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Monitoring Dehydration and Clearing in Tissue Processing for High-Quality Clinical Pathology.

Author(s): Lerch ML, Bauer DR, Theiss A, Chafin D, Otter M, Baird GS

Publication: Biopreserv Biobank, 2019, Vol. 17, Page 303-311

PubMed ID: 31107113 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study utilized time-of-flight (TOF) ultrasound analysis as a method of determining the diffusion rate of reagents into 13 different tissue types during dehydration and clearing of formalin-fixed tissue specimens. A total of 270 tissue specimens representing 13 different tissue types were procured from surgically resected specimens.  The tissue types chosen were selected to reflect a range of inferred reagent diffusion rates based on tissue composition. The size of the specimen was standardized, as either a 6 mm punch biopsy or a 6 mm² cube were used and both had a depth of 4-7 mm.  Tissues were fixed in neutral buffered formalin for 10 h at 4°C, then for 2 h at 45°C prior to the experiment. Formalin-fixed tissue specimens were placed in divided mesh biopsy cassettes between a transmitting and a receiving transducer and underwent automated processing with a modified “dip and dunk” commercial processor. Specimens were sequentially immersed in the following stages at 20°C for 8 h per stage: 70% ethanol (in water), 90% ethanol (in water), 100% ethanol, and xylene. ∆TOF was determined by calculating the differential between high frequency (4 MHz) pulses that passed through the tissue and the reagent. Equilibrium was reached with complete diffusion of the reagent. The time that elapsed until a 90% change in signal was termed the Ƭ90 constant. The effects of a rapid tissue processing protocol and incomplete dehydration on histomorphology were also investigated in a subset of specimens to  assess the sensitivity of the TOF ultrasound assay by (1) reducing the length of the dehydration and clearing incubations for 15 kidney and 34 breast specimens (4 h in 70% ethanol, 3 h in 90% ethanol, 3 h in 100% ethanol, 3 h in xylene, all at 20°C), and (2) omitting a key step in dehydration (incubation in 100% ethanol) in one kidney and one breast specimen. Morphology was assessed in specimens processed with the rapid tissue processing protocol and those with incomplete dehydration after paraffin embedding, sectioning (4 µm thick), and hematoxylin and eosin staining.

   Summary of Findings:

   Sound velocities of the different processing reagents were unique but comparable: 70% ethanol = 1245 m/s, 90% ethanol = 1178 m/s, 100% ethanol = 1144 m/s, xylene = 1330 m/s. The time constant (Ƭ90), the time necessary for a 90% change in signal which passed through the tissue (and changed as reagent penetrated the tissue and approached equilibrium), was influenced by both the tissue and reagent. Based on results obtained, the authors classified tonsil, colon, kidney, and lung as tissue types with fast reagent diffusion; and adipose, skin, and breast as tissues with slow reagent diffusion.  Using the complete tissue processing protocol (8 h in each reagent at 20°), Ƭ90 constants for fast diffusion tissue types were 2.86-3.04 h for 70% ethanol, 1.17-1.43 h for 90% ethanol, 0.97-1.75 h for 100% ethanol, and 1.06-1.61 h for xylene. Ƭ90 constants for slow diffusion tissue types were 3.82-7.25 h for 70% ethanol, 1.61-4.10 h for 90% ethanol, 1.43-5.87 h for 100% ethanol, and 1.20-2.14 h for xylene.  Based on Ƭ90 constants obtained with the complete tissue processing protocol, a rapid tissue processing protocol (4 h in 70% ethanol, 3 h in 90% ethanol, 3 h in 100% ethanol, 3 h in xylene) was developed and evaluated.  Ƭ90 constants obtained with the rapid tissue processing protocol with a subset of  kidney (fast diffusion) and breast (slow diffusion) specimens were slightly longer than those observed for the same tissue type with the complete tissue processing protocol: for 70% ethanol, 3.71 vs 2.97 for kidney, 4.95 h vs 3.85 for breast; for 90% ethanol, 1.59 vs 1.40 h for kidney, 2.67 h vs 2.26 h for breast; for 100% ethanol, 1.40 vs 1.08 h for kidney, 2.86 h vs 2.71 h for breast; and for xylene, 1.27 vs 1.06 h for kidney, 1.29 h vs 1.20 h for breast.  The authors stated that the slight increase in the Ƭ90 constant was expected given the reduction in dehydration and clearing incubation times.  Breast and kidney specimens processed under the rapid tissue protocol displayed normal morphological preservation.  Individual kidney and breast specimens that underwent incomplete dehydration via omission of the 100% ethanol incubation displayed a longer Ƭ90 constant for xylene compared to respective case-matched controls that underwent rapid tissue processing (kidney: 1.90 vs 1.27 h, breast: 0.91 vs 0.58 h, respectively), poor histomorphological preservation, and gross changes such as section fragility and brittleness.

   Biospecimens

   • Tissue - Kidney
   • Tissue - Breast
   • Tissue - Liver
   • Tissue - Colorectal
   • Tissue - Skin
   • Tissue - Tonsil
   • Tissue - Lung
   • Tissue - Adipose

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Morphology	Ultrasound
   Morphology	H-and-E microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Dehydration duration/condition	8 h per stage 3-4 h per stage
   Biospecimen Acquisition	Biospecimen location	Kidney Breast Liver Adipose Skin Tonsil Lung Colon
   Biospecimen Preservation	Clearing duration/condition	8 h per stage 3 h per stage

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