MSACL 2025 Abstract

HistoProbe: A Dual-Mode Thermally Assisted Microfluidic Platform for Rapid, Solvent-Minimal Mass Spectrometry Analysis of FFPE Tissues

Malek Hassan, Haidy Metwally, Martin Kaufmann, Rachel Theriault, Kevin Ren, Kaitlin Vanderbeck, Randy Ellis, John Rudan, Richard Oleschuk
Queen’s University, Kingston, Ontario, Canada

Malek Hassan, M.Pharm, M.Sc. (Presenter) Queen's University >> POSTER (PDF)

Presenter Bio: I am a PhD Candidate in analytical chemistry at Queen’s University, specializing in mass spectrometry method development for clinical and translational applications. My research focuses on automating sample preparation workflows and advancing ambient ionization techniques to support high-throughput molecular profiling in biobanking and histopathology. I am passionate about bridging analytical innovations with clinical utility and am committed to driving translational impact through interdisciplinary collaboration.

Relevant Financial Disclosures (within past 24 months, reported on Jun 09, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Formalin-fixed paraffin-embedded (FFPE) tissues are essential clinical resources, yet conventional deparaffinization methods are slow, laborious, and environmentally unfriendly, relying heavily on toxic solvents. Addressing these limitations, we introduce HistoProbe, a novel microfluidic platform enabling rapid, localized, and minimally solvent-dependent analysis and imaging of FFPE tissues by mass spectrometry (MS).

METHODS:
HistoProbe integrates a modified liquid microjunction surface sampling probe (LMJ-SSP) with precise thermal regulation adapted from 3D printer hardware. The platform operates in two distinct modes: Online and Offline. Online-HistoProbe enables direct metabolite extraction and real-time analysis from FFPE tissue sections at elevated temperatures (~60 °C) without any prior sample preparation or deparaffinization. It functions as a standalone tool by interfacing directly with the electrospray ionization (ESI) source of the mass spectrometer, offering a plug-and-play solution for rapid FFPE tissue analysis. Offline-HistoProbe enables targeted thermal deparaffinization using reduced volumes of solvents (i.e., ethyl acetate, toluene, or xylene) at controlled temperatures (up to 75 °C). This mode drastically lowers solvent usage compared to standard protocols while maintaining compatibility with high-resolution imaging. Both modes were coupled with desorption electrospray ionization mass spectrometry (DESI-MS) for metabolic profiling and hyperspectral molecular imaging. Data were analyzed using principal component analysis (PCA) for dimensionality reduction and unsupervised clustering of metabolic features.

RESULTS:
Optimization studies demonstrated that Online-HistoProbe eliminated the need for sample preparation and solvent use, reducing total analysis time to approximately 17 minutes per sample and enabling high-throughput analysis exceeding 80 samples per day. The practical spatial resolution of the online mode was approximately 1 millimeter, suitable for rapid tissue screening and metabolic profiling. Offline-HistoProbe, optimized with ethyl acetate at 75 °C, achieved comparable or superior metabolite recovery to traditional xylene-based deparaffinization, while reducing solvent consumption by over 99.5%. It supported a practical spatial resolution of 50–100 micrometers, allowing fine-grained molecular imaging. Both modes reliably distinguished neoplastic from non-neoplastic regions in melanoma FFPE tissues and enabled annotation of key biomolecules, including fatty acids, phosphatidylinositols, and oxidized lipids relevant to cancer metabolism.

CONCLUSION:
HistoProbe represents a significant advancement in mass spectrometry-based histopathology, combining speed, analytical precision, automation, and sustainability. Its dual-mode functionality addresses clinical demands by offering high-throughput direct analysis (Online mode) and high-resolution molecular imaging (Offline mode). HistoProbe substantially reduces solvent consumption, sample preparation time, and environmental impact, demonstrating immense potential for clinical diagnostics, biomarker discovery, and rapid intraoperative tissue assessments.