Chapter 12: Immunohistochemistry

Question 1

An antigen is a substance that triggers the production of:

Answer 1

Antibody (immunoglobulins).

Question 2

A light chain present in some antibodies is:

Answer 2

Kappa and Lambda.

Question 3

Different molecular sites on antigens are known as:

Answer 3

Epitopes.

Question 4

When an antibody labeled with a chromogen is reacted with tissue from a patient, the IHC technique is called:

Answer 4

Direct.

Question 5

What are two examples of a fluorochrome?

Answer 5

Rhodamine and Fluorescein Isothiocyanate (FITC).

Question 6

Please describe the indirect IHC method:

Answer 6

Patient serum is added to tissue sections containing known antigens.

Question 7

In the avidin-biotin methods, the primary Ab is followed by:

Answer 7

A biotinylated secondary Ab.

Question 8

A dye that absorbs light and then emits its own light at a longer wavelength is known as a:

Answer 8

A fluorochrome.

Question 9

In some IHC techniques, alkaline phosphate functions as the:

Answer 9

The enzyme.

Question 10

Horseradish peroxidase is used in some avidin-biotin methods as the:

Answer 10

The enzyme.

Question 11

The substrate for alkaline phosphatase labeled Ab is usually:

Answer 11

Napthol-AS-phosphate.

Question 12

What is a common chromogen that is insoluble in alcohol?

Answer 12

DAB (3,3’ diaminobenzidine tetrahydrochloride)

Question 13

If 3-amino-9-ethylcarbazole is used as the chromogen, the hemaxotylin used for the counterstaining must be:

Answer 13

Alcohol-free such as Mayer hematoxylin.

Question 14

Tissue for immunofluorescence MUST be:

Answer 14

Frozen and unfixed.

Question 15

A neoplasm is defined as:

Answer 15

A neoplasm is defined as a new growth of tissue in which cell multiplication is uncontrolled and progressive, it can either be malignant or benign.

Question 16

What is NOT the proper approach to validating a new Ab?

Answer 16

Validating it ONLY with the manufacturers instructions, the lab should never assume that the Ab is immediately ready for use.

Question 17

When the Vimentin stain is completely negative on formalin fixed tissues, this indicates that the tissue has been:

Answer 17

Unfixed.

Question 18

Besides the heat employed, another important factor in heat induced epitope retrieval (HIER) is:

Answer 18

The composition of the retrieval solution.

Question 19

How is Ficin used in IHC?

Answer 19

Ficin is used as the enzyme in induced epitope enhancement.

Question 20

0.5mL of a 1:50 dilution of Ab is needed. How many microliters of the primary would be needed?

Answer 20

10.0 microliters would be needed.

Question 21

What has been eliminated with polymeric IHC staining methods?

Answer 21

The serum and avidin-biotin blocking steps have been eliminated, which improves turnaround times.

Question 22

In the peroxidase-antiperoxidase (PAP) method of Ag detection, the PAP complex that is made is the same species as:

Answer 22

The primary Ab, or the secondary Ab won’t link them together.

Question 23

How is Imidazole used in the PAP method of Ag detection?

Answer 23

As an intensifier to the Dab kit.

Question 24

What link Ab would follow a monoclonal kappa primary Ab?

Answer 24

A rabbit anti-mouse because kappa is usually prepared in either mice or rabbits.

Question 25

What is CK20 an Ab for?

Answer 25

Colon cancer.

Question 26

Negative control slides that are run with each stain omit:

Answer 26

The primary Ab.

Question 27

T/F: Prediluted Ab should always be provided by the manufacturer.

Answer 27

False, they should always be validated before use.

Question 28

T/F: Negative tissue Ag controls may be run by substituting for the primary Ab: the diluent in which the Ab is prepared.

Answer 28

True, negative controls are those that emit the Ab during the primary incubation step and substitute for it.

Question 29

T/F: One standard staining protocol may be written to cover all specimens.

Answer 29

False, a detailed procedure must be written for each tissue type.

Question 30

T/F: Zinc formalin preserves immunoreactivity very well.

Answer 30

True.

Question 31

T/F: Multilink Ab can only be used with monoclonal Ab.

Answer 31

False, they can be used with both monoclonal and polyclonal Ab.

Question 32

T/F: Blocking reactions are used to block endogenous activity of the same enzyme

Answer 32

True.

Question 33

T/F: The enzyme label for immunoperoxidase methods contains horseradish peroxidase.

Answer 33

True.

Question 34

T/F: Monoclonal Ab are often preferred over polyclonal Ab because there is no batch to batch variability.

Answer 34

True.

Question 35

T/F: DAB is an alcohol soluble chromogen.

Answer 35

False, DAB is not soluble in alcohol.

Question 36

T/F: Harris hematoxylin should not be used with AEC because of the alcohol present.

Answer 36

True, AEC is soluble in alcohol.

Question 37

T/F: Regulations regarding predictive marker staining of tissue places responsibility for documenting fixation times on the lab.

Answer 37

True.

Question 38

T/F: The only method of heat induced epitope retrieval involves the use of the microwave oven.

Answer 38

False. Other acceptable methods are heating are: pressure cookers, vegetable steamers and a circulating water bath.

Question 39

T/F: Optimal dilutions for each Ab must be determined in your lab.

Answer 39

True.

Question 40

T/F: Metal salts involving nickel copper and osmium may be used to intensify 3-amino-9-ethylcarbazole reaction (AEC).

Answer 40

AEC is not intensified, but the DAB is.

Question 41

T/F: The Alkaline phosphatase label can be substituted for the peroxidase label in most IHC methods.

Answer 41

True.

Question 42

T/F: Precut control slides may be stored at room temperature indefinitely.

Answer 42

False, storing them at -20C is ideal.

Question 43

T/F: The procedure for staining cytology smears is the same as the one for staining parrafin sections.

Answer 43

False, they need different protocols.

Question 44

What does CD3 test for?

Answer 44

T cell lymphomas.

Question 45

What does CD20 test for?

Answer 45

B cell lymphomas.

Question 46

What does HMB45 test for?

Answer 46

Melanoma.

Question 47

What does HER2 test for?

Answer 47

Breast carcinoma.

Question 48

What does GFAP test for?

Answer 48

Glioblastoma.

Question 49

What does AE1/AE3 test for?

Answer 49

Carcinomas.

Question 50

In the bloody areas of a tissue section stained with the immunoperoxidase technique, there is a marked reaction of the RBCs. What happened?

Answer 50

Failure to use hydrogen peroxide.

Question 51

Paraffin sections stained with the immunoperoxidase technique show excess background staining. What happened?

Answer 51

The nonimmune serum was not applied.

Question 52

The skin control for S100 was stained with the immunoalkaline phosphatase using fast red TR as the chromogen. It shows negative staining. What happened?

Answer 52

The sections were dehydrated and cleared with the rest of the IHC slides.

Question 53

At the end of the immunoperoxidase staining procedure, it was realized that an anti-goat linking Ab was used with monoclonal HMB-45 Ab. Microscopic results show:

Answer 53

Negatively stained positive control and a negatively stained specimen because mice or rabbit would have to be used instead.

Question 54

Both the known positive control and the specimen are negative following immunoperoxidase staining with LCA primary Ab. Dab was used as the chromagen. What happened?

Answer 54

The wrong secondary Ab was used, it should have been targeted to the primary Ab.

Question 55

Excessive background staining is noted in both the control and the specimen stained with immunoperoxidase. What happened?

Answer 55

The slides weren't washed sufficiently with reaction buffer.

Question 56

Excessive background staining is noted on the specimen but not on the positive control stained with immunoperoxidase. What happened?

Answer 56

Free antigen in the patient tissue due to autolysis, necrosis or degeneration will cause this. Pam calls it mucin.`

Question 57

Both the positive control and the specimen stained with immunoalkaline phosphatase using CEA Ab shows very weak staining. What happened?

Answer 57

The primary Ab was incorrectly diluted.

Question 58

Why are acid-alcohol decolorizers generally recommended over aqueous decolorizers for use with AFB?

Answer 58

Acid-alcohol decolorizers allow for more uniform decolorization.

Question 59

What hematoxylin solution contains ethylene glycol and aluminum sulfate?

Answer 59

Gill hematoxylin.

Question 60

The bacterium Legionella pneumophilia can be demonstrated using which stain?

Answer 60

The Steiner technique.

Question 61

In the Jones and Gomori methenamine technique for basement membranes, sections are oxidized with what?

Answer 61

Periodic acid.

Question 62

Nissl substances can be demonstrated with what stain?

Answer 62

Thionin and cresyl echt violet stain.

Question 63

In the Wilder technique for demonstrating reticulin, uranyl nitrate acts as what?

Answer 63

The sensitizer.

Question 64

Microscopic evaluation of an H&E stained section reveals dark deposits of material dispersed irregularly over the tissue. What happened?

Answer 64

The hematoxylin was not filtered before use.

Question 65

Microscopic evaluation of a GMS stained section shows connective tissue fibers and RBCs stained black. What happened?

Answer 65

Overexposure to the hot methenamine silver.

Question 66

During microscopic evaluation of a fungus stain, extraneous fungal elements are seen above rather than in the section. What happened?

Answer 66

The staining solutions were cotaminated.

Question 67

Sections of lung stained with both GMS and PAS show positive staining of what appear to be fungal organisms. However, the same oval shaped bodies are seen in sections of the tissue where they shouldn't be. Polarization of the bodies reveal a Maltese cross configuration. What is the "positively" stained material?

Answer 67

Talcum powder from either the tech or the pathologist.

Question 68

What are spirochetes?

Answer 68

Filamentous bacteria that are known to be argyrophilic.

Question 69

Following H&E staining, slides are dehydrated through ascending strengths of alcohol and cleared in xylene, but the first xylene is milky white. What happened?

Answer 69

The xylene has been contaminated with water, all dehydrating and clearing solutions should be changed.

Question 70

If hematoxylin is unavailable, what dye would make an appropriate substitute in a routine H&E?

Answer 70

Celestine blue.

Question 71

The phosphotungstic acid-hematoxylin (PTAH) stain is useful for demonstrating what?

Answer 71

Muscle striations.

Question 72

Hemosiderin is thought to be composed of:

Answer 72

Protein.

Question 73

Whether collagen fibers are found in dense regular arrangement or in an irregular arrangements varies with the:

Answer 73

They vary due to the location and function of the organ or tissue.

Question 74

Microscopic review of sections stained with H&E reveals very pink cytoplasm and pale reddish-brown nuclei. What happened?

Answer 74

The hematoxylin has become oxidized and needs to be replaced.

Question 75

In silver impregnation techniques, the use of improperly cleaned glassware results in:

Answer 75

Nonselective silver precipitation, chemically cleaned glassware should be used with silver stains ONLY.