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INTSHP2 HISTOPATHOLOGY > CHAPTER 6 FIXATION > Flashcards

CHAPTER 6 FIXATION Flashcards

1
Q

first and most critical step in histotechnology

A

FIXATION

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2
Q

a process that preserves tissues from decay, thereby preventing autolysis or putrefaction

A

FIXATION

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3
Q

results from tissue digestion by intracellular enzymes that are released when organelle membranes rupture.

A

Autolysis

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4
Q

is brought about by microorganisms which may already be present in the specimen.

A

Bacterial decomposition or putrefaction

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5
Q

fixation in [?] will initially cause slight swelling of tissue specimens

A

10% buffered formalin

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6
Q

During processing, however, the specimen may shrink and lose [?] of its volume.

A

20% -30%

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7
Q

Leaving the tissue in water (?) will cause the cell to swell, while a strong salt (?) will cause the cell to shrink.

A

hypotonic solution

hypertonic solution

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8
Q

Every cell in the body has a structure or “?” within its cytoplasm called lysosome containing hydrolytic enzymes that are released when the integrity of the cell is destroyed.

A

suicide sac

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9
Q

occurs due to the action of these hydrolytic enzymes.

A

Postmortem decomposition (“autolysis”)

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10
Q

is rarely used on tissue specimens, its application being confined to smears of microorganisms.

A

Heat fixation

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11
Q

regarded as a form of heat fixation; is now widely practiced in routine laboratories.

A

microwave fixation

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12
Q

has some applications in histochemistry but is not usually applied to diagnostic tissue specimens.

A

cryo-preservation (freeze drying)

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13
Q

There are two basic mechanisms involved in fixation:

A
  1. Additive fixation
  2. Non-additive fixation
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14
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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15
Q

(Examples are formalin, mercury, and osmium tetroxide).

A

Additive fixation

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16
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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17
Q

(Examples are alcoholic fixatives)

A

Non-additive fixation

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18
Q

Traditionally, the amount of fixative used has been [?] the volume of tissue to be fixed.

A

10-20 times

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19
Q

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

A

6-8.

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20
Q

Commercial formalin is buffered with phosphate at a pH of

A

7

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21
Q

Many laboratories use tissue processors that work at [?] for regular tissue processing.

A

40°C

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22
Q

For electron microscopy and some histochemistry, the ideal temperature is

A

0-4°C.

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23
Q

bone marrow continues to undergo mitosis (growth) up to [?] after death when refrigerated

A

30 minutes

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24
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

60°C

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25
Q

for electron microscopy

A

1 to 2 mm2

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26
Q

wide for light microscopy

A

2 cm2

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27
Q

Brain is usually suspended whole in [?] for [?] to ensure fixation and some hardening prior to sectioning.

A

10% buffered formalin

2-3 weeks

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28
Q

[?] penetrate the best, and [?] the worst. [?] and others are somewhere in between. One way to get around this problem is by sectioning the tissues thinly (?).

A

Formalin and alcohol

glutaraldehyde

Mercurials

2 to 3 mm

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29
Q

penetrates tissues slowly so specimens may need to be opened, incised or sliced and left to fix for an adequate period of time prior to processing.

A

Formalin

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30
Q

It is not practical or within the realm of patient care to wait any longer than [?], since clinicians generally need prompt diagnosis.

A

1 or 2 days

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31
Q

To maintain an adequate fixation time of [?], the recommended size of the tissue is [?], and no more than [?]. thick.

A

4 to 6 hours

2 cm2

4 mm

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32
Q

A commonly quoted rate of penetration for aldehyde fixative is

A

two- to-three millimeter per hour.

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33
Q

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

A

10-15mm.

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34
Q

shrink; swell and burst

A

hypertonic; hypotonic

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35
Q

For that reason, we recommend using a [?] based fixative.

A

normal phosphate buffered saline (PBS)

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36
Q

give rise to cell shrinkage; cause cell swelling and poor fixation.

A

Hypertonic solutions; Isotonic and hypotonic fixatives

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37
Q

The best results are usually obtained using slightly hypertonic solutions (?).

A

400-450 mOsm; isotonic solutions are 340 mOsm

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38
Q

is commonly added to osmium tetroxide fixatives for electron microscopy.

A

Sucrose

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39
Q

Formaldehyde is normally used as a [?], and glutaraldehyde is normally used as a [?].

A

10% solution; 3% solution

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40
Q

The presence of a [?] causes polymerization of the aldehyde, with consequent decrease in its effective concentration.

A

buffer

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41
Q

Low concentrations of [?] have been found to be an ideal concentration for immuno-electron microscopy.

A

glutaraldehyde (0.25%)

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42
Q

Fibrous organs such as [?] take longer to fix than small or loosely textured tissues such as [?].

A

uterus or intestinal tract

biopsies or scrapings

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43
Q

Primary fixation in buffered formalin is usually carried out for [?] during the day when the specimen is obtained, but the tissue may remain in fixative over the weekend without much adverse effect.

A

2-6 hours

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44
Q

Most of the formalin can be washed out after fixation for

A

24 hours.

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45
Q

For electron microscopy, it is recommended that diced tissues be fixed for [?] and then placed in a holding buffer.

A

3 hours

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46
Q

Artefacts will be introduced by drying, so if the tissue is left out in the open, it should be kept moist with

A

saline.

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47
Q

There are four major groups of fixatives, namely the [?].

A

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

48
Q

act by cross-linking proteins.

A

aldehydes (formaldehyde, glutaraldehyde) and oxidizing agents (osmium tetroxide, potassium permanganate)

49
Q

are protein-denaturing agents.

A

Alcohol based fixatives (methyl alcohol, ethyl alcohol, acetic acid)

50
Q

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

A

Metallic fixatives

51
Q

According to COMPOSITION

A

A. Simple Fixatives

B. Compound Fixatives

52
Q

A. Simple Fixatives

A

I. Aldehydes
—a. Formaldehyde
—b. Glutaraldehyde
2. Metallic Fixatives
—a. Mercuric chloride
—b. Chromate fixatives
3. Picric acid
4. Acetic acid
5. Acetone
6. Alcohol
7. Osmium Tetroxide

53
Q
  1. According to ACTION
A

A. Microanatomical Fixatives

B. Cytological Fixatives

54
Q

A. 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

55
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

A. Microanatomical Fixatives

56
Q

B. Cytological Fixatives:

A
  1. Nuclear Fixatives
  2. Cytoplasmic Fixatives
  3. Histochemical Fixatives
57
Q
  1. Histochemical Fixatives:
A

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

58
Q

-are made up of only one component substance.

A

Simple Fixatives

59
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

60
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

61
Q
  • are those that preserve specific parts and particular microscopic elements of the cell itself.
A

Cytological Fixatives

62
Q
  • are those that preserve the nuclear structures (e.g., chromosomes) in particular.
A

Nuclear Fixatives

63
Q

Many fixatives have been used over the years, specifically for work with nucleic acids, but relatively few (including [?]) are known to react with them chemically.

A

mercury and chromium salts

64
Q

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

A

Mercuric chloride

65
Q
  • are those that preserve cytoplasmic structures in particular.
A

Cytoplasmic Fixatives

66
Q

They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm.

A

Cytoplasmic Fixatives

67
Q

They have a pH of more than 4.6.

A

Cytoplasmic Fixatives

68
Q

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

A

ethanol and acetone

69
Q
  • are those that preserve the chemical constituents of cells and tissues.
A

Histochemical Fixatives

70
Q

Secondary fixation may be done before dehydration and on deparaffinized sections before staining, usually with [?] as a primary fixative.

A

10% formalin or 10% formol saline

71
Q

For example, tissue fixed in 10% buffered neutral formalin may require secondary fixation with [?] (that acts as a mordant) prior to staining with [?] (for connective tissue), [?] (for collagen), or [?] (for striated muscle).

A

Zenker’s solution

Masson’s trichrome

Mallory’s aniline blue stain

phosphotungstic acid-hematoxylin (PTAH) stain

72
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

73
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

74
Q

[?] is used to remove:
a. excess chromates from tissues fixed in Kelly’s, Zenker’s, and Flemming’s solutions
b. excess formalin
c. excess osmic acid

A
  1. Tap water
75
Q

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

A
  1. 50-70% alcohol
76
Q

is used to remove excessive mercuric fixatives.

A
  1. Alcoholic iodine
77
Q

Slow freezing of unfixed tissues near [?] must be avoided since this may promote formation of ice crystal artefacts.

A

0°C

78
Q

Tissues should not be more than [?] thick except in lung edema (in which case tissue slices may be [?] thick), with minimum squeezing and handling.

A

5 mm

1-2 cm

79
Q

The amount of fixative must be adequate, approximately [?] times the volume of the tissue specimen except in osmium tetroxide which is very expensive, requiring only [?] times that of tissue volume for fixation.

A

10-20

5-10

80
Q

For prolonged fixation (e.g. museum preparation) volume of fixing fluid should not be less than [?] times that of the tissue.

A

50

81
Q

Human brains may be suspended by a cord tied under the [?] to prevent flattening.

A

Circle of Willis

82
Q

(washing out of blood with Ringer’s lactate) may lead to artifact formation with loss of blood content.

A

Intravascular perfusion

83
Q

Dense tissues such as brains are poorly penetrated, hence require long fixation (usually [?] prior to sectioning).

A

2 weeks

84
Q

must be injected before immersing the eye in the fixative.

A

Formal-alcohol

85
Q

Frozen sections may lead to formation of

A

ice crystal artifacts.

86
Q

Hard tissues (e.g. cervix, uterine, fibroids, hyperkeratotic skin, fingernails, etc.) may be washed out in running water overnight and immersed in 4% aqueous phenol solution for 1-3 days (?). This will soften the tissue and allow easier sectioning without producing any marked distortion of the inner structures.

A

Lendrum’s method

87
Q

An adequate volume is vital (at least [?]).

A

20:1

88
Q

Some formulated fixatives should be prepared from stock solutions immediately before use because they are unstable (e.g.,).

A

Helly’s fluid

89
Q

Avoid metal lids. Some fixatives are highly corrosive and will attack metals (e.g.,).

A

mercury salts

90
Q

Some fixatives require that specimens be washed in water prior to processing (e.g.,) or some other requirement may exist ([?] may precipitate from buffer in concentrations of alcohol of greater than 70%).

A

Zenker’s or Helly’s

phosphate

91
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”

92
Q

This may be due to partial coagulation of partially fixed protein by ethanol or by incomplete wax impregnation during subsequent histological processing.

A

“Crush artifact”

93
Q

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

A

Organic solvents

94
Q

form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens.

A

Cross-linking reagents (such as paraformaldehyde)

95
Q

Acetone Fixation
Fix cells in [?] acetone for 5-10 minutes.

A

-20°C

96
Q

No permeabilization step needed following acetone fixation.

A

Acetone Fixation

97
Q

Methanol Fixation
Fix cells in [?] methanol for 5-10 minutes.

A

-20°C

98
Q

Permeabilization step is needed following methanol fixation.

A

Methanol Fixation

99
Q

Ethanol Fixation
Fix cells in cooled [?] for 5-10 minutes.

A

95% ethanol, 5% glacial acetic acid

100
Q

Methanol-Acetone Fixation
Fix in cooled methanol, 10 minutes at [?].

A

–20 °C

101
Q

Remove excess methanol.

A

Methanol-Acetone Fixation

102
Q

Permeabilize with cooled acetone for 1 minute at –20 °C.

A

Methanol-Acetone Fixation

103
Q

1:1 methanol and acetone mixture.

A

Methanol-Acetone Mix Fixation

104
Q

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

A

Methanol-Acetone Mix Fixation

105
Q

1:1 methanol and ethanol mixture.

A

Methanol-Ethanol Mix Fixation

106
Q

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

A

Methanol-Ethanol Mix Fixation

107
Q

Fix cells in 10% neutral buffered formalin for 5-10 minutes.

A

Formalin Fixation

108
Q

Rinse briefly with PBS.

A

Formalin Fixation

109
Q

Permeabilize with 0.5% Triton X-100 for 10 minutes.

A

Formalin Fixation

110
Q

Fix in 3-4% paraformaldehyde for 10-20 minutes.

A

Paraformaldehyde-Triton Fixation

111
Q

Rinse briefly with PBS.

A

Paraformaldehyde-Triton Fixation

112
Q

Permeabilize with 0.5% Triton X-100 for 10 minutes.

A

Paraformaldehyde-Triton Fixation

113
Q

Rinse briefly with PBS.

A

Paraformaldehyde-Methanol Fixation

114
Q

Fix in 4% paraformaldehyde for 10-20 minutes.

A

Paraformaldehyde-Methanol Fixation

115
Q

Permeabilize with cooled methanol for 5-10 minutes at –20 °C.

A

Paraformaldehyde-Methanol Fixation

INTSHP2 HISTOPATHOLOGY (14 decks)

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