerboard” pattern.

Question 14

An immunoglobulin molecule can be divided into two fragments known as the antigen-binding fragment (Fab) and the crystalizable fragment (Fc). What is the function of the crystalizable fragment?

Answer 14

To be involved in biologic functions other than antigen binding. The crystalizable fragment of an immunoglobulin molecule is involved in a variety of non-antigen binding biologic functions that are part of the hosts immune response

Question 15

What are the two principal defense mechanisms of the body’s immune system?

Answer 15

humoral and cell-mediated

Question 16

With regard to the avidin-biotin- complex (ABC) immunohistochemistry method:
A. the primary antiserum is avidin-labeled
B. no conjugation steps are involved
C. the ABC complex binds to biotin-labeled secondary antibody
D. bridging antiserum is added in excess

Answer 16

C. The ABC complex binds to the biotin-labeled (biotinylated) secondary Ab.

Question 17

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?

Answer 17

Vimentin.

Question 18

Polyclonal antibody serum often labels non-specific antigens in the tissue because:

Answer 18

Polyclonal antiserum contains many Ab.

Question 19

Where do B cells mature?

Answer 19

The bone marrow.

Question 20

Which of the cells is responsible for immunoglobulin production?

Answer 20

Plasma cells.

Question 21

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:
A. Staining an unstained slide with a positive control
B. Staining over a negative control
C. Staining a previous HE of the same specimen
D. Staining over the IHC slide that did not work

Answer 21

D. Staining over the IHC slide that did not work.

Question 22

Light chain monoclonality is the hallmark of:

Answer 22

B-cell non-Hodgkin’s lymphoma

Question 23

Individuals who are hypersensitive to environmental allergens usually have above normal circulating levels of:

Answer 23

IgE.

Question 24

What is IgE?

Answer 24

IgE is the immunoglobulin responsible for immediate hypersensitivity reactions to environmental allergens such as food, pollen and insect bites. On initial exposure to an allergen, T-cells induce B-cells to produce IgE, which circulates in the blood and binds by its Fc tail to basophils and mast cells. When the IgE incorporated in basophil or mast cell binds its specific antigen, it stimulates the release of histamine and other mediators from the basophils or mast cells.

Question 25

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:

Answer 25

Vimentin, cytokeratin, S-100 and HMB45

Question 26

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:

Answer 26

Cells represent avidin-containing mast cells

Question 27

The most useful and specific panel for neuroendocrine tumors is:

Answer 27

Chromogranin and synaptophysin

Question 28

What does Neurofilament test for?

Answer 28

Neurofilament is an intermediate filament present in neural cells and variably present in neuroendocrine tumors, but is poorly visualized in paraffin sections, even with epitope retrieval techniques, and requires frozen sections.

Question 29

Sensitized T-cells respond to antigenic challenge by secreting a series of biological mediators termed:

Answer 29

Cytokines.

Question 30

A whole-antibody serum shows background staining despite extensive trials to eliminate the background. It is decided to purchase a new antibody. Which of the following would most likely provide the cleanest staining?
A. hybridoma supernatant
B. monoclonal Fab fragments
C. hyperimmune ascites fluid
D. polyclonal F(ab’)2 fragments

Answer 30

B. monoclonal Fab fragments.

Question 31

What are FAB fragments?

Answer 31

Fab fragments are the two separated hypervariable regions of the antibody. These are unlikely to show any non-specific staining.

Question 32

What are F(ab’)^2 fragments?

Answer 32

F(ab’)2 fragments are the two hypervariable regions still attached by a short Fc segment, which could contribute to non-specific staining (although very little).

Question 33

Following immunoperoxidase staining, it is observed that the positive control stains well, but there is no staining of a specimen expected to be positive. The most likely cause is that:

Answer 33

The specimen was not properly fixed.

Question 34

Monoclonal antibodies are highly specific but may show low sensitivity because:

Answer 34

The specific binding site in the tissue may be altered

Question 35

T/F: Monoclonal antibodies, although very specific, are more susceptible to alteration of their specific binding site (epitope) that would result in less binding and so less sensitivity.

Answer 35

True.

Question 36

Staining of a blood or marrow smear for peroxidase activity is sometimes helpful in the diagnosis of myeloid leukemia, but intensity of staining is sometimes low. How can you ensure that the incubation is adequate?

Answer 36

Incubate until some staining is seen, because there is always some stainable peroxidase activity, even in normal leukocytes

Question 37

Polyclonal antibodies usually show greater sensitivity than monoclonals because polyclonal antibodies:

Answer 37

Detect more distinct epitopes than monoclonals. Polyclonals are generally more sensitive than monoclonals because they detect hundreds of distinct epitopes, while monoclonals will detect only one. Polyclonals are less susceptible to blocking of epitopes by fixatives.

Question 38

Which of the following combinations would yield the best double-stain?
A. DAB as chromogen with ER and new fuchsin as the chromogen with
PR
B. DAB as chromogen with kappa and new fuchsin as chromogen with
lambda
C. Fast red as chromogen with high molecular weight keratin and new
fuchsin as chromogen with pan keratin
D. Fast Blue as chromogen for CD3 and BCIP-NBT as chromogen for
CD20

Answer 38

B. DAB as chromogen with kappa and new fuchsin as chromogen with lambda. The combination of the brown-stained kappa and the red new fuchsin-stained lambda provide clear, distinct staining that enables you to easily determine whether or not the specimen exhibits kappa or lambda monoclonality.

Question 39

Is the dual stain: DAB as chromogen with ER and new fuchsin as the chromogen with PR a good idea?

Answer 39

A double stain for ER and PR can be problematic in that it might be difficult to distinguish if one of the markers were weakly positive, especially if the PR positive cells were weaky positive, while the ER was strongly positive.

Question 40

Is a dual stain with Fast red as chromogen with high molecular weight keratin and new fuchsin as chromogen with pan keratin a good idea?

Answer 40

Fast red and new fuchsin are similar enough in color that it would be nearly impossible to distinguish between the two, especially since the pan keratin should stain the same areas as the high molecular weight keratin.

Question 41

Is a dual stain with Fast blue as the chromogen for CD3 and BCIP-NBT as chromogen for CD20 a good idea?

Answer 41

Fast blue and BCIP-NBT are both blue, and would make it very difficult to distinguish the CD3 postive cells from the CD20 positive cells.

Question 42

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:

Answer 42

Has antibodies to something in the tissue, while the primary serum does not

Question 43

Which of the following panel results are improbable?
A. Keratin +, vimentin -, LCA -, S100-
B. Keratin +, Vimentin +, LCA +, S100 +
C. Keratin -, Vimentin +, LCA -, S100 +
D. Keratin -, Vimentin +, LCA - S100 -

Answer 43

B. Keratin +, Vimentin +, LCA +, S100 +. There is no normal tissue or tumor that is routinely positive for all four of these markers.

Question 44

A section labeled with an antibody has no background but the antigen of interest stains very weakly. This is an example of:

Answer 44

High specificity, low sensitivity

Question 45

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:

Answer 45

Glial fibrillary acid protein (GFAP), cytokeratin AE1/3, synaptophysin

Question 46

What will GFAP stain?

Answer 46

Glial fibrillary acid protein (GFAP) will stain most glial neoplasms including the common glioblastoma multiforme as well as ependymal tumors that could resemble metastatic carcinomas. It stains only 1-4% of carcinomas, usually in less than 10% of the cells.

Question 47

Immunologic 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:

Answer 47

Metastatic adenocarcinoma

Question 48

Immunoperoxidase-stained tissues show reaction of red blood cells and granulocytes with the chromogenic substrate. The most likely explanation is that:

Answer 48

Endogenous peroxidase was not blocked

Question 49

S-100 protein shows reactivity with all of the following except:
A. Schwann cells and glial tissue
B. chrondrocytes and adipocytes
C. smooth muscle and skeletal muscle
D. Langerhans’ histiocytes and interdigitating reticulum cells

Answer 49

C. smooth muscle and skeletal muscle.

Question 50

What are some uses for S-100?

Answer 50

S-100 is a demeric calcium binding protein, and a widely used screening antibody for malignant melanoma. It is not expressed in normal or neoplastic muscle cells. My epithelial cells in normal breast, salivary and sweat glands and their tumors do express S-100, however. The wide reactivity of S-100 limits its use it’s use to that of a screening reagent.