EP2932233A1

EP2932233A1 - Method for enriching and isolating cells having concentrations over several logarithmic steps

Info

Publication number: EP2932233A1
Application number: EP14700851.0A
Authority: EP
Inventors: Michael Johannes Helou; Lukas RICHTER; Oliver Hayden
Original assignee: Siemens AG
Current assignee: Siemens AG
Priority date: 2013-01-22
Filing date: 2014-01-15
Publication date: 2015-10-21
Also published as: CN105190286A; WO2014114530A1; DE102013200927A1; US20150355072A1

Abstract

The invention relates to a device (10) for flow cytometry, which has a chamber (12), a channel (14) and a magnetic sensor (18). The channel (14) and the chamber (12) are present in a closed system, an axis (A) of the channel (14) extending along a flow direction (P) of the channel (14). A magnet (22) and a deflection means (20) are arranged on a predefined side of the channel (14). The deflection means (20) is divided into at least one segment (20-1, 20-2, 20-3) and each segment (20-1, 20-2, 20-3) is arranged in a concentration region (C1, C2, C3) of the channel (14). The deflection means (20) has guide elements (24) as means for guiding cells towards the axis (A). The invention further relates to a method for flow cytometry with the aid of such a device (10). Cells from complex samples can be fully or partially marked for this purpose.

Description

description

Method for enriching and separating cells with concentrations over several logarithmic stages

The present invention relates to magnetic flow cytometry.

In today known methods for flow cytometry cells are focused by means of additional fluid streams in a channel in the middle of the main channel and isolated in this way (Sheath-flow principle). This singulation makes it possible to count the cells in a subsequent measurement step (e.g., optically or impedometrically). By means of magnetic marking of the particles to be quantified, it is possible to isolate and enrich the particles from a complex sample before the actual quantification.

To th sustain conservation, the functionality of this technology, however, a significant amount of additional flues ^¬ sigkeit is necessary which generates the sheath-flow. This method is also associated with further processing with eg dilution steps and centrifugation steps, which is also associated with a risk of contamination. The supply dünnungsschritte are necessary to a count rate to the mi ^¬ nimale and maximum count rate of a magnetic sensor (ie the counter) of the cytometer adapt.

The problem to be solved is the provision of a device and a method by means of which a cytometric measurement, eg determination of the concentration of cell samples, can be carried out faster and at low risk of contamination. The object is achieved by the device according to the invention for flow cytometric measurement according to the patent claim 1. Furthermore, the object of the inventive method according to claim 10 is achieved. Advantageous Developments of the invention are given by the dependent claims.

The invention is based on the idea of carrying out several enrichment ^steps within a closed system. This is made possible by the constructional design of a channel of the device according to the invention and the use of a deflection device in the channel. The invention allows a targeted enrichment and separation of cells with concentrations over several orders of magnitude (of, for example, 10 to 10,000 cells / microliter). Above all, the invention makes it possible to measure several orders of magnitude of possible cell concentrations in a single measurement process. Even with limited dynamics of the counter may be as a large dynamic range in a single microfluidic channel abge ^¬ covers within a complex sample without the need of previous dilution, isolation or enrichment steps. For this purpose, the device according to the invention for flow cytometric measurement comprises a chamber and the channel, wherein the channel comprises a magnetic sensor and is arranged downstream of the chamber. The chamber and channel form a closed system with an axis of the channel extending along a flow direction of the channel. A closed system exists when the chamber passes directly into the channel. The device is characterized by a magnet, in particular a permanent magnet, and a deflection device, both of which are arranged on a predetermined side of the channel. In this case, the Umlenkeinrich ^¬ tion on at least one segment. Each segment is located in a concentration range of the channel. Each segment has means for guiding cells toward the axis. The deflection device thus makes it possible to accumulate magnetically marked cells, which have been pulled onto the channel side by the magnet in the respective concentration range, on the axis and to guide these cells along the axis to the magnetic sensor. The above object is also solved by the Invention ^¬ process according to enrich for cells of an animal to be detected ones of the cell type of a cell sample for a Durchflusszyto- geometry. Initially, magnetically labeled cells of the cell type to be detected are provided. The labeled cells are enriched in the channel of an embodiment of the device according to the invention. For this purpose, the marked cells are pulled by the magnet in the channel to the predetermined side of the channel. There, in lamina ^¬ rer flow in the channel at least a part of the cells of the deflection on the axis, which extends along the flow direction of the channel, steered and here ^¬ enriched by on the axis. This enables an efficient accumulation of cells. Even complex samples, ie non-purified samples containing a variety of different cells and other particles, such as proteins, can thus be used without intermediate steps, in particular undiluted, for a flow cytometry.

The provision of the magnetically labeled cells may include labeling the cells. Two marking variants can be used. In a first variant, the cells to be detected in an incubation step by mixing and / or stirring markers, in particular of at least one specific for de ^¬ tektierenden cell type antibody which is connected to at least one magnetic marker, and the cell sample in be marked in the chamber of the device. These

Variant is preferably used at small total cell concentrations of up to 10 ⁴ cells / microliter. A magnet arranged on one side of the chamber can assist the mixing and / or stirring process.

In one embodiment of the device, a magnet is arranged correspondingly on a predetermined side of the chamber, which supports the mixing and / or stirring process. In the case of cell samples with a greater total cell concentration, eg of up to 10 ⁶ cells / microliter, alternatively at the labeling, the at least one antibody specific for the cell type to be detected, which is associated with at least one magnetic marker, can be provided in the channel. Upon creation of a magnetic field in the channel with a magnet located on the side of the channel, the antibody is accumulated on the side of the channel. Thereafter, the introduction of the cell sample in the channel and thus the in-

Contacting the at least one antibody specific to that proportion of the cell sample, the laminar flows past the side of the Ka ^¬ Nals. This results in a partial labeling of the cells contained in this portion, without dilution or purification steps are necessary. In a laminar flow system, a layered marking of the desired cells is thus achieved. Alternatively, such a partial marking can also be carried out in the chamber by means of the magnet arranged there.

As a result, with fluidic flow in the chamber, only one layer, ie a defined fraction of the cell sample, e.g. 1% of the desired cells, magnetically marked in the closed system.

The deflection means may comprise one or as a means for guiding of cells in a segment, a plurality of guide elements on ^¬, for example, form a channel that directs the labeled cells to the axis. In a particularly advantageous embodiment of the device according to the invention, at least one segment of the deflection device comprises guide elements arranged as the means for guiding cells but arranged obliquely to the axis. Together or alone, these form at least one in the flow direction tapered funnel shape. This allows the deflection of magnetically labeled cells onto the axis of the channel and thus enrichment. At least one guide element may, for example if it consists wholly or partly of a ferromagnetic material, form in a further embodiment of the device a V-shape tapering in the flow direction, which favors a magnetophoretic guidance of magnetically marked cells.

Additionally or alternatively, at least two of the guide members may be configured as walls for the cells and staggered along the axis in the flow direction, thereby providing mechanical guidance to the axis by forming bands for the path of movement of the cells. In addition, in particular between two guide elements at the same axis height, a magnetic web can be arranged along the axis of the channel. This favors the guidance of the magnetically marked cell on the axis of the channel.

Preferably, at least two concentration ranges of the channel are configured differently. A cell sample can thus be enriched in several logarithmic stages. Thus it can be achieved that an enrichment is present, which can be measured with a dynamically limited counter.

For example, the at least two concentra ^¬ onsbereiche distinguish opposite side at a predetermined height of the channel between the predetermined page and the predetermined side. As a result, a defined volume is determined in each concentration range. Different volumes in different concentration ranges ^¬ effect a first enrichment of the cells by the magnet of the channel before enrichment in the deflecting element, so that a statistically significant and a defined number of cells in each Anrei ^¬ cherungsstufe is adjustable at a given concentration. The channel width, however, is preferably constant for all concen ^¬ tion areas. With a constant channel width, the at least two concentration ranges may additionally or alternatively differ by a predetermined width of the deflection device (measured perpendicular to the axis) and / or by a length of the respective segment of the deflection device along the axis of the channel. These dimensions determine the catchment area of the deflection device, thus influencing the degree of enrichment.

In one embodiment of the method according to the invention, a cytometry, in particular a cell count, is carried out downstream of the deflection device on the axis by means of the magnetic sensor. In order to have a concentration, for example ^¬ determination of the cell sample.

A concentration determination of the cell sample by means of the cytometry downstream of the deflection device preferably comprises the counting of the cells flowing through and enriched on the axis from at least one of the concentration ^ranges by means of the magnetic sensor. To at least one of the concentration ranges the concentration of the cell sample is determined using the average in counting the enriched cells count value of the concentration range, the Volu ^¬ men of the concentration range, and the width of the segment of the baffle in the concentration range. This is an efficient and time-saving measurement whose evaluation is quickly available. Of course, in this case at least the concentration range is selected, for which a count value results which is large enough for a statistically relevant statement and smaller than the maximum counting rate that can be reliably detected by the counter. May need to be determined, from which concen ^¬ tion area which at any given time just counted cells belong. In a further embodiment of the method, the respective concentration range, to which a count is determined based on a determined time ^¬ point of the counting of the magnetic sensor determined. In a calibrated device, an assignment of time intervals to the concentration range can thus take place. Under certain circumstances, the flow rate must be considered.

The invention will be explained in more detail below with reference to the accompanying drawings by concrete embodiments. This shows:

1 shows a sketch of an embodiment of a device according to Inventive ^¬ in cross-section,

Sketches of a channel of a device according to the invention, wherein the figure 1A shows a schematic cross section and the figure 1B shows a schematic on the channel,

Sketches of various embodiments of deflecting devices, wherein the 3A shows a perspective view of a deflection with mecha African leadership and the 3B and FIG 3C shows schematic plan views of each one um ^¬ steering device with magnetophoretic guidance, a sketch of a cell sample in the channel immedi ^¬ bar after entering the cell sample (FIG 4A) and after applying a magnetic field (FIG 4B) in of one embodiment of a method according to the invention, a sketch illustrating the enrichment of the cells is illustrated in one embodiment of a method according to the invention in a channel where ^¬ 5A shows a plan view of the channel at a time and FIG. 5B shows a plan view of a segment of the deflection device at different times, and FIG 6 shows a sketch in which the determination of a cell concentration in an embodiment of a method according to the invention and four sections of the channel at different times.

The embodiments described in more detail below represent preferred embodiments of the present invention. Functionally identical elements have the same reference numerals in the figures. The coordinate systems K and K 'are used in the figures as a guide, wherein a ver ^¬ tical axis "z" and a vertical horizontal axis "x" facilitate the orientation in cross section (FIG 1A), and wherein the horizontal axis "x" and an axis "y" perpendicular to the horizontal axis "x" and to the vertical axis "z" facilitates orientation in the plan view of the exemplary channel 14 (FIG. 1B). The flow direction P points here in the x direction. 1 shows a schematic diagram of an embodiment of a device 10 according to the invention for flow cytometric measurement. The device 10 comprises a chamber 12 and a channel 14. The flow direction of the laminar flow given during the cytometry is marked with the arrow P both in FIG. 1 and in the following figures. The chamber 12 may comprise on one side (S2) a magnet 16, which is here attached, for example, outside the chamber 12. The channel 14 is, according to the direction of flow through ^¬ P, downstream of the chamber 12 of the chamber 12 and the passage 14 form a closed system. The Ka ^¬ nal 14 further comprises a deflecting device 20 and a magnet 22, in particular a permanent magnet, on a common channel side (Sl), for example, the channel bottom. A sensor 18 or a sensor array for cytometric measurement, in particular with a sensor chip, is attached to the channel 14 downstream of the deflection device 20 and is designed to count magnetically marked cells. In particular, in the magnetoresistive resistors Sen ^¬ sor 18 may be used, preferably a GMR sensor. The sensor 18 and / or the sensor array is connected to an electronic evaluation device 23 which, for example, comprises a processor of a computer for evaluating the measurement results.

With the device, e.g. Blood samples undiluted to a concentration e.g. the white blood cells are examined.

The channel 14 comprises on the side of the channel 14 where accumulation of magnetically labeled cells is desired, the deflector 20, here e.g. is arranged at the bottom of the channel 14. Below the deflecting device 20, the magnet 22 is arranged. The channel 14 is in e.g. four concentration ranges Cl, C2, C3, C4 divided. However, the number of concentration ranges of four shown here is not mandatory, i. the channel 14 with any

Be configured number of concentration ranges. The height h of the channel can differ in the different concentration ^ranges C1, C2, C3, C4 of the channel 14. In the example of FIG. 2A, the height of the channel is for example the largest in the concentration range Cl, for example 100 micrometers, and decreases with each concentration range C2, C3, C4 following downstream. In the case of partial labeling of cells (see below), the height h is preferably the same in all concentration ranges C1, C2, C4, C4, for example, in stages.

FIG. 2B illustrates the structure of the deflecting device 20. In the exemplary channel 14, the deflecting device 20 is divided into, for example, three segments 20-1, 20-2, 20-3, where ^¬ of each in a different one of the concentration ranges ^C1 , C2, C3 lies. Each segment 20-1, 20-2, 20-3 may comprise as a means for guiding a plurality of cells is oblique to the axis ^¬ guide elements 24 here. For the sake of clarity, only a few guide elements 24 are shown in FIG. mark provided. The individual segments 20-1, 20-2, 20-3 each have a width b and a length a, which depending ^¬ differ weils in segments 20-1, 20-2, 20-3 of different concentration ranges of Cl, C2, C3 can. Preferably, the width b decreases downstream, so that wide segments upstream and narrow segments downstream angeord ^¬ net are. The width b of the deflection device 20 is that distance which is perpendicular to an axis A of the channel (gestri ^¬ smiled line), which extends along the direction of flow P of the channel 14 extends. The width b is thus the A ^¬ catchment area of the deflection device 20 within the channel 14 along the horizontal "Y" axis. Accordingly, the length a is the length of each segment of the baffle 20 along the axis A of the channel. The channel width, for example 100 Micrometer, is constant in each concentration range Cl, C2, C3, C4, for example, a 10 micrometer-wide segment 20-3 then enriches the marked cells 26 by 1/10 of the channel width, ie a logarithmic step less than, for example, the segment 20- 1, if it has a width b of 100 microns.

The guide elements 24 may be arranged obliquely to the axis A, preferably at an acute angle between 0 ° and 90 ° to the axis A, in particular between 0 ° and 45 °, and alone or together form at least one in the flow direction P tapered funnel shape. Such a guide ^¬ element 24 includes, for example, a wall which is mounted here in the example on the inside of the outer wall of the channel 14 and projects into the channel 14.

The guide elements 24 can be, for example, barriers made of, for example, photoresist, which mechanically guide the marked cells 26 and thus accumulate on the axis A, or, for example, ferromagnetic "herringbone structures" which magnetically focus and enrich the marked cells, or a combination of the two structures in one or different of the segments 20-1, 20-2, 20-3 of the deflection device 20. FIG. 2B shows the principle of the guide elements 24 which are shown in FIG are shown enlarged. A magnetophoretic guide element 24 is preferably wholly or partly formed from a ferromagnetic material and / or has a wall height of, for example, 10 nanometers to 100 nanometers. Preferably, the wall height is less than 20%, in particular less than

10% of the average cell diameter of the cells to be detected. The wall height of a guide member 24 to the me chanical ^¬ guide is preferably greater than 20%, in particular 10%, than the diameter of the selected cell 26. In this case, an average cell diameter of 3 microns is used as a basis in the example of platelets.

An example of guide elements 24, for example of a segment 20-1 of the deflection device 20, which form a mechanical guide, is shown in FIG. 3A. In this example, meh ^¬ eral of the guide elements 24 are offset in the direction of flow P to the axis A. Downstream of the deflection device 20, the sensor 18 is shown in FIG 3A. Furthermore, a magnetically marked cell 26 and the movement of the labeled cell 26, which is guided by the guide elements 24, are shown by the arrow Z.

Figures 3B and 3C each illustrate a further embodiment of the deflection device 20, a segment is formed from 20-1 guide elements 24 in the example, which cause a magnetophoresis guide the labeled cell h ^¬ le 26th The guide elements 24 are wholly or partially formed of a magnetic material, so are for example nickel strips. The wall height of such a guide element 24 is preferably 100 nanometers. The Führungsele ^¬ elements 24 are arranged obliquely to the axis A.

Two guide elements 24 of the shown in Figure 3B exporting approximately ^¬ example, which are located in respect to the axis A countertransference are not offset in the embodiment of FIG 3B ^¬ against each other. Between them, that is on the axis A, runs a magnetic web 28, a so-called soul, which consists wholly or partly of a magnetic material, for example nickel. disgust can be formed. In this case, the magnetic material is preferably not free, but instead is coated by a passivation layer which is eg 100 nanometers thick and thus separated from the cell sample 30. The distance of the web 28 to those ends of the guide elements 24 which point towards the axis A is preferably smaller than the diameter of the marked cell 26.

The guide elements 24 shown in the embodiment of FIG. 3C and adjacent to the axis A are offset in the direction of flow P.

FIGS. 4A to 6 illustrate movements of magnetically marked cells 26 in embodiments of the method according to the invention, e.g. a flow cytometry, by means of a device 10 according to the invention. In these examples, by means of the method according to the invention, e.g. determines the coagulability of a whole blood sample. For this purpose, e.g. prior to surgery on a patient, an assessment of the coagulation situation e.g. based on the platelet count of the whole blood sample to determine Haemostasestörun- gene.

To specific cells, e.g. Platelets or CD4 + cells, within a cell sample 30, in particular a complex

To quantify sample such as a whole blood sample, which ^¬ se must first be magnetically labeled with a cell-specific marker, as a complex sample contains different cell h ^¬ len and particles, such as proteins. The labeling is carried out by means of eg superparamagnetic markers, for example with magnetic particles (so-called micro-beads), which are bound to cell-specific or particle-specific antibodies. The magnetic particles may have a diameter of less than 500 nanometers, preferably less than 300 nanometers or between 40 and 300 nanometers. To connect such markers

Antibodies as well as the production of cell- or particle-specific antibodies can be used by techniques familiar to the person skilled in the art. By means of the device 10 according to the invention, a cell-specific labeling can be carried out within a complex sample having a total cell concentration of, for example, 1 to 10 ⁶ cells / microliter. For labeling, all cells of the desired cell type, the cell sample 30 and the Mark ^¬ ten antibodies are placed in the chamber 12 of the device 10 degrees. The antibodies then bind specifically to the cells to be enriched. By stirring the cell solution, this process, in which all cells to be enriched are marked, can be supported. , The magnet 16 can support the stirring and / or mixing process. This variant is advantageous for cell samples with a total cell concentration of up to 10 ⁴ cells / microliter

Alternatively, the labeling can also be carried out partially, eg only a fraction of 1% of all cells to be enriched can be labeled. This is advantageous, above all, in the case of a cell sample having a very high total cell concentration, for example 10 ⁶ cells / microliter, since otherwise a cell count could overtax the dynamic range of the sensor 18. In this case, the antibodies linked to the magnetic marker are first introduced into the channel 14 of the device 10. By the magnet 22 on the predetermined side Sl of the channel 14 (see FIG 1), a magnetic field can be generated whose magnetic ^¬ field attracts the magnetically labeled antibody to the predetermined side Sl of the channel 14 and enriched there. The Mag ^¬ net 16 is preferably on the outside of the duct 14 is introduced ^¬ so that it is not contaminated with the channel content. The cell sample 30 is then brought into the channel 14 ^¬ . The cells to be enriched, in this example the platelets, which are located in that portion of the cell sample 30 which flows past the predetermined side Sl of the channel 14 due to the laminar flow, thus comes into contact with the labeled antibodies. It is advantageous if a high number of markers is per cell ^¬. All other cells do not contact the label. Depending on eg shape and size of the channel 14 can be so a defined fraction of cells, eg 1% or 10% of the platelets, are magnetically labeled as these cells are present in a uniform spatial distribution in the channel 14 after the cell sample 30 is input. After labeling, the labeled cells 26 are present.

Whether one should mark mixing or only one fraction can be estimated before performing the method according to the invention via a blood picture.

In the process step of enrichment of the labeled cells 26, the cell sample 30 containing the magnetically-labeled cells 26 is replaced by the laminar flow e.g. With the aid of a suction device, this is conducted into the channel 14 and thus provided for enrichment, as shown in FIG. 4A (method step S10; for the sake of clarity, only individual marked cells 26 are marked with reference symbols in FIGS. The degree of enrichment (or focusing) in each concentration range C1, C2, C3, C4 of the channel 14 is determined by the volume of the concentration range C1, C2, C3, C4, ie by the height h of the channel 14, by the width b and / or the length a of the segment 20-1, 20-2, 20-3 of the deflection device 20 is determined in a concentration range C1, C2, C3.

FIG. 4A shows, by way of example, a stochastic distribution of the labeled cells 26 over the deflection device 20 immediately after introduction of the cell sample 30 into the channel 14. When a magnetic field is generated by the magnet 22, the marked cells in each concentration range C1, C2, C3, C4 pulled along the z-axis in the direction of the magnet 22 (FIG 4B and FIG 5A). The number of marked cells 26 drawn onto the respective segment 20-1, 20-2, 20-3 depends on the respective volume of the concentration range C1 (eg 140 to 200 microliters), C2 (eg 70 to 120 micro ^¬ liter), C3 (eg 30 to 60 microliters), C4 (eg 5 to 15 microliters), ie from the respective height h of the channel 14. As a result, a defined and statistically meaningful number of marked cells 26 per concentration range C1, C2, C3, C4 can be set. The sample 30 now flows in the flow direction P through the channel 14. The laminar flow causes the marked cells 26 on the deflection device 20 to be pulled toward the sensor 18.

The funnel shape of the deflector 20 directs the marked cells 26 to the axis A either by mechanical and / or by magnetophoretic guidance after enrichment. This is exemplified in FIG. 5B which shows the enlarged detail 32 of FIG. 5A at various successive times t1 , t2 and t3 shows. The marked cells 26 are thereby enriched on the axis A. The width b determines the Anreiche ^¬ magnification factor of the selected cells 26 of the cell sample 30 in the respective range of concentrations Cl, C2, C3, C4.

V-shaped magnetophoretic guide elements 24, as already described with respect to FIG. 2B, move the marked cells 26 along the axis A, due to the laminar flow of liquid, past the tapered ends of the V-shaped guide elements 24 while remaining due to the ferromagnetic Characteristics of these guide elements 24 adhere to the deflection device 20.

Alternatively or additionally, a guide element 24, as shown in FIG. 3B and FIG. 3C, magnetophores the marked cell 26 (see above). Through the gap between two guide elements 24, the cell 26 then slides in the direction of flow P in the direction of the sensor 18. The cell 26 in FIG. 3B is attracted by the web 28 as soon as it is close to the axis. The flow then presses the cell A in the

Flow direction on the web 28 further towards the sensor 18th Alternatively or additionally, a guide element 24 with a mechanical guide, for example a guide element as described for FIG. 3A, forms a barrier through which the marked cell 26 is directed to the respective next guide element 24 and finally to the axis A. Due to the flow in the channel 14, the cell 26 is then moved in the flow direction P on the axis A (FIG 3A, arrow Z).

The focused labeled cells 26 are directed directly to the sensor 18 on the axis A. Small magnetic strips, which may be mounted in front of the sensor, can additionally align the cells 26 with the sensor 18 and bind excess magnetic particles. As a result, enriched labeled cells 26 are not deflected by the sensor 18 and background noise due to free markers during the measurement is reduced.

FIG. 6 shows the channel 14 of the device 10 in a plan view during e.g. a concentration determination. In the example, the platelet count of the cell sample 30 is continuously measured. The section 32 is additionally shown enlarged at four different consecutive times t1 ("t = 1"), t2 ("t = 2"), t3 ("t = 3") and t4 ("t = 4"). In the enlargements of the cutout 32, the last guide element 24 of the segment 20-3 can be seen, which has a width b, e.g. 10 microns, has. The labeled cells 26, which were enriched in the respective concentration ranges C1, C2, C3 and C4, flow on the axis A via the sensor 18. At the time t1, marked cells 26 are led out of the concentration range C4 via the sensor 18. The concentration range C4 in this example does not include a segment of the deflection device 20. Thus, only those cells 26 which are incident on the axis A are detected by the sensor 18.

At the later time t2 ("t = 2"), the enriched labeled cells 26 become out of the concentration range C3 directed via the sensor 18 and counted by the evaluation device 23 by means of this.

The wider and / or longer a segment 20-1, 20-2, 20-3 of a concentration range C1, C2, C3, the larger the catchment area of the respective segment 20-1, 20-2, 20-3 and the more labeled cells 26 from the respective Konzentra ^¬ tion area Cl, C2, C3 are routed via the sensor 18 and counted from this. Accordingly, from time t3, as the enriched labeled cells 26 of the concentration range C2 flow across the sensor 18, the sensor 18 measures more counts than before. At time t4, the highly enriched fraction of the concentration range Cl is measured.

The volumes of the individual concentration ranges Cl, C2, C3, C4 and the width b of the corresponding segment 20-1, 20-2, 20-3 are known or can be easily measured. Using the determined cell count of each concentration range Cl, C2, C3, C4, the cell sample concentration for each

Concentration range Cl, C2, C3, C4 are calculated. Ideally, the values should be the same.

In addition, a time t1, t2, t3 or t4 of the counting process can be determined. It can thus be determined in a calibrated device from which concentration range C1, C2, C3, C4 even cells 26 are counted. In a calibrated device 10, the concentration range Cl, C2, C3, C4 can be determined on the basis of the measured time. A determined counting frequency f, ie the distance between the marked cells 26 to one another, depends on the magnetic force, the flow velocity and the cell concentration. The counting frequency f is lower, the lower the concentration of the sample. In FIG. 6, for example, the frequencies "f = 1", "f = 2", "f = 3" and "f = 4" are determined. The counting frequency f can also be used to determine the concentration of the cell sample 30. Additionally or alternatively, a time period t * can be determined, in which a predetermined count frequency f is present. The time duration t * is dependent on the selected length a of the respective segments 20-1, 20-2, 20-3 of the deflection device 20.

It is possible for the person skilled in the art by reference samples with a known cell concentration to calibrate the counting frequency f or the times t1, t2, t3, t4 for all concentration ranges C1, C2, C3, C4 to the flow velocity and the magnetic force.

In a possible calibration method can for example be a Be ^¬ rich set of flow rates. It is then determined during the measurement of the cell sample 30 in which time period t a defined volume and thus a specific concentration range Cl, C2, C3, C4 of the labeled cells 26 was performed.

Depending on the cell concentration, the frequency f sets in each concentration range Cl, C2, C3, C4 at the corresponding time tl, t2, t3 or t4. The calibration is preferably performed for each concentration range. Ideally, each concentration range for today's sensors is calibrated to 1000 counts to achieve stable statistics.

When measuring the desired cell sample 30 with an unknown concentration, it is then preferably possible to use the measurement of that concentration range C1, C2, C3, C4 for the determination in which the counting frequency is close to 1000, preferably additionally below 1000.

In summary, the invention can also be described with the following words:

In order to quantify specific particles, in particular cells, within a complex sample 30, they must first be labeled with a marker specific for the cells. The For example, required markers consist of a superparamagnetic material and are modified with antibodies on their surface. This label can specifically bind to the target particles. This step of the labeling is to be carried out, for example, within a complex sample 30 and does not require any subsequent purification of the sample. The labeling described here takes place, for example, with superparamagnetic markers.

The label can mark all cells, ie 100% of the cells, within a sample by stirring processes. However, the marking can also be partial (eg 1%). Here, the labeled antibody can be, for example, first introduced into the channel 14 and accumulated by an external Mag ^¬ netfeld on one side of the channel fourteenth If the sample 30 with particles or cells following in the channel

14 is introduced, only those cells or particles come into contact with the markers, which are just at the side of the channel 14. In the case of a stochastic distribution, a suitable proportion (for example 1%) can be marked by the appropriate design of the channel 14. All other particles do not contact the mark.

Focusing by magnetic and / or mechanical forces under laminar flow conditions:

Under the condition of a superparamagnetic labeling of the desired particles, the enrichment is carried out e.g. by means of a combination of magnetic forces (shown here in the z-direction: see FIG. 2A) and mechanical focusing by means of suitable structures (y-direction: see FIGS. 2A and 2B).

On the side at which an enrichment of the particles is desired, a permanent magnet 16 or an electromagnet 16 (FIG. 2) can be positioned. If a cell suspension 30 is introduced into the channel 14 (FIG. 4A), the labeled cells or particles 26 are stochastically distributed. Magnetic forces move the marked cells or particles 26 to one side of the channel 14 (FIG. 4B), here in the z-direction.

By a suitable design of the deflection device 20 (length a and width b) (FIG. 2B) on one of the inner sides of the In the case of the channel 14 used, it is possible to concentrate or focus the cells or particles 26 in the y-direction at any point on the channel wall (here: at y = 1/2).

The deflection device 20 can be, for example, barriers made of, for example, photographic paint, which mechanically guide the cells or particles 26, as well as ferromagnetic herringbone structures, which magnetically enrich and focus the marked cells or particles 26. A combination of both methods is possible. FIG. 5 shows an example of such an enrichment route. FIG 5A shows an example of the stochastic distribution mar ^¬ kierter cells 26 via the deflection device 20 30 immediately after introduction of the sample image 3B shows the principle of focusing of labeled cells 26 tl-t3 in the center of the channel 14 at the times.

Resolution of several powers of ten:

If the channel height h (FIG. 2A) is varied as desired over the different concentration ranges C1, C2, C3, C4 (FIG. 2B), the cell number in this area of the channel 14, for example, is also varied. This makes it possible to set a de ^¬ fined, minimum cell count per concentration range Cl, C2, C3, C4. This procedure allows the SET ^¬ development of a statistically significant number of cells.

In the case of a partial marking, the height h is preferably the same in all concentration ranges C1, C2, C3, C4. Only the layer which is on the side where the labeled antibodies are blocked by e.g. external magnetic field are attracted, is marked and enriched only by the concentration ranges Cl, C2, C3, C4 differently. Due to the freely selectable width b and length a of

Bypass 20 (FIG. 2B), it is possible to enrich a defined fraction of the sample 30 (e.g., 50% of the channel width at C3) in the center of the channel 14. Determination of cell or particle concentration:

The determination of the concentration of the particles within a sample 30 is possible by determining two parameters. The first parameter is the count frequency f in x-rich tion of the focused cells 26 or in other words, the distance between the particles 30 to each other. The count frequency f depends on the magnetic force, the flow velocity and the cell or particle concentration. Therefore, it can count the frequency f for all concentrations Cl, C2, C3, C4 on the flow velocity and the magnetic force to calib ^¬ Center. The second parameter is the time t at which a certain frequency f occurs. In addition, the duration ^¬ in which a specific frequency f is counted gives information about the present concentration range C1, C2, C3, C4. The time t and the duration t * are dependent on the selected length a of the respective deflection device 20. In general, the counting frequency f is lower, the lower the concentration of the sample 30 is.

Prerequisite is a calibrated system. That is, a range of flow rates {vi; vn} is set, which makes it possible to quantify ren in a subsequent step 26, the focused, vereinzel ^¬ ten cells. Thus, it can be determined, the period in which a defined volume and therefore a certain concentration range ^¬ Cl, C2, C3 , C4 of the cell sample 30 was performed. For a subsequent counting system to count, the application can be calibrated to a specific count frequency f. Depending on the cell or particle concentration, this frequency f sets itself at the concentration ranges C1, C2, C3 or C4 at the corresponding time t1, t2 t3 or t4. It is advantageous if the calibration area for each Konzentra ^¬ tion Cl, C2, C3, C4 is performed. So an optimal calibration allows a subsequent quantifi ^¬ authentication method resolves the concentration ranges C1-C4. Ideally, each calibration of a concentration range ^C1 , C2, C3 or C4 consists of up to 1000 counted cells 26 in order to achieve stable statistics.

Determination of concentration:

Over a certain counting frequency f and a time range t, the concentration range C1, C2, C3, C4 of the sample can be determined. be true. From the known geometry and of the resulting re sulting ^¬ liquid volume in this Konzentrationsbe ^¬ rich Cl, C2, C3, C4, it is possible to quantify cells or particles per volume. The counted cells or particles in this time window indicate a concentration of cells or particles throughout the sample 30.

Procedure for evaluation:

First, it can be determined after which time t a count frequency f has been reached. Thus, the concentration range Cl, C2, C3, C4, which was calibrated to 1000 counted cells 26 or particles per measurement, determined. The count value can also be determined for this concentration range C1, C2, C3, C4.

A further embodiment of the cytometric concentration measurement can be explained with reference to FIG. 6: A sample 30 labeled with superparamagnetic markers with an unknown concentration is present. By the described geometric parameters, the flow velocity and / or the strength of the magnetic field) it is possible to obtain a desired frequency f of e.g. labeled cells 26 for optimal quantification. 6 shows an example in which the system is calibrated to f = 4. It can be seen that the desired frequency f is set at time t = 3. There is therefore a sample 30 with a concentration range C2. If the frequency f is higher than 4, then the counting is completed, since one already counts the cells 26 in the enrichment area C1 at the time t = 4.

Some measurement examples in which the concentration ranges are calibrated to 1000 cells:

When calibrating the concentration range Cl, which has, for example, a volume of 10 microliters, eg a reference sample with a concentration of 10 ² cells / microliter is used. From the unknown sample 30, for example, 300 cells counted, therefore, the unknown sample has a concentration of 30 cells / microliter.

Calibration of the concentration range C2 (1 microliter, concentration of the reference sample: 10 ³ cells / microliter) is followed, for example, by a measurement of, for example, 400 cells. The unknown sample has a concentration of 400 cells / microliter.

When calibrating the concentration range C3 (eg 0.1 microliter), the reference sample has, for example, a concentration of 10 ⁴ cells / microliter. In the unknown sample 30 200 cells are counted, the required concentration is 2000 Zel ^¬ len / microliter. After calibration of the concentration range C4 (eg 0.01 microliter, concentration of the reference sample:

10 ⁵ cells / microliter) are counted 800 cells in a sample 30, for example, thus the unknown sample has a concent ration ^¬ of 80,000 cells / microliter.

Claims

claims

A flow cytometric measurement apparatus (10) comprising a chamber (12) and a channel (14), the channel (14) including a magnetic sensor (18) located downstream of the chamber (12) and communicating with the chamber (12 ) is present in a closed system, wherein an axis (A) of the channel (14) extends along a flow direction (P) of the channel (14),

characterized in that

on a predetermined side (Sl) of the channel (14) a magnet (22) and a deflection device (20) are arranged and where ^¬ in the deflection device (20) in at least one segment (20-

1. 20-2, 20-3) and each segment (20-1, 20-2, 20-3) in a concentration range (Cl, C2, C3) of the channel

(14) is arranged, in which the deflection device (20) comprises means for guiding cells (26) to the axis (A) out.

2. Device (10) according to claim 1, wherein on a predetermined side of the chamber (12), a magnet (16) is arranged.

3. Device (10) according to one of the preceding claims, wherein at least one segment (20-1, 20-2, 20-3) of the deflection device (20) as a means for guiding cells (26) obliquely to the axis (A ) arranged guide elements (24) sums ^¬ summarized or alone at least one in the

Flow direction (P) form a tapered funnel shape.

4. Device (10) according to claim 3, wherein

- At least one of the guide elements (24) one in the

Flow direction (P) forms a tapered V-shape and consists wholly or partly of a ferromagnetic material, and / or

- At least two of the guide elements (24) in the flow ^¬ direction (P) on the axis (A) are offset and thereby form a mechanical guide to the axis (A).

5. Device (10) according to any one of claims 3 or 4, wherein a magnetic web (28) along the axis (A) of the channel (14) is arranged.

6. Device (10) according to one of the preceding claims, wherein at least two concentration ranges (Cl, C2, C3, C4) are designed differently.

7. The apparatus (10) according to claim 6, wherein the at least two concentration ranges (Cl, C2, C3, C4) are at a predetermined height (h) of the channel (14) between the predetermined side (Sl) and the predetermined one Differentiate side (Sl) opposite side.

8. Device (10) according to claim 6 or 7, wherein a channel width is constant and the at least two Konzent ^¬ rationsbereiche (Cl, C2, C3) by a predetermined width (b) of the deflecting device (20) differ.

9. Device (10) according to one of claims 6 to 8, wherein each concentration range (Cl, C2, C3) by a length (a) of the respective segment (20-1, 20-2, 20-3) of the deflecting device (20 ) along the axis (A) of the channel (14).

10. A method of enriching cells of a cell type to be detected of a cell sample (30) for flow cytometry comprising the steps of:

Providing magnetically labeled cells (26) of the cell type to be detected, and

- Enriching the marked cells (26) in the channel (14) of a device (10) according to one of claims 1 to 9, where ^¬ in the marked cells (26) of the magnet (22) on the predetermined side (Sl) of the Channel (14) pulled there and there at least a part of the cells (26) of the deflecting device (20) on the axis (A) extending along the

Flow direction (P) of the channel (14) extends, steered and thereby enriched on the axis (A).

11. The method of claim 10, wherein providing the magnetically labeled cells (26) comprises labeling the cells, and wherein the labeling step comprises the following

Steps includes:

Providing at least one antibody specific for the cell type to be detected, which is connected to at least one magnetic marker, in the channel (14),

- Generating a magnetic field in the channel (14) with the on one side (Sl) of the channel (14) arranged magnet

(22) and thereby enriching the antibodies on the side of the channel (14),

- then introducing the cell sample (30) into the channel (14), contacting the at least one specific antibody with that portion of the cell sample which is at the side

(Sl) of the channel (14) flows past laminar, and thereby Mar ^¬ kieren of the cells contained in this fraction.

12. The method according to any one of claims 10 or 11, wherein downstream of the deflecting device (20) on the axis (A) ei ^¬ ne cytometry by means of the magnetic sensor (18) is performed.

13. The method of claim 12, wherein downstream of the environmental steering device (20) to determine the concentration of the cell sample (30) is performed by means of cytometry, umfas ^¬ send the steps of:

Counting the flowing on the axis (A) enriched cells (26) from at least one of the concentration ranges (Cl, C2, C3, C4) by means of the magnetic sensor (18),

for at least one of the concentration ranges (Cl, C2, C3, C4), determining the concentration of the cell sample (30) from the count of the concentration range (Cl, C2, C3, C4) determined during counting of the enriched cells (26), the volume of the Concentration range (Cl, C2, C3, C4) and the width (b) of the deflector (20).

14. The method of claim 13, wherein based on a determined ^¬ th time (tl, t2, t3, t4) of the counting of the magnetic sensor (18) is determined from which concentration range (Cl, C2, C3, C4) even cells are counted.