Standard pathology practice relies on automated processing of tissues fixed in 10% neutral buffered formalin followed by staining protocols that were optimized over the past century for microscopic visualization. In the last two decades, molecular assays are increasingly applied to formalin fixed, paraffin embedded tissues although this effort is hampered by lesser quantity and poorer quality of nucleic acid compared with that recovered from fresh or frozen tissue. Hypothesis to be tested: We propose that, in order to improve fixation technology that will be embraced by the pathology community, key steps of standard formalin fixation cannot be altered. On the other hand, addition of chemical stabilizers to standard reagents, and altering the temperature of the initial phase of formalin fixation, are realistic changes that could improve downstream molecular analysis without adversely impacting morphology and immunostain outcomes. Based on synthesis of a diverse literature, we present a two-part hypothesis to drive development of enhanced formalin fixation protocols: A). The irreversible damage to nucleic acid occurring during formalin fixation is mainly biochemical and can be largely prevented by inhibiting endogenous nuclease activity during formalin infusion. To address this, broad-spectrum nuclease inhibitors will be identified that are small enough to co-diffuse with formalin into tissue spaces, and these will be tested with or without refrigeration in an otherwise-standard, automated tissue processing protocol. B). Nucleic acid damage accrues after fixation, due mainly to slow, persistent, oxidation by reactive oxygen species (ROS) derived from atmospheric O2, trapped inside the tissue block. To address this, ROS scavengers will be identified that are water-soluble, inexpensive, and small enough to diffuse rapidly into tissue spaces during the first """"""""post-formalin"""""""" dehydration step, yet are poorly soluble in alcohol or xylene so that, upon tissue transfer into water-free solvents, the scavengers are embedded in the dehydrated tissue block matrix where they stand ready to quench newly-formed ROS during storage in situ. Relation to a follow-on R33: When this R21 is completed, procedural improvements will have been made which preserve DNA &RNA during ordinary 10% buffered formalin fixation and subsequent storage as paraffin embedded tissue. In R33 work, these compounds will be subjected to pilot scale manufacture as beta test kits, to be validated on diverse human cancer tissues at multiple sites.
IMAT Application: Enhanced Formalin Fixation to Improve Molecular Tests on Solid Tissues Margaret L. Gulley MD, 2-23-10 Project Narrative We describe a rational program of compound screening and early stage validation, with the goal of dramatically enhancing the practice of formalin fixation and tissue processing to better accommodate molecular methods. When completed, this R21 work will have identified stabilizers that can be added directly to the ordinary solvents used for fixation and automated processing that, along with thermal modification, improve archival tissue quality and are simple to use, inexpensive and compatible with conventional pathology laboratory protocols and automation. In the R33 phase of the work, the enhancements will be subjected to a rigorous program of beta testing in multiple CLIA-certified US pathology laboratories, both in academia and in reference laboratory settings. 1
