CHAPTER 6 FIXATION Flashcards
1
Q
first and most critical step in histotechnology
A
FIXATION
2
Q
a process that preserves tissues from decay, thereby preventing autolysis or putrefaction
A
FIXATION
3
Q
results from tissue digestion by intracellular enzymes that are released when organelle membranes rupture.
A
Autolysis
4
Q
is brought about by microorganisms which may already be present in the specimen.
A
Bacterial decomposition or putrefaction
5
Q
fixation in [?] will initially cause slight swelling of tissue specimens
A
10% buffered formalin
6
Q
During processing, however, the specimen may shrink and lose [?] of its volume.
A
20% -30%
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
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
9
Q
occurs due to the action of these hydrolytic enzymes.
A
Postmortem decomposition (“autolysis”)
10
Q
is rarely used on tissue specimens, its application being confined to smears of microorganisms.
A
Heat fixation
11
Q
regarded as a form of heat fixation; is now widely practiced in routine laboratories.
A
microwave fixation
12
Q
has some applications in histochemistry but is not usually applied to diagnostic tissue specimens.
A
cryo-preservation (freeze drying)
13
Q
There are two basic mechanisms involved in fixation:
A
- Additive fixation
- Non-additive fixation
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
15
Q
(Examples are formalin, mercury, and osmium tetroxide).
A
Additive fixation
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
17
Q
(Examples are alcoholic fixatives)
A
Non-additive fixation
18
Q
Traditionally, the amount of fixative used has been [?] the volume of tissue to be fixed.
A
10-20 times
19
Q
Hydrogen Ion Concentration: Fixation is best carried out close to neutral pH, in the range of
A
6-8.
20
Q
Commercial formalin is buffered with phosphate at a pH of
A
7
21
Q
Many laboratories use tissue processors that work at [?] for regular tissue processing.
A
40°C
22
Q
For electron microscopy and some histochemistry, the ideal temperature is
A
0-4°C.
23
Q
bone marrow continues to undergo mitosis (growth) up to [?] after death when refrigerated
A
30 minutes
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
25
for electron microscopy
1 to 2 mm2
26
wide for light microscopy
2 cm2
27
Brain is usually suspended whole in [?] for [?] to ensure fixation and some hardening prior to sectioning.
10% buffered formalin
2-3 weeks
28
[?] 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 (?).
Formalin and alcohol
glutaraldehyde
Mercurials
2 to 3 mm
29
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.
Formalin
30
It is not practical or within the realm of patient care to wait any longer than [?], since clinicians generally need prompt diagnosis.
1 or 2 days
31
To maintain an adequate fixation time of [?], the recommended size of the tissue is [?], and no more than [?]. thick.
4 to 6 hours
2 cm2
4 mm
32
A commonly quoted rate of penetration for aldehyde fixative is
two- to-three millimeter per hour.
33
For solid material (e.g., liver) the longest dimension should not exceed
10-15mm.
34
shrink; swell and burst
hypertonic; hypotonic
35
For that reason, we recommend using a [?] based fixative.
normal phosphate buffered saline (PBS)
36
give rise to cell shrinkage; cause cell swelling and poor fixation.
Hypertonic solutions; Isotonic and hypotonic fixatives
37
The best results are usually obtained using slightly hypertonic solutions (?).
400-450 mOsm; isotonic solutions are 340 mOsm
38
is commonly added to osmium tetroxide fixatives for electron microscopy.
Sucrose
39
Formaldehyde is normally used as a [?], and glutaraldehyde is normally used as a [?].
10% solution; 3% solution
40
The presence of a [?] causes polymerization of the aldehyde, with consequent decrease in its effective concentration.
buffer
41
Low concentrations of [?] have been found to be an ideal concentration for immuno-electron microscopy.
glutaraldehyde (0.25%)
42
Fibrous organs such as [?] take longer to fix than small or loosely textured tissues such as [?].
uterus or intestinal tract
biopsies or scrapings
43
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.
2-6 hours
44
Most of the formalin can be washed out after fixation for
24 hours.
45
For electron microscopy, it is recommended that diced tissues be fixed for [?] and then placed in a holding buffer.
3 hours
46
Artefacts will be introduced by drying, so if the tissue is left out in the open, it should be kept moist with
saline.
47
There are four major groups of fixatives, namely the [?].
aldehydes, oxidizing agents, alcohol based fixatives and the metallic group of fixatives
48
act by cross-linking proteins.
aldehydes (formaldehyde, glutaraldehyde) and oxidizing agents (osmium tetroxide, potassium permanganate)
49
are protein-denaturing agents.
Alcohol based fixatives (methyl alcohol, ethyl alcohol, acetic acid)
50
act by forming insoluble metallic precipitates like mercuric chloride and picric acid.
Metallic fixatives
51
According to COMPOSITION
A. Simple Fixatives
B. Compound Fixatives
52
A. Simple Fixatives
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
2. According to ACTION
A. Microanatomical Fixatives
B. Cytological Fixatives
54
A. Microanatomical Fixatives:
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
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
56
B. Cytological Fixatives:
1. Nuclear Fixatives
2. Cytoplasmic Fixatives
3. Histochemical Fixatives
57
3. Histochemical Fixatives:
Formal Saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer's Fluid
58
-are made up of only one component substance.
Simple Fixatives
59
- 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.
Compound Fixatives
60
-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.
Microanatomical Fixatives
61
- are those that preserve specific parts and particular microscopic elements of the cell itself.
Cytological Fixatives
62
- are those that preserve the nuclear structures (e.g., chromosomes) in particular.
Nuclear Fixatives
63
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.
mercury and chromium salts
64
has been found to react with viruses, and causes the loss of their infective power.
Mercuric chloride
65
- are those that preserve cytoplasmic structures in particular.
Cytoplasmic Fixatives
66
They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm.
Cytoplasmic Fixatives
67
They have a pH of more than 4.6.
Cytoplasmic Fixatives
68
For RNA, the precipitant fixatives -[?]- give the best quantitative results using frozen tissues as the standard.
ethanol and acetone
69
- are those that preserve the chemical constituents of cells and tissues.
Histochemical Fixatives
70
Secondary fixation may be done before dehydration and on deparaffinized sections before staining, usually with [?] as a primary fixative.
10% formalin or 10% formol saline
71
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).
Zenker's solution
Masson's trichrome
Mallory's aniline blue stain
phosphotungstic acid-hematoxylin (PTAH) stain
72
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.
Post-Chromatization
73
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.
Washing out
74
[?] 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
1. Tap water
75
is used to wash out excess amount of picric acid (Bouin's solution).
2. 50-70% alcohol
76
is used to remove excessive mercuric fixatives.
3. Alcoholic iodine
77
Slow freezing of unfixed tissues near [?] must be avoided since this may promote formation of ice crystal artefacts.
0°C
78
Tissues should not be more than [?] thick except in lung edema (in which case tissue slices may be [?] thick), with minimum squeezing and handling.
5 mm
1-2 cm
79
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.
10-20
5-10
80
For prolonged fixation (e.g. museum preparation) volume of fixing fluid should not be less than [?] times that of the tissue.
50
81
Human brains may be suspended by a cord tied under the [?] to prevent flattening.
Circle of Willis
82
(washing out of blood with Ringer's lactate) may lead to artifact formation with loss of blood content.
Intravascular perfusion
83
Dense tissues such as brains are poorly penetrated, hence require long fixation (usually [?] prior to sectioning).
2 weeks
84
must be injected before immersing the eye in the fixative.
Formal-alcohol
85
Frozen sections may lead to formation of
ice crystal artifacts.
86
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.
Lendrum's method
87
An adequate volume is vital (at least [?]).
20:1
88
Some formulated fixatives should be prepared from stock solutions immediately before use because they are unstable (e.g.,).
Helly’s fluid
89
Avoid metal lids. Some fixatives are highly corrosive and will attack metals (e.g.,).
mercury salts
90
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%).
Zenker’s or Helly’s
phosphate
91
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.
"Crush artifact"
92
This may be due to partial coagulation of partially fixed protein by ethanol or by incomplete wax impregnation during subsequent histological processing.
"Crush artifact"
93
such as alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture.
Organic solvents
94
form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens.
Cross-linking reagents (such as paraformaldehyde)
95
Acetone Fixation
Fix cells in [?] acetone for 5-10 minutes.
-20°C
96
No permeabilization step needed following acetone fixation.
Acetone Fixation
97
Methanol Fixation
Fix cells in [?] methanol for 5-10 minutes.
-20°C
98
Permeabilization step is needed following methanol fixation.
Methanol Fixation
99
Ethanol Fixation
Fix cells in cooled [?] for 5-10 minutes.
95% ethanol, 5% glacial acetic acid
100
Methanol-Acetone Fixation
Fix in cooled methanol, 10 minutes at [?].
–20 °C
101
Remove excess methanol.
Methanol-Acetone Fixation
102
Permeabilize with cooled acetone for 1 minute at –20 °C.
Methanol-Acetone Fixation
103
1:1 methanol and acetone mixture.
Methanol-Acetone Mix Fixation
104
Make the mixture fresh and fix cells at -20 C for 5-10 minutes.
Methanol-Acetone Mix Fixation
105
1:1 methanol and ethanol mixture.
Methanol-Ethanol Mix Fixation
106
Make the mixture fresh and fix cells at -20 C for 5-10 minutes.
Methanol-Ethanol Mix Fixation
107
Fix cells in 10% neutral buffered formalin for 5-10 minutes.
Formalin Fixation
108
Rinse briefly with PBS.
Formalin Fixation
109
Permeabilize with 0.5% Triton X-100 for 10 minutes.
Formalin Fixation
110
Fix in 3-4% paraformaldehyde for 10-20 minutes.
Paraformaldehyde-Triton Fixation
111
Rinse briefly with PBS.
Paraformaldehyde-Triton Fixation
112
Permeabilize with 0.5% Triton X-100 for 10 minutes.
Paraformaldehyde-Triton Fixation
113
Rinse briefly with PBS.
Paraformaldehyde-Methanol Fixation
114
Fix in 4% paraformaldehyde for 10-20 minutes.
Paraformaldehyde-Methanol Fixation
115
Permeabilize with cooled methanol for 5-10 minutes at –20 °C.
Paraformaldehyde-Methanol Fixation
