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Histology QIHC Exam > Detection Systems > Flashcards

Detection Systems Flashcards

1
Q

What are the two most common flourescent dyes?

A

FITC and TRICT.

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2
Q

What is photobleaching?

A

When a fluorophore loses its ability to fluorescence due to the buildup of reactive oxygen particles due to its natural activity.

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3
Q

How can photobleaching be reduced?

A

By either limiting the amount of free oxygen radicals in the environment or decreasing the excitation light in intensity and duration.

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4
Q

What are some common applications of IF in pathology?

A
  1. FISH to detect gene aberrations in cells.
  2. The detection of single monolayer organisms such as bacteria and parasites.
  3. The visualization of cell structures by super resolution microscopy.
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5
Q

What is ISH?

A

A common name for a technique used to identify gene amplifications, deletions and translocations as well as chromosomal copy number changes.

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6
Q

What are the three different ISH techniques?

A

FISH (fluorescent), CISH (chromogens) and SISH (silver). They are named for the system that is used to identify the probe.

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7
Q

Why is CISH used more commonly than FISH?

A
  1. The type of microscope (brightfield) used for CISH is more readily available than the microscope used for FISH (fluorescence).
  2. Brightfield CISH allows for better visualization of tissue structures.
  3. CISH signals do not fade overtime like FISH.
  4. Documentation by visual acquisition is simpler with brightfield than with fluorescent scopes decreasing the evaluation time for CISH vs FISH.
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8
Q

What are the two major categories of fixation?

A

Fixation by chemical means and fixation by drying.

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9
Q

What are the two most commonly used fluorochromes in IF?

A

FITC and rhodamine.

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10
Q

What are the two most common enzymes used for Ab visualization?

A

Horseradish phosphatase and alkaline phosphatase.

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11
Q

How are DAB and AEC commonly used?

A

To visualize the sites of Ab binding by forming a colored compound.

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12
Q

In Avidin-biotin methods, what follows the primary Ab?

A

The biotinylated secondary Ab.

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13
Q

How does alkaline phosphatase function in some IHC methods?

A

The enzyme.

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14
Q

Define fluorochrome.

A

A dye that absorbs light then emits its own light at a longer wavelength.

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15
Q

How is horseradish peroxidase used in some Avidin-biotin methods?

A

The enzyme.

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16
Q

What is the most common substrate used for alkaline phosphatase labeled Ab?

A

Alkaline phosphatase is demonstrated by the use of napthol-AS-phosphate.

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17
Q

What chromogen is commonly used to demonstrate alkaline phosphate?

A

Fast red-violet LB.

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18
Q

What chromogen is INSOLUBLE in alcohol?

A

DAB.

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19
Q

If 3-amino-9-ethylcarbazole (AEC) is used as the chromogen, what kind of hematoxylin should be used as the counterstain?

A

The hematoxylin following AEC must be alcohol free.

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20
Q

How must tissue for immunofluorescence be handled?

A

Frozen and unfixed because Ag activity will be least impacted.

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21
Q

What is a neoplasm?

A

A new growth of uncontrolled cell multiplication.

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22
Q

What does a completely negative Vimentin result indicate?

A

Overfixation in formaln.

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23
Q

Besides the heat employed, what is the other most important factor of HIER?

A

The composition of the retrieval solution used.

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24
Q

How is ficin used in IHC?

A

Ficin is one of the proteolytic enzymes used in IHC for EIER.

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25
Q

What is the main benefit of polymeric detection methods?

A

The serum and Avidin-biotin blocking steps have been eliminated and TAT has been decreased.

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26
Q

What is a “labeled” reagent?

A

An Ab that is conjugated with fluorophore, enzyme, or biotin.

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27
Q

How do you calculate the Volume of Ab concentrate required?

A

Volume of ‘Concentrate’ Required = `Volume of Diluted Antibody Desired/
Dilution Factor of Working Solution

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28
Q

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:
A. Kidney
B. Liver
C. Thyroid
D. Brain

A

Thyroid. Liver, kidney and brain are tissues that require a biotin block because of the endogenous biotin. If left unsuppressed stains would have a tremendous amount of background staining.

29
Q

What is the substrate of the enzyme peroxidase?

A

H2O2. A substrate is a substance upon which an enzyme acts; peroxidase therefore reacts with hydrogen peroxide (H2O2).

30
Q

Fixatives that tend to mask antigenic sites and hamper immunohistochemical localization of antigens contain:
A. mercury
B. phosphates
C. aldehydes
D. alcohol

A

Aldehydes. Aldehyde-containing fixatives, such as formaldehyde and glutaraldehyde, are thought to reduce or mask antigenic binding sites and hamper immunohistochemical staining

31
Q

What is the purpose of pretreatment with HIER or EIER?

A

Pretreatment of formalin-fixed sections with proteolytic enzymes or heat-induced antigen retrieval under controlled conditions frequently improves the immunohistochemical detection of tissue antigen

32
Q

An enzyme is a/an:
A. inorganic compound
B. lipid
C. protein
D. sugar

A

Protein. All enzymes are proteins but not all proteins are enzymes.

33
Q

Which of the following are enzymes commonly used in immunohistochemistry to unmask antigenic determinants?
A. papain, propane, chymase, lactase
B. pepsin, tryptophan, kinase, proteinase K
C. pronase, trypsin, proteinase K, pepsin
D. lactase, pepsin, trypsin, pepsin

A

C. Pronase, trypsin, proteinase K and pepsin.

34
Q

A common fluorochrome used in immunohistochemistry is:

A

fluorescein isothiocyanate

35
Q

. A common fluorochrome used in immunohistochemistry is:
A. fluorinated isotryptophan
B. fluorine isothiocyanate
C. fluorescein isothiocyanate
D. fluoroisothiocyanate

A

C. fluorescein isothiocyanate

36
Q

When using an alkaline phosphatase detection system:
A. It is always necessary to perform a hydrogen peroxidase quench
B. It is never necessary to perform a hydrogen peroxidase quench, but you must always perform an alkaline phosphatase quench.
C. It is recommended that an alkaline phosphatase quench be performed on frozen tissues
D. Using a quench of any type will result in very weak staining when using an alkaline phosphatase detection system

A

It is recommended that an alkaline phosphatase quench be performed on frozen tissues. It is recommended to perform alkaline phosphatase quenching when working with frozen section tissues that have the highest levels of endogenous alkaline phosphatase, such as placenta, lymphoid tissues, intestine, and kidney.

37
Q

Immunoglobulin antigens (IgA, IgD, IgG, IgE, IgM) are most likely to be demonstrated at maximum sensitivity with:

A

Frozen sections followed by acetone fixation.

38
Q

The specific substrate of horseradish peroxidase or HRP is:

A

Hydrogen peroxide.

39
Q

Long term fixation in formalin (weeks to months) will most likely result in:

A

Loss of Antigenicity.

40
Q

The best positive control tissue for IHC is one that is:
A. bought from a reputable vendor
B. as fresh as possible
C. processed identically to patient tissue
D. is available in large quantities

A

C. Processed identically to patient tissue.

41
Q

After fixation for 24 to 48 hours in formalin, tissue for IHC should be stored in which of the following fluids to preserve antigenicity?

A

70% alcohol.

42
Q

A chemical group which gives the property of color to a chromogen is a:

A

Chromophore

43
Q

The primary reason that frozen sections destined for immunohistochemistry are fixed in some way after sectioning is to:

A

Prevent diffusion of the tissue antigens of interest

44
Q

A cryostat may be defined as a/an:
A. device that maintains a controlled low temperature
B. instrument that cuts sections of frozen specimens
C. microtome contained in a freezing cabinet
D. section cut from a frozen specimen

A

C. Microtome contained in a freezing cabinet.

45
Q

Why is 70% ideal for long term storage?

A

70% ethanol will preserve antigenicity indefinitely. Formalin will continue to cross-link proteins and lead to loss of antigenicity. Acetone is never used for this purpose. Hanks buffered saline is used for fresh tissue transport for flow cytometry.

46
Q

To eliminate the problem of endogenous staining a particular IHC method is optimal. What method would that be?
A. LSAB+
B. ABC
C. Polymer-Based
D. PAP

A

Polymer-based. Polymer-based immunohistochemistry do not rely on biotin as part of the complex. The other methods all rely on some form of biotin linkage and though biotin blocks help they do not eliminate endogenous biotin therefore increasing the amount of background

46
Q

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?
A. Charged slides
B. Drying time of tissue on slides
C. Well fixed tissue
D. All of the above

A

D. All of the above.

47
Q

Tissue micro array (TMA) blocks are utilized in many ways in IHC. All of the following are ways a TMA block can be used except:
A. as a positive control tissue
B. to work up novel reagents
C. to identify reagent quality
D. study the interactions of tissue components

A

D. Study the interactions of tissue components.

48
Q

What is the main drawback of TMA blocks?

A

The problem with TMA blocks is that they only represent a small portion of the affected tissue. Because of this, it is not appropriate when trying to see the large picture of interactions.

49
Q

Antibodies must always be stored at:

A

According to manufacturers specifications. Although antibodies are normally best stored at 2-8o C, it is always best to follow manufacturers’ recommendations.

50
Q

What is a detection system?

A

Detection Systems are reagent products most often sold and used in a kit format that are designed to target a bound antibody directly or indirectly.

51
Q

What is Immunofluorescence (IF) used for?

A

In most labs today this is used in the diagnosis of skin lesions, autoimmune diseases and kidney biopsies.

52
Q

Describe direct IF.

A

In direct detection, the primary antibody specific for the target molecule is directly labeled.

53
Q

Describe indirect IF.

A

Indirect detection uses an unconjugated primary antibody. This can be done by hand or on an instrument.

54
Q

How is darkfield microscopy inferior to brightfield?

A

Nuclear details and the architecture of tissue are not as easily visualized as bright field .

55
Q

What are the three most commonly used detection systems?

A

A. Fluorescence labels
B. Enzyme labels
C. Polymer based detection

56
Q

What are the two most commonly used detection system?

A

In clinical labs the detection systems most widely used are HRP – Horseradish Peroxidase and/or AP - Alkaline Phosphatase based.

57
Q

What are enzymes?

A

Enzymes are a protein molecules that speed up a chemical reaction in an organism. For IHC we are talking about the main enzyme reaction in the detection system that you are using in your lab.

58
Q

What is the most widely used chromogen?

A

DAB (3,3’-Diaminobenzidine)is the most widely used chromogen for immuno-histochemical staining and immunoblotting. When in the presence of peroxidase enzyme, DAB produces a brown precipitate that is insoluble in alcohol and xylene.

59
Q

What is Permanent/Fast red?

A

Permanent Red is used with an Alkaline Phosphatase Detection System. This is often used in the diagnosis of Melanotic lesions so that the brown melanin pigment can be visualized next to the positive red staining end product

60
Q

What is blocking?

A

Blocking reactions are steps taken with different blocking solutions to help eliminate or reduce background staining . Most technology today, is based on a polymer detection kit, which contains blocking reagents, usually peroxide based.

61
Q

What is In-Situ Hybridization?

A

This is a sensitive and robust staining method. It is a type of hybridization that uses a labeled complimentary DNA, RNA or nucleic acid strand to localize specific DNA or RNA in a tissue section.

62
Q

What is FISH?

A

Fluorescent in situ hybridization (FISH) is a very sensitive cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes. This has long been considered the Gold standard for the detection of chromosomal abnormalities.

63
Q

What is FISH commonly used for?

A

Her 2 neu detection and in the diagnosis of lymphomas

64
Q

What are the main drawbacks of FISH?

A
  1. Requires a fluorescent microscope for review.
  2. The signal generated is not permanent and is often photographed for
    permanence.
  3. Requires various filters; Bulb life is short and instrumentation requires
    more maintenance than light microscopy
  4. More regulatory guidelines to be followed
65
Q

What is CISH?

A

Chromogenic In-Situ Hybridization (CISH) is a more robust and a more complicated and lengthy method than IHC with several
reagent and protocol steps that are critical to good results but is similar to FISH.

66
Q

What are the main pros for using CISH?

A
  1. Easier method of molecular testing than FISH, due to the ability to visualize under light
    microscopy.
  2. Kits are available from many vendors.
  3. Results are permanent.
67
Q

What is SISH?

A

Silver In-situ Hybridization (SISH) is a similar process to CISH except silver is used to visualize the probes and the coloration is black due to the silver precipitation.

Histology QIHC Exam (9 decks)

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