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Histopathologic & Cytologic Techniques (Lecture) > Chapter 6 - Fixation > Flashcards

Chapter 6 - Fixation Flashcards

1
Q

whereby the chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein.

A

additive fixation

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2
Q

examples of additive fixation

A

formalin
mercury
osmium tetroxide

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3
Q

whereby the fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H-bonds of certain groups within the protein molecule.

A

non-additive fixation

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4
Q

Specimens should be transferred to fixative quickly ______ after surgery as deterioration will commence with the loss of blood supply.

A

<1hour

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5
Q

Tissues should be fixed in a sufficient volume of solution; generally in a ratio of _____ or at least _____ fixative to specimen, for penetration to occur in the most efficient manner.

A

20:1 or at least 10:1

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6
Q

______ to fixation must be removed or incised (e.g. fascia, bone, feces, thick tissue)

A

anatomical barriers

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7
Q

large specimens must be sectioned or inflated with fixative (e.g. ____)

A

lung

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8
Q

opened and cleaned (________) to allow penetration.

A

gastrointestinal tract

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9
Q

Pinning specimens to a corkboard or inserting a ____ or _____ into tubular structures can improve fixation and reduce tissue distortion.

A

paper or gauze “wick”

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10
Q

The volume of fixative is important. Traditionally, the amount of fixative used has been _______ the volume of tissue to be fixed.

A

10-20 times

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11
Q

Hydrogen Ion Concentration: Fixation is best carried out close to neutral pH, in the range of ___

A

6-8

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12
Q

Common buffers

A

phosphate, bicarbonate, cacodylate, and veronal

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13
Q

Commercial formalin is buffered with phosphate at a pH of __

A

7

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14
Q

Temperature:
Many laboratories use tissue processors that work at ___ for regular tissue processing

A

40C

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15
Q

For electron microscopy and some histochemistry, the ideal temperature is ____

A

0-4C

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16
Q

Formalin heated to ____ is sometimes used for the rapid fixation of very urgent biopsy specimens, although the risk of tissue distortion is increased

A

60C

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17
Q

Thickness of section:
Tissue blocks should be small (e.g. ______ for electron microscopy and _____ wide for light microscopy) and thin (no more than ____ for light microscopy)

A

1 to 2mm^2 - EM
2cm^2 wide - LM
0.4cm - LM

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18
Q

Large solid tissue, such as _____, should be opened or sliced thinly to improve penetration of fixatives.

A

uterus

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19
Q

Brain is usually suspended whole in ___ buffered formalin for 2-3 weeks to ensure fixation and some hardening prior to sectioning.

A

10%

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20
Q

For solid material (e.g., liver) the longest dimension should not exceed ______

A

10-15mm

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21
Q

The best results are usually obtained using ________ (400-450 mOsm; isotonic solutions are 340 mOsm).

A

slightly hypertonic solutions

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22
Q

_____ is commonly added to osmium tetroxide fixatives for electron microscopy.

A

sucrose

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23
Q

Formaldehyde is normally used as a ___ solution

A

10%

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24
Q

glutaraldehyde is normally used as a ___ solution

A

3%

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25
Q

Primary fixation in buffered formalin is usually carried out for ______

A

2-6 hours

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26
Q

Most of the formalin can be washed out after fixation for ____

A

24 hours

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27
Q

For electron microscopy, it is recommended that diced tissues be fixed for _____

A

3 hours

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28
Q

There are four major groups of fixatives, namely the:

A

the aldehydes, oxidizing agents, alcohol based fixatives and the metallic group of fixatives

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29
Q

2 types of aldehydes

A

formaldehyde
glutaraldehyde

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30
Q

2 types of oxidizing agents

A

osmium tetroxide
potassium permanganate

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31
Q

acts by cross-linking proteins

A

oxidizing agents

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32
Q

alcohol based fixatives

A

methyl alcohol, ethyl alcohol, acetic acid

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33
Q

these are protein-denaturing agents

A

alcohol based fixatives

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34
Q

_____ act by forming insoluble metallic precipitates like mercuric chloride and picric acid.

A

metallic fixatives

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35
Q

The choice of the fixative is based on _______

A

tissue and anticipated ancillary tests

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36
Q

______ are made up of only one component substance.

A

simple fixatives

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37
Q

2 types of metallic fixatives

A

mercuric chloride
chromate fixatives

38
Q

these are the simple fixatives

A

Aldehydes
a. Formaldehyde
b. Glutaraldehyde

Metallic Fixatives
a. Mercuric chloride
b. Chromate fixatives

Picric acid
Acetic acid
Acetone
Alcohol
Osmium Tetroxide

39
Q

are those that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.

A

compound fixatives

40
Q

are those that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.

A

microanatomical fixatives

41
Q

7 types of microanatomical fixatives

A

10% formal saline
10% neutral buffered formalin Heidenhain’s Susa
Formal sublimate (formal corrosive)
Zenker ‘s solution
Zenker-formal (Kelly ‘s solution)
Bouin’s solution
Brasil’s solution

42
Q

are those that preserve specific parts and particular microscopic elements of the cell itself.

A

cytological fixatives

43
Q

are those that preserve the nuclear structures (e.g., chromosomes) in particular. They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin.

A

nuclear fixatives

44
Q

pH of nuclear fixatives

A

pH of 4.6 or less

45
Q

5 types of nuclear fixatives

A

Flemming’s fluid
Carnoy’s fluid
Bouin’s fluid
Newcomer’s fluid
Heidenhain’s Susa

46
Q

______ has been found to react with viruses, and causes the loss of their infective power.

A

mercuric chloride

47
Q

are those that preserve cytoplasmic structures in particular. They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm.

A

cytoplasmic fixatives

48
Q

pH of cytoplasmic fixatives

A

pH of more than 4.6

49
Q

5 types of cytoplasmic fixatives

A

Flemming’s fluid without acetic acid
Kelly’s fluid
Formalin with “post-chroming”
Regaud ‘s fluid (Muller ‘s fluid)
Orth ‘s fluid

50
Q

For RNA, the precipitant fixatives - ________ - give the best quantitative results using frozen tissues as the standard.

A

ethanol and acetone

51
Q

are those that preserve the chemical constituents of cells and tissues.

A

histochemical fixatives

52
Q

4 types of histochemical fixatives

A

Formal Saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer’s Fluid

53
Q

______ is the process of placing an already fixed tissue in a second fixative in order

A

secondary fixation

54
Q

______ may be done before dehydration and on deparaffinized sections before staining, usually with 10% formalin or 10% formol saline as a primary fixative.

A

secondary fixation

55
Q

tissue fixed in 10% buffered neutral formalin may require secondary fixation with ________ (that acts as a mordant)

A

zenker’s fixation

56
Q

stain used for connective tissue

A

masson’s trichrome

57
Q

stain used for collagen

A

mallory’s aniline blue stain

58
Q

stain used for striated muscle

A

phosphotungstic acid- hematoxylin (PTAH)

59
Q

is a form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues.

A

post-chromatization

60
Q

is the process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues. Several solutions may be used.

A

washing out

61
Q

Tap water is used to remove:

A

a. excess chromates from tissues fixed in Kelly’s, Zenker’s, and Flemming’s solutions
b. excess formalin
c. excess osmic acid

62
Q

used to wash out excess amount of picric acid (Bouin’s solution).

A

50-70% alcohol

63
Q

is used to remove excessive mercuric fixatives.

A

alcoholic iodine

64
Q

may be found in surgical
specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H&E stained sections.

A

crush artifact

65
Q

In conventional histopathological
techniques, lipids are largely removed
during preparation of tissues.

A

lipid fixation

66
Q

_______ or _______ should be used
for demonstrating lipid in tissues,
followed by a general lipid stain.

A

cryostat or frozen sections

67
Q

Fixatives containing ______ and ______ can be effective for preservation of lipids in cryostat sections.

A

mercuric chloride and potassium dichromate

68
Q

may be used to preserve phospholipids.

A

baker’s formal-calcium

69
Q

Carbohydrates are hydrophilic; they hold much water in the extracellular space by
hydrogen bonding.

A

carbohydrate fixation

70
Q

________ is a better
fixative in human skin compared with neutral buffered formaldehyde.

A

alcoholic formaldehyde

71
Q

The most useful fixatives for preserving glycogen are alcohol-based, such as:

A

Rossman’s fluid or cold absolute alcohol

72
Q

There is better retention of glycogen if the section is coated with ______

A

celloidin

73
Q

the most commonly used fixatives for amino acid histochemistry.

A

Neutral buffered formal saline or formaldehyde vapor

74
Q

_____, _____, and ______ with the whole procedure performed at 4°C.

A

osmium tetroxide, glutaraldehyde and paraformaldehyde

75
Q

For electron histochemistry and electron immunocytochemistry, ________ is useful

A

Karnovsky’s paraformaldehyde-glutaraldehyde

76
Q

FIXATION FOR ENZYME HISTOCHEMISTRY

A

4% formaldehyde or formal saline overnight

77
Q

pathology for the demonstration of
various antibodies.

A

FIXATION FOR IMMUNOFLUORESCENCE

78
Q

FIXATION FOR IMMUNOFLUORESCENCE:
In many instances, _____ and _____ may be used.

A

formalin-fixed and paraffin embedded sections

79
Q

In more sensitive cases, the tissue must be prepared as a cryostat section and fixation limited to a few seconds in:

A

absolute methanol or acetone

80
Q

To ensure free access of the antibody to its antigen, the cells must be fixed and permeabilized.

A

FIXATION FOR IMMUNOHISTOCHEMISTRY

81
Q

_______ such as alcohols
and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture.

A

organic solvents

82
Q

_______ (such as paraformaldehyde) form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens.

A

CROSS-LINKING REAGENTS

83
Q

Fix cells in -20°C acetone for 5-10 minutes. No permeabilization step needed following acetone fixation.

A

acetone fixation

84
Q

Fix cells in -20°C methanol for 5-10 minutes. Permeabilization step is needed following methanol fixation.

A

methanol fixation

85
Q

Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.

A

ethanol fixation

86
Q

Fix in cooled methanol, 10 minutes at –20 °C.
Remove excess methanol.
Permeabilize with cooled acetone for 1 minute at –20 °C.

A

methanol-acetone fixation

87
Q

1:1 methanol and acetone mixture. Make the mixture fresh and fix cells at -20 C for 5- 10 minutes.

A

Methanol-Acetone Mix Fixation

88
Q

1:1 methanol and ethanol mixture. Make the mixture fresh and fix cells at -20 C for 5- 10 minutes.

A

methanol-ethanol mix fixation

89
Q

Fix cells in 10% neutral buffered formalin for 5-10 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.

A

Formalin Fixation

90
Q

Fix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.

A

Paraformaldehyde-Triton Fixation

91
Q

Fix in 4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at –20 °C.

A

Paraformaldehyde-Methanol Fixation

Histopathologic & Cytologic Techniques (Lecture) (5 decks)

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