Microfluidic device and method for depleting leukocytes in a sample

Abstract

The invention relates to a microfluidic device (100) and to a method (500) for depleting leukocytes in a biological sample, in particular for a subsequent measurement or count of circulating tumour cells in the sample, comprising a region (110), in particular a chamber (110) or a channel, for receiving at least some of the sample, wherein the region (110) has a leukocyte-impairing medium (111), in particular a leukocyte-binding and / or leukocyte-damaging substance.

Description

[0001] Description

[0002] title

[0003] Microfluidic device and method for depleting leukocytes in a sample

[0004] State of the art

[0005] CTCs (abbreviated for circulating tumor cells) are circulating tumor cells released into the patient's bloodstream from a primary tumor or metastases from a primary tumor. They play a crucial role in tumor metastasis and are therefore of high diagnostic value, predictive of the further course of the disease. In the simplest case, the number of CTCs per unit volume of blood, for example, per milliliter of blood, is determined, the so-called CTC count.For this purpose, the CTCs in a patient's blood sample are labeled with fluorescently labeled antibodies after selective erythrocyte lysis and unmasking of their surface, for example, by enzymes. They are then preferably sedimented in a chip with a plurality of etched wells in a microfluidic cartridge. They are then counted using their fluorescent markers (CTC count), as described, for example, in published patent application DE 10 2021 203 897 A1. The CTC count alone is highly informative regarding the response to therapies and can therefore be used for therapy monitoring, which is particularly useful in the case of adjuvant therapies. An increase in the CTC count can be interpreted as a non-response or failure of the therapy used and thus serve as an indicator for a change in therapy. Disclosure of the invention.

[0006] Advantages of the invention

[0007] Against this background, the invention relates to a microfluidic device and a method for depleting leukocytes in a biological sample, in particular for subsequent isolation, measurement, or counting of circulating tumor cells in the sample. The microfluidic device comprises a region, in particular a chamber or a channel, for receiving at least a portion of the sample, wherein the region comprises a leukocyte-impairing medium, in particular a leukocyte-binding and / or leukocyte-damaging substance.

[0008] The biological sample may, in particular, be a sample comprising one or more body fluids, such as blood, urine, sputum, or a smear. In particular, the sample may comprise whole blood or blood components.

[0009] A leukocyte-impairing medium is understood, in particular, to be a medium or substance that reduces or even neutralizes the effect of leukocytes, preferably reducing the number of leukocytes. The device and method according to the invention thus advantageously enable an automated reduction of the effects of leukocytes in the portion of the sample collected in the cartridge, which facilitates processing and analysis of other components of the sample, in particular counting and / or examining the CTCs contained therein.

[0010] As stated above, the leukocyte-impairing medium can, in particular, be a leukocyte-binding and / or leukocyte-damaging substance. Binding the leukocytes advantageously limits their mobility, and the damage directly negatively impacts the effectiveness of the leukocytes. Preferably, the device comprises a filter for retaining the bound leukocytes. Due to the binding of the leukocytes to the substance and the resulting larger volume of the leukocyte-substance connection, these leukocytes can advantageously be filtered out of the sample portion and thus removed, particularly by a pore filter with a suitably selected pore size via size exclusion.This has the advantage of significantly reducing the high number of leukocytes, especially in a blood sample, especially before counting or further analysis of CTCs present in the sample, for example, extraction of CTCs by sedimentation and / or visualization of the CTCs by specific antibody staining. In the course of the invention, it was recognized that, particularly when counting the CTCs identified by EpCAM antibody labeling, a portion of the leukocytes would inevitably be falsely labeled as EpCAM-positive due to the finite specificity of the antibody labeling. For an assumed, otherwise excellent specificity error of, for example, only 10, 4 With EpCAM antibody labeling, one would select from typically available 10 7 Leukocytes / ml blood falsely approx. 10 3 / ml as "suspicious" or "false positive," which could exceed the number of "true" CTCs actually present by one to two orders of magnitude and consequently obscure possible correlations between therapy and CTC count. With a depletion of the leukocyte count by, for example, a factor of 100-1000, which is advantageously enabled by the invention, the ratio of CTCs to leukocytes in a blood sample is advantageously improved to typically approximately 1:1000, which significantly improves CTC detection after sedimentation and significantly reduces the risk of artifacts in antibody staining and thus errors in subsequent CTC identification and counting. The data quality and diagnostic significance of the data increase accordingly.Furthermore, detailed molecular genetic analysis methods such as downstream (q)PCR or RT-PCR can advantageously be performed immediately, either in situ or after removal of the cells from the cartridge, since these methods tolerate a background of the aforementioned magnitude. Furthermore, a dielectrophoresis array could also be used directly, for example, for the targeted selection of individual CTCs of particular interest. According to a preferred embodiment, the leukocyte-impairing medium comprises leukocyte-specific antibodies for binding to leukocytes. This has the advantage that the leukocytes' mobility is restricted and, preferably, they can be more easily separated from the remaining sample.For this purpose, the leukocyte-specific antibodies can be designed to bind to one or more structures on the cell surface of leukocytes, in particular to a characteristic protein on the surface of the cell wall such as CD45, CD3, CD4, CD8, CD14, CD19 and / or CD56.

[0011] Preferably, at least some of the leukocyte-specific antibodies are immobilized on a solid phase, in particular on a wall delimiting the region or on particles, especially on beads often used in microfluidics. By binding the antibodies to the solid phase on the one hand and to a leukocyte on the other, the latter is advantageously retained from the sample and thus removed, in particular due to an immobile solid phase or via filtration through a suitable pore filter, as described above.

[0012] The solid phase can be magnetizable. In particular, the particles are (ferro)magnetic particles, especially magnetic or ferromagnetic beads. This facilitates the retention of leukocytes bound to the antibodies by using a magnet. According to a particular embodiment, the device can have a magnet, especially a permanent magnet or an electromagnet, for retaining the magnetic particles. For example, the magnet is arranged adjacent to the region containing the leukocyte-specific antibodies. In addition, a pore filter as described above can preferably be provided to increase the overall effectiveness of leukocyte retention.

[0013] According to an advantageous embodiment of the invention, at least some of the leukocyte-specific antibodies are multifunctional and / or multispecific antibodies, in particular bispecific antibodies. Preferably, at least some of the antibodies each comprise several identical or different binding sites. According to a particular embodiment, at least some of these antibodies each comprise at least two, preferably more than two, binding sites for leukocytes, either similar binding sites for identical leukocyte structures, in particular for CD45 or other markers mentioned above, or different binding sites for different structures, in particular for different markers.This has the advantage that such an antibody can bind to at least two leukocytes and thus enables cluster formation of several leukocytes, whereby the clusters can then be easily removed from the sample via size exclusion (in particular using a pore filter).

[0014] In a particularly advantageous development of the invention, at least some of the multifunctional or multispecific, in particular bispecific antibodies, are designed to bind at least one leukocyte and at least one circulating tumor cell, in particular via the epithelial cell adhesion molecule (EpCAM for short). In particular, the antibody is thus designed to bind at least one leukocyte and at least one EpCAM-positive cell, i.e., a cell with an EpCAM on its surface. Specificity of the antibodies for EpCAM is particularly advantageous here, since circulating tumor cells from epithelial primary tumors typically contain multiple EpCAMs. For example, the commercially available trifunctional bispecific antibody catumaxomab or an antibody with the same or similar specificity can be used for this purpose.Thus, clusters consisting of one tumor cell and several leukocytes, each bound to the tumor cell via these antibodies, can advantageously be formed. This has the advantage that these clusters can be filtered out of the sample via size exclusion, i.e., via a pore filter with a suitable pore size. Thus, the large number of leukocytes in a blood sample is advantageously utilized to isolate CTCs from the sample. Furthermore, it is advantageous that these CTC clusters can subsequently be easily counted and / or subjected to further analysis, in particular PCR or gene sequencing or one or more other independent color markers.

[0015] According to a further advantageous embodiment of the invention, the leukocyte-damaging medium comprises, as leukocyte-damaging substances, in particular chemical substances for damaging leukocytes. The chemical substances can be, in particular, biochemical reagents such as enzymes or proteases, which selectively kill leukocytes and / or dissolve their membranes. This can advantageously utilize the fact that certain substances, such as Tween® or other detergents, damage leukocytes more quickly and / or more severely than CTCs. Furthermore, the leukocyte-damaging medium can comprise leukocyte-specific antibodies functionalized with biochemical agents toxic to cells, for example, cytostatics from the group of mustard compounds (cyclophosphamide, ifosphamide) or platinum compounds for damaging leukocytes.

[0016] In a further advantageous embodiment of the invention, the leukocyte-impairing medium comprises a dye for staining the leukocytes and / or leukocyte-specific antibodies. This allows for a simple optical count or estimation of the number of leukocytes present in that portion of the sample.

[0017] Short description of the drawings

[0018] Embodiments of the invention are illustrated schematically in the drawings and explained in more detail in the following description.

[0019] It shows

[0020] Figure 1 shows an embodiment of the device according to the invention and

[0021] Figure 2 shows a flow diagram of an embodiment of the method according to the invention.

[0022] Embodiments of the invention

[0023] Figure 1 shows an embodiment of the microfluidic device according to the invention

[0024] Device 100 in a schematic cross-sectional representation, which may in particular be a microfluidic cartridge, for example a microfluidic cartridge described in the above-mentioned published patent application DE 10 2021 203 897 A1, for example a cartridge for the Vivalytic® platform. Such a cartridge 100 can preferably be processed in an analytical device, in particular in the case of a passive cartridge whose microfluidic elements (membranes, valves, etc.) are controlled, for example, by pneumatic actuation via pressure supply by the analytical device. Figure 2 shows a flowchart 500 for an embodiment of the method according to the invention with, for example, such a device 100.

[0025] The device 100 has a region 110, in particular a chamber or a channel, for receiving at least a portion of a blood sample (hereinafter also referred to as sample liquid), wherein the region 110 is designed as a chamber 110 in this example. In a first step 501 of the method 500, the blood sample can first be introduced into the device 100 via an input chamber 105, which can be closed with a lid 106 and is fluidically connected to the chamber 110. The chamber 110 has a leukocyte-impairing medium 111, which in this example comprises leukocyte-specific antibodies 111, which bind to leukocytes in a second step 502 of the method 500. A leukocyte-specific antibody 111 is directed, for example, against the marker CD45, a characteristic protein on the surface of leukocytes. Other suitable markers may be CD3, CD4, CD8, CD14, CD19 or CD56, and / or combinations thereof.For the depletion of leukocytes, the fact that leukocytes are precisely known is exploited, i.e. one has a target with low variability against which the corresponding antibodies (or antibody mix) can be directed. This enables highly specific leukocyte binding and leukocyte separation from the sample fluid to be achieved. In contrast, CTCs typically represent a highly variable target that is more difficult to identify or extract due to mutations and dysregulation of surface proteins. As described in the published patent application DE 10 2021 203 897 A1, the device 100 can have a detection chamber 130 (also referred to as a sedimentation chamber or sedimentation cell) with a structured chip 135 (also referred to as a cavity array) with microcavities (also referred to as sedimentation cells) for sedimenting the CTCs, wherein the detection chamber 130 is connected to the region 110 orthe chamber 110 is preferably fluidically connected via one or more pore filters 140.

[0026] The leukocyte-depleting medium 111 and in particular the leukocyte-specific antibodies 111 can be immobilized on a surface 115 delimiting the region 110, for example, on an inner wall 115 of the chamber 110, for example, by being spotted there and bound by means of a suitable, well-known binding chemistry. During use of the device 100, leukocytes in the sample fluid come into contact with these antibodies and bind to them. This requires a certain waiting time, during which hybridization of the antibodies to the surface proteins of the leukocytes can take place. The latter are thus also immobilized via the antibodies to the inner channel surface and, in contrast to the CTCs in the sample fluid, are retained during further transport of the sample fluid in a third step 503 of the method 500.Thus, the number of leukocytes in the sample fluid is reduced accordingly in the desired manner on its way through the region 110 or through the chamber 110. In a subsequent fourth step 504, circulating tumor cells from the depleted sample in the detection chamber 130 can be deposited into the cavity array 135, preferably in a color-marked manner by sedimentation, and preferably counted, as described, for example, in the above-mentioned published patent application DE 10 2021 203 897 A1. The amount of antibodies stored in the region 110 depends on the number of expected leukocytes in the sample, i.e., in particular, on the amount of the portion of the sample to be introduced into the device 100, and also on the desired extent of leukocyte reduction. For example, 1-50 ng, preferably 1-10 ng of antibodies can be stored per milliliter of whole blood introduced.The antibodies 111 can either already be stored in the area 110 or alternatively be added after the sample has been introduced, for example from another area of ​​the device 110. According to a particular embodiment, multifunctional or multispecific leukocyte antibodies are used which have several identical or different binding sites, for example against CD3 or CD45 or other leukocyte surface proteins. Antibodies are made up of two “long chains” which form a Y. The two “short chains” are located in the V geometry of the Ypsilon. “Long chains” and “short chains” each form two binding sites, which can both be identically directed against CD3, CD45 or other leukocyte surface proteins, for example. In the case of multispecific antibodies, one binding site can be directed against CD3, CD45 or other leukocyte surface proteins, for example, and the other binding site against EpCAM on CTCs.It is also possible to functionalize the lower end of the Y in such a way that another binding site for surface proteins of immune cells is created there, for example in order to bind and / or activate dendritic cells or macrophages. This creates an antibody with several different specific binding sites for different targets, which is now known as "bispecific antibodies" and is part of the state of the art in biotechnology. This task is particularly simplified if both binding sites have the same target, e.g. CD45. If such antibodies are added to the sample fluid which contains leukocytes, for example by mixing the antibodies 111 which are preferably located upstream in region 110 with the sample fluid entering region 110, these antibodies 111 stick the leukocytes together to form cell clusters.The antibodies bind with one end to each leukocyte, resulting in binding between two leukocytes by an antibody. This creates large cell clusters with the antibodies as "glue" in between, which can then preferably be retained by one or more pore filters 140 with appropriately sized pores located in the device. The pore size is determined by the size of the leukocytes or CTCs and can be, for example, 20-100 pm, preferably 20-30 pm. CTCs in the sample fluid can easily penetrate these pore filters, while the much larger leukocyte clusters are retained by them, allowing the desired leukocyte depletion to occur. Alternatively or additionally, the leukocyte antibodies can be immobilized on particles 160 of a well-defined size, in particular on so-called "beads" used in microfluidics.These beads are suspended in the sample fluid, interact with the leukocytes present therein, and bind to them. For example, these beads can be selected so that their diameter is 5-10 times that of a typical cell diameter, for example 50-200 pm. The suspension of beads in the sample fluid, together with the bound and unbound leukocytes and the CTCs, then passes through a 140 pore filter with a pore diameter that is smaller than the bead diameter but larger than the cell diameter. A suitable pore diameter can be between 20-50 pm, depending on the size of the beads. If the bead diameter is selected to be 200 pm, the pore diameter can be 180 pm, for example. CTCs can pass through these pores unhindered along with the sample fluid, while the beads with the leukocytes bound to them are retained.This also reduces the leukocyte count on the way to the sedimentation cell in the desired manner. The particles 160 with the antibodies 111 immobilized thereon can already be stored in the region 110 or in another region of the device 100 before the sample is added.

[0027] Preferably, beads made of ferromagnetic materials, so-called magnetic beads, are used. These can also be ferromagnetic nanoparticles, meaning there is virtually no limit to their size. These magnetic beads are also functionalized with antibodies directed against leukocytes or against characteristic leukocyte surface proteins. The function is initially the same as described above. In the suspension of antibody-functionalized magnetic (nano)particles, CTCs, and leukocytes, the leukocytes bind to the magnetic beads, or the magnetic beads bind to the surface of the leukocytes, depending on the size ratio of the beads to the leukocytes. The CTCs remain unaffected by the magnetic beads.As the sample fluid containing the magnetic beads with bound leukocytes, free leukocytes, and CTCs is transported further, they pass through a channel or chamber region 110 in which a strong magnetic field acts. This magnetic field attracts the magnetic beads to an inner surface of the channel region of the microfluidic platform and holds them there, thereby also immobilizing the bound leukocytes. The aforementioned magnetic field can be generated by means of a small permanent magnet 150 integrated into the device, which can be inserted or positioned at a suitable location.by injection molding, where the magnetic beads and the leukocytes are to be retained (for example, arranged adjacent to the chamber 110, as shown in the figure), or by a permanent magnet in the analyzer, which is fixedly positioned at a suitable location and, due to its magnetic field, ensures that the corresponding area is impassable for magnetic beads in the device 100 lying thereon. Alternatively or additionally, an electromagnet is placed in the analyzer, which can be selectively switched on or off by the analyzer's control software. In this embodiment, too, the leukocytes are retained in the sample liquid on the way to the subsequent sedimentation cell 130, and the desired selective leukocyte depletion takes place.

[0028] The above-described "pore filter" and "magnetic filter" embodiments can also be combined by following the magnetic retention with a mechanical pore filter 140, which is impermeable to the magnetic beads due to their size but permeable to cells. In this case, a size match of the magnetic beads, the pores, and the cells is required, as described in more detail in the aforementioned embodiment. This combination ensures particularly effective retention of the beads and thus the leukocytes bound to them, by retaining them either in the magnetic zone or by the pore filter on their way to the sedimentation cell. Furthermore, the upstream magnetic depletion prevents the subsequent mechanical filter from becoming clogged by too many leukocytes.

[0029] Alternatively or additionally, the leukocyte count can be depleted by adding chemical substances such as Tween, or biochemical reagents such as enzymes or proteases that can selectively kill leukocytes or dissolve their membranes. It is known that, for example, Tween destroys leukocytes more quickly than CTCs. In addition, leukocyte-specific antibodies can be used that are functionalized with biochemical agents that have a toxic effect on cells and thus preferentially destroy leukocytes (because the antibodies carrying them bind specifically to them). Although a compromise between the highest possible leukocyte depletion while simultaneously preserving the highest possible number of intact CTCs is difficult to achieve with these methods, they are fundamentally suitable for this purpose and can, for example, be used to support one of the aforementioned methods."gentle" to slightly increase the discrimination of leukocytes from CTCs. This also achieves the desired depletion of leukocytes relative to CTCs on the way to sedimentation cell 130.

[0030] Alternatively or additionally, the leukocyte count can be reduced, and thus brought into a more favorable ratio to the EpCAM-positive CTCs, by first generating clusters of EpCAM-positive cells and leukocytes by adding or pre-storing a suitable bispecific antibody in the device. The commercial bispecific antibody catumaxomab, for example, is particularly suitable for this purpose and is described, for example, in Winter A, Zacharowski K, Meybohm P, et al. Removal of EpCAM-positive tumor cells from blood collected during major oncological surgery using the Catuvab device - a pilot study. BMC Anesthesiol. 2021 ;21 (1):261. Published 2021 Oct 29. doi:10.1186 / s12871-021-01479-3.The antibody catumaxomab binds to EpCAM on the CTC surface on the one hand, and to the CD3 antigen of leukocytes on the other, forming clusters of EpCAM-positive cells with surrounding leukocytes, with the antibodies acting as binding links or adhesive bridges. Further binding is possible at the lower end of the Y, the so-called Fc site, to dendritic cells or macrophages of the immune system, which can further enlarge the clusters around a CTC. Due to the high binding affinity of catumaxomab to EpCAM, all EpCAM-expressing parts of the surface of EpCAM-expressing cells are preferentially covered by antibodies. At least some of these antibodies also bind to CD3 of leukocytes with their second binding site, thus holding them to the EpCAM-positive cell. Accordingly, virtually all EpCAM-positive cells are connected to a manageable number of leukocytes.For example, between 1 and 2 nanograms of catumaxomab for each expected milliliter of blood can be stored in chamber 110 upstream or added after sample entry.

[0031] The clusters generated, each comprising at least one EpCAM-positive cell, in particular a CTC, and a manageable number of leukocytes surrounding it, can in turn be easily retained by size exclusion using a pore filter 140, since the cluster size is a multiple of a normal cell size. The retention rate of such cell clusters, each containing at least one EpCAM-positive cell, by suitably dimensioned pore filters is therefore preferably practically 100%, while non-clustered individual cells, for example individual unbound leukocytes, pass through them unhindered. The pore diameter for the pore filter can, for example, be a multiple of a typical cell diameter, e.g. 1.5 - 3 times a typical cell diameter of leukocytes of 8 pm.Thus, the EpCAM-positive CTCs and all artifacts surrounded by bound leukocytes remain trapped on the pore filter, while all unclustered leukocytes pass through the filter and are thus removed. The ratio of EpCAM-positive CTCs to artifacts and leukocytes is thus orders of magnitude more favorable than without this type of separation, while, on the other hand, very few EpCAM-positive cells are lost. This embodiment of the invention therefore not only represents a good leukocyte depletion but also a highly efficient isolation or enrichment method for EpCAM-positive cells, especially CTCs. Due to the high binding affinity of catumaxomab for EpCAM and the efficient physical retention of the cell clusters on the pore filters, the loss of CTCs is negligible, thus realizing a low-loss CTC isolation method.

[0032] For further analysis, the retained EpCAM-positive cells and the leukocytes bound to them can subsequently be separated again if necessary, for example by shifting the pH value into the acidic or alkaline range, thereby changing the isoelectric point of antibody binding so that the antibody bonds are broken again. Catumaxomab is designed for a neutral blood pH of around 7; the binding affinity is lost if the pH deviates significantly from pH 7 or if the blood is rebuffered. To subsequently label and visualize the CTCs, they can be stained again, for example, with fluorescently labeled EpCAM antibodies or with cytokeratin antibodies, etc. Due to the significantly reduced number of leukocytes, the number of artifacts generated is also significantly reduced.

[0033] Additionally or alternatively, in a further embodiment of the invention, leukocytes can be identified by optical methods based on their phenotypes and / or leukocyte-specific antibody staining and physically separated from the EpCAM-positive cells, e.g. in a cytometer.

[0034] As an alternative to physical isolation, a pure count of leukocytes can be performed, whereby the leukocytes can be identified based on their phenotypes and / or leukocyte-specific antibody staining using optical methods. If the number of leukocytes in the sample and the specificity or specificity error of the EpCAM-specific antibody staining are known, the "background" of artifacts produced by the antibody staining method, i.e., the number of leukocytes falsely labeled as CTCs, can be determined as a product of the specificity error (e.g., 10 4) and the number of leukocytes and subtract this value from the number of cells suspected of being CTCs. Instead of physically separating the leukocytes, a numerical subtraction of the resulting artifacts is performed.

Claims

Claims 1 . A microfluidic device (100) for depleting leukocytes in a biological sample, in particular for subsequent isolation, measurement or counting of circulating tumor cells in the sample, comprising a region (110), in particular a chamber (110) or a channel, for receiving at least a portion of the sample, wherein the region (110) comprises a leukocyte-impairing medium (111), in particular a leukocyte-binding and / or leukocyte-damaging substance.

2. Device (100) according to claim 1, wherein the leukocyte-affecting medium comprises leukocyte-specific antibodies (111) for binding leukocytes from the portion of the sample.

3. Device (100) according to claim 2, wherein at least some of the antibodies (111) are immobilized on a solid phase (115, 160), in particular on a wall delimiting the region or on in particular magnetic particles (160).

4. Device (100) according to claim 3, wherein the solid phase (115, 160) is magnetizable and the device (100) preferably comprises a magnet (150) for interacting with, in particular for immobilizing, the solid phase (115, 160), in particular the magnetic particles (160).

5. Device (100) according to any one of claims 2 to 4, wherein at least some of the antibodies (111) are multifunctional or multispecific antibodies (111).

6. Device (100) according to claim 5, wherein at least some of the multifunctional or multispecific, in particular bispecific antibodies (111), are designed to bind at least two leukocytes each, in particular via two similar binding sites.

7. Device (100) according to claim 5 or 6, wherein at least some of the multifunctional or multispecific, in particular bispecific antibodies (111), are designed to bind at least one leukocyte and at least one circulating tumor cell, in particular via the epithelial cell adhesion molecule (EpCAM), and / or an EpCAM-positive cell.

8. Device (100) according to one of the preceding claims, wherein the device (100) comprises a filter (140) for retaining the bound and / or damaged leukocytes.

9. Device (100) according to one of the preceding claims, wherein the leukocyte-impairing medium comprises, in particular, chemical substances for damaging leukocytes- 10. Device (100) according to one of the preceding claims, wherein the leukocyte-affecting medium comprises a dye for staining the leukocytes and / or the leukocyte-specific antibodies (111).

11. A microfluidic method (500) for depleting leukocytes in a biological sample, in particular for subsequent isolation, measurement or counting of circulating tumor cells in the sample, comprising a device (100) according to any one of the preceding claims, wherein at least a portion of the sample is received in a region (110), in particular a chamber (100) or a channel, and wherein leukocytes are depleted from the portion of the sample with the leukocyte-impairing medium located in the region (110).

12. The method (500) according to claim 11, wherein the bound and / or damaged leukocytes are removed from the part of the sample via a solid phase (115, 160) and / or a filter (140) of the device (100).

13. The method (500) according to claim 11 or 12, wherein in a subsequent step (504) cells, in particular circulating tumor cells, are separated from the part of the sample by sedimentation and preferably counted.

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