Breast cancer HER2 analysis by extra-short incubation microfluidics-assisted fluorescence in situ hybridization (ESIMA FISH)

Fluorescence in situ hybridization (FISH) is a well-recognized technique for evaluating the human epidermal growth factor receptor 2 (HERZ) gene status in breast cancer, despite its traditionally long and costly experimental procedure. While microfluidics was found to help reducing the experimental duration by improving the mass transport of the reactants, the slow reaction rate of the reagents used in the probe solution was still the rate limiting step. In particular, the use of formamide, serving as a double-helix destabilizing agent, slowed down the hybridization. This article presents a new approach, the extra-short incubation microfluidics-assisted FISH (ESIMA FISH), combining a different, highly reactive probe solution containing ethylene carbonate buffer for fast double helix destabilization (HER2 IQ-FISH pharmDx (TM) probe), with a microfluidic system for improved reactant delivery. As this fast FISH probe is radically different from the ones used in any previous microfluidic FISH studies, a complete redesign of the protocol is required. Therefore, we used a fractional factorial design of experiments to optimize the microfluidic, ethylene carbonate buffer-based FISH protocol. After optimization, the incubation time required for performing FISH can be as short as 15 min for a cell line sample and 35 min for a human tissue slide, corresponding respectively to a fourth and a half of the duration of the corresponding step in the classical IQ-FISH protocol. ESIMA FISH is overall the fastest and most economic microfluidic FISH technique for tissue analysis at the moment, at the expense of a weaker HER2 gene signal than that of a standard FISH protocol. However, this limitation does not affect the scoring of the FISH signals, as the HER2 assessments of adjacent slides of human breast cancer tissue by ESIMA FISH and standard IQ-FISH were similar. (C) 2017 Elsevier B.V. All rights reserved.