Tissue fixation
Tissue fixation, usually just fixation, is an important part of tissue preparation for histologic examination. It is typically done with formalin.
Implications
Pathologist have a great lifestyle 'cause tissue takes long to fix; the penetration of tissue by formalin is 1 mm/hour.[1]
Ratio
The dictum is:[2]
- The volume of fixative should be 10x the volume of specimen.
Tissue fixation
A list of fixatives:[3]
| Fixative | Comment |
|---|---|
| Formalin, neutral buffered | standard fixative |
| Formalin, unbuffered | ??? |
| Glutaraldehyde[4] | electron microscopy |
| Ethanol | cytopathology |
| Carnoy | ??? |
| Bouin | toxic ??? |
| B5 | used for lymphoma, superior morphology - but toxic, not good for IHC & DNA analysis[5] |
| IBF fixative | used bone marrow and lymphatic tissue;[6] gives better nuclear detail in prostate biopsies compared to formalin[7] |
Tissue fixation
Formalin
- May be written (incorrectly) as "formulin".
- Formaldehyde + methanol.
Formaldehyde is often supplied as a 37% w/v solution. To prevent polymerisation of formaldehyde, methanol is added. The resulting solution is often referred to as formalin. This can be further diluted and buffered to produce 10% formalin (equivalent to 4% formaldehyde). This concentration is often used as a fixative and preservative for histological specimens. It gives tissues a firmer texture, making subsequent cut-up easier and also stops autolysis and necrosis of the tissue by inactivating micro-organisms and proteolytic enzymes. Poorly fixed tissue stains unreliably and certain features may be lost. Mitotic figures, for instance, are easily lost in poorly fixed tissue. Formalin is also used to preserve prosected and dissected cadavers. As a rule of thumb, fixation occurs at about 1 mm per hour, but the reality is more complex as fixation lags behind diffusion and fixation also changes the tissue properties, slowing diffusion.[8]
Formalin cross-links sections of the peptide chains of proteins as well as nucleic acids. The effect on the former alters epitopes, which means that antibodies designed for fresh tissue may not work with fixed tissue. The cross-linking process can be partially reversed with various methods of antigen retrieval. The effect on nucleic acids is to reduce DNA quality.
Fixing marking dye
To fix marking dye:
- Formal-acetic alcohol (FAA):[9]
- Bouin's solution.
Formal-acetic-alcohol
General:
- Different recipes exist.
One recipe:[10]
- Ethanol.
- Acetic acid.
- Formaldehyde.
- Methanol.
See also
References
- ↑ Gross rounds. 14 August 2009.
- ↑ URL: http://www.cap.org/apps/docs/committees/cancer/cancer_protocols/2011/UvealMelanom_11protocol.pdf. Accessed on: 27 March 2012.
- ↑ Hunt, JL. (Feb 2008). "Molecular pathology in anatomic pathology practice: a review of basic principles.". Arch Pathol Lab Med 132 (2): 248-60. doi:10.1043/1543-2165(2008)132[248:MPIAPP]2.0.CO;2. PMID 18251585. http://www.archivesofpathology.org/doi/pdf/10.1043/1543-2165%282008%29132%5B248%3AMPIAPP%5D2.0.CO%3B2.
- ↑ Prentø, P. (Nov 1995). "Glutaraldehyde for electron microscopy: a practical investigation of commercial glutaraldehydes and glutaraldehyde-storage conditions.". Histochem J 27 (11): 906-13. PMID 8787969.
- ↑ Bonds, LA.; Barnes, P.; Foucar, K.; Sever, CE. (Aug 2005). "Acetic acid-zinc-formalin: a safe alternative to B-5 fixative.". Am J Clin Pathol 124 (2): 205-11. doi:10.1309/29DA-CY9K-BHNW-4BG6. PMID 16040290.
- ↑ URL: http://www.leicabiosystems.com/specimen-preparation/consumables/reagents-solutions/fixatives/details/product/ibf-tissue-fixative-1/. Accessed on: 26 September 2014.
- ↑ Trpkov, K.; Renault, P.; Yilmaz, A.; Ali-Ridha, N. (Jan 2006). "IBF as a formalin substitute fixative in prostate biopsy pathology.". Arch Pathol Lab Med 130 (1): 13-4. doi:10.1043/1543-2165(2006)130[13b:IAAFSF]2.0.CO;2. PMID 16390230.
- ↑ "How much formalin is enough to fix tissues?". Ann Diagn Pathol 16 (3): 202–9. June 2012. doi:10.1016/j.anndiagpath.2011.12.003. PMID 22483550.
- ↑ URL: http://www.brunelmicroscopes.co.uk/acatalog/Solvents___Reagents.html. Accessed on: 5 January 2011.
- ↑ URL: https://fscimage.fishersci.com/msds/45357.htm. Accessed on: 5 January 2011.