Abstract
Modern histopathology is built on the cornerstone principle of tissue fixation, however there are currently no analytical methods of detecting fixation and as a result, in clinical practice fixation is highly variable and a persistent source of error. We have previously shown that immersion in cold formalin followed by heated formalin is beneficial for preservation of histomorphology and have combined two-temperature fixation with ultra-sensitive acoustic monitoring technology that can actively detect formalin diffusing into a tissue. Here we expand on our previous work by developing a predictive statistical model to determine when a tissue is properly diffused based on the real-time acoustic signal. We trained the model based on the morphology and characteristic diffusion curves of 30 tonsil cores. To test our model, a set of 87 different tonsil samples were fixed with four different protocols: dynamic fixation according to our predictive algorithm (C/H:Dynamic, N = 18), gold-standard 24 hour room temperature (RT:24hr, N = 24), 6 hours in cold formalin followed by 1 hour in heated formalin (C/H:6+1, N = 21), and 2 hours in cold formalin followed by 1 hour in heated formalin (C/H:2+1, N = 24). Digital pathology analysis revealed that the C/H:Dynamic samples had FOXP3 staining that was spatially uniform and statistically equivalent to RT:24hr and C/H:6+1 fixation protocols. For comparison, the intentionally underfixed C/H:2+1 samples had significantly suppressed FOXP3 staining (p<0.002). Furthermore, our dynamic fixation protocol produced bcl-2 staining concordant with standard fixation techniques. The dynamically fixed samples were on average only submerged in cold formalin for 4.2 hours, representing a significant workflow improvement. We have successfully demonstrated a first-of-its-kind analytical method to assess the quality of fixation in real-time and have confirmed its performance with quantitative analysis of downstream staining. This innovative technology could be used to ensure high-quality and standardized staining as part of an expedited and fully documented preanalytical workflow.
Highlights
Clinical tissue processing techniques “fix” tissues with crosslinking agents that shut down metabolism within cells and preserve crisp and clear cellular morphology
Tissue morphology was improved with 5 hours in cold neutral buffered formalin (NBF) (C/H:5+1) but further cold soak times provided no additional benefit
We have shown the development of a first-of-its-kind tissue fixation system capable of determining in real-time when a biospecimen has adequate formaldehyde to guarantee high-quality fixation as demonstrated by ideal and spatially uniform functional staining from downstream IHC assays
Summary
Clinical tissue processing techniques “fix” tissues with crosslinking agents that shut down metabolism within cells and preserve crisp and clear cellular morphology. Proper fixation protocols are empirically determined by examining the histologically stained tissue for proper morphological features. There have been some published guidelines from the College of American Pathologists to provide better tissue handling standards, including suggested times of fixation for most tissues in room temperature formalin [2]. The result is a mixed bag of adequate and poor morphology depending on the operator, institution, tissue type, and biomarker of interest. By the time the morphology is interrogated, it is too late to improve the quality of the tissue so proper fixation the first time is critically important
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