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Assessing the Effects of a 3-Dimensional (3D) Pathology Tissue-Processing Workflow on Downstream Molecular Analyses.
Elena Baraznenok1, Huai-Ching Hsieh2, Lydia Lan3
1Department of Bioengineering, University of Washington, Seattle, Washington; Department of Mechanical Engineering, University of Washington, Seattle, Washington; Department of Bioengineering, University of California Berkeley, Berkeley, California.
Non-destructive 3D pathology preserves tissue integrity and biomolecules like DNA, RNA, and proteins for downstream molecular analysis. This approach enhances histopathology by enabling volumetric imaging and future molecular studies.
Area of Science:
- Biomedical Imaging
- Molecular Pathology
- Cancer Research
Background:
- Standard 2D histopathology has limitations in tissue sampling and volumetric context.
- Non-destructive 3D pathology offers enhanced tissue analysis and preserves tissue integrity for future studies.
- The impact of 3D pathology protocols on biomolecules requires systematic evaluation.
Purpose of the Study:
- To systematically evaluate the impact of 3D pathology protocols on DNA, RNA, and proteins.
- To assess the compatibility of 3D pathology processed tissues with downstream molecular assays.
- To compare biomolecule quality and assay performance between 3D pathology processed and control FFPE tissues.
Main Methods:
- Applied a 3D pathology protocol (deparaffinization, staining, clearing, light-sheet microscopy) to FFPE cancer specimens.
- Re-embedded processed tissues in paraffin and compared them with unprocessed controls.
- Extracted DNA and RNA, assessed quality, and tested amplifiability using PCR and RT-qPCR.
Main Results:
- Slightly decreased DNA/RNA yield and increased fragmentation were observed post-3D processing, but PCR amplifiability was largely preserved.
- Sanger sequencing confirmed accurate DNA sequence determination.
- Total RNA sequencing showed largely unchanged global transcriptomic profiles, and IHC staining indicated preserved protein biomarkers.
Conclusions:
- 3D pathology is feasible for integration with downstream molecular analyses.
- The study demonstrates the preservation of key biomolecules (DNA, RNA, proteins) after 3D processing.
- Further research is warranted to explore 3D pathology's integration with diverse advanced molecular assays.
