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

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.