Fixation Flashcards
What is fixation?
A process by which biological tissues are preserved from decay, thereby preventing autolysis or purification
How does fixation work?
It terminates any on-going biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues
Why is fixation the most important step?
It is the foundation of all subsequent processing through to final diagnosis
Why is it difficult to choose the best fixative?
Fixation involves a complex series of chemical events which differs for each chemical substance e.g. proteins vs lipids
It can be difficult to find an ideal fixative which can effectively preserve all compounds of a tissue
It is important to select the correct method for the necessary laboratory techniques e.g. glutaraldehyde for electron microscopy
What are the four features of a good fixative?
Leaves tissues as close to their living state as possible i.e. without the loss of rearrangement of components
Prevents autolysis and bacterial putrefaction
Does not change the shape or volume of a tissue
Leaves a tissue in a condition which allows for subsequent clear staining
What are the two main types of fixation?
Physical vs chemical
What are the different types of physical preservatives?
Thermal preservation (microwave fixation vs microwave chemical fixative)
Cryogenic (liquid nitrogen)
Write about thermal methods of preservation
Microwave fixation by heat coagulation using PBS @ 70-85 C -> even penetration and rapid fixation
Microwave chemical fixation uses 10% NBF - urgent biopsies
Write about liquid nitrogen as a preservative
Cryogenic preservation @ -196 C
For frozen sections where urgent diagnosis is required
For lipid stain controls
For storage of materials for further studies e.g. lymphoma studies
Used for the storage of frozen cells/tissue -70 degrees or -20, repeate freezing/thawing is damaging
Need to use cryoprotectant - without it the air around the outside of the tissue can prevent the edges of the tissue from freezing properly -> may get some components with improper preservation which may impair diagnosis
What is chemical preservation?
The immersion of cells/tissue in a chemical solution, which penetrates from the outside to achieve preserving effect throughout the tissue
What are the different chemical preservatives?
Cross linking
Protein denaturing agents
Write about formaldehyde as a preservative
The most commonly used fixative -> 10% neutral buffered formalin
Formalin is an aqueous solution of formaldehyde -> made up in water with added methanol which acts as a stabilising agent
Works by forming methylene bridges between proteins forming a solid mesh and thus trapping other components
Cross-linking of primary amino groups in proteins with other nearby nitrogen atoms in proteins or DNA through a -CH2 linkage
The work of 10% NBF causes the masking of antigens - should be taken into account when preforming IHC
It is highly toxic and carcinogenic
Can result in the deposition of formalin pigment in tissues
Write about glutaraldehyde as a preservative
A larger molecule than formaldehyde which penetrates more slowly
Provides a more rigid crosslinking of the proteins and a more stable fixation
Very toxic and deteriorates rapidly
Masks antigenicity
Only really used for fixation of specimens which require electron microscopy e.g. renal/muscle biopsies in Beaumont Hosp
Write about protein denaturing agents as preservatives
Non-aqeous chemicals like acetone and alcohols
Ethanol/methanol alters the structure of proteins due to disruption of the hydrophobic interactions that contribute to the maintenance of the tertiary structure of proteins
Hydrogen bonds are more stable in methanol and ethanol than in water so that while affecting the tertiary structure of proteins, these alcohols may preserve their secondary structure and any associated epitopes
Alcohol fixation can cause distortion of nuclear detail and shrinkage of cytoplasm
If fixation is prolonged, the alcohols can remove histones from the nuclei and later extract RNA and DNA
E.g. cytolyt and preservcyt for cytology
Write about oxidising agents as preservatives
Use of metallic ions like osmium tetroxide
