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QIHC maryjw79 @ gmail email 4 access > SPECIMEN HANDLING > Flashcards

SPECIMEN HANDLING Flashcards

1
Q

When does the pre-analytical stage of tissue handling begin?

A

Once the tissue leaves the body

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2
Q

What is autolysis?

A

Degeneration caused by enzymes within the cell

Begins immediately upon death and is accelerated by increased temperatures

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3
Q

What is the “Cold Ischemic Time”?

A

Time between when tissue is taken out of the body and when it’s placed in a fixative

Goal is to have this time be as little as possible and has to be less than one hour

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4
Q

Can epitope damage due to ischemia be recovered using antigen retrieval techniques?

A

No, this type of epitope damage is irreversible

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5
Q

How does formalin fix tissue?

A

By forming cross-linking methylene bridges between the reactive amino groups in proteins

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6
Q

What is the minimum time that non HER2 tissue can be formalin fixed?

A

24 hours

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7
Q

What is the formalin fixation window for HER2 tissue?

A

6-72 hours

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8
Q

How does alcohol fix tissue?

A

By coagulating and precipitating proteins

Dehydrates the tissue causing shrinking and hardening

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9
Q

What is a main advantage of alcohol fixation over formalin?

A

Alcohol fixation eliminates the need for antigen retrieval

Penetrates more easily than formalin and it is much more useful for molecular work

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10
Q

What kind of slides are made more effective by formalin fixation?

A

Charged slides because formalin fixation makes tissue more acidic (negative) to attract the positively charged slides

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11
Q

Why are nylon biopsy bags recommended above sponges during grossing?

A

Sponges soak up excess reagent and transfer reagent between solution containers during processing

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12
Q

What is one practical application for frozen fixed tissue over FFPE tissue?

A

Frozen tissue is necessary for DIF (Direct Immunofluorescence).

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13
Q

What is DIF used for?

A

To demonstrate the Immunoglobulins and complement in skin biopsies with a much shorter turnaround time than with FFPE tissue

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14
Q

What is a TMA?

A

A Tissue Microarray (TMA) is a method of control preparation in which multiple types of tumors and normal tissue are combined into a single block

a.k.a. “sausage blocks”

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15
Q

When should validation be performed?

A

Anytime there is a modification or the addition of a new: fixative, processing protocol, staining protocol, antibody, ISH protocol, decalcifying agent or control tissue type

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16
Q

When should validation be reassessed or revisited?

A

New antibody used ex: new lot #

New protocol or protocol adjustment

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17
Q

What are the two major categories of fixation?

A

Chemical

Physical

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18
Q

What is the ideal fixation protocol for frozen tissue?

A

Acetone
Zenker
Bouin
Methly-carnoy

Nonadditive coagulants

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19
Q

What is the most common 10% NBF alternative used in routine processing?

A

Zinc formalin

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20
Q

What IHC can be ordered to test for overfixation?

A

Vimentin

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21
Q

How does Vimentin test for overfixation?

A

Demonstrated in the intermediate filaments in stromal tissue.

Intermediate filaments are found in all tissue samples and the antigen is sensitive to over fixation

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22
Q

How does reprocessing affect IHC?

A

Excessive protein cross-linking and poor antigen retrieval

This can mask methylene bridges formed during aldehyde fixation

Can cause irreversible damaged and false negatives

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23
Q

Why specimens should not be
allowed to dry-out prior to fixation?

A

Drying can prevent penetration of formalin and affect immunoreactivity

Negatively impacts cellular morphology (nuclear detail/poorly defined chromatin)

Increases the risk for non-specific binding or staining artifacts

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24
Q

What pre-analytic factors may affect IHC results?

A

Cold Ischemic time

Time in fixative (too long or too short)

The type of fixative

Incorrect accessioning delays

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25
Q

What analytic factors ay affect IHC results?

A

Too strong antigen retrieval causing tissue damage

Antibody selection

Picking he correct chromogen (AEC vs DAB)

Does the protocol in the product insert match the digital program on the machine?

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26
Q

What fixatives may be used for IHC?

A

10% NBF

Bouin’s

B-5

Zenker

95% Alcohol fixatives for cytology may also be used

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27
Q

How does fixation affect immunoreactivity?

A

The protein confirmation of antigens are modified and antibodies may not recognize the epitopes.

a.k.a. cross-linking, masking, cloaking

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28
Q

What is the effect of decalcification on immunoreactivity?

A

Epitopes are retained and can be localized by IHC

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29
Q

What are the three types of decalcification?

A
  1. Acid (HCl, Formic)
  2. Chelating (EDTA)
  3. Ion exchange
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30
Q

Why is fixation the most important factor in IHC?

A

Fixation immobilizes antigens while retaining cellular and subcellular structures

Fixation causes denaturation of most molecules

Causes changes in the conformation of the proteins together with their epitopes

Modifies the epitopes directly by binding to them

Blocks access of antibodies or detection systems to these epitopes

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31
Q

How does the quality of fixation affect dilution of the antibody?

A

The optimal dilution will depend on the type and duration of fixation

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32
Q

How does the quality of fixation affect antibody incubation time?

A
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33
Q

How does the quality of fixation affect retrieval method?

A

It can affect the type and timing of antigen retrieval

It can affect the choice of control material

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34
Q

How does the quality of fixation affect the type of retrieval solution used?

A
35
Q

How does the quality of fixation affect pretreatments?

A
36
Q

Explain the action of 10% NBF on tissues used for IHC

A
37
Q

Explain the action of mercuric chloride based fixatives (ex: B-5) on tissues used for IHC

A
38
Q

Explain the action of zinc formalin on tissues used for IHC

A
39
Q

Explain the action of ethanol on tissues used for IHC

A
40
Q

Explain the action of acetone on tissues used for IHC

A
41
Q

What is protein denaturation?

A
42
Q

How does protein denaturation affect staining?

A
43
Q

What are the effects of under fixation?

A
44
Q

What are the effects of over fixation?

A
45
Q

What testing method can be used to see the effects of fixation on a tissue?

A
46
Q

How does inadequate dehydration of tissues affect staining?

A
47
Q

How does adequate dehydration of tissues affect staining?

A
48
Q

How does inadequate clearing of tissues affect staining?

A
49
Q

How does adequate clearing of tissues affect staining?

A
50
Q

How does inadequate infiltration of tissues affect staining?

A
51
Q

How does adequate infiltration of tissues affect staining?

A
52
Q

How does paraffin temperature affect antigens?

A
53
Q

How does reprocessing affect epitope sites?

A
54
Q

Describe microwave processing

A
55
Q

What are the advantages of microwave processing?

A
56
Q

What are the disadvantages of microwave processing?

A
57
Q

How does the waterbath temperature affect staining?

A
58
Q

How does the use of water bath adhesive usage affect staining?

A
59
Q

How does the use of optimal microscopic slides temperature affect staining?

A
60
Q

How do folds/wrinkles affect staining?

A
61
Q

How does tissue thickness affect staining?

A
62
Q

What is the optimal tissue section thickness for IHC?

A
63
Q

How does optimal placement of tissue on the slide affect staining?

A
64
Q

What is the optimal temperature and time for slide drying?

A
65
Q

What are the effects of drying slides at a high temperature?

A
66
Q

What epitopes experience weak/false negative staining due to excessively high drying temperatures?

A
67
Q

How does using the snap frozen method affect specimen antigenicity?

A
68
Q

How does the slide air drying time prior to fixation affect specimen antigenicity and morphology?

A

Drying causes
- morphological changes
- poorly defined chromatin
- alter the structure of the target protein along the edge of tissue
- inhibit ligand binding
- tissue to be more adsorbent increasing nonspecific adsorption of reagents during staining

69
Q

How does the slide air drying time post fixation affect specimen antigenicity and morphology?

A

Can result in artifactual staining or loss of staining

70
Q

How does using an optimal fixative affect specimen antigenicity?

A
71
Q

How does long term storage affect antigenicity?

A
72
Q

What is the best fixative for IHC on cytology smears?

A
73
Q

What method is used to optimize morphology prior to staining a prepared smear?

A
74
Q

What is the proper short-term storage of unstainted smears to be used for IHC?

A
75
Q

What is the proper long-term storage of unstained smears to be used for IHC?

A
76
Q

What are the effects of decolorization of cytology smears on the staining results?

A
77
Q

How are cell blocks treated prior to staining?

A
78
Q

How do you properly decolorize H&E stained slides so that IHC can be performed?

A
79
Q

How do you create additional slides from a single cell source?

A
80
Q

What technique is used to stain for multiple epitopes on one slide?

A
81
Q

What are the two methods used to stain for multiple epitopes on one slide?

A
82
Q

What are the four steps of IHC staining?

A
  1. Fixation and processing
  2. Blocking non-specific background staining
  3. Detection systems
  4. Antigen retrieval
83
Q

What is the purpose of fixation?

A

To preserve tissue bc this affects morphology and IHC results

QIHC maryjw79 @ gmail email 4 access (16 decks)

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