Maintaining Optimal Fixation Part 1 – The Histochemistry of Fixation

The preservation and study of tissue and cells is dependent upon one’s ability to stop all cellular degradation and autolysis once tissues have been removed from the body. Cells must be suspended or ‘fixed’ to stop enzymatic breakdown and allow normal and abnormal tissue to be viewed in its present state. As critical and essential as this process is in anatomic pathology, even more vital in clinical (hospital) histopathology is the ability to maintain this process on a day-to-day basis. With the constant daily flow of surgery, it is imperative that histology laboratories set up systems which will provide consistency in fixation procedures, management of solution integrity, and quality results. Fixation is a process involving the stabilization of proteins and protoplasmic substances. To establish and maintain this process on a daily basis one must first understand the histochemical nature of a fixative’s effect on tissue. 10% neutral buffered formalin is the preferred fixative used in most routine histopathology operations. The graphic below illustrates the chemical formula of formaldehyde, and the additive effect it has on tissue.

At the molecular level, in Step 1, you can see formaldehyde binding with the reactive hydrogen atoms in tissue, thus forming a still reactive hydroxy methyl compound. In Step 2 we see the continuing additive effect of the hydroxy methyl compound binding with other reactive hydrogen atoms, now forming a methylene bridge crosslink. This crosslinking effect stabilizes the protein and renders the cells insoluble; fixing or locking it in to its existing state. There are two primary goals that must be achieved and controlled to provide consistency in optimal fixation: 1) Penetration Rate and 2) Binding Time. With respect to both goals, the efficacy of the histochemical reaction shown above is grossly determined by the size and thickness of the tissue, and the time given for the molecular additive process to happen.

Penetration Rate – the average rate of penetration for 10% neutral buffered formalin in tissue is rapid for the first 1-2 cell layers. It is 3.6 mm. per hour, however, this rate of penetration progressively and rapidly slows down with time. In 4 hours, the rate of penetration has slowed down by half to 1.8 mm/hour; in 9 hours to 1.2 mm/hour; and in 16 hours to 0.9 mm/hour. Without some form of external manipulation such as solution agitation, formalin has a tendency to progressively slow to a point and stop, depending on the size and thickness of the specimen.

Therefore, it is important to have consistent optimal grossing at no more than 3-5 mm. thickness. In addition to agitation, additional steps such as mild heat can be used to enhance the penetration of fixatives, but these methods should be developed and validated as a part of the routine fixation procedures. It is impossible to optimize your day-to-day maintenance of optimal fixation if these additional measures are adopted to circumvent the acceptance of poor fixation practices such as inconsistent grossing quality.

Binding Time – the formation of the methylene bridges takes time to happen, even in the presence of optimally grossed tissues (thickness). The inconsistency in time given for fixation, i.e., the molecular binding or additive effect, will grossly affect the quality of cellular image presented at the end of the process. Image slides will progressively become blurry instead of sharp and distinct, and the histochemical staining with routine hematoxylin & eosin as well as other special stains will be altered because tissue will have less of an affinity for the dyes.

In this brief treatment we have discussed maintaining optimal fixation by first understanding the histochemistry of fixation. In part 2 we will continue this discussion with a focus on consistent process, procedure, and reagent management, from a daily operational standpoint and quality assurance practices. As mentioned in the early part of this article, laboratories must develop and be consistent with daily fixation protocol procedures. Achieving optimal fixation is easy. Maintaining it requires constant focus and commitment.

References:
Brown, S., “The Cost of Reprocessing”, Professional Development Series, Lab Management Consultants 2018.
Carson, FL, Histotechnology – A Self-Instruction Text, Chicago, IL., ASCP Press, 1997.
Hewlett, B., “NBF Penetration Rate”, Histonet Server, 2002.