High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research

Area of Science:

• Biomedical Imaging
• Molecular Pathology
• Immunohistochemistry

Background:

• Multiplex immunofluorescence (mIF) enables spatial analysis of multiple protein biomarkers in single tissue sections.
• Existing mIF technologies often compromise sensitivity or throughput, hindering the detection of low-abundance biomarkers.
• This limitation is critical for comprehensive analysis of complex biological samples.

Purpose of the Study:

• To introduce a new mIF approach overcoming sensitivity-throughput tradeoffs.
• To enable high-quality, high-multiplexing, and high-throughput tissue analysis.
• To facilitate the detection of multiple biomarkers for deeper biological insights.

Main Methods:

• Utilized DNA barcodes conjugated to antibodies for amplification via single-molecule amplification.
• Employed sequential detection cycles of fluorescently labeled oligonucleotides to visualize biomarkers.
• Integrated automated tissue preparation, signal amplification, iterative imaging, and AI-enhanced spatial analysis.
• Applied the protocol to formalin-fixed, paraffin-embedded (FFPE) multi-tissue micro-arrays (TMAs).

Main Results:

• Achieved staining quality comparable to clinical-grade immunohistochemistry (IHC).
• Enabled detection of eight or more biomarkers per sample with high multiplexing.
• Demonstrated high-throughput whole-slide imaging capabilities.
• Successfully applied to FFPE TMAs including tonsil, melanoma, colon, and lymph node tissues.

Conclusions:

• The developed DNA-barcoded mIF assay overcomes critical limitations in sensitivity, multiplexing, and throughput.
• This advanced technique provides deeper insights into tumor microenvironment and immune landscape.
• Facilitates understanding of molecular signatures and therapeutic response biomarkers, advancing precision medicine.