Miniaturized FISH for Screening of Onco-Hematological Malignancies

Abstract

Fluorescence in situ hybridization (FISH) represents a major step in the analysis of chromosomal aberrations in cancer. It allows the precise detection of specific rearrangements, both for diagnostic and prognostic purposes. Here we present a miniaturized FISH method performed on fresh and fixed hematological samples. This procedure has been developed together with a microfluidic device that integrates cluster-assembled nanostructured TiO₂ (ns-TiO₂) as a nanomaterial promoting hematopoietic cell immobilization in conditions of shear stress. As a result of miniaturization, FISH can be performed with at least a 10-fold reduction in probe usage and minimal cell requirements, creating the possibility of using FISH in genetic screening applications. We developed the protocol on tumor cells and bone marrow (BM) from a normal donor using commercially sex-specific and onco-hematology probes. The procedure was then validated using either BM or peripheral blood (PB) from six patients with hematological diseases, each associated with different genetic lesions. Miniaturized FISH demonstrated comparable performance to standard FISH, indicating that it is suitable for genetic screenings, in research, and in clinical settings for the diagnosis of samples from onco-hematological malignancies.

FISH and Chips

Fluorescence in situ hybridization (FISH) is frequently used in basic research and clinical settings to resolve complex chromosomal rearrangements. While the technique is sensitive and effective, the probes needed for FISH can be costly and samples with reduced numbers of cells may not yield useful results. The engineering of polymeric microchannels within microfluidic devices offers possible solutions to these problems since smaller volumes of reagents are required and cell samples can be concentrated, but their implementation for FISH experiments has been limited due to the shearing forces generated by solutions moving through the channels. Immobilization of cells within the channels is therefore necessary to ensure that meaningful FISH results can be obtained from cell samples in microfluidic devices. A. Zanardi et al. at Tethis S.R.L. (Milan, Italy) recently found that cluster-assembled nanostructured TiO₂ (ns-TiO₂) coatings are able to rapidly immobilize living and fixed hematopoietic cells and hold them stably, even in the presence of prolonged shear stresses. Using this coating, the authors have engineered a microfluidic device capable of immobilizing cell samples for miniature FISH. The goals of their design were to decrease the cost of FISH by reducing the required volume of reagents and fluorescent probe, increase the throughput of the experiments by allowing parallel processing of samples and automated microscopic evaluation of the results, and enable FISH with cell-sparse samples by concentrating the cells within a confined space. The device, which successfully fulfilled each of the authors' goals, consists of a single straight microchannel in a polymeric microfluidic pad, which adheres to a standard glass slide coated with ns-TiO₂. The device was validated in a series of FISH experiments conducted on cultured hematopoietic tumor cells, bone marrow, and peripheral blood samples and run in parallel with a standard FISH protocol. Using miniaturized FISH, the authors could detect hematological malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, as well as a case of sex chromosome chimerism following bone marrow transplant, all with similar accuracy and reproducibility to standard FISH.

See “Miniaturized FISH for screening of onco-hematological malignancies” on page 497.

Keywords: :

• FISH
• titanium dioxide
• onco-hematology
• nanomaterial

Acknowledgments

We wish to thank Elena Belloni and Omar Malazzi (IFOM-IEO Campus, Milan, Italy) for technical support and Marzia Quaglio (Politecnico di Torino, Italy) and Matteo Cocuzza (CNR, Torino, Italy) for technical advice in microfluidics.

Competing interests

A.Z., D.B., and R.C. are currently employees of Tethis S.R.L.; and E.B. and P.M. are share-holders of Tethis S.R.L.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2144/000113445