Active monitoring of formaldehyde diffusion into histological tissues with digital acoustic interferometry

The preservation of certain labile cancer biomarkers with formaldehyde-based fixatives can be considerably affected by preanalytical factors such as quality of fixation. Currently, there are no technologies capable of quantifying a fixative's concentration or the formation of cross-links in tissue specimens. This work examined the ability to detect formalin diffusion into a histological specimen in real time. As formaldehyde passively diffused into tissue, an ultrasound time-of-flight (TOF) shift of several nanoseconds was generated due to the distinct sound velocities of formalin and exchangeable fluid within the tissue. This signal was resolved with a developed digital acoustic interferometry algorithm, which compared the phase differential between signals and computed the absolute TOF with subnanosecond precision. The TOF was measured repeatedly across the tissue sample for several hours until diffusive equilibrium was realized. The change in TOF from 6-mm thick ex vivo human tonsil fit a single-exponential decay ([Formula: see text]) with rate constants that varied drastically spatially between 2 and 10 h ([Formula: see text]) due to substantial heterogeneity. This technology may prove essential to personalized cancer diagnostics by documenting and tracking biospecimen preanalytical fixation, guaranteeing their suitability for diagnostic assays, and speeding the workflow in clinical histopathology laboratories.