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Optimizing Human Tissue Fixation for High‐Quality Downstream Analysis Using Real‐Time Fixation Monitoring

Molecular markers are invaluable to characterizing and treating disease. The pathologist routinely uses immunohistochemistry of formalin‐fixed paraffin embedded tissues to determine diagnosis or molecular subtypes in cancer and other diseases. Most markers that have been developed to date are robustly detected after varied fixation conditions. A greater challenge will be to develop diagnostics for more difficult markers, such as phosphoproteins, that are highly labile yet are critical “companion diagnostic” biomarkers that indicate sensitivity to selected chemotherapeutics.. Meeting this challenge with formalin fixation, the standard clinical tissue preservation process, will be difficult because of the overall poor understanding of formalin's ability to preserve all tissue biomarkers. We have demonstrated that labile phosphoproteins are better preserved using a rapid fixation protocol of 2 hours cold formalin followed by 2 hours hot formalin, which allows the cold formalin to penetrate tissue specimens completely while minimizing other enzymatic activity and the subsequent hot formalin step to crosslink the tissue proteins rapidly and thoroughly. To aid in studying this problem as well as to provide a potential metric for assessing fixation, we have invented a real‐time ultrasound time‐of‐flight (TOF) instrument that is capable of monitoring and imaging the critical step in formalin fixation, diffusion of the fixative into tissue, thereby creating a quantifiable quality metric for tissue fixation. This technology has been applied to most human tissues types and tissue from multiple disease states to demonstrate a broad range of diffusion, and thus fixation, behaviors. Our measurements can be converted into tissue diffusivity constants that correlate with the apparent diffusion constant calculated using MRI, despite the differences in the approaches, indicating that our approach is biophysically plausible. Using data collected from TOF analysis of many tissues, we have therefore developed a novel rapid fixation program that will ensure high quality downstream analysis for a broad range of tissues that correspond to the thickness of a full histology cassette (6 mm).