Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.

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...

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Autores principales:	Daniel R Bauer, Torsten Leibold, David R Chafin
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f058443f3b3a4efeb2da853ecc879f2e
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spelling	oai:doaj.org-article:f058443f3b3a4efeb2da853ecc879f2e2021-12-02T20:19:11ZMaking a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.1932-620310.1371/journal.pone.0258495https://doaj.org/article/f058443f3b3a4efeb2da853ecc879f2e2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0258495https://doaj.org/toc/1932-6203Modern 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.Daniel R BauerTorsten LeiboldDavid R ChafinPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10, p e0258495 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daniel R Bauer Torsten Leibold David R Chafin Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.
description	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.
format	article
author	Daniel R Bauer Torsten Leibold David R Chafin
author_facet	Daniel R Bauer Torsten Leibold David R Chafin
author_sort	Daniel R Bauer
title	Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.
title_short	Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.
title_full	Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.
title_fullStr	Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.
title_full_unstemmed	Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.
title_sort	making a science out of preanalytics: an analytical method to determine optimal tissue fixation in real-time.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/f058443f3b3a4efeb2da853ecc879f2e
work_keys_str_mv	AT danielrbauer makingascienceoutofpreanalyticsananalyticalmethodtodetermineoptimaltissuefixationinrealtime AT torstenleibold makingascienceoutofpreanalyticsananalyticalmethodtodetermineoptimaltissuefixationinrealtime AT davidrchafin makingascienceoutofpreanalyticsananalyticalmethodtodetermineoptimaltissuefixationinrealtime
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Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time.

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