Rapid micro fluorescence in situ hybridization in tissue sections

This paper describes a micro fluorescence in situ hybridization ({\mu}FISH)-based rapid detection of cytogenetic biomarkers on formalin-fixed paraffin embedded (FFPE) tissue sections. We demonstrated this method in the context of detecting human epidermal growth factor 2 (HER2) in breast tissue sections. This method uses a non-contact microfluidic scanning probe (MFP), which localizes FISH probes at the micrometer length-scale to selected cells of the tissue section. The scanning ability of the MFP allows for a versatile implementation of FISH on tissue sections. We demonstrated the use of oligonucleotide FISH probes in ethylene carbonate-based buffer enabling rapid hybridization within < 1 min for chromosome enumeration and 10-15 min for assessment of the HER2 status in FFPE sections. We further demonstrated recycling of FISH probes for multiple sequential tests using a defined volume of probes by forming hierarchical hydrodynamic flow confinements. This microscale method is compatible with the standard FISH protocols and with the Instant Quality (IQ) FISH assay, reduces the FISH probe consumption ~100-fold and the hybridization time 4-fold, resulting in an assay turnaround time of < 3 h. We believe rapid {\mu}FISH has the potential of being used in pathology workflows as a standalone method or in combination with other molecular methods for diagnostic and prognostic analysis of FFPE sections.

💡 Research Summary

This paper introduces a rapid micro‑fluorescence in situ hybridization (μFISH) platform that leverages a non‑contact microfluidic scanning probe (MFP) to perform HER2 status assessment on formalin‑fixed paraffin‑embedded (FFPE) breast tissue sections. Conventional FISH, while accurate, suffers from long hybridization times (often overnight), high probe consumption (10–16 µL per assay), and the need for specialized equipment and skilled personnel. The authors address these limitations by integrating microfluidic flow confinement with precise spatial control, enabling probe delivery and washing directly over selected microscopic regions of the tissue without physical contact.

The MFP head is a silicon‑glass hybrid containing four micro‑channels (two inner injection apertures of 100 × 100 µm² and two outer aspiration apertures of 100 × 200 µm²). By simultaneously injecting a probe solution through the inner pair and a wash buffer through the outer pair, a hierarchical hydrodynamic flow confinement (hHFC) is generated. This creates a micron‑scale “liquid pocket” that contacts the tissue surface, allowing rapid diffusion of probes into the nuclei. After a short incubation, the inner flow is stopped and the outer flow washes away unbound probe, after which imaging is performed. The footprint of each assay is only 0.096 mm², and the probe volume required is as low as 0.5 µL.

Key methodological innovations include: (1) the use of short 18‑nt oligonucleotide probes for the centromere of chromosome 17 (Cen17) and a FITC‑labeled HER2 probe; (2) a formamide‑free hybridization buffer based on 15 % ethylene carbonate, 20 % dextran sulfate, 0.67× SSC and 600 mM NaCl. This EC‑based buffer dramatically accelerates hybridization kinetics while preserving specificity. As a result, centromere signals appear within ~30 seconds, and HER2/Chr17 ratio determination is completed in 10–15 minutes, compared with several hours in standard protocols.

The authors demonstrate the system on FFPE MCF‑7 and BT‑474 cell blocks and on 5 µm thick breast cancer tissue microarrays. Probe recycling is achieved by recirculating the same probe volume through successive hHFC cycles, enabling 5–10 consecutive tests from a single loading, which translates to a ~100‑fold reduction in probe consumption relative to conventional microfluidic FISH devices that typically require 2–16 µL per assay.

Performance comparison with the commercial Instant Quality (IQ) FISH assay shows that μFISH yields identical HER2 amplification classifications (HER2/CEN17 ratio >2 for positive, ≤2 for negative) while cutting total assay turnaround time to under 3 hours (including sample pretreatment, hybridization, washing, and imaging). The method is fully compatible with existing FISH protocols, allowing laboratories to adopt it without major workflow changes.

Beyond speed and cost, the non‑contact nature of the MFP eliminates mechanical stress on delicate FFPE sections, and the ability to move the liquid pocket across the slide enables targeted analysis of heterogeneous tumor regions—a critical advantage for precision oncology. The authors suggest that coupling μFISH with multiplexed probe sets and AI‑driven image analysis could further enhance diagnostic throughput and accuracy.

In summary, the study presents a robust, scalable, and clinically relevant microfluidic approach that transforms FISH from a labor‑intensive, reagent‑heavy assay into a rapid, low‑volume, and highly adaptable diagnostic tool, poised for integration into routine pathology workflows for HER2 and potentially other gene‑level biomarkers.