Histotechnology: Fixation and Fixatives
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1
Primary definition and nature of Fixation are
Process by which constituents of cells/tissues are fixed in a physical and partly chemical state to withstand subsequent treatment with minimal distortion
2
Methods of Fixation include:
Physical
- Heating
- Microwave
- Cryopreservation
Chemical
- Immersion
- Perfusion
- Vapor form
- Heating
- Microwave
- Cryopreservation
Chemical
- Immersion
- Perfusion
- Vapor form
3
Two major groups of chemical fixatives
1. Cross linking Fixatives
2. Precipitating/ Drnaturing
2. Precipitating/ Drnaturing
4
Two classes of fixation methods
Organic Solvents
Cross linking
Cross linking
5
Critical designation of Fixation in histotechnology is
The first and most critical step in histotechnology
6
Primary aim of fixation is
Preserve the morphologic and chemical integrity of the cell in as life-like a manner as possible
7
Secondary goal of fixation is
Harden and protect tissue from trauma of further handling for easy cutting during gross examination
8
Most important chemical reaction involved in fixation is
Stabilization of proteins
9
Protein stabilization is achieved physically/chemically by
Forming cross-links between proteins
10
Morphologic distortion outcome of leaving a tissue specimen in air is
Dry out (distortion of morphologic appearance)
11
Cellular outcome of leaving a tissue specimen in water (hypotonic solution) is
Cell will swell (swell of RBCs - ghost cells)
12
Cellular outcome of leaving a tissue in strong salt (hypertonic solution) is
Cell will shrink (crenated RBCs)
13
Mechanism and features of Additive Fixatives are
Chemical constituent of fixative is taken in and becomes part of tissue through cross-links formation or molecular complexes
14
Chemical examples of Additive Fixatives include
Formaldehyde, Osmium tetroxide, Mercuric chloride
15
Mechanism and features of Non-additive Fixatives are
Fixing agent is not incorporated into tissue but alters tissue composition and stabilizes it through water removal
16
Chemical examples of Non-additive Fixatives include
Alcoholic fixatives
17
Optimal pH range for general fixatives using normal phosphate buffered saline is
pH 6.0 - 8.0 (neutral pH around 7.0)
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Traditionalsual temperature parameter for general fixation is
Room Temperature / RT (18-30°C)
19
Temperature parameter used for tissue processors is
40°C
20
Temperature parameter used for Electron Microscopy and Histochemistry is
0-4°C
21
Temperature parameter used for Mast cells for EM is
Room Temperature (RT)
22
Temperature parameter used for nucleic acid fixation is
Rapid at higher temperatures
23
Temperature and purpose of heating formalin to 60°C are
Formalin heated to 60°C = rapid fixation of very urgent biopsy specimens
24
Temperature and purpose of heating formalin to 100°C are
Formalin heated to 100°C = to fix tissues with tuberculosis
25
Tissue section thickness requirements for Electron Microscopy vs Light Microscopy are
Electron Microscopy: 1-2 mm²; Light Microscopy: 2 cm² (thin: ≤0.4 cm or as prescribed)
26
Preparation method required for large solid tissues (e.g., uterus) during fixation is
Should be opened or sliced thinly
27
Fixation preparation protocol required for brain specimens is
Suspended whole in 10% NBF for 2-3 weeks
28
Osmolality condition yielding best results in fixation is
Slightly hypertonic solutions (400-450 mOsm)
29
Concentration parameter for Formaldehyde is
10%
30
Concentration parameter for Glutaraldehyde is
3%
31
Concentration parameter for Glutaraldehyde in Immuno-electron microscopy is
0.25%
32
Standard duration of fixation for most formalin fixatives is
24 hours (washed out)
33
Duration of fixation for Buffered Formalin is
2-6 hours up to 1 week
34
Duration of fixation for Electron Microscopy (EM) is
3 hours
35
Initial speed requirement for placing specimens in fixative is
Placed in fixative as soon as it is removed from the body to prevent autolysis and putrefaction
36
Condition where fixation is NOT required is
Microbiological and Toxicological studies
37
General penetration rate of Formalin into tissue is
Approximately 1 mm/hour (aldehydes: 2 to 3 mm/hr)
38
Fixative agents with BEST penetration rate are
Formalin, Alcohol
39
Fixative agent with POOR penetration rate is
Glutaraldehyde
40
Traditional vs recently recommended volume ratio of fixative to tissue is
Traditionally 10-20x the volume of tissue; recently 20x is maximum effective concentration
41
Duration of fixation for biopsy specimens vs standard samples vs immunohistochemistry is
Biopsy specimens: 6-18 hrs; Standard samples: 24-72 hrs; Immunohistochemistry: 12-24 hrs
42
Key characteristics of a good fixative include
Cheap, stable, safe to handle, kill cells quickly, produce minimum shrinkage, harden tissues properly, inhibit bacterial decomposition & autolysis, permit rapid/even penetration, make cellular components insoluble
43
Three main objectives of fixation are
1. To preserve the tissue (stop all cellular activities), 2. To prevent breakdown of cellular elements (prevent autolysis & putrefaction), 3. To coagulate or precipitate protoplasmic substances
44
Physical methods of fixation include
Heating (smears), Microwaving (routine lab practice), Cryopreservation (freeze drying)
45
Chemical methods of fixation include
Immersion fixation (commonly done in lab), Perfusion fixation, Vapor form (paraformaldehyde & osmium tetroxide)
46
Mechanism of Crosslinking Fixatives is
Form covalent bonds between proteins, anchoring them to cytoskeleton (e.g., aldehydes)
47
Mechanism of Precipitating / Denaturing Fixatives is
Decrease protein solubility and disrupt hydrophobic interactions (e.g., alcoholic fixatives)
48
Action of Organic Solvents (alcohol and acetone) as fixatives is
Remove lipids, dehydrate cells, and precipitate proteins
49
Action of Cross-linking Reagents (aldehydes and paraformaldehyde) is
Form intermolecular bridges creating a network of linked antigens
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