#3 FIXATION Flashcards
1
Q
What is the first and most critical step inside the laboratory?
A
Fixation/Preservation
2
Q
It is the process of preserving cells and tissue constituents
A
Fixation/Preservation
3
Q
This is to preserve the morphologic and chemical integrity of the cell as life-like manner as possible.
A
Primary Aim of Fixation/Preservation
4
Q
To harden and protect the tissue from the trauma of further handling
A
Secondary aim of fixation/preservation
5
Q
This is the most important reaction for maintaining tissue morphology
A
Stabilization of proteins
6
Q
The tissue is preserved by preventing?
A
Degeneration
Putrefaction
Decomposition
Distortion
7
Q
T or F: Fixatives have the property of forming cross-links between proteins
A
True
8
Q
T or F: Fixation must be done later upon removal of the tissue from oatients body.
A
FALSE: Later - immediately
9
Q
What is the process when tissue is submitted to the laboratory and examined by a pathologist, assisted by medtech?
A
Gross Examination
10
Q
What are the external appearance needed to observe for gross examination?
A
Size, shape, consistency, number, and color
11
Q
What is the measurement of sample in gross examination?
A
3D MEASUREMENT in CM (l,w,h)
12
Q
T or F: Effects of fixatives in general
-Harden soft and friable tissue
A
True
13
Q
T or F: Effects of fixatives in general
-Make tissue easy to damage and distortion
A
False: easy - resistant
14
Q
T or F: Effects of fixatives in general
Inhibit bacterial decomposition
A
True
15
Q
T or F: Effects of fixatives in general
Decrease optical differentiation of cells
A
False: decrease-increase
16
Q
T or F: Effects of fixatives in general
Act as mordants/accentuators
A
True
17
Q
T or F: Effects of fixatives in general
Increase the risk of infection
A
False: Increase - reduced
18
Q
How many hours will you observe changes in the tissues after fixation?
A
8 hours
19
Q
What is the standard fixation time in laboratory?
A
24-72 hours; overnight
20
Q
What are the 2 mechanism of action of fixative?
A
- Additive
- Non-additive
21
Q
It becomes part of the tissue by formation of cross links or complexes
A
Additive fixation
22
Q
It is not incorporated in the tissue, stabilizes tissue by removing of the bound water
A
Non-additive fixation
23
Q
Give examples of additive fixation
A
Formalin, Hg, Osmium tetroxide
24
Q
Give examples of additive fixation
A
Formalis, Hg, Osmium tetroxide
25
Give an examples of non-additive fixation?
Alcoholic Fixatives
26
What is the standard fixative pH?
6-8
27
What is the fixation temperature in autotech?
40C
28
What is the fixation temperature in Traditionally?
Room temperature
29
What is the fixation temperature in electron microscopy and histochem?
0-4C
30
What is the fixation temperature in Rapid examination?
60C
31
What is the fixation temperature in tissue w/ TB?
100C
32
What is the thickness in fixation of Light Microscopy?
2cm²
33
What is the thickness in fixation of brain tissue?
suspended whole 2-3 weeks (to preserve circle of willis)
34
What is the thickness in fixation of large solid tissue?
open or sliced thinly
35
What is the osmolality standard in fixation?
slightly hypertonic solution around 400-450mOsm
36
What are the factors involved in fixation?
1. pH
2. Temperature
3. Thickness
4. Osmolality
5. Time duration
37
What are the factors involved in fixation?
1. pH
2. Temperature
3. Thickness
4. Osmolality
5. Time duration
38
pertonic solution gives rise to?
cell shrinkage
39
In osmolality, what fixative causes cell swelling and poor fixation?
Isotonic and hypotonic
40
What are the 3 common concentration in fixation?
1. 10% formalin
2. 3% glutaraldehyde
3. 0.25% glutaraldehyde
41
What is the time duration for primary and EM fixation?
Primary: 2-6 hours
EM: 3 hours
42
What are the practical considerations in fixation?
1. Speed
2. Rate of penetration
3. Volume
4. Duration
43
T or F: Specimen should be placed in fixative as soon as it is removed from body to prevent autolysis and putrefaction
True
44
Tissue to fixative ratio?
1:20
45
What is the maximum effectiveness volume of tissue?
20:1
46
What is the Osmium tetroxide volume of tissue?
5-10:1
47
What is the volume of a prolonged fixation (museum preparation) tissue?
50-100:1
48
T or F: Fibrous organs (uterus) is faster than small tissues (biopsies or scraping)
False: faster-longer
49
What are the types of fixative?
Simple and compound fixative
50
What are the types of fixative?
Simple and compound fixative
51
What fixative are made up of only ONE component substance?
Simple Fixative
52
Give an example of simple fixative
Formaldehyde
Glutaraldehyde
Chromate fixatives
Lead fixatives
Picric acid
Acetic acid
53
These are made up of two or more fixatives which have been added together
Compound fixative
54
What are the types of fixative according to ACTION?
1. Microanatomical Fixative
2. Cytological fixative
a. Nuclear fixative
b. Cytoplasmic fixative
3. Histochemical fixative
55
These fixatives are those that permit the **general microscopic study** of tissue structures without altering the structural pattern.
Microanatomical fixative
56
What are the microanatomical fixatives?
1. 10% Formol saline
2. 10% Neutral buffered formalin (NBF)
3. Heidenhain's SuSa
4. Formol sublimate
5. Zenker’s solution
6. Zenker’s formol
7. Bouin’s solution
8. Brasil's solution
57
These preserve specific parts and particular microscope elements of the cell
Cytological fixative
58
it contains **glacial Acetic acid** and is the primary component due to its affinity for nuclear chromatin
Nuclear fixative
59
What is the pH of a nuclear fixative?
4.6 or less
60
What are the fixatives under nuclear fixative?
1. Bouin’s fluid
2. Flemming's fluid
3. Newcomers fluid
4. Carnoy’s fluid
5. Heidenhain's SuSa (BFNCH)
61
This fixative must never contain glacial Acetic acid cause it destroys **mitochondria and golgi bodies**
Cytoplasmic fixatives
62
What is the pH of Cytoplasmic fixatives?
more than 4.6
63
What are the examples of Cytoplasmic fixative?
1. Helly's fluid
2. Orth's fluid
3. Regaud's fluid
4. Flemming fluid w/o acetic acid
5. Formalin with post chroming (HORFF)
64
What are examples of histochemical fixative?
1. 10% Formol saline
2. Absolute Ethyl Alcohol
3. Newcomers fluid
4. Acetone (FANA)
65
What type of fixation has fixatives containing mercuric chloride and potassium dichromate in **cryostat section**?
Lipid fixation
66
In lipid, phospholipids can be stained with what aldehyde?
Bakers formol calcium
67
This fixation uses alcoholic fixative for glycogen
Carbohydrate Fixation
68
Give an example of carbohydrate fixation
Rossman's fluid or cold absolute alcohol
69
Protein fixation uses what type of fixative?
Neutral buffered formalin
70
Protein fixation uses what type of fixative?
Neutral buffered formalin
71
What are the different fixation techniques under histochemical fixative?
1. Lipid fixation
2. Carbohydrate fixation
3. Protein fixation
72
It is a gas produced by the oxidation of methyl alcohol
Formaldehyde (Folin)
73
T or F: In using formaldehyde (formalin), concentrated solutions should be neutralized (explosion)
False - concentrated solutions should **NOT** be neutralized.
74
What is the percentage of **stock solution**?
37-40%
75
If the 10% working solution has no buffer, what will happen?
It will become unstable
76
What are the formalin pigments?
a. Paraformaldehyde
b. Acid formaldehyde hematin
77
White crystalline precipitates, due to prolonged standing
Paraformaldehyde
78
What are the different aldehyde fixatives?
1. Formaldehyde (Formalin)
a. Paraformaldehyde
b. Acid formaldehyde hematin
2. 10% Formol Saline
3. 10% Neutral Buffered Formalin (NBF) or PO4 buffered formalin
4. Formol corrosive (formol sublimate)
5. Glutaraldehyde
6. Karnovsky's paraformaldehyde - glutaraldehyde
7. Acrolein
8. Formol-calcium
79
Paraformaldehyde is removed by?
10% METOH/filtration
80
It has brown/black granular deposits that may obscure microscopic details
Acid formaldehyde hematin
81
It is diluted in 10% NaCl and use for CNS
10% Formol saline
82
What may tend to overharden the outer layer of the tissue?
Pure stock solution (40%)
83
this is for routine post mortem tissues with **HgCl2**
Formol corrosive (formol sublimate)
84
this aldehyde fixative is used for EM
Glutaraldehyde
85
used for EM but mainly electron histochemistry & electron immunocytochemistry
Karnovsky's paraformaldehyde - glutaraldehyde
86
A mixture with **formaldehyde/formaldehyde**
acrolein
87
This aldehyde fixative is use for lipids (frozen section)
Formol-calcium
88
It refers to metallic chloride as the most common metallic fixative, used in saturated aqueous solution of 5-7%
DEZENKERIZATION
89
T or F: Tissues fixed with mercuric chloride contain black precipitates of mercury, but is removed by **0.5% iodine solution in 70% ethanol for 5-10mins**
True
90
for BONE MARROW (BM Biopsies), with fixation time of **1 half - 2 hours**
B5 fixative
91
B5 fixative FT?
1 half - 2 hours
92
For tumor biopsies esp of the skin, FT: 3-12hours
Heidenhain Susa
93
FT of Heidenhain SuSa?
3-12 hours
94
This metallic fixative fix small pieces of liver, spleen, CT fibers and nuclei, FT: 12-24 hours
Zenker
95
What is the FT of Zenker?
12-24 hours
96
Metallic fixative for pituitary gland, BM and blood containing organs.
Zenker's formol (helly's solution)
97
to demonstrate chromatin, mitochondria, mitotic figures, golgi bodies, RBC
Regaud's (mollers) (molliflex)
98
to study early degenerative processes and tissue necrosis, preserves myelin better
Orth's fluid
99
Preserves CHO
Chromic acid
100
Preserves lipids and mitochondria
Potassium Dichromate
101
What is the pH of Potassium dichromate?
4.5-5.2
102
it fixes Connective Tissue (CT) mucin and is recommended for acid mucopolysaccarides
Lead fixatives
103
This is highly explosive when dry, and has excessive **yellow staining** of tissues
Picric acid fixative
104
T or F: In picric acid, never wash in water before dehydration
True
105
This is a picric acid fixative recommended for embryos and pituitary biopsies
Bouin's
106
An excellent fixative for glycogen and less messy than Bouin's solution (excellent)
Brasil's alcoholic picroformol fixative
107
Glacial acetic acid solidifies at?
17C
108
What are the alcohol fixatives?
1. Methyl alcohol
2. Ethanol
3. Isopropyl alcohol
4. Carnoy's fluid
5. Alcoholic formalin (Gendre's fixative)
6. Newcomer's
109
for fixing dry and wet smears (PBS and BM)
Methyl alcohol
110
simple fixative with compound fixative for better results
Ethanol
111
for fixing touch preparation
Isopropyl alcohol
112
for fixing touch preparation
Isopropyl alcohol
113
for fixing chromosomes, lymph glands, and urgent biopsies and is **most rapid: 1-3 hours**
Carnoy's fluid
114
To preserve sputum
Alcoholic formalin (Gendre's fixative)
115
for fixing mucopolysacchardies and nuclear proteins
Newcomer's
116
Pale yellow powder which dissolves in water (up to 6% at 20C) to form a strong oxidizing solution
Osmium Tetroxide
117
MOST COMMON chrome-osmium acetic acid fixative used, permanently fixes fat for nuclear structures
Flemmings solution
118
this may cause conjunctivitis or blindness and black osmic oxide crystals
Osmium tetroxide
119
Precipitates proteins with **swelling effect**, poor penetrating agent. weak agent.
Thrichloroacetic acid
120
for fixing **brain tissue** rabies diagnosis
Acetone
121
microwave temperature
45-55C
122
Failure to arrest early autolysis of cells
Cause: failure to fix immediately
123
Removal of substances soluble in fixing agent
Cause: wrong choice of fixative
124
have presence of artifact pigments and tissues are soft and feather like
cause: incomplete fixation
125
Loss or inactivation of enzymes
cause: wrong choice of fixative
126
shrinkage and swelling of cells
cause: overfixation
127
tissue blocks are brittle and hard
cause: prolonged fixation
128
process of placing an already fixed tissue in a second fixative
secondary fixation
129
Fixation whereby a primarily fixed tissue is places in aqueous solution
Post-chromatization
130
a term for removing excess fixative
Washing out
131
Give a factor that affect fixation of tissues
1. size and thickness of tissue specimen
2. presence of mucus
3. presence of fat
4. presence of blood
5. cold temp
