WO2010052471A1

WO2010052471A1 - Apparatus and method for the detection of cells

Info

Publication number: WO2010052471A1
Application number: PCT/GB2009/002623
Authority: WO
Inventors: Zhigang Wang
Original assignee: The University Of Dundee
Priority date: 2008-11-06
Filing date: 2009-11-06
Publication date: 2010-05-14
Also published as: GB0820300D0

Abstract

An apparatus and method for in-vivo detection of magnetically labelled biological molecules or cells, located in a flowing medium in a human or animal body such as the blood stream (13). The apparatus provides a magnetic field to a region of interest inside the body in order to attract the magnetically labelled biological molecules or cells (15) to the region of interest. A second magnetic field provides a variable magnetic field to the region of interest and which transfers energy to the magnetically labelled biological molecules or cells. A detector detects changes in the electromagnetic energy emitted by the labelled cells in response to the variable magnetic field generated by the second magnetic field source and provides a quantitative or qualitative measure of the magnetically labelled biological molecules or cells. The energy detected may be infra-red energy.

Description

Apparatus and Method for the Detection of Cells

Xn.tjCQdu,ction.

The present invention relates to an apparatus and method for the detection of cells such as those labelled with or connected to a biologically compatible marker and in particular the detection of circulating tumour cells (CTCs) .

Background to the Invention

CTCs are cells that have detached from a primary tumor and circulate in the bloodstream. CTCs may constitute ^Λseeds' for subsequent growth of additional tumors (metastasis) in different tissues. As CTCs circulate in the bloodstream, collection of these cells from blood samples represents a potential alternative to invasive biopsies as a source of tumour tissue for the detection, characterization and monitoring of cancers. Therefore the detection of CTCs may have important prognostic and therapeutic implications. However because their numbers can be very small they are not easily detected.

In general, detection of malignancy can be achieved either in vivo or in vitro. In vivo diagnostic imaging techniques include computer tomography, magnetic resonance imaging and positron emission tomography. These techniques can detect micro- metastases down to a size of 2-3 mm.

Earlier detection of metastatic disease typically uses an in vitro diagnostic test which combines the techniques of immunomagnetic separation, immunostaining, fluorescence imaging and/or flow cytometry. Immunomagnetic separation (IMS) is a laboratory tool that can efficiently isolate cells out of body fluid or cultured cells. The technique involves coating paramagnetic beads with antibodies which can bind to antigens present on the surface of cells. Once the cells have been labelled in this way, a magnetic field is used to concentrate the cells in a predetermined location.

Fluorescence Microscopy works on the principle that the light incident on a subject will have a different wavelength to the light emitted by the subject when it fluoresces. Accordingly, the subject can be manipulated to include labeling materials that emit a predetermined wavelength of light which provides an image of a particular feature of interest on the subject. This has led to the widespread use of fluorescence light microscopy in biomedical research and the development of different fluorescent dyes to stain different biological structures, which can then be detected simultaneously.

Flow cytometers operate by directing a beam of light (usually laser light) of a single wavelength onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes^' through the light beam and reflected light is collected by one or more fluorescence detectors. Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a wavelength different from that of the incident light. This combination of scattered and fluorescent light is picked up by the detectors, and by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is possible to derive various types of information about the physical and chemical structure of. individual particles.

One example of Immunomagnetic separation is described in an article in the Journal of Clinical Oncology, Volume 23 No. 7 of 1 March 2005 entitled "Circulating Tumour Cells: A Novel Prognostic Factor for Newly Diagnosed Metastatic Breast Cancer". This paper mentions the use of an immunomagnetic analytical technique where samples of blood were removed from a patient and immunomagnetically enriched using ferrofuids coated with antibodies targeting the epithelial cell adhesion molecule. The cells were fluorescently labelled with a nucleic acid dye and analyzed using the CellSpotter™ system.

International patent application WO 2006/102233 describes the CellSpotter™ system as one which operates on blood samples removed from a subject and which utilizes a fluorescence microscope image analysis system which permits through visualization of the cells, an assessment of morphological features to allow objects to be identified. The fluorescence microscope of the CellSpotter™ system is computer controlled and further comprises an automated stage with a magnetic yoke that is designed to enable ferro-fluid labelled candidate tumour cells to be magnetically localized to the upper viewing surface of the sample cartridge for microscope viewing.

The paper entitled "In vivo quantitation of rare circulating tumour cells by multiphoton intravital flow cytometry" discloses the detection of CTCs in the peripheral vasculature using a tumour specific fluorescent ligand. WO2005/106480 discloses a method for the collection of magnetically- labelled particles in vitro where there is no fluid movement and the fluid is contained in a test tube or the like. This approach uses antibodies pre-fixed on the test (tube) channel inner wall which capture the antigens, and subsequently the captured antigen captures another antibody which is coated with magnetic particle. This requires multiple procedures to fix the magnetic particle (and antigen) and is only suitable for ex vivo test in a test chip (or tube) .

JP 200302880 uses AC magnetic heating to heat a magnetic particle and its coated layer (capturing layer, e.g., with antibodies) to a pre-determined temperature in order to better capture a substance for ex vivo (or in vitro) immunoassay in a container. The purpose of heating the magnetic particle is to better capture a substance (e.g., antigen) through a magnetic particle coated antibody layer.

The advantages of JP 200302880 over other immunoassay methods are that no conventional heater and the sensor for temperature control are needed. They achieve this pre-determined temperature via ferromagnetic particles self-temperature regulating function around its Curie temperature. In order to achieve this they engineer particles which are made to have a specific Curie temperature suitable for the above purpose. It is an object of the present invention to provide an improved apparatus and method for the detection of circulating tumour

Summary of the Invention

In accordance with a first aspect of the invention there is provided an apparatus for in-vivo detection of magnetically labelled biological molecules or cells, located in a flowing medium in a human or animal body, the apparatus comprising: a first external magnetic field source which provides a magnetic field to a region of interest inside the human or animal body in order to attract the magnetically labelled biological molecules or cells to the region of interest; a second external magnetic field source which provides a variable magnetic field to the region of interest and which transfers energy to the magnetically labelled biological molecules or cells; and a detector adapted to detect changes in the electromagnetic energy emitted by the labelled cells in response to the variable magnetic field generated by the second magnetic field source to provide a quantitative or qualitative measure of the magnetically labelled biological molecules or cells.

A quantitative measure will count the number of detected magnetically labelled biological molecules or cells and a qualitative measure will provide information on their presence or absence.

Preferably, the second magnetic field source acts to heat the magnetically labelled molecules or cells. Preferably, the second magnetic field source acts to heat the -mag-ne-t-iσally labeJ.led..jDΩ.lecules or cells to a temperature below that which would induce necrosis in cells (e.g., below 42 ⁰C).

Preferably, the first magnetic field source provides a high- gradient magnetic field at or near the region of interest.

Preferably, the high-gradient magnetic field has a field gradient in the range of 8 to 10 Tesla/M to provide a penetration depth of 1 to 2 cm.

The second magnetic field may act to heat the magnetically labelled particles by hysteretic heating and/or domain relaxation heating.

The magnetic field may act to heat the magnetically labelled particles by magnetocaloric (MC) heating. In this special case of using MC heating, preferably, just one magnetic source (switchable) is needed for both attraction and heating.

Preferably, the flowing medium is the bloodstream.

Preferably, the apparatus of the present invention is contained in a housing that is adapted to be placed on the human or animal body.

Preferably, the second magnetic field source comprises an electromagnet. Optionally, the second magnetic field source comprises a source of electromagnetic waves.

Preferably, the first magnetic field source comprises a magnet which is shaped to concentrate the magnetic field gradient at the region of interest.

Preferably, the first magnetic field source comprises a switchable permanent magnet.

Optionally, the first magnetic field source comprises an electromagnet .

Preferably, the detector is an infra-red detector.

Preferably, the apparatus of the present invention is adapted to measure the difference between the infra red signal when the second magnetic field is on and off.

Preferably, the apparatus of the present invention holds the magnetically labelled biological molecules or cells in the region of interest, measure a first infra red signal, apply the second magnetic field, measure a second infra-red signal and determine the difference between the first and second signal.

Preferably, the infra-red detector is a single infra-red detector.

Alternatively, the infra-red detector is an infra-red detector array. Alternatively, the infra-red detector is an infra-red camera.

Alternatively, the infra-red detector is a thermal infra-red microscope.

Preferably, the apparatus is adapted to detect energy emitted from paramagnetic iron oxide magnetic labels biological molecules or cells.

In accordance with a second aspect of the invention there is provided a method for in-vivo detection of magnetically labelled biological molecules or cells located in a flowing medium in a human or animal body, the method comprising the steps of: Applying a first external magnetic field in order to attract the magnetically labelled cells to a region of interest inside the human or animal body; applying a variable magnetic field which transfers energy to the magnetically labelled biological molecules or cells; and detecting the energy emitted by the magnetically labelled biological molecules or cells to provide a quantitative or qualitative measure of the magnetically labelled biological molecules or cells.

Preferably, the second magnetic field source acts to heat the magnetically labelled cells.

Preferably, the second magnetic field source acts to heat the magnetically labelled molecules or cells to a temperature below that which would induce necrosis in the molecules or cells. Preferably, the first magnetic field provides a high-gradient magnetic field at or near the region of interest.

Preferably, the detector is an infra-red detector.

Preferably, the second magnetic field source comprises an electromagnet .

Optionally, the first magnetic field source comprises an electromagnet. Preferably, infra-red radiation is detected. Preferably, the, difference between the infra red signal when the second magnetic field is on and off is measured. Preferably, the method of the present invention holds the magnetically labelled biological molecules or cells in the region of interest, measures a first infra red signal, applies the second magnetic field, measures a second infra-red signal and determines the difference between the first and second signal .

Preferably, the infra-red detector is a single infra-red detector.

Alternatively, the infra-red detector is an infra-red detector array.

Alternatively, the infra-red detector is an infra-red camera.

Alternatively, the infra-red detector is a thermal infra-red microscope.

Brief Description of the Drawings

The present invention will now be described by way of example only with reference to the accompanying drawings in which:

Figure Ia is schematic diagram of an embodiment of a device in accordance with the present invention; Figure Ib is schematic diagram of another embodiment of a device in accordance with the present invention

Figure 2 shows an embodiment of the invention located in a housing;

Figure 3 is a flow diagram which illustrates an embodiment of the method of the present invention; and

Figure 4 is a flow diagram which illustrates another embodiment of the method of the present invention.

Detailed Description of the Drawings

The present invention provides an apparatus and method in which cells or molecules are labelled with a magnetic particle. The labelled cells or molecules are attracted by a magnetic field to a region of interest and a second magnetic field is applied to heat the magnetic particle. Therefore, the magnetic particle had a dual function, firstly by being attracted to the region of interest and secondly by being heated. The invention provides for the in vivo detection of single circulating tumour cells (CTC) or magnetic labelled cells for non-invasive monitoring/diagnosis of cancer metastasis before or after cancer treatment/therapy) .

One important aspect of the present invention is that it provides a means for trapping the magnetic particles or magnetic labelled cells which are situated in a flowing liquid medium (blood) . This dynamic capture is achieved by using a high gradient magnetic field where the magnetic attraction force is between the external magnetic source at the skin and the magnetic particle internalized into or attached to the cell at the region of interest (ROI) of the blood vessel.

The magnetic force Fm on a cell can be obtained using an ^effective' dipole moment method in which the cell is replaced by an ^equivalent' point dipole with a moment m_c, eff

where μf is the permeability of the fluid, and H_a is the applied magnetic field intensity and vv is the gradient operator.

For a single magnetic particle, with volume V_p and permeability μ_p, and relative permeability μ_pr, (μ_p=μoμ_Pr, and μo is the permeability of free space (4π x 10-7 T*m*A-l) .

Magnetization m_p, its magnetic force under applied magnetic field can be further simplified as below

(2)

Equation (2) shows that the higher the magnetic gradient H_a, the larger the magnetic force F_m.

Magnetic labelled cell motion under externally applied magnetic field can be predicted using Newton' s law

m =F_m+F_f+F_g where m_c and v_c are the mass and velocity of the cell, and F_m , F_f -and F_g are th_e_magnetic, flμidic, and gravitational force (including buoyancy) , respectively.

Magnetic field gradient requirement

In order to drag and hold the magnetic particles (and the magnetized CTC cells), a field gradient in the range of 8 T/m to 10 T/m, together with magnetic flux density range of 200 mT is preferred for a penetration depth of 1-2 cm into the body. This can be achieved by specific designs of magnet which are shaped to concentrate the magnetic field at a predetermined spatial location. For example, a maximum B-field of 1.43 T could be obtained with a magnet having a pointed tip at which the magnetic flux density is 588 mT . With the distance from the tip the flux density falls then rapidly. The field gradient ranges from 27.08 T/m for z = 1 mm to 10.37 T/m at a distance of 2 cm from the magnet surface. Such field gradients are suitable for use in the present invention.

The capture of magnetic labelled cells is temporary but long enough for detection and then releases into the blood stream. In some cases, the trapping of the magnetically labelled species may simply be deflecting them into the region of interest for a sufficient time to allow their presence to be detected.

Detection of the labelled cells can be via, thermal imaging such as temperature mapping of the region of interest and detection of thermally significant differences between the heat absorbed by the magnetic labelled cells in comparison with heat absorbed by the background flowing blood and its contents. The dynamic flow of the blood will assist in providing contrast because heat -w-ill be- tranSLfejcxjacL ,t.Q a moving liquid which will mean that, overall, the region of interest will not experience a significant temperature rise whereas the magnetically labelled particles will.

Heating of magnetic nanoparticles using -external AC magnetic fields of various strengths and frequencies (10 kHz to 300 kHz) has been achieved using three commercially available magnetic nanoparticles, i.e. Fe₂O₃, Fe₃O₄, and Fe. Ferrofluids based on aminosilane coated superparamagnetic iron oxide nanoparticles (core diameter: 15 nm) were used in thermotherapy for patients with recurrent glioblastoma multiforme [J Neurooncol, 81:53-60 (2007)], and the particles generated heat in an alternating magnetic field (at frequency of 100 kHz) by (Brownian and Neel) relaxation processes.

The heating mechanisms involves hysteretic (magnetic domain change) heating and domain relaxation heating. All the above- mentioned magnetic nanoparticles and heating mechanisms can be easily used in the present invention, which requires much lower thermal energy in detection than is required in hyperthermia treatment. For SPIO nanoparticles, the heating mechanisms are relaxation heating (Neel and Brownian relaxations of the magnetic momentl) . Heat dissipation can be due to rotation of the entire magnetic particle within a surrounding liquid (Brownian relaxation) and/or to rotation of the magnetic moment within the magnetic core (Neel relaxation) . Generally, heating power is dominated by the faster regime of relaxation which is Neel relaxation for magnetic cores less than 12 nm or Brownάan relaxation for larger size (> 15 nm) . For micron sized paramagnetic ir.on_ oxide (MPIO), there could be mixed heating mechanisms involving hysteretic heating and/or domain relaxation heating.

Micro-scale iron oxide magnetic particles, such as those microspheres (0.9 μm mean diameter, from Bangs Laboratories, Fishers, IN) used in MRI contrast agents [Blood. 2003; 102:867- 872] , can be used in the present invention, which would enhance magnetic attraction and increase heating due to its larger size when compared with nanoparticles .

In addition, another type of magnetic material which exhibits the magnetocaloric effect (MCE) can be detected in the present invention. In a material displaying the MCE, the alignment of randomly oriented magnetic moments by an external magnetic field results in heating, and gadolinium (and its alloy) and manganese compounds are such magnetocaloric materials.

Cells may be magnetically labelled using different methods including (a) in vivo labelling in which magnetic particles are pre-coated with antibody (e.g., Folate) and injected into blood stream, where CTCs for example with folate receptor (FR) will be magnetically labelled, resulting in a compound (CTC + Antibody_Magnetic particle) ; or (b) in vivo local magnetic labelling of cancer cells such as local injection of magnetic fluid into a primary or lymphatic cancer, with or without using additional cell transfection methods; subsequently, magnetic labelled metastasis cancer cells spread into blood stream and are to be detected in a compound (CTC Magnetic particle) ; (c) in vitro cell culture cancer cells can be magnetic labelled without using a specific antibody and the magnetically labelled cancer cells can be injected into an animal model for cancer metastasis study. Subsequently the magnetic labelled cancer cells become magnetic labelled CTCs in the blood stream when cancer metastasis occurs and can then to be detected using the present invention.

Figure Ia shows an embodiment of the apparatus in accordance with the present invention comprising a first magnetic field source provided by a permanent magnet 3 which provides a high- gradient magnetic field in an area of interest 4.

The permanent magnet comprises a pair of ferromagnets separated by a cavity 6. The ferromagnets may be of any suitable shape and the magnetic field be of a suitable strength and intensity to allow the CTCs 15 to be attracted to the area of interest. The first magnetic field source may alternatively be provided by an electromagnet. In addition, the strength and homogeneity of the field may vary temporally and spatially.

Figure 1 also shows an electromagnetic coil which, in this example of the invention is situated inside the cavity 6 at or near the surface of the skin 11. The purpose of the coil is to provide an oscillating magnetic field which excites the magnetically labelled CTCs. In this example of the present invention, the coil produces radio waves at a frequency of around 150 kHz; this frequency has been selected to provide an oscillating magnetic field which generates heating (i.e., infra red radiation) from the magnetically labelled CTCs. The RF frequency is, preferably, chosen from 100 kHz to 300 kHz, since too high-frequency magnetic fields are more likely to cause direct tissue heating.

The detector 9 includes a wave guide 7 which channels the radiated infra red waves to detection means . In this example the detection means comprises an infra-red detector such as an IR camera which detects the difference between the normal background infra red signal and that which is emitted when the coil is switched on.

The apparatus of the present invention shown in figure Ia is shown in close proximity to the body of a patient where there are CTCs present. The skin 11, is shown with the area 13 representing superficial blood vessels within which magnetically labelled CTCs 15 are circulating. Tissue below the superficial blood vessel is also shown.

Operation of the apparatus of figure Ia is described as follows. The first step is to inject pre-prepared ferrofluids, in which magnetic particles, preferably nanoparticles, already coated with tumor-specific ligand such as folate or antibodies, into the bloodstream 13 such that these particles become attached to circulating tumour cells 15 and act as labels. The magnetic nanoparticles may be an MRI contrast agent such as ferumoxides (magnetite) , gadolinium nanoparticles, super paramagnetic nanoparticles (e.g. iron oxides, iron platinum). The size of the magnetic particle, together with its surface coating characteristics, are selected to provide long enough blood half- life for in vivo labelling of the CTCs, but not too long in the blood circulation since magnetic particles, uncleared (from circulation) and uncaptured by CTCs, would cause background noise (by being heated as well) for the detection.

In general, ultrasmall superparamagnetic iron oxides (USPIO) with diameter up to 10-30 nm are less prone to phagocytosis due to their smaller size versus ferumoxides (around 150 nm) , and will have longer blood half-lives. The blood half-lives of the various iron oxide nanoparticles administered in patients vary from 1 hour to 24-36 hours. Also, coating magnetic particles with tumor-specific ligand is preferable to coating with tumor- specific antibodies, since, in vivo, tumor-specific antibodies were found to promote phagocytic clearance of the CTCs to which they bound, thereby causing significant underestimation of CTC counts .

The second step involves using the permanent magnet 3 to attract the magnetically labelled CTCs 15 to a predetermined area of interest 4 in the superficial blood vessel 13 at or near the surface of the skin 11. The coil 5 is then switched on and an electromagnetic signal of a frequency that is suitable for the generation of heat from the magnetic CTC labels is applied.

The detector 9 detects thermal irradiation from the heated magnetic nanoparticles thereby obtaining a quantitative or qualitative measure of the CTCs in the subjects blood stream.

To increase signal-to-background ratio, the RF coil itself should not be heated up, and preferably a circulating water pad or other cooling arrangement is included to cool it down. Heating of the permanent magnets (by the coil) should be avoided, preferably by RF coil directional design and focusing at underneath blood vessel, and by magnetic separation and insulation design between the RF coil and magnets. Additionally, -a -eirculating—water—pad or other cooling arrangement may be coupled to the magnet.

In addition, a differential thermal imaging (DTI) method can be used to improve detection. A DTI method can be simply described as taking two thermal images and subtracting one image from the other image. A simple case in this application is to take the first (hot) thermal image when the EM field is switched on, and to take the second (cold) thermal image after EM field is switched off. Normally, only the first image is enough to identify and locate the hotter spots/area of the heated magnetically labelled CTCs. However, the difference of the two imaging data can improve detection because only the heated tracers are highlighted. Thermal noise and interferences from background and surrounding tissues can be reduced or eliminated. Repeatedly taking hot (EM switched on) and cold (EM switched off) images and finally averaging the differential thermal images will further improve the system performance.

DTI may be of particular benefit when an MCE material (e.g. gadolinium) is used as a label. For MCE material, when the magnetic field is subsequently turned off, the magnetic moments randomize again, which leads to cooling of the material below the ambient temperature. Therefore, the temperature difference between hot and cold MCE tracers would be more significant and result in higher sensitivity for detection.

This embodiment of the invention describes the concentration, excitation and detection of magnetically labelled CTCs. It will be appreciated that the present invention may also be used to measure other magnetically labelled species including but not Jj.mit£d_to. tumour _ce_lls_. other than CTCs, stem cells (for monitoring their trafficking/homing) .

It might be desirable to release concentrated CTCs into blood circulation in order not to cause significant bloodstream flow disturbance, e.g., causing blockage of the blood vessel. This can be done by switching off or reducing the magnetic attraction field at certain time interval or automatically threshold- control based on the detected IR signal strength (i.e., numbers of CTCs detected) . It might also be desirable to increase RF radiation to heat CTCs over 42 ⁰C (i.e., cell necrosis) before releasing them into blood circulation.

Other arrangements of the components shown in figures Ia and Ib which fall within the scope of the invention are contemplated. For example, the coil may be located outside the permanent magnets. Other designs or arrangements of components may be used to optimise factors such as the signal to noise ratio of the apparatus, detection sensitivity and spatial resolution.

It .will be appreciated that this embodiment of the invention is designed to be worn by or attached to a patient and is capable of collecting data which allows the quantitative and qualitative measurement of CTCs over an extended period of time.

Figure Ib shows another embodiment of the present inventin with features similar to those of figure Ia. In this example, the first magnetic source 3a is shaped to have a pointed tip in order to increase the magnetic field gradient in the region of interest 10a. In this example the first magnetic source is positioned above the region of interest, consequently, the -position of the seconcLmagnetic source 5a and the detector 9a are slightly different to those shown in figure Ia. In use, the device operates to deflect magnetically labelled cells 15a in the bloodstream 13a towards the region of interest where they are heated and detected.

Figure 2 shows another embodiment of the present invention. In this case, the components of the apparatus are functionally similar to those described with reference to figure Ia notwithstanding any design modifications required to optimise or enhance performance.

The device 21 comprises a housing 23 which contains the first magnetic field source, a second magnetic field source and a detector. The housing 23 is mounted on a strap 25 which is used to attach the device 21 to the skin of the patient.

Figure 2 also shows the skin 27 and superficial blood vessels

29.

This embodiment of the present invention can be worn by the patient in order to detect the presence of CTCs in the superficial blood vessels over an extended period, e.g., continuously monitoring up to 1 - 2 hours each day. Interrogation of the patient's entire blood volume is likely to take more than one hour In which case, the patient might, preferably, monitor his/her CTCs at home for a prescribed days.

Figures 3 and 4 are flow diagrams which show the steps of the method of the present invention. In figure 3 the method 31 comprises injecting magnetic particle 1-abe-ls—i-n-to the blood stream of the patient which attach themselves to CTCs. The device of the present invention is then positioned on or near the skin of the patient 35 and the magnet, (in this case an electromagnet) is switched on 37. The magnet attracts the labelled CTCs to a region of interest 39 and the oscillating magnetic field provided by a radio frequency electromagnetic wave is switched on 41. Heat generated in the magnetic labels by the oscillating magnetic field is detected using an infra-red detector 43 and the signal is measured 45 in order to give a measure of the quantity of CTCs present.

In figure 4 the method 51 comprises injecting 33 magnetic particle labels into the blood stream of the patient which attach themselves to CTCs. The device of the present invention is then positioned on or near the skin of the patient 55 and the magnet, (in this case an electromagnet) is switched on 57. The magnet attracts the labelled CTCs to a region of interest 59 a first measurement of the infra red signal is taken 61. Thereafter, the oscillating magnetic filed provided by a radio frequency electromagnetic wave is switched on 63. Heat generated in the magnetic labels by the oscillating magnetic field is detected using an infra-red detector 65 and the signal is measured 67 in order to give a measure of the quantity of CTCs present. The first infra red measurement is subtracted from the second to give a measure of the magnetically labelled CTCs present in the region of interest.

In other embodiments of the invention, the second magnetic source may be chosed to have a field strength and frequency which excited the magnetically labelled species at different parts of the electromagnetic spectrum such as visible light or _ultrayiolet.._

Improvements and modifications may be incorporated herein without deviating from the scope of the invention.

Claims

-1- An apparatus....for in_-vivp detection of magnetically labelled biological molecules or cells, located in a flowing medium in a human or animal body, the apparatus comprising: a first external magnetic field source which provides a magnetic field to a region of interest inside the human or animal body in order to attract the magnetically labelled biological molecules or cells to the region of interest; a second external magnetic field source which provides a variable magnetic field to the region of interest and which transfers energy to the magnetically labelled biological molecules or cells; and a detector adapted to detect changes in the electromagnetic energy emitted by the magnetically labelled biological molecules or cells labelled in response to the variable magnetic field generated by the second magnetic field source to provide a quantitative or qualitative measure of the magnetically labelled biological molecules or cells.

2. An apparatus as claimed in claim 1 wherein, the second magnetic field source acts to heat the magnetically labelled molecules or cells.

3. An apparatus as claimed in claim 1 or claim 2 wherein, the second magnetic field source acts to heat the magnetically labelled molecules or cells to a temperature below that which would induce necrosis in cells.

4. An apparatus as claimed in any preceding claim wherein, the first magnetic field source provides a high-gradient magnetic field at or near the region of interest.

5. An apparatus as claimed in claim 4 wherein the high- gradient magnetic field has a field gradient in the range of 8 to 10 Tesla/M to provide a penetration depth of 1 to 2 cm.

6. An apparatus as claimed in any preceding claim, wherein the flowing medium is the bloodstream.

7. An apparatus as claimed in any preceding claim, wherein the apparatus of the present invention is contained in a housing that is adapted to be placed on the human or animal body.

8. An apparatus as claimed in any preceding claim, wherein the second magnetic field source comprises an electromagnet.

9. An apparatus as claimed in claims 1 to 7, wherein the second magnetic field source comprises a source of electromagnetic waves.

10. An apparatus as claimed in any preceding claim, wherein, the first magnetic field source comprises a magnet which is shaped to concentrate the magnetic field gradient at the region of interest.

11. An apparatus as claimed in any preceding claim, wherein, the first magnetic field source comprises a switchable permanent magnet .

12. An apparatus as claimed in claims 1 to 11 wherein, the first magnetic field source comprises an electromagnet.

13. An apparatus as claimed in any preceding claim, wherein the detector is an infra-red detector.

14. An apparatus as claimed in any preceding claim, wherein the apparatus of the present invention is adapted to measure the difference between the infra red signal when the second magnetic field is on and off.

15. An apparatus as claimed in any preceding claim, wherein, the apparatus of the present invention holds the magnetically labelled biological molecules or cells in the region of interest, measures a first infra red signal, applies the second magnetic field, measures a second infra-red signal and determines the difference between the first and second signal.

16. An apparatus as claimed in claim 13, wherein, the infra-red detector is a single infra-red detector.

17. An apparatus as claimed in claim 13 wherein, the infra-red detector is an infra-red detector array.

18. An apparatus as claimed in claim 13, wherein the infra-red detector is an infra-red camera.

19. An apparatus as claimed in claims 13, wherein the infra-red detector is a thermal infra-red microscope.

20. An apparatus as claimed in any preceding claim wherein the apparatus is adapted to detect energy emitted from paramagnetic

-i-r-o-n oxide mag-n-e-tic labels biological molecules or cells.

21. A method for in-vivo detection of magnetically labelled biological molecules or cells located in a flowing medium in a human or animal body, the method comprising the steps of: applying a first external magnetic field in order to attract the magnetically labelled cells to a region of interest inside the human or animal body; applying a variable magnetic field which transfers energy to the magnetically labelled biological molecules or cells labelled; and detecting the energy emitted by the magnetically labelled biological molecules or cells to provide a quantitative or qualitative measure of the magnetically labelled biological molecules or cells. labelled.

22. A method as claimed in claim 20, wherein the second magnetic field source acts to heat the magnetically labelled cells .

23. A method as claimed in claims 21 and 22 wherein, the second magnetic field source acts to heat the magnetically labelled molecules or cells to a temperature below that which would induce necrosis in the molecules or cells.

24. A method as claimed in claims 21 to 23 wherein, the first magnetic field provides a high-gradient magnetic field at or near the region of interest.

25. A method as claimed in claim 24 wherein the high-gradient magnetic -fie-l-el—-has a field gradient in the. range of 8 to 10 Tesla/M to provide a penetration depth of 1 to 2 cm.

26. A method as claimed in claims 21 to 25, wherein the detector detects infra red radiation.

27. A method as claimed in claims 21 to 26 wherein, the first magnetic field source comprises a magnet which is shaped to concentrate the magnetic field gradient at the region of interest.

28. A method as claimed in claims 21 to 27 wherein, the first magnetic field source is provided by a switchable permanent magnet.

29. A method as claimed in claims 21 to 27, wherein the first magnetic field source is provided by an electromagnet.

30. A method as claimed in claims 21 to 29 wherein, infra-red radiation is detected.

31. A method as claimed in claim 30wherein, the difference between the infra red signal when the second magnetic field is on and off is measured.

32. A method as claimed in claim 30 or 31 comprising the steps of holding the magnetically labelled biological molecules or cells in the region of interest, measures a first infra red signal, applies the second magnetic field, measures a second infra-red signal and determines the difference between the first and second signal.

33. A method as claimed in claims 21 to 32 wherein energy emitted from paramagnetic iron oxide magnetic labels biological molecules or cells are detected.