GB2447255A

GB2447255A - Preparation of enriched target cell samples for use in a chemosensitivity assay

Info

Publication number: GB2447255A
Application number: GB0704067A
Authority: GB
Inventors: David Woolley
Original assignee: ONCOPROBE Ltd
Current assignee: ONCOPROBE Ltd
Priority date: 2007-03-02
Filing date: 2007-03-02
Publication date: 2008-09-10
Also published as: GB0704067D0

Abstract

Methods for preparing enriched target cell samples for a chemosensitivity assay by removal of contaminants. The depletion of contaminants is performed in either of two manners: A) Binding of contaminant to primary contaminant specific antibody which is coupled to a magnetic bead allowing for direct magnetic separation, or B) Binding of contaminant (1) to primary contaminant specific antibody (4) which is bound to a magnetic bead via a secondary antibody (3). The contaminant-antibody-antibody-magnetic bead is removed by a magnet (5) leaving the target cells e.g., cancer cells (2) enriched. The contaminant specific cell markers of choice are CD45, CD31 and CD90. The enriched population of target cells are applied in a chemosensitivity assay that can measure the electrochemical response of, for example, cancer cells to a potential chemotherapeutic agent and thereby determine the sensitivity towards the agent.

Description

PREPARATION OF A TEST SAMPLE

FOR USE IN A CHEMOSENSITIVITY ASSAY

The present invention relates to a method for preparing a chemosensitivity assay test sample and an in-vitro chemosensitivity assay using such a test sample.

Cancer patients with malignant neoplasms commonly undergo surgery to remove the cancerous tissue. A large proportion of those patients will subsequently receive chemotherapy, i.e. be treated with recognised anti-cancer drugs, often in combination.

Of those drugs some may be therapeutically effective and others non-therapeutically effective. Typically, a patient will be administered several different chemotherapeutic agents and their response monitored to help the medical practitioner identify the most appropriate therapeutically effective agent or mixture of agents. The selection of the most appropriate agent or mixture of agents depends upon a number of different factors, not only the relative levels of cancer cell cytotoxicity exhibited by the different agents under test. It is also often important, for example, to balance the level of cancer cell cytotoxicity against the cytotoxic effect of the potential agent(s) on non-cancer cells, which, if too high, could be harmful to the patient.

In view of the adverse side effects often associated with chemotherapy, and in particular combination chemotherapy, many different in-vitro bioassays have been developed to monitor changes in the behaviour of a patient's cancer cells andlor non-cancer cells resulting from exposure to potential chemotherapeutic agents and/or other ligands (e.g. histamine, cytokines, growth factors, prostanoids etc). Observing these changes can be used to assess the chemosensitivity' or chemoresistance' of a patient's cells (cancer cells and/or non-cancer cells) to potential chemotherapeutic agents and ligands.

Conventionally, a chemosensitivity assay' highlights agents which are likely to be therapeutically effective in-vivo, whereas a chemoresistance assay' highlights agents which are unlikely to be therapeutically effective in-vivo.

A refinement of the conventional chemoresistance assay has been developed which seeks to identify extreme drug resistance' (EDR) specifically in cancer cells by administering to a specimen containing cancer cells a very high concentration of a potential chemotherapeutic agent for several days and assessing cell growth rates.

Cancer cells which survive this exposure are charactensed as exhibiting extreme drug resistance' and so the particular chemotherapeutic agent(s) under test can be eliminated from the patient's potential treatment regimen.

For conciseness the term chemosensitivity' will be used hereafter in an inclusive sense and should be understood as relating, wherever appropriate, to both chemosensitivity' and chemoresistance'. References hereafter to a chemosensitivity assay' should be interpreted, wherever appropriate, to relate to an assay used to determine the chemosensitivity or chemoresistance of a target cell' towards a particular agent or ligand (e.g. histamine, cytokines, growth factors, prostanoids and the like), and it will be appreciated that, unless otherwise stated, the target cell' is the type of cell under test and can be a cancer cell or a non-cancer cell.

Unfortunately, the in-vitro assays currently available have limitations andlor associated problems which have resulted in low acceptance of such tests by medical practitioners.

Examples include ultrasensitive pH meters (microphysiometer) developed to measure small changes in extracellular acidification (Jrotons) resulting from cellular metabolic responses to drugs or ligands. Techniques have also been developed which use bioluminescent detection of adenosine triphosphate (ATP) and adenosine diphosphate (A.DP) to assess cell viability, apoptosis and proliferation. Such techniques generally only measure intracellular metabolites at one moment in time and require the use of expensive instrumentation such as a luminometer or liquid scintillation counter.

Sensors have been developed which can detect changes in cellular respiration arising from the reduced oxygen consumption observed for dead or dying cells compared to healthy viable cells. While such sensors may find application in certain types of cytotoxicity testing, it is well recognised that many cancerous cells exhibit an enhanced glycolytic potential (the alternative to respiration).

Indicator dyes can be used to assess in-vitro cell growth and cytotoxicity. Fluorometnc and colourimetric indicators are available which change colour upon reduction of a cellular growth medium as a result of cell growth. Although tests based on such reagents are relatively simple and inexpensive, they are restricted to an assessment of cell growth at one moment in time, usually after several days.

Further chemosensitivity assays are available, for example electric cell-substrate impedance sensing (ECIS) and cell-corpse' testing, which, while showing certain advantages, still do not provide the level of reliable, technical information that the medical community requires in order to more fully accept the predictive value of chemosensitivity assays.

An object of the present invention is to obviate or mitigate one or more of the above problems.

According to a first aspect of the present invention there is provided a method for preparing a chemosensitivity assay test sample containing target cells, said method comprising enrichment of the target cells in an initial sample containing said target cells.

Enrichment of the target cells may be considered as a type of sample purification in which the proportion of target cells in the sample is increased compared to other cell species present in the sample.

It is recognised that tumour tissue samples are typically a mixture of cancer cells and many different normal' healthy cells, such as stromal fibroblasts, endothelial, lymphocytes, monocyte-macrophages and others. By way of example, most tumour tissues contain only a minor proportion of cancer cells relative to the total number of cells in the tissue. For the first time, the devisors of the present invention have appreciated that in a chemosensitivity assay, where the target cells often represent a minor proportion of the total cell content, the non-target cells are likely to swamp the chemosensitivity assay test sample over the ensuing hours/days during testing and adversely affect the test result. For example, if the target cells are cancer cells and the other contaminating' species are the normal cells, since the target cancer cells are present in significantly smaller numbers than the normal cells and the target cancer cells generally proliferate much more slowly in-vitro compared to the normal cells, the normal cells are likely to swamp the chemosensitivity assay test sample during testing and confound the assay result. This effect has not previously been recognised in earlier chemosensitivity assays, which explains many of the poor results reported for such tests which have hindered the acceptance of chemosensitivity assays by medical practitioners.

A second aspect of the present invention provides an in-vitro chemosensitivity assay comprising preparation of a test sample containing target cells and analysis of said test sample to determine the sensitivity of said target cells to at least one potential chemotherapeutic agent, wherein preparation of said test sample comprises enrichment of the target cells from an initial sample containing said target cells.

A third aspect of the present invention relates to use of an in-vitro target cell enrichment procedure in the preparation of a chemosensitivity assay test sample containing target cells.

A fourth aspect of the present invention provides a chemosensitivity assay test sample containing target cells, wherein said sample has been subject to an in-vitro target cell enrichment procedure.

As mentioned above, the target cell' can be any type of cell which is present in the tissue sample under test. It will be appreciated that the type of cell which represents the target cell' is dependent upon the particular test being carried out. In a preferred embodiment of the present invention the target cells undergoing chemosensitivity testing are cancer cells. In this case, the target cancer cells are preferably obtained from a tissue sample that has been surgically excised from the cancer cell tissue of a primary or secondary tumour, or blood borne leukaemic cells.

The initial target cell-containing sample is preferably prepared by enzymatic digestion of a tissue sample, preferably using a collagenolytic enzyme. Preparation of the initial sample may also involve centnfugation of the tissue sample following enzymatic digestion to separate the dissociated cells from unwanted tissue remnants.

Any appropriate procedure may be employed to achieve enrichment of the target cells, although it is preferred that the target cells are enriched by depletion of at least a first contaminant present in said initial sample. In a first preferred example, the first contaminant represents healthy non-cancerous cells, such as fibroblastic stromal cells.

In a further preferred example, the first contaminant represents cancer cells.

In order to enrich the target cells by depletion of the first contaminant, the target cells and the first contaminant should be separated within the initial sample. A number of suitable methods may be employed to separate the target cells from the contaminant.

For example, the contaminant may be immobilised and the target cells removed from the initial sample, alternatively, the target cells may be immobilised and the contaminant removed. Immobilisation of one species generally involves modifying that species so that it can be immobilised. It will be appreciated that any changes to the physical, chemical or biological structure of the target cells may affect the results of the chemosensitivity assay. Consequently, it is preferred that such changes are avoided as far as possible. It is therefore preferred to inimobilise the first and any additional contaminants present in the initial sample so that the unaffected target cells can be separated and tested, i.e. it is preferred that the method comprises negative depletion of the target cells.

A convenient method of imniobilisation is to bind the contaminant(s) to a magnetic material and then use a magnetic field to immobilise said contaminant(s) whilst the target cells are separated. In a preferred embodiment, negative depletion of the first contaminant comprises binding the first contaminant to a magnetic species, such as a magnetic bead, and separating said magnet contaminant complex from the target cells.

Any convenient method may be used to bind the contaminant(s) to the magnetic material. Where the contaminant(s) is/are biological species, it is preferred to use contaminant-specific antibodies to recognise and bind to specific contaminants and then use these antibodies to attach the contaminant(s) to the magnetic species.

It is therefore preferred to bind said first contaminant to a first contaminant-specific antibody to form a contaminant-antibody complex and separate said contaminant-antibody complex from the target cells. Separating said contaminant-antibody complex from the target cells preferably comprises binding said contaminant-antibody complex to a magnetic bead and exposing the resulting contaminant-magnetic bead complex to a magnetic field. The contaminant-antibody complex may be bound to the magnetic bead via a further antibody attached to the magnetic bead.

The selection of appropriate contaminant-specific antibodies is dependent upon the nature of the contaminant(s) present in the initial sample which it is desired to remove.

As described above, the contaminant(s) may be healthy non-cancerous cells or, in certain applications, cancer cells. A very large number of different cell-specific antibodies are available which, when attached to the specific cell type, can bind to further antibodies attached to magnetic species and thereby facilitate depletion of the contaminant(s) in the manner described above. Each cell-specific antibody is preferably specific to a cell type selected from the group consisting of various cancer cells (e.g. renal carcinoma, epithelial carcinoma cells, melanoma etc), monocytes, macrophages, granulocytes, B-cells, T-cells, endothelial cells, pan leucocytes, stromal fibroblasts, and haematopoietic precursor cells. It has been determined, for example, that gp200 is eminently suitable for use as a renal carcinoma cell-specific antibody in the method of the present invention, and that BerP4 is eminently suitable for use as an epithelial carcinoma cell-specific antibody in the method of the present invention. It has been further determined (by checking responsiveness to enzyrnically dissociated cells using standard methods) that the first contaminant-specific antibody is a functionally effective antibody specific for a particular type of cancer cell (e.g. gp200 or BerP4, which are specific for renal cancer cells), CD31, CD45 or CD9O. Suitable functionally effective antibodies can be purchased from Invitrogen Ltd, UK.

In a preferred embodiment, the initial sample contains a second contaminant and enrichment of the target cells in the initial sample comprises depletion of the second contaminant employing a second contaminant-specific antibody. It is preferred that the second contaminant-specific antibody is a functionally effective antibody specific for any one of various types of cancer cells (e.g. gp200 or BerP4), CD3 1, CD45 and CD9O, subject to the proviso that the second contaminant-specific antibody is different to the first contaminant-specific antibody.

In a still further preferred embodiment, the initial sample contains a third contaminant and enrichment of the target cells in the initial sample comprises depletion of said third contaminant employing a third contaminant-specific antibody. The third contaminant-specific antibody is preferably selected from the group consisting of functionally effective antibodies specific for various types of cancer cells (e.g. gp200 or BerP4), CD31, CD4S and CD9O, subject to the proviso that the third contaminant-specific antibody is different to the first and second contaminant-specific antibodies.

While the initial sample may contain any number of contaminants it is preferred that the initial target cell-containing sample is contacted with at least two, more preferably three of the above-mentioned functionally effective contaminant-specific antibodies: a functionally effective antibody specific for a particular type of cancer cell (e.g. gp200 or BerP4, which are specific for renal cancer cells); CD3 1; CD45; and CD9O. It is further preferred that the initial sample is contacted with CD3 1 and CD45, CD3 I and CD9O, or CD45 and CD9O so as to remove two of the contaminants most commonly observed in initial target cell-containing samples which may affect the accuracy and relevance of the chemosensitivity assay data where the target cells are cancer cells. It is still further preferred to contact the initial sample with CD3I, CD45 and CD9O to remove three of the most common contaminants. In a further preferred embodiment where the target cells are monocytes, macrophages, granulocytes, B-cells, T-cells or endothelial cells it is preferred that the or each contaminant-specific antibody is selected from the group consisting of an appropriate antibody specific for the type of cancer cell present in the sample (e.g. gp200 or BerP4 for renal cancer cells), CD45 and CD9O. Where the target cells are pan leucocytes it is preferred that the or each contaminant-specific antibody is selected from the group consisting of an appropriate antibody specific for the particular type of cancer cell present (e.g. gp200 or BerP4), CD3 1 and CD9O. Where the target cells are stromal fibroblasts, haematopoietic precursor cells or capillary endothelial cells it is preferred that the or each contaminant-specific antibody is selected from the group consisting of a suitable antibody specific for the type of cancer cell present (e.g. gp200 or BerP4), CD31 and CD45.

While the above-described procedure is suitable to prepare a test sample for use in any appropriate chemosensitivity assay, a particularly preferred in-vitro chemosensitivity assay comprises electrochemical analysis of the test sample to determine the sensitivity of the target cells to at least one potential chemotherapeutic agent.

The electrochemical analysis preferably comprises attaching, preferably by natural adherence, said target cells to a chemically inert electrically conductive surface immersed in a culture medium which is in electrical contact with a reference electrode, and analysing variations and/or fluctuations in electrochemical signals generated between said electrically conductive surface and the reference electrode by processes occurring at the target cell surfaces.

Preferably the electrochemical signals are detected by an electrochemical signal detector connected between the electrically conductive surface and the reference electrode. The electrochemical analysis may comprise detection and analysis of variations and/or fluctuations in electrochemical voltage and/or current between two or more electrically conductive electrodes at least one of which is in direct contact with the target cell surfaces.

The culture medium preferably retains the viability of the target cells throughout the testing period, which may be a few seconds or minutes, or possibly longer, such as 24 to 48 hours or more. If the tests are to be conducted over an extended period of time, such as 1 to 2 days, then it is preferred that the culture medium is replenished periodically as required. To help maintain the viability of the target cells it is preferred that the cells are placed in a 5 % C02, 37 C incubator during testing.

To ensure a constant and reliable electrochemical signal (referred to as an open circuit potential', OCP) throughout the testing period, target cell adhesion and confluence should be achieved and this typically takes up to a few hours after first seeding the target cells on to the electrically conductive surface. Following this initial settling period a potential chemotherapeutic agent or other ligand (e.g. histamine, cytokines, growth factors, prostanoids) is added to the test sample and changes in the open circuit potential signal monitored to determine whether the chemotherapeutic agent or ligarid will be effective (i.e. whether it changes cell behaviour as registered as a change in OCP signal) against the target cells.

The chemosensitivity assay is not limited to any specific form(s) of cancer, although it is envisaged that it is eminently suitable for chemosensitivity testing in relation to solid' cancers, such as ovarian, renal, colorectal, breast, prostate etc. The second aspect of the present invention provides a chemosensitivity assay comprising enrichment of the target cells in an initial sample containing said target cells to provide a test sample enriched in target cells for use in the chemosensitivity assay. It will be appreciated that enrichment of the target cells may be achieved using a method according to any embodiment of the first aspect of the present invention.

Aspects of the present invention will be further described by way of example only with reference to the following non-limiting Example and Figures in which: Figure 1 is a schematic illustration of a negative enrichment procedure; and Figure 2 is a photograph of an OncoprobeTM device.

EXAMPLE

Details relating to an in-vitro chemosensitivity assay in accordance with the second aspect of the present invention are set out below. The assay comprises preparation of a test sample in accordance with the first aspect of the present invention followed by analysis of the test sample to determine the sensitivity of cancer cells to at least one potential chemotherapeutic agent. It will be appreciated that the same methodology as set out below can be applied to tests in which the sensitivity of non-cancer cells is to be examined, and also where the sensitivity of cancer cells or non-cancer cells to other ligands (e.g. histamine, cytokines, growth factors, prostanoids), not potential chemotherapeutic agents/drugs, is to be tested.

Preparation of Chemosensitivity Test Sample A patient tissue sample containing target cancer cells and contaminating normal healthy cells is surgically excised from a primary or secondary tumour, or an ascites or haematological sample. The tumour tissue sample is then digested using a collagenolytic enzyme and the resulting cell suspension partially purified by centrifugation.

In order to enhance the accuracy and relevance of the chemosensitivity assay the cancer cells in the sample are enriched. The present example employs a new negative depletion process to separate the contaminating normal cells from the cancer cells, which is related to a commercially available process developed by Dynal Biotech Ltd. The partially purified cell suspension is incubated with mouse monoclonal antibodies to CD31, CD45 and CD9O which bind antigens on the surface of the contaminating cells.

Excess, unbound antibodies are removed by centrifugal washing. Magnetic Dynabeads (Dynal Biotech Ltd) conjugated to anti-mouse IgG are then added to the cell suspension with mixing. The Dynabeads bind to the CD3 1, CD45 and CD9O antibodies attached to the contaminating cells thereby forming a magnetic contaminant-containing complex.

A suitably configured magnet can then be used to immobilise the contaminant cells within the sample to facilitate separation of the cancer cells.

Figure 1 is schematic illustration of the negative depletion of CD9O+ contaminant cells (1) (e.g. fibroblasts) from a sample containing CD9O-tumour cells (2). The sample is incubated with magnetic Dynabeads conjugated to anti-mouse IgG (3) which binds functionally effective CD9O antibodies (4), which bind the CD9O antigen on the surface of the contaminant cells (1). A magnet (5) is then placed sufficiently close to the sample to attract and thereby immobilise the contaminant cells (1).

Chemosensitivity Analysis of the Test Sample The cancer cell-enriched test sample prepared as described above is then analysed using technology developed by Oncoprobe Limited, which is the subject of the following patents: EP0783690; GB2307990; and US 6,010,889.

The OncoprobeTM technology provides a means for detecting and analysing electrochemical signals expressed at the surface of viable cancer cells immersed in a culture medium placed on a chemically inert electrically conductive sensor surface.

With reference to Figure 2, the OncoprobeTM device (6) comprises a disposable assay probe (7) comprising a plurality of separate wells (8), each containing three working electrodes (not visible in Figure 2) and an integral reference electrode (not visible in Figure 2).

Equal aliquots of the enriched cancer cell sample are seeded into the wells (8) of the OncoprobeTM device (6) containing a suitable culture medium. The device (6) is then placed in a 5 % C02, humidified, 37 C incubator (not shown in Figure 2). Cell adhesion and confluence is achieved within a few hours to produce a constant open circuit potential signal. Chemotherapeutic agents or other ligands (e.g. histamine, cytokines, growth factors, prostanoids etc) may then be added to separate wells (8) and the culture medium replenished, as required, throughout the testing penod, which may be up to 24 to 48 hours.

Within each well (8) variations and/or fluctuations in electrochemical signals generated between the working electrodes and the reference electrode by processes occurring at the cancer cell surfaces in response to exposure to the different chemotherapeutic agents and/or ligands are constantly monitored in real time to provide an indication of the cellular response to each agent. Such tests facilitate the identification of cellular responses to such agents and/or ligands.

The OncoprobeTM technology offers several advantages over other chemosensitivity assays. For example, the OncoprobeTM technique directly detects electrochemical signals reflecting physiological processes occurring at cell surfaces, most of which are sensitive to even minor changes in cell behaviour. The OncoprobeTM technique thus provides the ability to passively listen in' on cell surface activities in real time for any desirable period of time, from a few minutes to up to 48 hours, or longer. Moreover, the technique continuously monitors the effect of a chemotherapeutic agent on the cancer cells over the testing period rather than providing a cell-count at a single point in time.

Since the OncoprobeTM technique does not require cell culturing to increase the cell sample quantum, the time required for cell adaptation is minimised and thereby reduces any variance between in-vivo and in- vitro indices. Additionally, the technique is essentially passive' and in this way avoids potentially traumatising influences, such as intracellular invasion, clamping and exposure to voltage or current.

Claims

1. A method for preparing a chemosensitivity assay test sample containing target cells, said method comprising enrichment of the target cells in an initial sample containing said target cells.

2. A method according to claim 1, wherein enrichment of the target cells comprises depletion of a first contaminant present in said initial sample.

3. A method according to claim 2, wherein depletion of said first contaminant comprises binding said first contaminant to a first contaminant-specific antibody to form a contaminant-antibody complex and separating said contaminant-antibody complex from the target cells.

4. A method according to claim 3, wherein separating said contaminant-antibody complex from the target cells comprises binding said contaminant-antibody complex to a magnetic bead and exposing the resulting contaminant-magnetic

bead complex to a magnetic field.

5. A method according to claim 4, wherein the contaminant-antibody complex is bound to the magnetic bead via a further antibody attached to the magnetic bead.

6. A method according to claim 3, 4 or 5, wherein the first contaminant-specific antibody is selected from the group consisting of functionally effective antibodies to specific cancer cells, CD3 1, CD45 and CD9O.

7. A method according to claim 6, wherein the initial sample contains a second contaminant and enrichment of the target cells in the initial sample comprises depletion of said second contaminant employing a second contaminant-specific antibody.

8. A method according to claim 7, wherein the second contaminant-specific antibody is selected from the group consisting of functionally effective antibodies to specific cancer cells, CD3I, CD45 and CD9O, subject to the proviso that the second contaminant-specific antibody is different to the first contaminant-specific antibody.

9. A method according to claim 7 or 8, wherein the initial sample contains a third contaminant and enrichment of the target cells in the initial sample comprises depletion of said third contaminant employing a third contaminant-specific antibody.

10. A method according to claim 9, wherein the third contaminant-specific antibody is selected from functionally effective antibodies to specific cancer cells, CD3 1, CD45 and CD9O, subject to the proviso that the third contaminant-specific antibody is different to the first and second contaminant-specific antibodies.

11. A method according to any one of claims I to 5, wherein said target cells are cancer cells.

12. A method according to any preceding claim, wherein said chemosensitivity assay comprises electrochemical analysis of said test sample to determine the sensitivity of said target cells to at least one potential chemotherapeutic agent or ligand.

13. A method according to claim 12, wherein said electrochemical analysis comprises attaching said target cells to a chemically inert electrically conductive surface immersed in a culture medium which is in electrical contact with a reference electrode and analysing variations and/or fluctuations in electrochemical signals generated between said electrically conductive surface and the reference electrode by processes occurring at the target cell surfaces.

14. A method according to claim 13, wherein said electrochemical signals are detected by an electrochemical signal detector connected between the electrically conductive surface and the reference electrode.

15. A method according to claim 12, 13 or 14, wherein said electrochemical analysis comprises detection and analysis of variations and/or fluctuations in electrochemical voltage and/or current between two or more electrically conductive electrodes at least one of which is in direct contact with the target cell surfaces.

16. A method according to any preceding claim, wherein said initial sample is prepared by enzymatic digestion of a tissue sample.

17. A method according to claim 16, wherein said enzymatic digestion employs a collagenolytic enzyme.

18. Use of an in-vitro target cell enrichment procedure in the preparation of a chemosensitivity assay test sample containing target cells.

19. Use of an in-vitro target cell enrichment procedure according to claim 18, wherein said target cells are cancer cells.

20. A chemosensitivity assay test sample containing target cells, wherein said sample has been subject to an in-vitro target cell enrichment procedure.

21. A sample according to claim 20, wherein said target cells are cancer cells.

22. An in-vitro chemosensitivity assay comprising preparation of a test sample containing target cells and analysis of said test sample to determine the sensitivity of said target cells to at least one potential chemotherapeutic agent or ligand, wherein preparation of said test sample comprises enrichment of the target cells from an initial sample containing said target cells.

23. An assay according to claim 22, wherein said assay comprises electrochemical analysis of said test sample to determine the sensitivity of said target cells to at least one potential chemotherapeutic agent or ligand.

24. An assay according to claim 23, wherein said electrochemical analysis comprises attaching said target cells to a chemically inert electrically conductive surface immersed in a culture medium which is in electrical contact with a reference electrode and analysing variations andlor fluctuations in electrochemical signals generated between said electrically conductive surface and the reference electrode by processes occurring at the target cell surfaces.

25. An assay according to claim 24, wherein said electrochemical signals are detected by an electrochemical signal detector connected between the electrically conductive surface and the reference electrode.

26. An assay according to claim 23, 24 or 25, wherein said electrochemical analysis comprises detection and analysis of variations and/or fluctuations in electrochemical voltage and/or current between two or more electrically conductive electrodes at least one of which is in direct contact with the target cell surfaces.

27. An assay according to any one of claims 22 to 26, wherein said enrichment of the target cells is achieved using a method according to any one of claims 1 to 17.