Fixation formalin

To study the role of lysine residues in susceptibility to formalin fixation, the amino acid composition of immunoreactive peptides (to various monoclonal antibodies) was studied. Each peptide was evaluated to determine if immu-noreactivity was lost after formalin fixation. Formalin sensitivity was correlated with the peptides amino acid composition. The first step in the method is biopanning from a peptide combinatorial library with a monoclonal antibody. The peptides that bind to the antibody were tested for their sensitivity to formalin fixation. Some peptides remain immunoreactive whereas others do not. The peptides were then sequenced to look for differences between those that were sensitive to formaldehyde versus those that were not. The goal was to find whether there is a particular amino acid that is present in formalin-sensitive epitopes but absent in formalin-resistant epitopes, or vice versa. An advantage of this approach is that it is open-ended, without excluding any amino acids. 

The fixative system generally used is a two-vial technique with one vial containing 5 to 10% buffered Formalin and the other vial containing polyvinyl alcohol (PVA) fixative. A portion of the specimen is added to the fixative in a ratio of approximately 3 parts fixative to 1 part specimen and thoroughly mixed to ensure adequate fixation. An alternative to Formalin is Merthiolate-iodine-formaldehyde (MIF), which fixes and stains at the same time. If unfixed specimens are processed in the laboratory, fecal films may be prepared and immediately fixed in Schaudinn fixative. 

Namimatsu S, Ghazizadeh M, Sugisaki Y. Reversing the effects of formalin fixation with citraconic anhydride and heat a universal antigen retrieval method. J. Histochem. Cytochem. 2005 53 3-11. 

Gown AM. Unmasking the mysteries of antigen or epitope retrieval and formalin fixation. Am. J. Clin. Pathol. 2004 121 172-174. 

Rait VK, Xu L, O Leary TJ, et al. Modeling formalin fixation and antigen retrieval with bovine pancreatic RBase A II. Interrelationship of cross-linking, immuno-reactivity, and heat treatment. Lab. Invest. 2004 84 300-306. 

A simple and effective AR technique of boiling archival paraffin-embedded tissue sections in water to enhance the signal of IHC was developed to circumvent the deleterious effects of formalin fixation, which had previously... 

In general, the mechanism of heat and alkaline solution for DNA extraction may be based upon a hypothesis, previously proposed for the AR technique.32 Strong alkaline solution may denature and hydrolyze proteins, resulting in breaking cell and nuclear membranes as well as disrupting cross-linkages due to formalin fixation. It is no surprise to observe the similarity between retrieval of nucleic acid and retrieval of protein (antigen) based on a similar chemical reaction of formaldehyde with these two kinds of macromolecules (Fig. 3.1).15"19... 

Some investigators described artifactual DNA sequence alterations after formalin fixation, when testing DNA samples extracted from FFPE tissues. Williams et al.46 reported that up to one mutation artifact per 500 bases was found in FFPE tissue. They also found that the chance of artificial mutations in FFPE tissue sample was inversely correlated with the number of cells used for DNA extraction that is, the fewer cells, the more the artifacts. However, they mentioned that these artifacts can be distinguished from true mutations by confirmational sequencing of independent amplification products, in essence comparing the product of different batches. Quach et al.47 documented that damaged bases can be found in DNA extracted from FFPE tissues, but are still readable after in vitro translesion synthesis by Taq DNA polymerase. They pointed out that appropriate caution should be exercised when analyzing small numbers of templates or cloned PCR products derived from FFPE tissue samples. 

The quantity of RNA extracted from FFPE cell/tissue sections by the heating and nonheating methods, and extracted from fresh cell/tissue embedded in OCT without fixation, was comparable, showing no significant difference for all yields of RNA by Student s f-test, with the exception of one sample, MDA cells fixed in formalin for 24 h (p < 0.05). 

Williams C, Ponten F, Moberg C, et al. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am. J. Pathol. 1999 155 1467-1471. 

Quach N, Myron GF, Shibata D. In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BioMed Central 2004 4 1-11. http //www.biomedcentral.eom/1472-6890/4/l. 

Koshiba M, Ogawa K, Hamazaki S, et al. The effect of formalin fixation on DNA and the extraction of high-molecular-weight DNA from fixed and embedded tissues. Pathol. Res. Pract. 1993 189 66-72. 

Castiglione F, Degl Innocenti DR, Taddei A, et al. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues effects of the fixation on outcome reliability. Appl. Immunohistochem. Mol. Morphol. 2007 15 338-342. 

Figure 5.1 Diagrammatic explanation of standardization of IHC via AR and test battery to achieve a maximal retrieval level by an optimal protocol of AR. The intensity of IHC (axis y) is inversely correlated with the time of formalin fixation (axis x) as indicated by a reduced slope. Three arrows indicate a potential maximal retrieval level that may equalize the intensity of IHC to a comparable result for routinely processed, paraffin-embedded tissues with various time of fixation. Reproduced with permission from Shi et alHistotechnol. 1999 22 177-192.

To test further this hypothesis, a simulated cell/tissue model system has been devised using quantitatively comparable cell fines, in which the amount of selected antigen (potential reference standard) can be measured accurately on a cell-to-cell basis in fresh and FFPE specimens that are processed under clearly defined but variable conditions, including periods of formalin fixation, delay times of fixation (prefixation time or warm ischemic time), storage conditions, and other technical issues such as thickness of each tissue section, in... 

Figure 5.3 Diagram depicts the further-designed studies to test our hypothesis with respect to standardization of immunohistochemistry based on the antigen retrieval technique exemplified in a multiple direction to draw a conclusion, (a) Periods of formalin fixation, (b) Variable delay of fixation, (c) Storage of FFPE tissue blocks or sections, (d) Variable thickness of FFPE tissue sections, (e) Other variable conditions of processing FFPE tissue blocks. The stereoscopic frame of a cube represents the reliable limitation of quantitative IFIC demonstrated by serial studies as recommended in the text. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2007 55 105-109.

This is important when considering your subsequent period of fixation. For example, suspended cells in fixative for 24h are not being subjected to the same effects as a 5-mm biopsy for the same period. The formalin penetration and fixation effects are greater across a 50-100p cell versus a 5-mm piece of tissue. Penetration and the actual fixation of the tissue, cells, and protein are two different things. 

Figure 6.5 After formalin fixation the centrifuge tube is cut open to gain access to the cells with the syringe.

Morgan JM. A protocol for preparing cell suspensions with formalin fixation and paraffin embedding which minimises the formation of cell aggregates. J Cell. Pathol. 2001 5 171-180. 

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