Abstract:
A flow cytometer has a flow chamber in which labeled cells are highly likely to be detected by a corresponding sensor as a medium carrying the magnetically labeled cells flows through the flow chamber. The flow chamber has at least one sensor positioned on an inner surface thereof to detect the cells. The flow chamber also has a magnetic or magnetizable cell guiding device which can be positioned upstream of the sensor in the direction of flow to guide the flowing, magnetically labeled cells directly across the sensor, so that only a small percentage of labeled cells pass outside of the reach of the sensor.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is the U.S. national stage of International Application No. PCT/EP2010/061931, filed Aug. 17, 2010 and claims the benefit thereof. The International Application claims the benefit of German Application No. 10 2009 047 801.9 filed on Sep. 30, 2009; both applications are incorporated by reference herein in their entirety. 
     
    
     BACKGROUND 
       [0002]    Described below is a flow chamber of a flow cytometer, in which labeled cells may be detected with a high level of probability with the assistance of an appropriate sensor. 
         [0003]    In a magnetic flow cytometer, labeled cells which are to be detected with the assistance of appropriate sensors must be conveyed close above the surface of a sensor in a flow chamber. For example, GMR (giant magnetoresistance) sensors or optical fluorescence or scattered light sensors are used for this purpose. The cell must be close to the sensor, since for example in the case of a GMR sensor the magnetic scatter field of the magnetic labels, which is ultimately utilized by the GMR sensor for detection, declines with the cube of distance from the sensor. The same applies to optical measurement methods. 
         [0004]    In order to ensure that a labeled cell passes by in the immediate vicinity of the sensor, it is in principle conceivable to make the diameter of the channel through which the medium carrying labeled cells flows as small as possible, i.e. in an extreme case the diameter of the channel is just big enough for individual cells to be able to pass through. The drawback of this approach is of course that the presence of impurities or disruptive particles very rapidly results in the channel being blocked. On the other hand, if the channel is made larger, there is also a greater probability that individual labeled cells will pass by outside the range of the sensor and will thus not be detected. This drawback may be countered by providing a larger number of sensors, but this entails more complex electronics. 
       SUMMARY 
       [0005]    Described below is a flow chamber in which there is an elevated probability of detecting a labeled cell with a sensor of the flow chamber. Through the flow chamber in a flow cytometer flows a medium carrying magnetically labeled cells. The flow chamber has at least one sensor for cell detection positioned on an internal surface of the flow chamber, and is equipped with a magnetic or magnetizable cell-guiding device. The latter is positioned upstream of the sensor in the direction of flow and arranged and constructed there such that it guides the flowing, magnetically labeled cells over the sensor. 
         [0006]    The cell-guiding device is advantageously arranged on the internal surface of the flow chamber and includes a number n, with n≧1, of magnetic or magnetizable flow strips oriented substantially parallel to the direction of flow, wherein
       the number n of flow strips corresponds to the number of sensors,   one flow strip is in each case assigned to one sensor and   a magnetically labeled cell guided by a flow strip is guided over the assigned sensor.       
 
         [0010]    In a first embodiment, a flow strip is of a width which remains constant throughout in the direction of flow. 
         [0011]    In a second embodiment, a flow strip tapers in the direction of flow, in particular in the manner of a funnel or half funnel. 
         [0012]    In a third embodiment, an individual, wide flow strip divides, in the direction of flow, into a plurality of narrower, substantially parallel flow sub-strips, wherein the number of flow sub-strips corresponds to the number of sensors. 
         [0013]    In a fourth embodiment, the flow strips are arranged in a herringbone pattern. 
         [0014]    In an advantageous embodiment, part of a flow strip, in particular the downstream part in the direction of flow, is subdivided into a plurality of portions lying downstream of one another and spaced apart from one another. 
         [0015]    In an advantageous embodiment, a magnet is provided which is arranged in such a manner on the flow chamber that a force directed towards the internal surface is generated which acts on the magnetically labeled cells. 
         [0016]    In a further advantageous embodiment, the sensor is a GMR sensor. 
         [0017]    In a further embodiment, a further magnetic or magnetizable cell-guiding device is provided which is positioned downstream of the sensor in the direction of flow. 
         [0018]    In the method, magnetically labeled cells in a medium flowing through a flow chamber of a flow cytometer are detected with a sensor by guiding the flowing, labeled cells over the sensor with a magnetic or magnetizable cell-guiding device, which is positioned upstream of the sensor in the direction of flow. 
         [0019]    In an advantageous further embodiment of the method, a further cell-guiding device is used, which is arranged downstream of the sensor in the direction of flow. The medium is guided over the sensor alternately in a first direction and in a second direction, which is contrary to the first direction. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]    These and other aspects and advantages will become more apparent and more readily appreciated from the exemplary embodiments described below with reference to the accompanying drawings of which: 
           [0021]      FIG. 1  is a cross-section of a flow chamber, 
           [0022]      FIG. 2  is a plan view of a first embodiment of the cell-guiding device, 
           [0023]      FIG. 3  is a plan view of a second embodiment of the cell-guiding device, 
           [0024]      FIG. 4  is a plan view of a third embodiment of the cell-guiding device, 
           [0025]      FIG. 5  is a plan view of a fourth embodiment of the cell-guiding device, 
           [0026]      FIG. 6  is a plan view of a fifth embodiment of the cell-guiding device, 
           [0027]      FIG. 7  is a plan view of a further embodiment of the cell-guiding device, 
           [0028]      FIGS. 8A-8C  and  8 A′- 8 C′ are plan views and side views, respectively, of three embodiments of the flow strip and 
           [0029]      FIGS. 9A-9C  are side views illustrating the principle of cell concentration. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0030]    In the figures, identical or mutually corresponding zones, components, and component assemblies are designated with the same reference numerals. 
         [0031]      FIG. 1  shows a flow chamber  10  of a flow cytometer in cross-section. A medium  70 , which contains the magnetically labeled cells  20  to be detected as well as unlabeled cells  30 , passes in the direction of flow  130  through an orifice  40  into the flow chamber  10 . The medium  70  flows through a microfluidic channel  11  of the chamber  10  and, after detection, leaves the latter through a further orifice  50 . The magnetically labeled cells  20  are detected with the aid of a sensor  60 . The sensor  60  may for example be a GMR sensor or an optical fluorescence or scattered light sensor. By way of example below, it is assumed that a GMR sensor  60  is used. 
         [0032]      FIG. 1  likewise shows an optional permanent magnet  140 , which is located below the microfluidic channel  11  and which generates a magnetic field (not shown). This field on the one hand attracts the magnetically labeled cells  20 , so ensuring that they brush over the sensor  60  close to the surface thereof. On the other hand, the magnet  140 , especially in the case assumed here of a sensor  60  of the GMR type, may be used in order to generate the gradient field required for operation of this type of sensor; when the magnetic cells  20  pass over the GMR sensor  60  they influence the magnetic field prevailing at the location of the sensor. This is recorded by the GMR sensor and utilized for detection. Alternatively, a corresponding energized coil may of course also be used instead of the permanent magnet  140 . In the event that the sensor  60  is an optical fluorescence or scattered light sensor or the like, a magnetic field is, of course, not required for sensor operation. Nevertheless a magnet may also be provided in order, as mentioned, to ensure that the labeled cells  20  pass close over the surface of the sensor  60 . 
         [0033]    When dimensioning the magnet  140 , care must be taken to ensure that the strength of the magnetic field is matched to the flow velocity of the medium. If the magnetic field and thus the retention force is too strong, disruption to flow cannot be ruled out as individual cells  20  may possibly be immobilized. Conversely, if the magnetic field is too weak, it is to be assumed that some of the labeled cells  20  will pass by the sensor  60  outside the range thereof, i.e. that they will not be detected. 
         [0034]    By way of the interplay between the strength of the magnetic field of the magnets  140  and the flow  130 , generated for example by pumps (not shown), or the velocity thereof, it is possible purposefully to adjust the retention force for magnetically labeled cells  20  in order, on the one hand, to remove cells with low labeling density, i.e. “false positive” cells, and, on the other hand, only to convey cells with sufficiently strong immunomagnetic labeling to the sensor  60 , with any unbound labels, for example superparamagnetic particles, not being conveyed to the sensor due to the lower retention force. 
         [0035]    In a concentration device not shown in  FIG. 1 , which is described in greater detail in  FIG. 8 , the medium  70  may initially be concentrated before the actual detection, i.e. the concentrated medium  70  leaving the concentration device would enter the flow chamber  10  via the orifice  40 . 
         [0036]    The flow chamber  10  includes a cell-guiding device  120 . This device  120  ensures that the magnetically labeled cells  20  which are still stochastically distributed at the inlet  40  to the flow chamber  10 , (cf.  FIGS. 2 to 6 ) can be purposefully guided over the sensor  60 . This has the advantageous consequence that a substantially larger number of cells  20  may be detected, since distinctly fewer cells flow past, for example to the side of, the sensor  60 . It is accordingly no longer left to chance whether a labeled cell  20  comes within the range of the sensor  60  and is detectable. 
         [0037]    To this end, magnetic or magnetizable metal tracks are arranged in the direction of flow on or in that internal surface  12  of the flow chamber  10  on which the sensor  60  is also arranged. As is explained below with reference to the figures, these metal tracks or “flow strips” may for example be of constant width, taper in the manner of a funnel or half funnel, converge in a fan shape or also be arranged in a herringbone pattern. Others arrangements which likewise ensure that the labeled cells  20  are guided over the sensor  60  are, of course, likewise conceivable. The flow strips may furthermore be of continuous or alternatively of discontinuous design. A discontinuous design (cf.  FIG. 8B ,  8 C) singulates the cells  20 , i.e. it is ensured that a plurality of cells  20  do not brush over the sensor  60  simultaneously or immediately one after the other. Because individual cells  20  now brush over the sensor  60 , it is ensured that individual cell analysis may be carried out more efficiently. 
         [0038]      FIG. 2 , like  FIGS. 3 ,  4 ,  5  and  6 , shows a plan view of the interior of a flow chamber  10 , the unlabeled cells  30  not being shown for the sake of clarity. For the same reason, only a few of the cells  20  are provided by way of example with reference numerals. In this exemplary embodiment, the cell-guiding device  120  has four flow strips  121  made of a magnetic or a magnetizable material. The flow strips  121  are arranged parallel to one another and are oriented in the direction of flow  130  of the medium. The width of the flow strips  121  may be substantially in line with the diameter of the cells  20 , but is however generally less than the width of the sensors  60 . 
         [0039]    The interaction between the magnetic cells  20  and the magnetic flow strip  121  ensures that the cells  20 , as they flow past the strips  120  with the medium  70 , leave their stochastic distribution and arrange themselves on the strips  121 :
       in a first zone I, the cells  20  are stochastically distributed.   in a second zone II, the cells  20  align themselves with the flow strip  121 .   in a third zone III, the cells  20  arranged on the flow strip  121  are conveyed to the sensors  60 .   in a fourth zone IV, (individual) cell detection takes place.       
 
         [0044]    The boundaries of zones I to IV are here not sharply defined, but are instead variable, for example, as a function of the field of the magnet  140  and the flow velocity. In other words, the zones shown in the figures should be understood as examples. 
         [0045]    Because the magnetic gradient is steepest at the edge of the respective flow strip  121 , it is to be assumed that the cells  20  will not arrange themselves centrally on the respective flow strip  121 , but instead on the edge thereof. 
         [0046]    In the direction of flow downstream of each flow strip  121 , i.e. as an extension of the strip  121 , there is located a sensor  60 , such that the labeled and ordered cells  20  may be purposefully guided over the sensor  60  with the assistance of the cell-guiding device  120 . Apart from a few exceptions, which were not caught by the magnetic flow strip  121  and were therefore not guided to the sensors  60 , it may be assumed that a large proportion of the labeled cells  20  in the medium  70  will come within the range of the sensors  60 , such that a substantially higher yield may be achieved with the arrangement, which is for example manifested, with constant statistics, in a shorter measurement time or, with a constant measurement time, in improved statistics. 
         [0047]    The flow strips may for example be made of nickel and be ≦10 μm wide and 100-500 nm thick. Thicknesses of an order of magnitude of 1 μm are, however, likewise conceivable. The microfluidic channel  11  is typically 100-400 μm wide, 100 μm high and approx. 1 mm long. The GMR sensors  60  are approx. 25-30 μm long (in a direction perpendicular to the direction of flow  130 ). 
         [0048]      FIG. 3  shows a further exemplary embodiment of a cell-guiding device  120 . In this case, the cell-guiding device  120  has only one flow strip  122 , which however tapers in the manner of a funnel in the direction of flow  130  until it is ultimately of a width which approximately corresponds to the diameter of the cells  20 . At its wide end, the strip  122  covers the entire width of the flow cell  10  or of the microfluidic channel  11 . This wide zone of the strip virtually acts as a collector with which the cells  20  may be led towards the narrow flow strip. 
         [0049]    In this exemplary embodiment too, the flow strip  122  may be made of a magnetic or a magnetizable material, such that here too the initially stochastically distributed, magnetically labeled cells  20  may be ordered and finally guided over the sensor  60 . 
         [0050]    The advantage of the arrangement of  FIG. 3  over that of  FIG. 2  is, for example, that in this case only one sensor  60  is required. This permits simplification of the readout electronics. 
         [0051]    In a third exemplary embodiment of the cell-guiding device  120  which is shown in  FIG. 4 , the latter is formed of two magnetic or magnetizable flow strips  123 , which in each case taper in the manner of a half funnel in the direction of flow  130 . As in the other exemplary embodiments, in this case too a sensor  60  is assigned to each flow strip  123 , which sensor is located in the direction of flow  130  downstream of the flow strip  123  and over which the labeled cells  20  are guided. 
         [0052]      FIG. 5  shows a fourth exemplary embodiment. The flow strip  124  shown here is, like the examples of  FIGS. 2 and 3 , of comparatively wide construction on the input side, i.e. in zone I. The single, wide flow strip  124  is, however, divided into four flow sub-strips  124 / 1  to  124 / 4 , over which the cells  20  are guided to the sensors  60 , as in the previous exemplary embodiments. 
         [0053]      FIG. 6  shows a fifth exemplary embodiment of the cell-guiding device  120 . In this case, the flow strips  125  are arranged in a herringbone pattern, i.e. a central flow strip  125 / 1  is on the one hand provided which extends to the sensor  60 . Further flow strips  125 / 2 ,  125 / 3  are on the other hand provided, which are arranged at an angle of for example ±45° to the direction of flow  130 , such that the magnetically labeled cells  20  are initially guided to the central flow strip  125 / 1  and thence over the sensor  60 . 
         [0054]      FIG. 7  shows an embodiment which, with regard to the arrangement of the flow strips  121 , corresponds in principle to that of  FIG. 2 . Unlike  FIG. 2 , however, flow strips  121 ,  121 ′ are in this case arranged both upstream and downstream of the sensors  60  in the direction of flow. In a corresponding detection method, the medium and thus the labeled cells  20  would be conveyed alternately in a first direction of flow  130  and in the opposite direction  130 ′, for example in order to improve the statistics. The cells  20  accordingly brush repeatedly over the sensors  60 . 
         [0055]    In principle, the embodiment of  FIG. 7  with a cell-guiding device arranged on both sides of the sensors  60  may, of course, also be constructed in accordance with the embodiments of the cell-guiding devices of  FIGS. 3 to 6 . However, since the cells  20  passing over the sensor  60  are generally already ordered, i.e. no longer stochastically distributed, it is generally sufficient to construct the further cell-guiding device  120 ′ as shown in  FIG. 7 . A kind of “collector”, as the cell-guiding devices  120  in particular of  FIGS. 3 ,  4  and  5  in zone I which primarily serve to guide the stochastically distributed cells  20  towards the individual tracks, would only be necessary in the case of the further cell-guiding device if it were possible to supply a medium to the flow chamber  10  both via the orifice  40  and via the orifice  50 . 
         [0056]    FIGS.  8 A to  8 C′ show various embodiments of individual flow strips. The figures provide a side view and a plan view of the flow strip of each embodiment with magnetically labeled cells  20  arranged thereon. 
         [0057]    The flow strip  126  of  FIG. 8A  is of continuous construction, as also shown in  FIGS. 1 to 7 . 
         [0058]      FIG. 8B , in contrast, shows discontinuous flow strip  127 . In the upstream part  127 / 1  in the direction of flow  130 , the strip is likewise of continuous construction. The downstream part  127 / 2  of the flow strip  127  is, however, discontinuous, i.e. the strip is here divided into a plurality of portions  127 / 3  arranged downstream of one another. As described above, this has an advantageous effect on the possibility of individual cell detection. The length of the individual portions  127 / 3  may for example correspond to the width of the strip and/or approximately to the diameter of the cell. 
         [0059]    The flow strip  128  of  FIG. 8C  substantially corresponds to that of  FIG. 8B , i.e. an upstream, continuous part  128 / 1  and a downstream, discontinuous part  128 / 2  with individual portions  128 / 3  are provided. In addition, however, a continuous strip  128 / 4  is applied onto the portions  128 / 3 , which continuous strip for example prevents cells  20  being diverted into the zones between the portions  128 / 3  by any turbulence in the flow. 
         [0060]      FIG. 9  illustrates the principle of concentration in simplified manner.  FIG. 9A  here shows a plan view of the concentration device  80 , while  FIGS. 9B and 9C  show two side views or cross-sections of the device  80  at successive points in time t 1 , t 2  (t 2 &gt;t 1 ). Typically, the concentration of the magnetically labeled cells  20  is comparatively low in the original medium, for example whole blood. Analysis would be very time-consuming. The original medium, which flows through a channel  100  in the concentration device  80 , is therefore concentrated before detection, the intention being to increase the proportion of labeled cells  20  in the medium relative to the proportion of unlabeled cells  30 . 
         [0061]      FIG. 9  illustrates “semi-continuous” concentration, in which the concentration proceeds first at time t 1  (cf.  FIG. 9B ) and then the concentrated medium is conveyed to the flow chamber at time t 2  ( FIG. 9C ). Further concentration (not shown) would then proceed etc. 
         [0062]    Concentration is performed using a magnet  90  which generates a first magnetic field (not shown) of an order of magnitude of approx. 100-1000 mT. This attracts the magnetically labeled cells  20  onto the side of the channel  100  on which the magnet  90  is arranged. Accordingly, the concentration of labeled cells  20  is distinctly increased on this side of the channel  100 . It is specifically on this side that a further channel  110  is furthermore provided, via which the now concentrated medium reaches the flow chamber  10 , which is shown only symbolically in  FIG. 9 . In order to keep the magnetically labeled cells  20  also in the channel  110  and finally in the flow chamber  10  on the side on which the sensor  60  is also positioned, a further magnet  91  is provided, which however generates a weaker magnetic field than the magnet  90 , for example of an order of magnitude of up to 100 mT. 
         [0063]    The method which may be performed with the flow chamber described above is intended for use for example for mammalian cells, microorganisms or magnetic beads. Magnetic flow cytometry may be used in combination with optical (for example fluorescence, scattered light) or other non-magnetic detection methods (for example radiochemical, electrical) in order to perform in situ observations or carry out further analyses. 
         [0064]    A description has been provided with particular reference to preferred embodiments thereof and examples, but it will be understood that variations and modifications can be effected within the spirit and scope of the claims which may include the phrase “at least one of A, B and C” as an alternative expression that means one or more of A, B and C may be used, contrary to the holding in  Superguide v. DIRECTV,  358 F3d 870, 69 USPQ2d 1865 (Fed. Cir. 2004).