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Histology QIHC Exam > General Immunology > Flashcards

General Immunology Flashcards

1
Q

What is immunohistochemistry?

A

The microscopic localization of specific Ag in tissues by staining with Ab labeled with fluorescent or pigmented material to test for disease, cancer or inflammation.

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2
Q

What is an Ag?

A

A molecule in the patient tissue that are localized by the primary or secondary Ab for cancer diagnosis.

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3
Q

What is an Ab?

A

Ab conjugate to enzymes that catalyze reactions to form detectable compounds to visualize and localize specific antigens in a tissue sample.

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4
Q

What are the two main types of Ab?

A

Monoclonal and Polyclonal. Additionally, Ab can either be primary or secondary.

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5
Q

What is a monoclonal Ab?

A

A homogenous population of immunoglobin detected against a single epitope. The Ab are generated by a single B-cell clone from one animal and are thus immunochemically similar.

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6
Q

What is a polyclonal Ab?

A

A heterogenous mixture of Ab directed against various epitopes of the same Ag. Due to their multiclonality, polyclonal Ab are more robust and are more likely to survive in the tissue processor.

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7
Q

What are the Ab classes?

A

There are five classes of antibodies: IgG, IgA, IgM, IgD, and IgE with Ig standing for immunoglobulin.

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8
Q

What are the benefits of monoclonal Ab preparation?

A

Lot-to-lot consistency is possible and there is less variability than with polyclonal Ab. Monoclonal Ab can also be produced infinitely.

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9
Q

What are the benefits of polyclonal Ab preparation?

A

Since a polyclonal Ab can recognize multiple epitopes on a single molecule, it is less susceptible to the deleterious effects on tissue during the pre-analytical stages of IHC.

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10
Q

What are the pre-analytical stages of IHC?

A

Anything that can happen before the staining process such as: processing, embedding, microtomy and fixation.

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11
Q

What is the analytical stage of IHC?

A

The staining process.

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12
Q

What is the general staining process of IHC?

A
  1. Ag retrieval is performed to recover Ag lost by processing.
  2. Endogenous enzymes are blocked.
  3. The primary Ab is applied
  4. The secondary Ab is applied to bind to the primary Ab.
  5. A chromogen is added to visualize the Ab/Ag complex.
  6. Counterstaining is performed to visualize the nuclei and overall tissue architecture.
  7. Slides are dehydrated, cleared and mounted with coverslip.
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13
Q

What are the post-analytical steps of IHC?

A

Interpretation and QC with known positive tissue controls and submission to the pathologist.

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14
Q

What is the general makeup of an Ag?

A

Two identical heavy chains (K) and two identical light chains (L). The two H chains differ in antigenic and structural properties which distinguishes each member of the immunoglobulin class. The two L chains are always either Kappa or Lambda.

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15
Q

What kind of bonds adhere H-H and H-L chains?

A

Covalent bonds.

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16
Q

What are the most frequently utilized Ab class in IHC?

A

IgG and IgM.

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17
Q

What is an epitope?

A

The part of the Ag which is specifically recognized by the host’s immune system, also called the Ag determinant.

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18
Q

What three factors can affect Ab affinity to tissue?

A

High temperature, high salt concentration, excessive agitation and very low pH during the washing process.

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19
Q

Which type of Ab is ideal for forming a precipitate in tissue?

A

Polyclonal Ab because monoclonal are unable to unless they have a “high affinity” version which costs $$$.

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20
Q

Define Ab cross-reactivity.

A

An immunochemical activity in which the Ab reacts with the Ab. This can be several Ab with an Ag or multiple Ag with the same Ab with the common denominator being a common epitope.

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21
Q

Why is the formation of a precipitate in tissue important?

A

The formation of a precipitate denotes strong IHC staining, it’s literally the point.

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22
Q

What is a negative aspect of cross-reactivity?

A

The accidental induced changes between one or more epitopes through Ag retrieval that causes the specimen to lose specificity toward certain monoclonal Abs.

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23
Q

What is an epitope?

A

A molecular, reactive site on an Ag.

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24
Q

T/F: An Ab can target more than one Ag, but only one epitope.

A

True.

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25
Q

What is polyclonal Antiserum?

A

The result of Antigenic stimulation (human IgM Ab injected into a rabbit and isolated into serum) and the production of several lines of lymphocytes.

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26
Q
  1. Commercial monoclonal antibodies are produced by all of the following except:
    A. synthesized utilizing automated instrumentation in a factory
    B. a single plasma cell and its clones
    C. mice in a lab
    D. cell culture
A

D: there are no synthetic antibodies commercially available.

27
Q

What chromogen produces a brown end product that is insoluble in alcohol and other organic substances?

A

Diaminobenzidine. (DAB)

28
Q

What chromogen produces a red end product that is soluble in alcohol and other organic substances?

A

Amino ethylcarbazole or AEC has a red end product and is soluble in alcohol.

29
Q

What chromogen produces a blue end product and is soluble in alcohol?

A

Chloronapthol or CN has a blue end product and is also soluble in alcohol.

30
Q

What chromogen has a black/blue end product and is insoluble in alcohol?

A

Hanker Yates is insoluble in alcohol and other organic substances but it has a blue/black end product.

31
Q

A molecule which stimulates an immune response is called a(n):

A

Immunogen. An antigen is any substance that may be specifically bound by an antibody, but is not necessarily capable of stimulating an immune response.

32
Q

What is a determinant?

A

An epitope, also called a determinant, is the specific binding site of an antibody, and is not capable of stimulating an immune response by itself.

33
Q

Endogenous peroxidase activity can be diminished by the use of:

A

Hydrogen Perioxide.

34
Q

How does hydrogen peroxide block endogenous peroxidase?

A

Endogenous peroxidase activity is an enzymatic reaction that results in the decomposition of H2O2. When allowed to occur without quenching, this can yield background staining of hemoproteins such as hemoglobin, myoglobin, cytochrome, and catalases. This activity can be quenched by allowing the sections to incubate in a solution of H2O2.

35
Q

All enzymes:
A. change the rate of chemical reactions
B. catalyze oxidation-reduction reactions
C. act upon extracellular substrates
D. have enhanced activity at 60°C

A

A. Catalyze the rate of chemical reactions.

36
Q

Formalin fixation causes cross-linkages, what affect might this have on IHC staining?

A

It masks epitopes. In the presence of high concentrations of environmental proteins that cross-link another molecule directly to the epitope of interest, hence masking the epitope and reducing access of the antigen to the epitope.

37
Q

Paraffin sections for IHC should be cut at:

A

3-5 microns

38
Q

Besides peroxidase, the second most frequently used enzyme for labeling antibodies is:

A

Alkaline phosphatase.

39
Q

What is Glucose Oxidase?

A

Glucose oxidase is sometimes used as a label, but not nearly as commonly as alkaline phosphatase. Glucose oxidase is occasionally used in histochemistry as a source of hydrogen peroxide, which is generated by enzyme-catalyzed oxidation of glucose by oxygen.

40
Q

What is Trypsin?

A

Trypsin is sometimes used in immunohistochemistry to permeabilize tissues, but not as a labeling enzyme.

41
Q

Why is the size of tissue sample important to IHC?

A

The fixative must penetrate the entire sample. If fixative cannot reach the center of the sample. IHC labeling will be artifactually deficient.

42
Q

An immunohistochemistry labeling method in which a molecule of avidin is directly labeled with an enzyme is called:

A

Labeled avidin-biotin.

43
Q

What is the difference between Labeled avidin-biotin and the avidin-biotin complex?

A

In the labeled avidin-biotin method, a molecule of avidin is directly labeled with an enzyme. This complex binds with a biotinylated antibody (primary or secondary) on the tissue section. This is in contrast to the avidin-biotin complex method in which an enzyme is bound to biotin molecules, which are in turn bound to avidin molecules in a large complex. The entire complex then binds to a biotinylated antibody on the tissue section.

44
Q

The tissues most often tested for diagnostics using fluorescent-labeled antibodies are:

A

Kidney and skin.

45
Q

The primary advantage for using non-formalin fixatives in immunochemistry it to:
A. reduce exposure to toxic chemicals
B. prevent loss of antigenicity
C. better preserve tissue morphology
D. reduce cost

A

B. Prevent loss of antigenicity. Some antigenic sites cannot survive any formalin exposure and require non-formalin fixative. There are many types of non-formalin fixatives which are often formulated for specific epitomes.

46
Q
  1. Which statement is true concerning pH?
    A. a liquid is more acidic if its pH is higher
    B. adding sodium hydroxide will make the pH higher
    C. indicator paper always turns blue if the pH is high
    D. the pH increases by 2 units for a 10°C temperature rise
A

B. Adding Sodium hydroxide will make the pH higher

47
Q

What is pH?

A

The pH of a liquid is the logarithm of the reciprocal of the concentration of hydrogen ions (as expressed in gram-ions or moles per liter). This is the same as the log of the concentration of H+ multiplied by minus-one. A strongly acidic solution which has a high concentration of H+ has a low pH. This is less than zero for concentrated hydrochloric nitric and sulfuric acids and their tenfold dilutions.

48
Q

Which statement about the pH of a solution is true?
A. the pH number is the concentration of hydrogen ions in gram-ions or
moles per liter
B. a concentrated acid can have a negative pH, such as -1.5
C. the pH is a meaningful value for all liquids, even those that contain
no water
D. a solid object, such as a crystal, a hair or a section of tissue has a pH
value

A

B. A concentrated acid can have a negative pH, such as -1.5.

49
Q

Which of the following is a very useful positive control for lymphatic IHC stains?
A. stomach
B. tonsil
C. kidney
D. brain

A

Tonsil. Tonsil is a part of the lymphatic system, and as such contains many of the cells required for a immune response, including T and B cells.

50
Q

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?

A

The most likely is that AEC chromogen was used to make the stain visible and when it was counterstained, the alcohol dissolved the product. AEC is also the most likely chromogen used on the pigments in skin.

51
Q

What is immunology?

A

The branch of science that deals with the immune
system and the cell-mediated and humoral aspects of immunity
and immune responses

52
Q

What is cell-mediated immune response?

A
  1. Macrophages engulf foreign antigen and present the antigen to T-Helper cells.
  2. Certain T-Helper cells secrete cytokines which activate Cytotoxic T-Cells.
  3. Cytotoxic T-Cells will kill the infected cell through initiation of apoptosis
53
Q

What is humoral (Ab mediated) immune response?

A
  1. Macrophages engulf foreign antigen and present the antigen to T-Helper cells
  2. Certain activated T-Helper cells interact with B cells, causing the B cells to
    proliferate, differentiate and produce antibodies.
54
Q

What is a biomarker?

A

Proteins that may exist on the surface of a cell, in the cytoplasm, in the nucleus or be transmembrane part of the antigen that is specifically recognized by the immune system.

55
Q

What are the two types of epitopes?

A

Linear and conformational epitopes.

56
Q

Define linear epitope.

A

Defined by primary amino acid sequence; typically 5-20 amino acids in length. Chain of amino acids is called a peptide

57
Q

Define conformational epitope.

A

Defined by spatial structure of the protein, brought together via folding

58
Q

Define affinity.

A

The strength with which an antibody binds to its epitope

59
Q

Define avidity.

A

Overall strength of an antibody-antigen complex

60
Q

What is the FAB fragment?

A

The Fab fragment is comprised of the entire light chain, as well as the heavy chain above the hinge region

61
Q

What is the FC fragment?

A

The Fc fragment is comprise solely of the heavy chain beneath the
hinge region

62
Q

What is the half life of IgM?

A

4-6 days.

63
Q

What is the half life of IgG?

A

3 weeks.

Histology QIHC Exam (9 decks)

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