MIDTERM LEC: FIXATION Flashcards
1
Q
First and most critical step in tissue processing because if fixation is inadequate, the succeeding tissue processing steps will also
be inadequate
A
FIXATION
2
Q
Primary purpose:
Preserve morphological & chemical integrity of cell in a lifelike manner as possible by stopping all cellular activities
A
FIXATION
3
Q
Performed as soon as tissue is removed from the body
A
FIXATION
4
Q
If tissues/cells are exposed to:
a. Air
A
drying of tissue
5
Q
If tissues/cells are exposed to:
b. Water
A
swelling of cells
6
Q
If tissues/cells are exposed to:
c. Saline
A
shrinkage of cell
7
Q
Effects of Fixatives:
A
■ Hardens soft tissues in preparation for further tissue processing
■ Render cells resistant to damage caused by chemicals used in further processing
■ Inhibit decomposition caused by bacteria and fungi
■ Minimize the risk of occupational infection
■ Act as mordant for certain stains, thus promoting or hastening staining, or inhibit certain dyes
8
Q
Characteristics of Ideal Fixative:
A
- Cheap
- Stable
- Safe to handle
- Kill cells quickly to minimize cell distortion
- Inhibit bacterial decomposition and autolysis
- Permit rapid and even penetration of tissues
- Must harden tissues thus easier cutting of tissues
- Must make cellular components insoluble to hypotonic solutions, and insensitive to subsequent processing
- Permit application of staining procedures
9
Q
Fixative will forms cross-links between
soluble molecules, thus gluing them
together into an insoluble meshwork
Mechanism of Fixation
A
Additive Fixation
10
Q
Fixative will not chemically bind with tissue
but removes water from tissue protein
groups thus causing denaturation of cell
proteins
Mechanism of Fixation
A
Non-additive Fixation
11
Q
FACTORS AFFECTING FIXATION:
A
- Fixative of Choice
- Time
- Tissue-to-fixative ratio
- Penetration time
- Thickness of section
- Tissue components
- Hydrogen ion concentartion (ph)
- Temperature
- Osmolality
- Agitation, Vacuum
12
Q
Fixative of Choice
Factors Affecting Fixation
A
10% Neutral Buffered Formalin (NBF)
13
Q
Morphologic criteria for diagnosis have been established based on______
A
FormalinFixed Paraffin Embedded Specimen (FFPES)
14
Q
Fixation must be done ________ after
cutting off blood supply (to shorten
warm ischemia time)
A
20-30mins
15
Q
Prolonged fixation ->
A
shrinkage
16
Q
Tissue-toFixative Ratio
A
1:10 or 1:20
17
Q
common Tissue-toFixative Ratio
A
1:20
18
Q
Tissue-toFixative Ratio Osmic acid fixatives:
A
1:5
19
Q
Penetration Rate Formalin:
A
1 mm/hr (but slows down as
it goes deeper into the tissue)
20
Q
Larger->
Thickness of setion
A
Longer fixation time, more
fixative
21
Q
Light Microscopy:
Thickness of setion
A
2cm2 x 0.4cm
22
Q
Electron Microscopy:
Thickness of setion
A
1-2 mm2
23
Q
Longer fixation time:
Tissue Components
A
- Fibrous tissues
- Presence of Mucus (wash with NSS)
- Fat (cut into thin slices fixed longer)
- Blood (flushed out with saline)
24
Q
Shorter fixation time:
Tissue Components
A
Small or loosely textured tissues
25
Optimal pH:
| Hydrogen Ion Concentration (pH)
6 to 8
26
If outside this pH, ultrastuctural
changes may occur
Hydrogen Ion Concentration (pH)
27
May require the use of buffers
Hydrogen Ion Concentration (pH)
28
For Electron microscopy:
| Hydrogen Ion Concentration (pH)
pH should match physiologic pH
29
Higher temp ->
faster fixation rate and
autolysis
30
Cold temp ->
enzyme inactivation
31
Optimal Temperature (routine):
Room temp to 45C
32
Tissue processors:
40 C
33
Microwave processing:
Up to 65C
34
Electron microscopy:
0-4C
35
Tuberculosis:
100C
36
Rapid biopsy:
60C
37
Hypertonicity ->
| Osmolality
cell shrinkage
38
Isotonicity and hypotonicity ->
| Osmolality
cell swelling
39
Thus, maintain tissues at slightly
hypertonic solution (400-450 mOsm)
Osmolality
40
Hastens fixation
Agitation, Vacuum
41
TYPES OF FIXATIVES
According to Composition:
* simple
* compound
42
According to Composition:
made of one component
| TYPES OF FIXATIVES
SIMPLE
43
According to Composition
SIMPLE:
| TYPES OF FIXATIVES
i. Aldehyde
a. Formaldehyde
b. Glutaraldehyde
ii. Metallic Fixatives
a. Mercuric chloride
b. Chromate
c. Lead
iii. Picric acid
iv. Glacial acetic acid
v. Alcohol
vi. Osmium Tetroxide
vii. Trichloroacetic acid
viii. Acetone
ix. Heat
44
According to Composition:
2 or more components or fixatives
| TYPES OF FIXATIVES
COMPOUND
45
According to Action:
| TYPES OF FIXATIVES
* Microanatomical
* Cytological
* Histochemical
46
permits general study of tissues without altering the structure of the subjects of interest
Microanatomical
47
Microanatomical:
(10,10,HFZZBB)
1. 10% NBF
2. 10% Formol-Saline
3. Heidenhain’s Susa
4. Formol-Sublimate/Corrosive
5. Zenker’s
6. Zenker-formol (Helly’s)
7. Bouin’s
8. Brasil’s
48
preserve specific parts of the cell
Cytological
49
Cytological 2 TYPES:
1. Nuclear Fixatives
2. Cytoplasmic Fixatives
50
o Preserve nucleus
Nuclear Fixatives
51
o pH ≤ 4-6
Nuclear Fixatives
52
o Glacial acetic acid has affinity to nuclear chromatin
Nuclear Fixatives
53
Nuclear Fixatives:
(FCBNH)
a. Flemming’s with glacial acetic acid
b. Carnoy’s
c. Bouin’s
d. Newcomer’s
e. Heidenhain’s
54
o Other organelles aside
from nucleus
Cytoplasmic Fixatives
55
o pH > 4-6
Cytoplasmic Fixatives
56
o HAc destroys
mitochondria and Golgi
bodies
Cytoplasmic Fixatives
57
Cytoplasmic Fixatives:
(HORFF)
a. Helly’s
b. Orth’s
c. Regaud’s/Moller’s
d. Formalin with Post-chroming
e. Flemming’s without glacial acetic acid
58
Preserves chemical constituents of cells & tissues
Histochemical
59
Histochemical:
(10FANA)
1. 10% Formol Saline
2. Absolute ethanol
3. Newcomer’s
4. Acetone
60
RANGE TISSUE-TO-FIXATIVE RATIO:
1:15-1:20
61
RECOMMENDED TISSUE-TO-FIXATIVE RATIO:
1:10
62
For routine HP techniques
| I. ALDEHYDES
Formaldehyde AKA Formalin
63
Produced from oxidation of methanol
| I. ALDEHYDES
Formaldehyde AKA Formalin
64
Usually buffered to pH 7 with phosphate buffer
| I. ALDEHYDES
Formaldehyde AKA Formalin
65
Formaldehyde AKA Formalin
Concentrations:
100%
| I. ALDEHYDES
GAS FORM
66
Formaldehyde AKA Formalin
Concentrations:
37-40%
| I. ALDEHYDES
stock concentration (causes overhardening
of the external surfaces of tissues)
67
Formaldehyde AKA Formalin
Concentrations:
10%
| I. ALDEHYDES
working solution; most commonly used
68
Formaldehyde AKA Formalin ADVANTAGES:
| I. ALDEHYDES
cheap, readily available, easy to prepare, stable, compatible with most stains
69
Formaldehyde AKA Formalin DISADVANTAGES:
| I. ALDEHYDES
nose and eye-irritant, may cause allergic dermatitis
70
Formalin diluted with 10% NaCl
| I. ALDEHYDES
10% Formol-Saline
71
Traditionally, the most common fixative
| I. ALDEHYDES
10% Formol-Saline
72
Recommended for CNS tissue and general post-mortem tissues for histochemical examination
| I. ALDEHYDES
10% Formol-Saline
73
# ```
10% Formol-Saline ADVANTAGES:
| I. ALDEHYDES
ideal for Silver impregnation staining technique
74
10% Formol-Saline DISADVANTAGES:
| I. ALDEHYDES
tissue shrinks during alcohol dehydration [Remedy: Secondary fixation]
75
pH 7
| I. ALDEHYDES
10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin
76
# ```
Best general tissue fixative
| I. ALDEHYDES
10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin
77
Best for iron-containing pigments and elastic fibers which do not stain well after Susa, Zenker or Chromate fixation,
| I. ALDEHYDES
10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin
78
# ```
10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin DISADVANTAGES:
| I. ALDEHYDES
longer to prepare, inert to phospholipids and neutral
fats
79
Has HgCl2
| I. ALDEHYDES
Formol-Sublimate/Corrosive
80
Formol-Sublimate/Corrosive ADV:
| I. ALDEHYDES
Excellent for silver reticulum staining method, does not need washing, fixes lipids
81
Formol-Sublimate/Corrosive DISADVANTAGES:
| I. ALDEHYDES
forms mercuric chloride deposits
82
Has 95% ETOH, Picric acid, and GHAc
| I. ALDEHYDES
Gendre’s (Alcoholic Formalin)
83
# ```
Gendre’s (Alcoholic Formalin) ADV:
| I. ALDEHYDES
good for microincineration techniques, fixes sputum
84
For gastrointestinal (GI) tissues, prostate biopsies, and bone marrow (BM)
| I. ALDEHYDES
Hollande’s
85
Made up of 2 formaldehyde resides linked by three carbon
chains
| I. ALDEHYDES
Glutaraldehyde
86
# ```
For enzyme histochemistry and electron microscopy
| I. ALDEHYDES
Glutaraldehyde
87
Glutaraldehyde ADVANTAGES:
| I. ALDEHYDES
more pleasant and less irritating compared to formalin
88
Glutaraldehyde DISADVANTAGES:
| I. ALDEHYDES
Less stable and more expensive than formalin
89
Container must be refrigerated
| I. ALDEHYDES
Glutaraldehyde
90
Glutaraldehyde
Concentrations:
for immunoEM
| I. ALDEHYDES
0.25%
91
Glutaraldehyde
Concentrations:
for small TSE fragments
| I. ALDEHYDES
2.5%
92
Glutaraldehyde
Concentrations:
most common
| I. ALDEHYDES
3%
93
Glutaraldehyde
Concentrations:
for large TSE
| I. ALDEHYDES
4%
94
Polymer of formalin
| I. ALDEHYDES
Paraformaldehyde
95
Powder in form, used in 4%
| I. ALDEHYDES
Paraformaldehyde
96
Plastic embedding
| I. ALDEHYDES
Paraformaldehyde
97
For ultrathin and electron microscopy
| I. ALDEHYDES
Paraformaldehyde
98
Acrolein in glutaraldehyde or formalin
| I. ALDEHYDES
Karnovsky’s Paraformaldehyde/Glutaraldehyde
99
P: for Electron Histochemistry and Electron Immunocytochemistry
| I. ALDEHYDES
Karnovsky’s Paraformaldehyde/Glutaraldehyde
100
ADV: no smudging of nuclei and distortion of staining compared with formalin
| I. ALDEHYDES
40% Aqueous Glyoxal
101
D: reduced staining capacity
[Remedy: increase staining time]
40% Aqueous Glyoxal
102
METALLIC FIXATIVES:
1. Mercuric chloride
2. Chromates
3. Lead
103
Mercuric chloride:
| II. METALLIC FIXATIVES
(ZZCHBS)
Zenker
Zenker-Formol (Helly’s)
Carnoy-Lebron
Heidenhain’s Susa
B5
Schaudinn’s
104
Chromates:
| II. METALLIC FIXATIVES
(CROP)
Chromic acid
Regaud’s/Muller’s
Orth’s
Potassium dichromate
105
Most common metallic fixative
| II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
106
A: penetrates and hardens tissue rapidly
| II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
107
Routine fixative of choice for preservation of cell detail in tissue photography
| II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
108
Conc. 5-7%
| II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
109
Mostly incorporated in compound fixatives
| II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
110
DADV: Banned worldwide d/t extreme toxicity, marked cell shrinkage [Remedy: add acid]
| II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
111
May produce black granular deposits except in Heidenhain’s Susa
| II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
112
May produce black granular deposits except in Heidenhain’s Susa
Mercuric Chloride (HgCl2) Remedy:
| II. METALLIC FIXATIVES
Dezenkerization
0.5% Iodine + 70% ETOH H20 5% Na thiosulfate H20
113
HgCl2 + potassium dichromate + glacial acetic acid
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Zenker’s
114
Good general fixative for adequate preservation of all kinds of tissues
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Zenker’s
115
Good for Trichrome staining
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Zenker’s
116
HgCl2 + potassium dichromate + strong formalin (40%)
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Zenker-formol/Helly’s
117
For piituitary gland, BM, blood-containing organs, preserves cytoplasmic granules
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Zenker-formol/Helly’s
118
Brown pigments are removed with saturated alcoholic picric acid or NaOH
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Zenker-formol/Helly’s
119
Susa: Su = sublimate ; Sa = saure (acid)
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Heidenhain Susa
120
HgCl2 + NaCl + TCA + glacial acetic acid + formalin
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Heidenhain Susa
121
Skin tumor biopsy
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Heidenhain Susa
122
ADV: minimum cell shrinkage and tissue hardening due to counter-balance effect of acids and mercury:
Acids : swelling
Mercury: shrinkage
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Heidenhain Susa
123
Does not produce black pigments
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Heidenhain Susa
124
DADV: Weigert’s staining of elastic fibers not possible
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
Heidenhain Susa
125
# ```
HgCl2 + Anhydrous Na acetate
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
B5 Fixative
126
BM biopsies
| II. METALLIC FIXATIVES
## Footnote
Mercuric Chloride (HgCl2)
B5 Fixative
127
Conc.: 1-2% aqueous solutions
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
Chromic acid
128
Precipitates all proteins, and preserves carbohydrates
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
Chromic acid
129
P: chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid-containing TSEs
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
Regaud’s/Muller’s
130
DADV: prolonged fixation may lead to blackening of tissue pigment
[Remedy: Wash in running tap water before
dehydration]
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
Regaud’s/Muller’s
131
P: early degenerative processes and necrosis,
demonstration of Rickettsia and other bacteria
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
Orth’s
132
E: preserves myelin
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
Orth’s
133
E: preserves lipids, mitochondria, at pH4.5-5.2,
cytoplasm, chromatin and chromosome are fixed
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
3% Potassium dichromate
134
Corrosive, thus avoid skin contact
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
3% Potassium dichromate
135
P: for acid mucopolysaccharides and mucin
| II. METALLIC FIXATIVES
## Footnote
Chromate Fixatives
4% Aqueous Lead
136
DADV: Prolonged standing formation of insoluble lead carbonate
[Remedy: add drops of acetic acid to dissolve residue]
| II. METALLIC FIXATIVES
4% Aqueous Lead
137
Used in strong saturated aqueous solution (1%)
PICRIC ACID
138
For Glycogen preservation
PICRIC ACID
139
ADV: may be used as a stain as yellowing of tissue will prevent small fragments from being overlooked; suitable also with Aniline stains
PICRIC ACID
140
PICRIC ACID DISADVANTAGES:
1. Explosive when dry
[Remedy: add distilled H2O or 0.5-1% saturated alcohol]
2. Yellowing of tissues excessive staining
[Remedy: immerse in Li2CO3 with 70%ROH water
70% ethanol 5% Na thiosulfate water]
3. RBC hemolysis
(PBB)
141
PICRIC ACID:
(PBB)
1. Bouin’s
2. Brasil’s
142
P: for embryo and pituitary biopsies, and tissues to be stained with Masson’s Trichrome
| III. PICRIC ACID
Bouin’s
143
ADV: minimum cell shrinkage and tissue hardening due to counter-balance effect of glacial acetic acid (swelling) and picric acid (shrinking)
| III. PICRIC ACID
Bouin’s
144
DADV: poorly penetrates large tissue, thus limited to small fragments of tissues
| III. PICRIC ACID
Bouin’s
145
C: TCA
| III. PICRIC ACID
Brasil’s
146
ADV: Better and less messy than Bouin’s
| III. PICRIC ACID
Brasil’s
147
Incorporated in compound fixatives
GLACIAL ACETIC ACID
148
Solidifies at 17CI
GLACIAL ACETIC ACID
149
Important for nuclear fixatives (precipitates nucleoproteins, chromatins)
GLACIAL ACETIC ACID
150
Destroys mitochondria and Golgi elements, thus not for cytoplasmic fixation
GLACIAL ACETIC ACID
151
ADV: good for glycogen
ALCOHOL FIXATIVES
152
DADV: never for FATs and LIPOPROTEINS (dissolves); causes polarization of glycogen (granules will move towards the poles
or ends of the cells)
ALCOHOL FIXATIVES
153
Effect: rapidly denatures and precips CHONs, preserves nuclear
stains
ALCOHOL FIXATIVES
154
ALCOHOL FIXATIVES:
(CEMING)
1. Carnoy’s Fixative
2. 70-100% Ethanol
3. 100% Methanol/Wood alcohol
4. 95% Isopropyl Alcohol/Rubbing Alcohol
5. Newcomer’s
6. Gendre’s (Alcoholic Formalin)
155
Most rapid tissue fixative
Carnoy’s Fixative
156
Fixing brain tissues for rabies diagnosis
Carnoy’s Fixative
157
E: fixes Nissl granules (Tigroid substance) and cytoplasmic
granules
Carnoy’s Fixative
158
# 1.
Enzyme studies
70-100% Ethanol
159
Does not fix but preserves glycogen
70-100% Ethanol
160
Dry and wet smears, BM smears, bacterial smears
100% Methanol/Wood alcohol
161
Touch prep smears to be Wright-stained
95% Isopropyl Alcohol/Rubbing Alcohol
162
Mucopolysaccharides and nuclear CHONs
Newcomer’s
163
# ```
Better reaction in Feulgen stain than Carnoy’s
Newcomer’s
164
Pale yellow powder in water (6% in 20C)
OSMIUM TETROXIDE / OSMIC ACID
165
Ultrathin sections in Electron Microscopy
OSMIUM TETROXIDE / OSMIC ACID
166
E: Fixes and stains conjugated fats and lipids blac
OSMIUM TETROXIDE / OSMIC ACID
167
DADV: very expensive, very volatile, inhibits hematoxylin
OSMIUM TETROXIDE / OSMIC ACID
168
OSMIUM TETROXIDE / OSMIC ACIDTissue-to-fixative ratio:
1:5
169
OSMIUM TETROXIDE / OSMIC ACID:
(OFF)
1. Flemming’s
2. Flemming’s w/o acetic acid
170
Most common osmic acid fixative
Flemming’s
171
P: nuclear structures
Flemming’s
172
Effect: permanently fixes fat
Flemming’s
173
ADV: needs less amount of fixative
Flemming’s
174
Cytoplasmic structures
Flemming’s w/o acetic acid
175
Incorporated also in compound fixatives
TRICHLOROACETIC FIXATIVES
176
Marked swelling effect on tissues
TRICHLOROACETIC FIXATIVES
177
Poor penetrating agent thus for small pieces of tissues or bones
TRICHLOROACETIC FIXATIVES
178
Weak decalcifying agent, thus has softening effect on dense fibrous tissues
TRICHLOROACETIC FIXATIVES
179
# 1.
Used at cold temp -5-4C
ACETONE
180
For water-diffusible enzymes (Phosphatase, Lipase)
ACETONE
181
For brain tissues (such as in Rabies)
ACETONE
182
DADV: Dissolves fat, evaporates rapidly
ACETONE
183
Principle: Thermal coagulation of tissue proteins
HEAT
184
For rapid diagnosis: frozen tissue sections and Bacterial smear
prep
HEAT
185
PCPL: Increases movement of molecules to accelerate fixation, staining, decalcification
Microwave Technique
186
ADV: Tissue is heated right through the block in a very short time; preserves neurochemical substances (acetylcholine)
Microwave Technique
186
Electron Microscopy and immunohistochemistry
Microwave Technique
187
DADV: Penetrates at 10-15mm thickness; spores and pathogen may remain in tissues
Microwave Technique
188
SECONDARY FIXATION:
* Refixation” with another fixative
* Post-Chromatization
188
Microwave Technique Optimum Temp:
45-55C
189
Done before dehydration or restaining of deparaffinized
TSEs
Refixation” with another fixative
190
Improve demonstration of substance
Refixation” with another fixative
191
Make special staining techniques possible (with the next fixative as mordant)
Refixation” with another fixative
192
Ensure further and complete handling
Refixation” with another fixative
193
Use of 2.5-3% aqueous K2Cr2O7 that will act as mordant
Post-Chromatization
194
Removal of excess fixative to improve staining and remove
artifacts
WASHING OUT
195
WASHING OUT:
1. Tap Water
2. 50-70% ROH
3. Alcoholic iodine
196
for excess formalin, osmic acid, and chromates
Tap Water
197
for excess picric acid fixatives and
Gendre’s
50-70% ROH
198
for excess mercuric chloride
Alcoholic iodine
