FIXATION 2012 GREGORIOS Flashcards
It is the art and science performed by the histotechnologist to produce a tissue section of good quality
Histotechnology
What is the first and most critical step in histotechnology
fixation
What the primary aim of fixation?
to preserve the morphologic and chemical integrity of the cell in as life-like manner as possible
what is the cell composed of?
- proteins
- lipids
- carbohydrates
- organic
- inorganic
these are loosely held together in the cellular protoplasm by hydrogen bonds or salt linkages that are in constant state of chemical and biological equilibrium
cellular substances
in fixation, what are being preserved?
- shape
- structure
- intercellular relationships
- chemical constituents
Fixation prevents the what of tissues after removal from the body?
- degeneration
- putrefaction
- decomposition
- distortion
What happens to all vital cellular processes when the tissue is placed in a fixative?
they stop
The weak chemical associations are changed into what that will not break down with further histologic handling
changed into stable complexes
What is the secondary goal of fixation?
to harden and protein the tissue from the trauma of further handling, so that it is easier to cut during gross examination
How does fixation preserve the tissue?
by stopping all cellular activities
leaving the tissue in water, a hypertonic solution, will cause the cell to swell
True or false
false
hypotonic
leaving the tissue in water, a hypertonic solution, will cause the cell to swell
True or false
false
hypotonic
What happens to the cell if the tissue is left in water (hypotonic solution)?
the cell will swel
What happens to the cell if the tissue is left in water (hypotonic solution)?
the cell will swell
a strong salt (hypertonic solution) will the cell to swell
True or false
false
cell will shrink
a strong salt (hypertonic solution) will the cell to swell
True or false
false
cell will shrink
The surigcal removal of the tissue from the body will lead to what to the tissue?
leading to a degradative chemical process or cell death
cell death is caused by the deprivation of what?
deprivation of oxygen and nutrition
Every cell in the body has a structure or “suicide sac” within its cytoplasm called what?
lysosome
this contains hydrolytic enzymes that are released when the integrity of the cell is destroyed
lysosome
postmortem decomposition is called …
autolysis
this occurs due to the action of these hydrolytic enzymes
autolysis
Fixation prevents autolysis
true or false
true
How does fixation prevent autolysis?
by inactivating the lysosomal enzymes, or by chemically altering, stabilizing, and making the tissue components insoluble
Decomposition is also called …
putrefaction
Decomposition is also called …
putrefaction
Decomposition is also called …
putrefaction
Decomposition is also called …
putrefaction
Decomposition is also called …
putrefaction
Fixation also protects the tissue from further putrefaction after death due to what?
due to bacterial or fungal colonization and overgrowth
Fixation also protects the tissue from further putrefaction after death due to what?
due to bacterial or fungal colonization and overgrowth
What are the two basic mechanisms involved in fixation?
- additive fixation
- non additive fixation
This is the mechanism involved in fixation 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
ADDITIVE FIXATION
This is the mechanism involved in fixation whereby the chemical constituent of the fixative is taken in and BECOMES PART of the tissue by forming what?
by forming cross-links or molecular complexes
What are examples of additive fixation?
- formalin
- mercury
- osmium tetroxide
This is a mechanism involved in fixation 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
NON ADDITIVE FIXATION
What is an example of a non additive fixation?
alcoholic fixatives
What are the 6 MAIN FACTORS INVOLVED IN FIXATION
CTTHOD
- Concentration
- Temperature
- Thickness of section
- Hydrogen Ion concentration
- Osmolality
- Duration of Fixation
Satisfactory fixation occurs between what pH levels?
pH 6 and pH 8
Fixation of surgical specimens is traditionally carried out at what temperature?
at room temperature
Many laboratories use tissue processors that work at what temperature?
40C
For electron microscopy and some histochemistry the ideal temperature is what?
0-4C
What kind of cells are best fixed at room temperature even for electron microscopy?
mast cells
nucleic acids do not react with fixatives to any extent at 4C
True or false
FALSE
at room temperature
nucleic acids do not react with fixatives to any extent at 4C
True or false
FALSE
at room temperature
Nucleic acids do not react with chemical reactions including those involved in fixation are more rapid at higher temperatures
true or false
true
Formalin is heated at what temperature is sometimes used for rapid fixation of VERY URGENT biopsy specimens, although the risk of tissue distortionis increased?
60C
Formalin at what temperature can be used to fix tissues with tuberculosis?
100C
What must be the thickness of the section for electron microscopy?
1 to 2 mm^2
what is the thickness of the section in light microscopy?
2 cm^2
if the tissue must be thin, the measurement must be how thin for light microscopy?
0.4 cm
Large solid tissue, such as UTERUS, should not be opened or sliced thinly
True or false
false
should be opened or sliced thinly
Large solid tissue, such as UTERUS, should not be opened or sliced thinly
True or false
false
should be opened or sliced thinly
Brain is usually suspended in parts
True or False
FALSE
suspended whole
Brain is usually suspended in parts
True or False
FALSE
suspended whole
Brain is usually suspended whole in what fixative?
10% buffered formalin
Brain is usually suspended whole in 10% buffered formalin for how long?
2-3 weeks
What kind of solution gives rise to cell shrinkage?
Hypertonic solution
these fixatives causes cell swelling and poor fixation
Isotonic as well as hypotonic fixatives
Regarding osmolality, best results are usually obtained using what solution?
SLIGHTLY HYPERTONIC SOLUTIONS
Regarding osmolality, best results are usually obtained using what solution?
SLIGHTLY HYPERTONIC SOLUTIONS
How many mOsm is a slightly hypertonic solution?
400 - 450 mOsm
Isotonic solutions are 340 mOsm
True or false
TRUE
What is commonly added to osmium tetroxide fixatives for electron microscopy?
Sucrose
Sucrose is commonly added to what fixative for electron microscopy?
osmium tetroxide
Sucrose is commonly added to osmium tetroxide fixative for what?
electron microscopy
The vehicle osmolality is generally more important than the total osmolality fixative, and ideally should be more or less hypertonic with tissues in their normal living environment
True or false
TRUE
For glutaraldehyde fixatives, the vehicle osmolality should be more or less 300 mOsm
True or false
true
This is normally used as a 10% solution
Formaldehyde
Glutaraldehyde is normally used as a 3% solution
True or false
true
The presence of a what causes polymerization of the aldehyde, with consequent decrease in its effective concentration
The presence of a buffer
The presence of a buffer causes what?
Polymerization of the aldehyde
High concentrations of glutaraldehyde have been found to be an ideal concentration for immunoelectron microscopy
True or false
FALSE
Low concentrations
High concentrations of glutaraldehyde have been found to be an ideal concentration for immunoelectron microscopy
True or false
FALSE
Low concentrations
What is the ideal concentration of glutaraldehyde in immunoelectron microscopy?
low concentrations of glutaraldehyde (0.25%)
Primary fixation in buffered formalin is usually carried out for how long?
2-6 hours during the day the specimen is obtained
A specimen in primary fixation in buffered formalin may remain in fixative over the weekend without much adverse effect
True or false
true
Most of the formalin can be washed out after fixation in how many hours?
24 hours
Prolonged fixation may cause what?
- shrinkage
- hardening of tissue
- may severely inhibit enzyme activity and immunological reactions
Prolonged fixation may cause what?
- shrinkage
- hardening of tissue
- may severely inhibit enzyme activity and immunological reactions
Washing of the tissue in running water considerably restores the activity of some enzymes
true or false
true
For electron microscopy, it is recommended that diced tissues be fixed for how many hours?
3 hours
For electron microscopy, after fixing the diced tissues for 3 hours, where should it be placed?
in a holding buffer
What are the fixatives according to COMPOSITION?
SC
- Simple fixatives
- Compound fixatives
These are made up of only one component substance
Simple fixatives
These 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
What are the fixatives under Simple fixatives?
MA
- Aldehydes
- Metallic Fixatives
What are the aldehyde fixatives?
FG
- Formaldehyde
- Glutaraldehyde
What are the metallic fixatives?
MR. LEAD HEAT CHROMATE
- mercuric chloride
- heat
- lead fixatives
- chromate fixatives
What are the chromate fixatives?
CP
- Chromic acid
- Potassium dichromate
What are the lead fixatives?
AAAPO
- Acetic acid
- acetone
- alcohol
- Picric acid
- Osmium tetroxide (osmic acid)
What are the fixatives according to ACTION?
MC
- MICROANATOMICAL FIXATIVES
- CYTOLOGICAL FIXATIVES
These 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
These fixatives are those that preserve specific parts and particular microscopic elements of the cell itself
cytological fixatives
What are the MICROANATOMICAL FIXATIVES?
10 -10, B-B, Z-Z, H-F
- 10% Formol saline
- 10% neutral buffered formalin
- Brasil’s solution
- Bouin’s solution
- Heidenhain’s Susa
- Formol sublimate (formol corrosive)
What are the MICROANATOMICAL FIXATIVES?
10 -10, B-B, Z-Z, H-F
- 10% Formol saline
- 10% neutral buffered formalin
- Brasil’s solution
- Bouin’s solution
- Heidenhain’s Susa
- Formol sublimate (formol corrosive)
What are the cytological fixatives?
CHN
- Nuclear fixatives
- Cytoplasmic fixatives
- histochemical fixatives
These are those that preserve the NUCLEAR structures (e.g., chromosomes) in particular
Nuclear fixatives
Nuclear fixatives usually contain what as their primary component?
glacial acetic acid
Glacial acetic acid is a primary component in nuclear fixatives due to what?
due to its AFFINITY for nuclear chromatin
These fixatives have a pH of 4.6 or less
nuclear fixatives
What are the nuclear fixatives?
FBNCCH
- Flemming’s fluid
- Carnoy’s fluid
- Bouin’s fluid
- Newcomer’s fluid
- Heidenhain’s Susa
- Clarke’s fixatives
This nuclear fixative has been found to react with viruses, and cause the loss of their infective power
MERCURIC CHLORIDE
At temperatures normally used for fixation (20C), DNA and RNA do not react with formaldehyde
true or false
true
If reaction mixtures are heated, to about 45C in case of DNA and 65C in case of RNA, reaction begins to take place
TRUE OR FALSE
FALE
45C RNA; 65C DNA
If reaction mixtures are heated, to about 45C in case of DNA and 65C in case of RNA, reaction begins to take place
TRUE OR FALSE
FALE
45C RNA; 65C DNA
If reaction mixtures are heated, to about 45C in case of RNA and 65C in case of DNA, reaction begins to take place due to what?
due to the coiling of DNA and RNA
Coiling is also observed in glutaraldehye and nucleic acids
true or false
true
What percent is lost during usual fixation when aldehydes continue to react with nucleic acids longer than after they are placed in fixative?
30%
These fixatives are commonly used fixatives for nucleic acids
m-etha-noy
- methanol
- ethanol
- carnoy’s
The presence of WHAT is known to be essential for the maximum precipitation of nucleic acids FROM ALCOHOL?
SALTS
In this state, there is no significant difference between fixation in neutral buffered formaldehyde, B5, Hollande, and zinc formaldehyde
IN SITU HYBRIDIZATION
This fixative appears to give the most usable DNA fragments for polymerase chain reactions (PCR)
ETHANOL
This fixative limits the size of fragments which can be retrieved
formaldehyde
These are limited in its use for PCR
BA
- Bouin’s sol’n
- acid fixed tissue
These fixatives are those that preserve cytoplasmic structures in particular
cytoplasmic fixatives
These fixatives must never contain glacial acetic acid
cytoplasmic fixatives
Using glacial acetic acid in cytoplasmic fixatives destroys what?
destroys mitochondria and Golgi bodies of the cytoplasm
ctyoplasmic fixatives have a pH of 4.6
true or false
false
more than 4.6
What are the cytoplasmic fixatives?
FFORH
- Flemming’s fluid withoud HAC
- Formalin with “post-chroming”
- Orth’s fluid
- Regaud’s fluid
- Helly’s fluid
Regaud’s fluid is also called …
Muller’s fluid
For RNA, what precipitant fixatives give the best QUANTITATIVE results using FROZEN tissues as the standard
ethanol, acetone
These are the fixatives that preserve the chemical constituents of cells and tissues
histochemical fixatives
What are the histochemical fixatives?
FANA
- Formol saline 10%
- Absolute Ethyl Alcohol
- Newcomer’s Fluid
- Acetone
What should be used for demonstrating lipid in tissues?
Cryostat or frozen section followed by a general lipid stain
Fixatives containing these fixatives can be effective for preservation of lipids in cryostat sections
- mercuric chloride
- potassium dichromate
in general, phospholipids contain what?
amino groups
Phospholipids are fixed by what?
by aldehydes
This fixatives reacts with unsaturated fatty acids during histological fixations
formaldehyde
This can be used to preserve phospholipids
Baker’s formol-calcium
Improved ultrastructural demonstration of lipids has been achieved by what?
by post-fixing in IMIDAZOLE OSMIUM TETROXIDE
This fixes cholesterol for ultrastructural demonstration
DIGITONIN
What is fixed by digitonin for ultrastructural demonstration
cholesterol
These fixatives are generally recommended for glycogen fixation
alcoholic fixatives
This can be demostrated satsfactorily for diagnosis, although losses of this can be high (60%-805) in aqueous solution
glycogen
This is a better fiative in HUMAN SKIN compared with NETRUAL BUFFERED FORMALDEHYDE
Alcoholic formaldehyde
Alcoholic formaldehyde is a better fixative in human skin compared with neutral buffered formaldehyde
true or false
true
These are the most commonly used fixatives for AMINO ACID HISTOCHEMISTRY
- neutral buffered formol saline
- formaldehyde vapor
The most useful fixatives for preserving glycogen are alcohol-based
true or false
true
These are alcohol-based fixatives that are the most useful for preserving glycogen
- Rossman’s fluid
- cold absolute alcohol
This is essential when processing tissues from patients with glycogen storage diseases.
- Rossman’s fluid
- cold absolute alcohol
- glycogen fixatives
There is better retention of glycogen if the section is coated with what?
celloidin
These aldehyde fixatives have been particularly useful for electron cytochemistry
- Karnovsky’s paraformaldehyde-glutaraldehyde
- acrolein
This is another aldehyde which has been introduced as a mixture with glutaraldehyde and formaldehyde
Acrolein
This fixative penetrates tissues rapidly, preserves morphology and enzyme activity at low concentrations
Acrolein
This may be useful for immersion fixation of surgical biopsies
acrolein
These fixatives are satisfactory for routine paraffin sections, electron microscopy, histochemical and enzyme studies
Aldehyde
Formalin is also called
formaldehyde
One of the most widely used fixatives
10% formalin
10% formalin is made from what
formaldehyde
This is a gas produced by the oxidation of methyl aalcohol
10% formalin
10% FORMALIN IS SOLUBLE IN WATER TO THE EXTENT OF 37-40% weight in volume
true or false
true
The commercially available solution of formaldehyde contains 35-40% gas by weight
true or false
true
This is unsatisfactory for routine fixation since high formaldehyde concentrates tend to overharden the outer layer of the tissue and affecting staining adversely
Pure stock solution of 40% formalin
Formaldehyde is commonly used as a 2% solution, giving 10% formalin for tissue fixation
true or false
false
4%
Formaldehyde is commonly used as a 2% solution, giving 10% formalin for tissue fixation
true or false
false
4%
dilution for formalin
1:10 or 1:20
What is the usual fixation time for formalin
24 hours
Formaldehyde is usually buffered to what pH?
pH 7 with phosphate buffer
It is a “tolerant’ fixative, used for mailing specimen
formalin
This is a simple microanatomical fixative made up of saturdated formaldehyde (40% by weight volume)
10% formol saline
Dilution of 10% formol saline
10% with sodium chloride
this is recommended for fixation of central nervous tissues
10% formol saline
this is recommended for general post mortem tissues for histochemical examinations
10% formol saline
Fixation time of 10% formol saline at 35C
24 HOURS
Fixation of 10% formol saline at 20-25C
48 hrs
It penetrates and fixes tissues evenly
10% formol saline
It preserves microanatomic and cytologic details with minimum shrinkage and distortion
10% formol saline
It preserves enzymes and nucleoproteins
10% formol saline
10% Neutral Buffered Formaline is also called
Phosphate Buffered formalin
10% NBF has a pH of 7
true or false
true
This is recommended for preservation and storage of surgical, post-mortwm, and research specimens
10% NBF
wHAT IS THE FIATION TIME OF PHOSPHATE BUFFERED FORMALIN?
4-24 hours
It is the best fixative for tissues containing iron pigments and for elastic fibers
10% NBF
Formol corrosive is also called …
formol sublimate
This solution is recommended for routine post mortem-tissues
formol-mercuric chloride
What is the ixation time of formol corrsive?
3-24 hours
It penetrates small pieces of tissues rapidly
formol sublimate
Alcoholic formalin is also called …
Gendre’s fixative
This fixative is good for preservation of glycogen and micro-incineration
Gendre’s fixation
It is used to fix sputum
Alcoholic formalin
It causes partial lysis of RBC
ALCOHOLIC FORMALIN
This is most effective at cross linking
glutaraldehyde
Made up of two formaldehyde residues, linked by three carbon chains
glutaraldehyde
it acts in a manner similar to formaldehyde
glutaraldehyde
It is followed by secondary fixation in osmium tetroxide. It is satisfactory for electron microscopy
Buffered glutaraldehyde
It is the most common metallic fixative
Mercuric chloride
It is widely used as a secondary fixative reacting with a number of amino acid residues and accompanied by spectroscopic changes
Mercuric chloride
It penetrates poorly and produces shrinkages of tissues
mercuric chloride
Contains black precipitates of mercury
mercuric chloride
these are removed from deparaffinized sections before staining
mercuric chloride
Trichrome staining is excellent with this fixative
mercuric choride
It is recommended for renal tissues, fibrin, connective tissues, and muscles
merucric chloride
Is made up of mercuric chloride stock solution to which glacial acetic acid has been added just before its use to prevent turbidity and formation of a dark precipitate
ZENKER’S FLUID
It is a good general fixaive for adequate preservation of all kinds of tissues
Zenker’s fluid
It is recommended for fixing small pieces of liver, spleen, connective tisse fibers and nuclei
Zenker’s fluid
What is the fixation time of Zekner’s fluid?
12-24 hours
what is the fixation time of Zenker-formol?
12-24 hrs
zenker-formol is also called
Helly’s solution
It is an excellent microanatomic fixative for pituitary gland, bone marriw, and blood containing organs such as spleen and liver
Zenker-formol
Brown pigment are produced if tissues are allowed to stay in the fixative for more than 24 hrs due to RBC lysis
Zenker-formol
The brown pigments produced in Zenker-formol may be removed by immersing the tissue in what?
- saturated alcoholic picric acid
- sodium hydroxide
This is recommended mainly for tumor biopsies especially of the skin; it is an excellent ctyologic fixative
Heidenhan’s Susa Solution
What is the fixation time of Heidenhan’s Susa sol’n?
3-12 hrs
This is commonly used for bone marrow biopies
B-5 Fixative
It precipitates all proteins and adequately preserves carbohydrates
chromic acid
It is a strong oxidizng agent, hence, a strong reducing agentmust be added to chrome-containing fixatives before use in order to prevent counteracting effects
chromic acid
It preserves lipids
Potassium dichromate
It preserves mitochondria
Potassium dichromate
It fixes but does not precipitate cytoplasmic structures
potassium dichromate
It is recommended for demonstration of chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid-containing tissues
Regaud’s/Muller’s fluid
What is the fixation time of Regud’s
12-48 hrs
it is recommended for tudy of early degenerative processes and tissue necrosis
Orth’s fluid
It demonstrates rickettsiae and other bacteria
Orth’s fluid
It preserves myelin better than buffered formalin
orth’s fluid
What is the fixation time of orth’s fluid
36-72 hrs
It is recommended for acid mucopolysaccharides
Lead fixatives
It fixes connective tissue mucin
lead fixatives
It takes up CO2 to form insolube lead carbonate
lead fixatives
This dyes the tissues, but the yellow color may be removed by treatment with another acid dye or lithium carbonate
picric acid
It is highly explosve when dry
picric acid
