Biochemical Use of Polyamines in Tissue Processing

Saturday, February 13, 2016
Jacob Neff, Northwest Nazarene University, Nampa, ID
In an effort to minimize artifacts created during biochemical tissue staining procedures a naturally occurring, non-toxic and relatively odorless polyamine was added to the aqueous solutions used in standard staining procedures. The purpose of the polyamine pretreatment in this novel approach to tissue staining is to react it with residual formaldehyde to prevent displacement of current methylene bridges, and reoccurrence of them. In turn, cross-linking among the bridges is eliminated. Such bridge structures are called artifacts and greatly distract from microscopic clarity. Incorporating polyamines into the staining procedure assists in the overall effort to enhance photomicrographic detail and provides the opportunity for more accurate disease diagnosis. In the absence of artifacts, clarity of microscopic detail is now preserved up to 1000X magnification, as compared to the universal standard of 200X. Tissue preparation can be discussed in two processes: fixation and staining. In fixation the tissue is immersed in a 5% aqueous formaldehyde solution. While in solution, formaldehyde forms methylene bridges between positively charged amino acid sites in the tissue. These bridges that are initially formed provide rigidity, enabling the tissue to maintain its structural integrity throughout the fixation.  Once the tissue has spent a sufficient amount of time in the formaldehyde solution to form essential methylene bridges, it is removed from the solution. Following this procedure, the tissue is washed with 100% ethanol followed by a xylene wash to remove any non-bonded formaldehyde from the tissue sample. Fixed tissue is stained by two different histological stains, hematoxylin and eosin. In order to stain the tissue, the staining dyes must bind to positively charged in situ amino acids sites. These sites are the same ones which formaldehyde bonds to when forming methylene bridges in the fixation process. Because of this, there is nucleophilic competition between the stains and formaldehyde for bonding sites. Therefore, as the stains bond to the tissue, methylene bridges are displaced and formaldehyde is released. As a result, the tissue loses its rigidity and the displaced formaldehyde forms new methylene bridges at different tissue sites in distorted positions that change the sample geometry. Such spurious methylene bridge structures constitute the majority of artifacts created in currently accepted tissue staining procedures. The focus of this research is to eliminate these artifacts by binding formaldehyde as it is released from the tissue, thereby preventing the formation of methylene bridges in improper positions. Sequestering formaldehyde during this procedure is accomplished via chemical reaction. By placing the polyamine in the aqueous staining solutions, a bond forms between formaldehyde and amino groups of the polyamine, rather than relocate to the inappropriate amino acid site. The artifacts are therefore eliminated, enabling pathologists to make diagnoses with greater confidence.