GB2330907A

GB2330907A - A karyotyper and methods for producing karyotypes

Info

Publication number: GB2330907A
Application number: GB9722771A
Authority: GB
Inventors: Shiddhartha Nandy; Mark Gregson
Original assignee: Applied Imaging International Ltd
Current assignee: Leica Microsystems Gateshead Ltd
Priority date: 1997-10-28
Filing date: 1997-10-28
Publication date: 1999-05-05
Also published as: AU1367499A; WO1999022026A1; GB9722771D0

Abstract

A method of producing a karyotype from a metaphase comprising the steps of: labelling chromosomes in the metaphase to produce discrete colour bands within each chromosome; acquiring an image of the metaphase showing the discrete colour bands; segmenting the image into objects comprising individual chromosomes; and generating an intensity profile of each chromosome in the image.

Description

"A KARYOTYPER AND METHOD FOR PRODUCING KARYOTYPES" THIS INVENTiON relates to a karyotyper and method for producing karyotypes and for detecting regions of interchromosomal aberration or intiachromosomal rearrangements.

During mitosis, the 23 pairs of human chromosomes condense and are visible with a light microscope. A karyotype analysis usually involves blocking cells in mitosis and staining the condensed chromosomes to produce a chromosome banding pattern that can be analysed. The analysis involves comparing chromosomes for their length, the placement of centromeres (areas where the two chromatids are joined), and the location and sizes of bands.

A band is defined as that part of a chromosome which is clearly distinguishable from its adjacent segments by being made to appear darker or brighter with one or more banding techniques. The chromosomes are visualised as consisting of a continuous series of bright and dark bands.

The banding techniques fall into two principal groups: those resulting in bands distributed along the length ofthe wliole chromosome, such as G-, Q and R-bands; and those that stain a restricted number of specific bands or structures. These latter techniques include methods which reveal centromeric bands (C-bands), and nucleolus organiser regions (NORs) at terminal regions of acrocentric chromosomes. C-banding methods do not permit identification of evely chromosome in the somatic cell complement, but can be used to identity specific chromosomes. G- and R- bands can be bright field or fluorescent.

G-bands are most commonly used. They take their name from the Giemsa dye, but can be produced with other dyes. In G-bands, the dark regions tend to be heterochromatic, late-replicating and AT rich, The bright regions tend to be euchromatic, early-replicating and GC rich.

A number of karyotypers are available but their performance is limited and it is necessary for highly trained personnel to complete the karyotype analysis usually to the extent of requiring the trained personnel to classify many chromosomes manually.

A recently developed altemative to karyotyping metaphases with a recognisable banding pattern, is to identify the twenty-four different human chromosomes by the simultaneous hybridisation of a set of chromosomespecific DNA probes, each labelled with a different combination of fluorescent dyes. These DNA probes are visualised with an image analysis system and can be displayed as twenty four different colours - one for each chromosome.

Wheíeas such techniques may prove useful in detecting interchromosomal rearrangements such as translocations, they cannot identify intrachromosomal rearrangements such as inversions, amplifications and deletions.

Recently, the University of Cambridge (UK Patent Application 9704054.7, Cambridge University Technical Services Ltd.) has developed an approach whereby chromosomes may be stained in discrete colour bands provided by respective fluorochromes where bands occur as a result of crossspecies hybridisation.

It is an object of the present invention to seek to provide a method of automatic karyotyping which leaves few, if any, chromosomes to be manually classified and which is also capable of easily detecting intrachromosomal rearrangements and interchromosomal aberrations.

One aspect of the present invention provides a method of producing a karyotype from a metaphase comprising the steps of: labelling chromosomes in the metaphase to produce discrete colour bands within each chromosome; acquiring an image of the metaphase showing the discrete colour bands; segmenting the image into objects comprising individual chromosomes; and generating an intensity profile for each chromosome in the image.

A further aspect of the present invention provides a karyotyper for producing a karyotype of a metaphase including chromosomes labelled to show discrete colour bands within each chromosome, which karyotyper comprises: means to acquire an image of the metaphase showing the discrete colour bands; means to segment the image into objects comprising individual chromosomes; and means to generate an intensity profile for each chromosome in the image.

In order that the present invention can be more readily understood, embodiments thereof will now be described, by way of example, with reference to the accompanying figures, in which: FIGURE 1 shows a metaphase of the chromosomes from a cell; and FIGURE 2 shows a karyotype produced by a method embodying the present invention.

This invention relates to a karyotyper and a method for producing a karyotype from a metaphase in which chromosomes have been stained in discrete colour bands so as to produce an accurate karyotype and to identify where chromosomal rearrangements occur.

Referring to the metaphase shown in Figure 1, chromosomes from a single cell are labelled by, in this example, four fluorochromes (fluorescent dyes) which stain the chromosomes in the metaphase. The four fluorochromes comprise: a counterstain fluorochrome DAPI (4'-6-diamidino-2-phenylindole) which attaches to all chromosomes present in the metaphase; and three aligned fluorochromes FITC (fluoroescein isothiocyanate), Cy3 and Cy5 which attach and stain only specific palms of the respective chromosomes. In order to generate an appropriate image from the metaphase, the karyotyper embodying the present invention provides means for illuminating the fluorochromes attached to the chromosomes, each fluorochrome being excited to fluoresce upon illumination by a specific wavelength of light. In this example, an excitation filter wheel in combination with a multi-wavelength emission block are utilised to generate the pre-determined wavelengths for fluorescing the respective fluorochromes thiough a fluorescence microscope.

The karyotyper and method is designed to work with a variety of fluorescence microscopes. The excitation filter wheel and the multiwavelength emission block are both controlled by a computer system of the karyotyper. The computer system selects light of an appropriate wavelength to illuminate the metaphase to be imaged thereby fluorescing one of the fluorochromes.

The four fluorochromes are sequentially excited so that four sequential images of the four respective fluorochromes can be obtained. Image acquisition is achieved using a monochrome, integrating charge coupled device (CCD) video camera. Camera integration is controlled in order to obtain signals of suitable brightness. A series of four images is thus acquired, one image per fluorochrome of interest.

The kajyotyper is flexible so that (by selection of appropriate sets of filters from the excitation filter wheel and/or selection of appropriate wavelengths of light from the emission block) it may handle a wide variety of fluorochrome combinations. Each image is digitised for later processing and analysis, by y feeding the video signal into an image acquisition card, housed in the computer system of the karyotyper and stored, for example, in TIFF format.

Because tlie four images are acquired under different optical conditions, the fluorochrome excitation wavelengths being different, there is a certain degree of optical shift between the four images. Since the counterstain fluorochrome illuminates all the chroinosomes, the counterstain image is used as a reference point for the other three fluorochroile images. The karyotyper aligns and registers the other "raw" fluorochrome images to the counterstain image, to adjust for optical shift, using conventional 2-dimensional correlation methods. Examples of such techniques are disclosed in "Numerical Recipes in C" (1988) W.H. Press, B.P. Flannery, S.A. Teukolosky, W.T. Vetterling published by Cambridge University Press.

Background fluorescence is then removed from each fluorochrome image using standard molphological operators to allow accurate quantitation of the intensity of each fluorocilrorne image. Examples of morphological operators are given in "The Image Processing Handbook", J.C. Russ, published by CRC Press.

An example of a resultant composite image created by the three aligned fluorochromes with background fluorescence removed is shown in Figure I.

The counterstain image is not shown. In this example, the three aligned fluorochromes FITC, Cy3 and Cy5 are respectively labelled as blue, green and red. It should be noted that each chromosome has a unique banding pattern, the numbers, sizes and locations of the bands providing identification information about respective chromosomes.

The counterstain image is automatically thresholded to produce a binary mask which identifies all the objects in the sample, i.e. all clusters of chromosomes and individual chromosomes. Each of the three aligned fluorochrome images are then sampled under this mask to calculate normalisation factors based on median fluorochrome intensities.

Automatic segmentation of the counterstain image separates clusters of touching chromosomes into individual chromosomes. A binary mask is then created for each individual chromosome which is known as a chromosome mask. The central axis or chromosome axis of each chromosome is generated by applying skeletonisation techniques to the cllromosome mask of each individual chromosome. One such skeletonisation technique is disclosed in "Digital Image Processing" (1987) R.C. Gonzalez, P. Wintz, published by Addison-Wesley.

For each chromosome, intensity profiles are then generated for each fluorochrome image by averaging the image data along slices perpendicular to the chromosome central axis. These profiles are intensity normalised using the normalisation factors calculated earlier ensuring profiles of the same chromosome in different metaphases have similar characteristics.

Segmentation of any remaining chromosome clusters is performed semiautomatically by user interaction with the metaphase images. For example, in some circumstances, two chromosomes in the metaphase are in contact with one another along their longer sides and are therefore difficult to segment. In such a case, a user would apply a dividing line to separate the images of the two chromosomes. Other techniques can be used.

The karyotyper then utilises the fiuorochrome intensity profiles to automatically classify each chromosome into a conventional karyogram layout.

A user can view any combination of the fluorochrome images overlaid onto the chromosomes and compare the colour banding patterns to colour ideograms. The colour ideograms are gellerated from normal chromosome samples and allow the user to quickly identify possible abnonnalities such as amplifications, deletions and insertions.

In another embodiment of the invention, the system also provides for multi-cell analysis, where chromosomes of a particular class, from a number of karyotyped cells, are displayed together. This allows identification of recurrent abnormalities visible throughout a number of cells.

A classifier containing "models" of chromosomes or characteristic data describing respective chromosomes automatically identifies a chromosome based on its intensity profiles where each profile corresponds to a different fluorochrome. This provides the advantage that classification is independent of chromosome morphology. The classifier is trained using conventional statistical methods on intensity profiles from manually classified chromosomes.

Intensity normalised profiles from these chromosomes are length normalised to a predefined reference length. This takes account of the variation in chromosome length between metaphases due to factors such as microscope magnification setting, variations in the preparations etc.

All length and intensity nonnalised profiles from manually classified chromosomes of the same type are then aligned using conventional 1dimensional correlation techniques. From these profiles various pointwise statistics including mean profiles are collated. These are collectively known as the classifier data.

Once the classifier data is available, automatic karyotyping proceeds by the karyotyper measuring profiles from a set of images produced with the same fluorochromes, preparations and probes as were used for the manually classified chromosomes. Classification of a chromosome against a chromosome type involves comparing its intensity and length noimalised profiles with the classifier data. Using statistical techniques the chromosome type displaying the best match provides the identification thereby producing the automatic karyotype display shown in Figure 2.

It should be appreciated that the use of a trainable classifier allows the method of the invention to adapt itself to the specific banding characteristics produced by respective labelling techniques.

The profiles of a classified chromosome can be compared with classifier data, again using statistical techniques, to identify ranges of inconsistent intensities. In this way regions of interchromosomal aberration such as translocations or intrachromosomal rearrangements such as amplifications, deletions and inversions can be easily detected.

Whilst the above examples of the present invention are based on the use of multiple fluorochrome images, it should be appreciated that other methods of labelling chromosomes so as to produce discrete colour banding patterns can also be used.

Another labelling technique involves labelling regions of a chromosome with different ratios of fluorochromes, e.g. one band of a chromosome may be labelled with 100% FITC and another with only 50% FITC, to allow labelling of more bands without using more fluorochlomes. Such examples can be readily differentiated by analysis of the intensity profiles.

The use of neural networks and artificial intelligence systems is envisaged in place of or in addition to the aforementioned statistical techniques.

The invention can be practised on both human and non-human cell lines.

Fluorochromes which may be used comprise: FITC, TRITC tetramethyl rhodamine-isothiocyanate, Cy3, Cy5, Texas Red, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, Cy2, Cy3.5 (also known as Cy3++) Cy5.5 (also known as Cy5++), Cy7.

Counterstain fluorochromes which may be e used comprise: DAPI, PI - propidium iodide, rhodamine.

Claims

CLAIMS: 1. A method of producing a karyotype from a metaphase comprising the steps of: labelling chromosomes in the metaphase to produce discrete colour bands within each chromosome; acquiring an image of the metaphase showing the discrete colour bands; segmenting the image into objects comprising individual chromosomes; and generating an intensity profile of each chromosome in the image.
2, A method according to Claim 1, comprising the further step of classifying chromosomes into a karyotype by comparing the intensity profile of each chromosome in the image with the intensity profile of a classified chromosome, said classification thereby being independent of chromosome molpholog.
3. A Inethod according to Claim 1, comprising tlie further step of comparing the intensity profile of a chromosome in the image with characteristic data obtained from one or more intensity profiles from one or more classified chl omosomes to identify interchromosomal and intrachromosomal rearrangements.
4. A method according to any preceding claim, comprising the further step of training a classifier on intensity profiles from manually classified chromosomes, the classifier thereby y being adaptable to specific banding characteristics of different labelling techniques.
5. A method according to any preceding claim, comprising the further steps of producing a mask to identify all the chromosomes in the metaphase and sampling the image under the mask to calculate nonnalisation factors.
6. A method according to Claim 5, wherein the intensity profile of each chromosome in the image is normalised using the normalisation factors.
7. A method according to any preceding claim, wherein the intensity profile is generated from an image by averaging the image data along slices perpendicular to the chromosome central axis.
8. A method according to Claim 7, wherein the chromosome central axis is generated by using skeletonisation techniques.
9. A method according to any preceding claim, wherein background noise is removed from each image to allow accurate quantitation of the intensity levels from the image.
10. A method according to any preceding claim, comprising the further step of comparing an image of a chromosome to a colour ideogram of a given chromosome to identify possible abnolmalities.
11. A method according to Claim 10, comprising the further step of overlaying a plurality of images of a chromosome and comparing the overlaid images of the chromosome to a colour ideogram of a given chromosome to identify possible abnormalities.
12. A method according to any preceding claim, wherein the chromosomes are labelled with at least one fluorochrome to produce a fluorochrome image of the metaphase showing discrete colour bands within each chromosome.
13. A method according to Claim 12, comprising the further step of labelling chromosomes ill a metaphase with a counterstain fluorochrome attachable to all chromosomes in the metaphase and acquiring a counterstain image of the metaphase.
14. A method according to Claim 12 or 13, wherein the fluorochrome is excited to fluoresce by light of a specific wavelength generated by an emitter block.
15. A method according to Claim 14, wherein the light from the emitter block is filtered prior to illuminating the fluorochrome.
16. A method according to Claim 15, wherein the wavelength of light illuminating the fluorochrome is selectable by controlling the wavelength of light provided by the emitter block and/or connolling the wavelengths of light filtered.
17. A method according to any one of Claims 12 to 16, comprising the further steps of labelling chromosomes in the metaphase with one or more further fluorochromes and generating one or more further intensity profiles of each chromosome in respective fluorochrome images.
18. A method according to Claim 17, wherein three fluorochromes are used.
19. A method according to any one of Claims 12 to 18, wherein the or each fluorochrome is selectable from the group consisting of: FITC, Cy3, Cy5, TRITC, Texas Red, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, Cy2, Cy3.5(Cy3++), Cy5.5(Cy5++) and Cy7.
20. A method according to any one of Claims 13 to 19, wherein the counterstain fluorochiome is selectable from the group consisting of: DAPI, PI and Rhodamine.
21. A method according to any preceding claim, wherein the chromosomes are stained by the at least one fluorochrome in discrete colour bands as a result of cross-species hybridisation.
22. A method according to any one of Claims 1 to 11, wherein the chromosomes are labelled with different ratios of fluorochromes such that chromosomes can be identified by analysis of the intensity profiles.
23. A method according to any preceding claim for use with human and non-human cell lines.
24. A karyotyper for producing a karyotype of a metaphase including chromosomes labelled to show discrete colour bands within each chromosome, which karyotyper comprises: means to acquire an image of the metaphase showing the discrete colour bands; means to segment the image into objects comprising individual chromosomes; and means to generate an intensity profile for each chromosome in the image.
25. A method substantially as hereinbefore described with reference to and as shown in the accompanying drawings.
26. A karyotyper substantially as Ilereinbefore described with reference to the accompanying drawings.
27. Any novel feature or combination of features disclosed herein.