MIDTERM LEC: FIXATION Flashcards
First and most critical step in tissue processing because if fixation is inadequate, the succeeding tissue processing steps will also
be inadequate
FIXATION
Primary purpose:
Preserve morphological & chemical integrity of cell in a lifelike manner as possible by stopping all cellular activities
FIXATION
Performed as soon as tissue is removed from the body
FIXATION
If tissues/cells are exposed to:
a. Air
drying of tissue
If tissues/cells are exposed to:
b. Water
swelling of cells
If tissues/cells are exposed to:
c. Saline
shrinkage of cell
Effects of Fixatives:
■ 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
Characteristics of Ideal Fixative:
- 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
Fixative will forms cross-links between
soluble molecules, thus gluing them
together into an insoluble meshwork
Mechanism of Fixation
Additive Fixation
Fixative will not chemically bind with tissue
but removes water from tissue protein
groups thus causing denaturation of cell
proteins
Mechanism of Fixation
Non-additive Fixation
FACTORS AFFECTING FIXATION:
- Fixative of Choice
- Time
- Tissue-to-fixative ratio
- Penetration time
- Thickness of section
- Tissue components
- Hydrogen ion concentartion (ph)
- Temperature
- Osmolality
- Agitation, Vacuum
Fixative of Choice
Factors Affecting Fixation
10% Neutral Buffered Formalin (NBF)
Morphologic criteria for diagnosis have been established based on______
FormalinFixed Paraffin Embedded Specimen (FFPES)
Fixation must be done ________ after
cutting off blood supply (to shorten
warm ischemia time)
20-30mins
Prolonged fixation ->
shrinkage
Tissue-toFixative Ratio
1:10 or 1:20
common Tissue-toFixative Ratio
1:20
Tissue-toFixative Ratio Osmic acid fixatives:
1:5
Penetration Rate Formalin:
1 mm/hr (but slows down as
it goes deeper into the tissue)
Larger->
Thickness of setion
Longer fixation time, more
fixative
Light Microscopy:
Thickness of setion
2cm2 x 0.4cm
Electron Microscopy:
Thickness of setion
1-2 mm2
Longer fixation time:
Tissue Components
- Fibrous tissues
- Presence of Mucus (wash with NSS)
- Fat (cut into thin slices fixed longer)
- Blood (flushed out with saline)
Shorter fixation time:
Tissue Components
Small or loosely textured tissues
Optimal pH:
Hydrogen Ion Concentration (pH)
6 to 8
If outside this pH, ultrastuctural
changes may occur
Hydrogen Ion Concentration (pH)
May require the use of buffers
Hydrogen Ion Concentration (pH)
For Electron microscopy:
Hydrogen Ion Concentration (pH)
pH should match physiologic pH
Higher temp ->
faster fixation rate and
autolysis
Cold temp ->
enzyme inactivation
Optimal Temperature (routine):
Room temp to 45C
Tissue processors:
40 C
Microwave processing:
Up to 65C
Electron microscopy:
0-4C
Tuberculosis:
100C
Rapid biopsy:
60C
Hypertonicity ->
Osmolality
cell shrinkage
Isotonicity and hypotonicity ->
Osmolality
cell swelling
Thus, maintain tissues at slightly
hypertonic solution (400-450 mOsm)
Osmolality
Hastens fixation
Agitation, Vacuum
TYPES OF FIXATIVES
According to Composition:
- simple
- compound
According to Composition:
made of one component
TYPES OF FIXATIVES
SIMPLE
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
According to Composition:
2 or more components or fixatives
TYPES OF FIXATIVES
COMPOUND
According to Action:
TYPES OF FIXATIVES
- Microanatomical
- Cytological
- Histochemical
permits general study of tissues without altering the structure of the subjects of interest
Microanatomical
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
preserve specific parts of the cell
Cytological
Cytological 2 TYPES:
- Nuclear Fixatives
- Cytoplasmic Fixatives
o Preserve nucleus
Nuclear Fixatives
o pH ≤ 4-6
Nuclear Fixatives
o Glacial acetic acid has affinity to nuclear chromatin
Nuclear Fixatives
Nuclear Fixatives:
(FCBNH)
a. Flemming’s with glacial acetic acid
b. Carnoy’s
c. Bouin’s
d. Newcomer’s
e. Heidenhain’s
o Other organelles aside
from nucleus
Cytoplasmic Fixatives
o pH > 4-6
Cytoplasmic Fixatives
o HAc destroys
mitochondria and Golgi
bodies
Cytoplasmic Fixatives
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
Preserves chemical constituents of cells & tissues
Histochemical
Histochemical:
(10FANA)
1. 10% Formol Saline
2. Absolute ethanol
3. Newcomer’s
4. Acetone
RANGE TISSUE-TO-FIXATIVE RATIO:
1:15-1:20
RECOMMENDED TISSUE-TO-FIXATIVE RATIO:
1:10
For routine HP techniques
I. ALDEHYDES
Formaldehyde AKA Formalin
Produced from oxidation of methanol
I. ALDEHYDES
Formaldehyde AKA Formalin
Usually buffered to pH 7 with phosphate buffer
I. ALDEHYDES
Formaldehyde AKA Formalin
Formaldehyde AKA Formalin
Concentrations:
100%
I. ALDEHYDES
GAS FORM
Formaldehyde AKA Formalin
Concentrations:
37-40%
I. ALDEHYDES
stock concentration (causes overhardening
of the external surfaces of tissues)
Formaldehyde AKA Formalin
Concentrations:
10%
I. ALDEHYDES
working solution; most commonly used
Formaldehyde AKA Formalin ADVANTAGES:
I. ALDEHYDES
cheap, readily available, easy to prepare, stable, compatible with most stains
Formaldehyde AKA Formalin DISADVANTAGES:
I. ALDEHYDES
nose and eye-irritant, may cause allergic dermatitis
Formalin diluted with 10% NaCl
I. ALDEHYDES
10% Formol-Saline
Traditionally, the most common fixative
I. ALDEHYDES
10% Formol-Saline
Recommended for CNS tissue and general post-mortem tissues for histochemical examination
I. ALDEHYDES
10% Formol-Saline
```
10% Formol-Saline ADVANTAGES:
I. ALDEHYDES
ideal for Silver impregnation staining technique
10% Formol-Saline DISADVANTAGES:
I. ALDEHYDES
tissue shrinks during alcohol dehydration [Remedy: Secondary fixation]
pH 7
I. ALDEHYDES
10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin
```
Best general tissue fixative
I. ALDEHYDES
10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin
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
```
10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin DISADVANTAGES:
I. ALDEHYDES
longer to prepare, inert to phospholipids and neutral
fats
Has HgCl2
I. ALDEHYDES
Formol-Sublimate/Corrosive
Formol-Sublimate/Corrosive ADV:
I. ALDEHYDES
Excellent for silver reticulum staining method, does not need washing, fixes lipids
Formol-Sublimate/Corrosive DISADVANTAGES:
I. ALDEHYDES
forms mercuric chloride deposits
Has 95% ETOH, Picric acid, and GHAc
I. ALDEHYDES
Gendre’s (Alcoholic Formalin)
```
Gendre’s (Alcoholic Formalin) ADV:
I. ALDEHYDES
good for microincineration techniques, fixes sputum
For gastrointestinal (GI) tissues, prostate biopsies, and bone marrow (BM)
I. ALDEHYDES
Hollande’s
Made up of 2 formaldehyde resides linked by three carbon
chains
I. ALDEHYDES
Glutaraldehyde
```
For enzyme histochemistry and electron microscopy
I. ALDEHYDES
Glutaraldehyde
Glutaraldehyde ADVANTAGES:
I. ALDEHYDES
more pleasant and less irritating compared to formalin
Glutaraldehyde DISADVANTAGES:
I. ALDEHYDES
Less stable and more expensive than formalin
Container must be refrigerated
I. ALDEHYDES
Glutaraldehyde
Glutaraldehyde
Concentrations:
for immunoEM
I. ALDEHYDES
0.25%
Glutaraldehyde
Concentrations:
for small TSE fragments
I. ALDEHYDES
2.5%
Glutaraldehyde
Concentrations:
most common
I. ALDEHYDES
3%
Glutaraldehyde
Concentrations:
for large TSE
I. ALDEHYDES
4%
Polymer of formalin
I. ALDEHYDES
Paraformaldehyde
Powder in form, used in 4%
I. ALDEHYDES
Paraformaldehyde
Plastic embedding
I. ALDEHYDES
Paraformaldehyde
For ultrathin and electron microscopy
I. ALDEHYDES
Paraformaldehyde
Acrolein in glutaraldehyde or formalin
I. ALDEHYDES
Karnovsky’s Paraformaldehyde/Glutaraldehyde
P: for Electron Histochemistry and Electron Immunocytochemistry
I. ALDEHYDES
Karnovsky’s Paraformaldehyde/Glutaraldehyde
ADV: no smudging of nuclei and distortion of staining compared with formalin
I. ALDEHYDES
40% Aqueous Glyoxal
D: reduced staining capacity
[Remedy: increase staining time]
40% Aqueous Glyoxal
METALLIC FIXATIVES:
- Mercuric chloride
- Chromates
- Lead
Mercuric chloride:
II. METALLIC FIXATIVES
(ZZCHBS)
Zenker
Zenker-Formol (Helly’s)
Carnoy-Lebron
Heidenhain’s Susa
B5
Schaudinn’s
Chromates:
II. METALLIC FIXATIVES
(CROP)
Chromic acid
Regaud’s/Muller’s
Orth’s
Potassium dichromate
Most common metallic fixative
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
A: penetrates and hardens tissue rapidly
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Routine fixative of choice for preservation of cell detail in tissue photography
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Conc. 5-7%
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Mostly incorporated in compound fixatives
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
DADV: Banned worldwide d/t extreme toxicity, marked cell shrinkage [Remedy: add acid]
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
May produce black granular deposits except in Heidenhain’s Susa
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
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
HgCl2 + potassium dichromate + glacial acetic acid
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Zenker’s
Good general fixative for adequate preservation of all kinds of tissues
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Zenker’s
Good for Trichrome staining
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Zenker’s
HgCl2 + potassium dichromate + strong formalin (40%)
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Zenker-formol/Helly’s
For piituitary gland, BM, blood-containing organs, preserves cytoplasmic granules
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Zenker-formol/Helly’s
Brown pigments are removed with saturated alcoholic picric acid or NaOH
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Zenker-formol/Helly’s
Susa: Su = sublimate ; Sa = saure (acid)
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Heidenhain Susa
HgCl2 + NaCl + TCA + glacial acetic acid + formalin
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Heidenhain Susa
Skin tumor biopsy
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Heidenhain Susa
ADV: minimum cell shrinkage and tissue hardening due to counter-balance effect of acids and mercury:
Acids : swelling
Mercury: shrinkage
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Heidenhain Susa
Does not produce black pigments
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Heidenhain Susa
DADV: Weigert’s staining of elastic fibers not possible
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
Heidenhain Susa
```
HgCl2 + Anhydrous Na acetate
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
B5 Fixative
BM biopsies
II. METALLIC FIXATIVES
Mercuric Chloride (HgCl2)
B5 Fixative
Conc.: 1-2% aqueous solutions
II. METALLIC FIXATIVES
Chromate Fixatives
Chromic acid
Precipitates all proteins, and preserves carbohydrates
II. METALLIC FIXATIVES
Chromate Fixatives
Chromic acid
P: chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid-containing TSEs
II. METALLIC FIXATIVES
Chromate Fixatives
Regaud’s/Muller’s
DADV: prolonged fixation may lead to blackening of tissue pigment
[Remedy: Wash in running tap water before
dehydration]
II. METALLIC FIXATIVES
Chromate Fixatives
Regaud’s/Muller’s
P: early degenerative processes and necrosis,
demonstration of Rickettsia and other bacteria
II. METALLIC FIXATIVES
Chromate Fixatives
Orth’s
E: preserves myelin
II. METALLIC FIXATIVES
Chromate Fixatives
Orth’s
E: preserves lipids, mitochondria, at pH4.5-5.2,
cytoplasm, chromatin and chromosome are fixed
II. METALLIC FIXATIVES
Chromate Fixatives
3% Potassium dichromate
Corrosive, thus avoid skin contact
II. METALLIC FIXATIVES
Chromate Fixatives
3% Potassium dichromate
P: for acid mucopolysaccharides and mucin
II. METALLIC FIXATIVES
Chromate Fixatives
4% Aqueous Lead
DADV: Prolonged standing formation of insoluble lead carbonate
[Remedy: add drops of acetic acid to dissolve residue]
II. METALLIC FIXATIVES
4% Aqueous Lead
Used in strong saturated aqueous solution (1%)
PICRIC ACID
For Glycogen preservation
PICRIC ACID
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
PICRIC ACID DISADVANTAGES:
- Explosive when dry
[Remedy: add distilled H2O or 0.5-1% saturated alcohol] - Yellowing of tissues excessive staining
[Remedy: immerse in Li2CO3 with 70%ROH water
70% ethanol 5% Na thiosulfate water] - RBC hemolysis
(PBB)
PICRIC ACID:
(PBB)
1. Bouin’s
2. Brasil’s
P: for embryo and pituitary biopsies, and tissues to be stained with Masson’s Trichrome
III. PICRIC ACID
Bouin’s
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
DADV: poorly penetrates large tissue, thus limited to small fragments of tissues
III. PICRIC ACID
Bouin’s
C: TCA
III. PICRIC ACID
Brasil’s
ADV: Better and less messy than Bouin’s
III. PICRIC ACID
Brasil’s
Incorporated in compound fixatives
GLACIAL ACETIC ACID
Solidifies at 17CI
GLACIAL ACETIC ACID
Important for nuclear fixatives (precipitates nucleoproteins, chromatins)
GLACIAL ACETIC ACID
Destroys mitochondria and Golgi elements, thus not for cytoplasmic fixation
GLACIAL ACETIC ACID
ADV: good for glycogen
ALCOHOL FIXATIVES
DADV: never for FATs and LIPOPROTEINS (dissolves); causes polarization of glycogen (granules will move towards the poles
or ends of the cells)
ALCOHOL FIXATIVES
Effect: rapidly denatures and precips CHONs, preserves nuclear
stains
ALCOHOL FIXATIVES
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)
Most rapid tissue fixative
Carnoy’s Fixative
Fixing brain tissues for rabies diagnosis
Carnoy’s Fixative
E: fixes Nissl granules (Tigroid substance) and cytoplasmic
granules
Carnoy’s Fixative
1.
Enzyme studies
70-100% Ethanol
Does not fix but preserves glycogen
70-100% Ethanol
Dry and wet smears, BM smears, bacterial smears
100% Methanol/Wood alcohol
Touch prep smears to be Wright-stained
95% Isopropyl Alcohol/Rubbing Alcohol
Mucopolysaccharides and nuclear CHONs
Newcomer’s
```
Better reaction in Feulgen stain than Carnoy’s
Newcomer’s
Pale yellow powder in water (6% in 20C)
OSMIUM TETROXIDE / OSMIC ACID
Ultrathin sections in Electron Microscopy
OSMIUM TETROXIDE / OSMIC ACID
E: Fixes and stains conjugated fats and lipids blac
OSMIUM TETROXIDE / OSMIC ACID
DADV: very expensive, very volatile, inhibits hematoxylin
OSMIUM TETROXIDE / OSMIC ACID
OSMIUM TETROXIDE / OSMIC ACIDTissue-to-fixative ratio:
1:5
OSMIUM TETROXIDE / OSMIC ACID:
(OFF)
1. Flemming’s
2. Flemming’s w/o acetic acid
Most common osmic acid fixative
Flemming’s
P: nuclear structures
Flemming’s
Effect: permanently fixes fat
Flemming’s
ADV: needs less amount of fixative
Flemming’s
Cytoplasmic structures
Flemming’s w/o acetic acid
Incorporated also in compound fixatives
TRICHLOROACETIC FIXATIVES
Marked swelling effect on tissues
TRICHLOROACETIC FIXATIVES
Poor penetrating agent thus for small pieces of tissues or bones
TRICHLOROACETIC FIXATIVES
Weak decalcifying agent, thus has softening effect on dense fibrous tissues
TRICHLOROACETIC FIXATIVES
1.
Used at cold temp -5-4C
ACETONE
For water-diffusible enzymes (Phosphatase, Lipase)
ACETONE
For brain tissues (such as in Rabies)
ACETONE
DADV: Dissolves fat, evaporates rapidly
ACETONE
Principle: Thermal coagulation of tissue proteins
HEAT
For rapid diagnosis: frozen tissue sections and Bacterial smear
prep
HEAT
PCPL: Increases movement of molecules to accelerate fixation, staining, decalcification
Microwave Technique
ADV: Tissue is heated right through the block in a very short time; preserves neurochemical substances (acetylcholine)
Microwave Technique
Electron Microscopy and immunohistochemistry
Microwave Technique
DADV: Penetrates at 10-15mm thickness; spores and pathogen may remain in tissues
Microwave Technique
SECONDARY FIXATION:
- Refixation” with another fixative
- Post-Chromatization
Microwave Technique Optimum Temp:
45-55C
Done before dehydration or restaining of deparaffinized
TSEs
Refixation” with another fixative
Improve demonstration of substance
Refixation” with another fixative
Make special staining techniques possible (with the next fixative as mordant)
Refixation” with another fixative
Ensure further and complete handling
Refixation” with another fixative
Use of 2.5-3% aqueous K2Cr2O7 that will act as mordant
Post-Chromatization
Removal of excess fixative to improve staining and remove
artifacts
WASHING OUT
WASHING OUT:
- Tap Water
- 50-70% ROH
- Alcoholic iodine
for excess formalin, osmic acid, and chromates
Tap Water
for excess picric acid fixatives and
Gendre’s
50-70% ROH
for excess mercuric chloride
Alcoholic iodine
