An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH)

Abstract

Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes—the 22 autosomes and the X and Y sex chromosomes—can be stained or “painted” with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.

Identification of a subtle t(16;19)(p13.3;p13.3) in an infant with multiple congenital abnormalities using a 12-colour multiplex FISH telomere assay, M-TEL

“…Analysis of the combinatorially labelled probes was performed using two software analysis programmes: (i) a modified version of MacProbe v 4.1, which allowed karyotyping of the inverted DAPI image and sequential viewing of the individual fluorochrome channels in karyotyped format, and (ii) a modified version of the spectral imaging approach originally devised for M-FISH. 11 This conceptually new analysis approach (goldFISH™) performs classification of telomeres according to their fluorochrome combination after identification of telomeres by anisotropic non-linear diffusion filtering. 12 24-colour whole chromosome painting and M-FISH analysis M-FISH was performed using a set of combinatorially labelled whole chromosome paints.…”

“…12 24-colour whole chromosome painting and M-FISH analysis M-FISH was performed using a set of combinatorially labelled whole chromosome paints. 11 The hybridisation conditions and image analysis were as previously described. 11 Images were collected using a motorised epifluorescence microscope with an eight-position filter wheel (Leica DMRXA-RF8) and a Sensys CCD camera (Photometrics, Tucson, USA).…”

“…11 The hybridisation conditions and image analysis were as previously described. 11 Images were collected using a motorised epifluorescence microscope with an eight-position filter wheel (Leica DMRXA-RF8) and a Sensys CCD camera (Photometrics, Tucson, USA). M-FISH analysis was performed using Leica MCK software (Leica Microsystems Imaging Solutions).…”

Translocation t(5;14)(q35;q32) in three cases of childhood T cell acute lymphoblastic leukemia: a new recurring and cryptic abnormality

“…Chromosomes were counterstained with DAPI. For image acquisition, an epifluorescence microscope Leica DMR-XA equipped with Leica special filters (Leica, Wetzlar, Germany) was used, according to Speicher et al 1 and Eils et al 2 Images were acquired using a cooled charged device (CCD) camera (Sensys, Photometric Perceptive Scientific, Chester, UK) on a vario-TV adapter with a magnification factor of 0.8 to 1.63. Image analysis was made using M-FISH software from Leica (Leica Q-FISH and Leica MCK).…”

“…[1][2][3] These methods are based on the cohybridization of 24 fluorescently labeled chromosome painting probes to metaphase chromosomes, which allows simultaneous vizualization of each chromosome pair by a unique color. We performed analysis on 60 patients with various hemopathies using a new M-FISH technique: IPM-FISH (IRS-PCR multiplex FISH).…”