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.

Introduction

Considerable effort is currently being concentrated toward the development of analytical tools with improved performance, sensitivity, and information throughput through integrated approaches of engineering and miniaturization at affordable costs (1,2); new disciplines such as microfluidics, biotechnology, and nanotechnology, are expected to play a key role in driving this innovation (3).

Among analytical assays based on fluorescence read-out, fluorescence in situ hybridization (FISH) is a widespread and informative tool, utilized in both basic research and in diagnostics (4,5). Through fluorescence detection by hybridization with DNA probes of chromosomal sequences in fixed nuclei on slides, FISH represents a robust method able to resolve complex genetic rearrangements that would have remained unresolved by conventional cytogenetics, relying on the analysis of chromosome structure in metaphase. This consideration particularly applies to tumors showing poor chromosome morphology in metaphase preparations, therefore preventing accurate diagnostic evaluation. In fact, using appropriate probes, FISH can also be successfully performed on interphase nuclei to detect specific chromosomal rearrangements, such as the 9/22 translocation in chronic myeloid leukemia (6), offering an essential tool for cancer diagnosis and prognosis.

FISH technology has remained substantially unchanged since its introduction approximately 20 years ago (7,8), and its widespread utilization was mainly hampered by its cost. Indeed, in many laboratories, FISH is only utilized as a second step on a selected number of cases previously screened using classical cytogenetic techniques. Additionally, this technique may suffer severe limitations whenever samples with poor cell content have to be managed.

To reduce costs and improve assay performance, microfluidics (9,10) can provide a means for miniaturization through the engineering of polymeric microchannels in devices wherein reagents can be loaded in small volumes, and cellular samples can therefore be concentrated. However, relevant technical challenges have to be overcome: due to the micrometric section of channels in such tools, flowing fluids cause intense shear stress on cells. This can cause them to be easily disrupted or detached, which can therefore compromise the assay (11).

In this context, miniaturization of FISH through microfluidic methods could be a promising solution only if cell immobilization inside the microchannel is appropriately provided.

Sieben and coworkers recently proposed a “lab-on-a-chip” approach for FISH miniaturization based on microfluidic technology (12,13). They engineered a fully integrated chip in which hematopoietic cells were immobilized by heating inside a microwell together with an automated FISH protocol; however, the complexity of the chip (which required a sophisticated fabrication facility), and its consequent cost were indicated by the authors as key challenges that needed to be addressed in order to make this technique accessible.

We have focused our research on the characterization of biomaterials and coatings with properties promoting cell adhesion (14), and discovered that the cluster-assembled nanostructured TiO₂ coating (ns-TiO₂) is able to trigger a rapid and efficient immobilization of both living and fixed hematopoietic cells, even in the presence of prolonged shear stress (unpublished data). To fully exploit this feature, we have engineered a simple device, based on microfluidics, to set up a miniaturized FISH approach. The device consists of a polymeric microfluidic pad, with a single straight microchannel, adhering to a standard glass slide coated with ns-TiO₂ that enables rapid immobilization of cells in a small and confined space.

The efficiency of the approach was tested by performing FISH on a panel of cultured hematopoietic tumor cells as well as on bone marrow (BM) from normal donor, prepared from fresh samples. Its performance versus the standard FISH protocol was also evaluated in either BM or peripheral blood (PB) of different cases of hematological malignancies, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and a case of sex chromosome chimerism after BM transplantation.

By comparing classic versus miniaturized FISH, we obtained a similar degree of accuracy, quality, and reproducibility with respect to the standard protocol. The procedure is simple, and the analysis is performed on a standard fluorescence microscope both at low and high resolution. In addition, automation of the procedure can be envisaged when genetic screening programs are planned.