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Assessing the effects of a 3D pathology tissue-processing workflow on downstream molecular analyses.

Elena Baraznenok1,2,3, Huai-Ching Hsieh4,5, Lydia Lan2,6

  • 1Department of Bioengineering, University of Washington, Seattle, WA, USA.

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Summary

Non-destructive 3D pathology enhances tissue analysis by preserving samples for molecular studies. This study confirms that 3D pathology protocols maintain DNA, RNA, and protein integrity for downstream molecular assays.

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Area of Science:

  • Biomedical Imaging
  • Pathology
  • Molecular Biology

Background:

  • Standard 2D histopathology limits tissue sampling and morphological context.
  • Non-destructive 3D pathology offers volumetric data and sample preservation for future analyses.
  • The impact of 3D pathology on biomolecules for downstream assays is not well understood.

Purpose of the Study:

  • To evaluate the effect of a 3D pathology protocol on DNA, RNA, and protein integrity.
  • To assess the compatibility of 3D pathology processed tissues with downstream molecular assays.
  • To determine if 3D pathology can be combined with molecular analyses.

Main Methods:

  • A 3D pathology protocol (deparaffinization, staining, optical clearing, light-sheet microscopy) was applied to FFPE cancer specimens.
  • Tissues were re-embedded and compared with non-processed controls.
  • DNA/RNA extraction, quality assessment, PCR, RT-qPCR, Sanger sequencing, RNA sequencing, and IHC were performed.

Main Results:

  • A slight decrease in DNA/RNA yield and increased fragmentation were observed, but amplifiability was largely preserved.
  • Sanger sequencing confirmed accurate DNA sequences, and RNA sequencing showed largely unchanged transcriptomic profiles.
  • Immunohistochemistry (IHC) staining for common biomarkers yielded comparable results, indicating good protein preservation.

Conclusions:

  • The 3D pathology workflow minimally impacts DNA, RNA, and protein integrity.
  • 3D pathology is compatible with downstream molecular analyses, including sequencing and IHC.
  • This study demonstrates the feasibility of integrating 3D pathology with molecular applications for comprehensive cancer analysis.