EP2841940A1 - Method and arrangement for detecting cells in a cell suspension - Google Patents

Method and arrangement for detecting cells in a cell suspension

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Publication number
EP2841940A1
EP2841940A1 EP13726515.3A EP13726515A EP2841940A1 EP 2841940 A1 EP2841940 A1 EP 2841940A1 EP 13726515 A EP13726515 A EP 13726515A EP 2841940 A1 EP2841940 A1 EP 2841940A1
Authority
EP
European Patent Office
Prior art keywords
cell
pair
sensor elements
platelets
signal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13726515.3A
Other languages
German (de)
French (fr)
Inventor
Michael Johannes Helou
Oliver Hayden
Lukas RICHTER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens AG
Original Assignee
Siemens AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Siemens AG filed Critical Siemens AG
Publication of EP2841940A1 publication Critical patent/EP2841940A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
    • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
    • G01R33/09—Magnetoresistive devices
    • G01R33/093—Magnetoresistive devices using multilayer structures, e.g. giant magnetoresistance sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
    • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
    • G01R33/09—Magnetoresistive devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0092—Monitoring flocculation or agglomeration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the invention relates to a method and an arrangement for detecting and in particular counting cells in a cell suspension.
  • the detection of cells and cell interactions within one and the same blood sample using magnetoresistive methods has been an unsolved problem.
  • changes ⁇ effects are important for medical Diagnos ⁇ policy to close as quickly as possible to a particular clinical picture.
  • thrombocytopenia ie the low number of platelets or blood platelets in the blood.
  • Thrombocytopenia may be due to a clotting disorder or increased activity of the immune system against the body's own platelets (immunothrombocytopenia) entste ⁇ hen.
  • a Immunothrombozytopenie can Autoimmunerkran ⁇ kung (immunthrombozytician purpura or idiopathic thrombogenic ⁇ bozytopenische purpura, ITP) occur, then, in the tracking's own immune system platelets and removed.
  • immunothrombocytopenia may also occur if the number of platelets decreases dramatically during an infectious disease.
  • platelets perform tasks within the Pro ⁇ zesses the immune defense.
  • the platelets occur either in direct interaction with immune cells (monocytes) and thereby form immune cell / platelet aggregates or in direct interaction with the invaded microorganisms (bacteria, viruses, yeasts / fungi).
  • the platelets are detected by monocytes and removed.
  • Monocytes are circulating in the blood cells of the immune system and the precursor ⁇ among other things in the tissues lenti ⁇ overbased macrophages as well as a portion of the dendritic cells. Platelets within such aggregates are required for ben during blood clotting or hemostasis is no longer available.
  • the resulting reduction in platelet count by acute immune responses may be confused with a coagulation disorder.
  • the rapid differentiation of these two clinical pictures can accelerate the diagnosis.
  • the present invention makes it possible to count immune cell / platelet aggregates in whole blood.
  • the arrangement according to the invention for the quantification of cells distinguishing at least two different sizes of cell types and / or cell conglomerate species in a cell suspension with a magnetic field sensitive sensor comprising at least a first and second pair of sensor elements, wherein the sensor elements of the first pair have a first distance of between half and twice a first average size of a first type of cell or cell conglomerate to be measured,
  • the sensor elements of the second pair have a second distance of between half and twice a second average size of a second type of cell or cell conglomerate to be measured
  • a third distance of the closest sensor elements of the pairs is greater than the larger of the two middle
  • the assembly includes an evaluation device for evaluating a first signal of the first and a second signal of the second pair, wherein the evaluation device is designed from ⁇ , zueben both the time interval between the first and second signal and the amplitude of the two signals.
  • FIG. 1 shows a measuring system with fluid channel and GMR sensor
  • Figure 2 is a conglomerate of monocyte and platelet over the sensor and the associated measurement signal
  • Figure 3 shows a platelet over the sensor and the associated
  • FIG. 4 shows a medium-sized conglomeration of thrombocytes above the sensor and the associated measurement signal
  • FIG. 5 shows a large conglomeration of platelets over the sensor and the associated measurement signal
  • Figure 6 is a schematic of a GMR sensor in parallel arrangement in a Wheatstone bridge
  • FIG. 7 is a schematic of a GMR sensor in a diagonal arrangement in a Wheatstone bridge.
  • 1 schematically shows the basic structure of an exemplary sensor 10 according to the invention: a fluid channel 20 serves to guide and guide a cell suspension via sensor elements 11 of a GMR sensor (Giant Magnetoresistve). The delivery of the cell suspension is carried out by microfluidic channel systems as known from US 20110315635 AI.
  • the Sen ⁇ sorium thereby form a first pair 12 and a second pair 13. Both pairs 12, 13 are made in manner known per se in a parallel arrangement as shown in Figure 6 together in each of a Wheatstone bridge.
  • the first pair 12 generates a first sensor signal and the second pair 13 generates a second sensor signal.
  • Both signals were ⁇ generated when magnetically marked cells or conglomerates in the fluid passage 20 Wennbe ⁇ because of the sensor elements, because the sensor elements 11 are able to detect magnetic fields in their immediate vicinity.
  • the sensor element 11 can also be used directly for the measurement, without interconnecting it in a Wheatstone bridge.
  • Figures 6 and 7 show the connection to a Wheatstone bridge in parallel arrangement, as used in the following examples or in a diagonal arrangement. In this case, the actual sensor elements 11 are electrically connected by means of conductor tracks 61.
  • the first embodiment deals with the specific counting of aggregates of monocytes 21 and / or platelets 22 within a whole blood sample.
  • the platelets 22 are marked in advance with superparamagnetic nanoparticles 23, which in turn are associated with a specific antibody.
  • antigens eg CD154
  • platelets 22 interact with monocytes 21, they present antigens (eg CD154) on their surface which they would not present during the process of hemostasis. Be distinguished in this way Kings ⁇ nen this platelet 22 with the help of specifically labeled nanoparticles 23 of platelets 22, which are involved in blood clotting. Platelets 22, which are involved in blood clotting, are therefore not labeled.
  • the individual cells and aggregates are detectable by means of GMR sensor technology.
  • monocyte / platelet aggregate or platelet aggregate 41, 51 is passed over the sensor, then characteristic signals are produced. If Thrombo cytes ⁇ 22 react on specific antigen-antibody interactions with monocytes 21, located cell / cell aggregates of an average size of about 25ym form.
  • the sensor geometry of the sensor shown in Figure 1 is adapted to the measuring task before ⁇ geous.
  • 2 ym is used as the distance of the sensor elements 11 of the first pair 12, furthermore as the distance of the sensor elements 11 of the second pair 13 25 ym and as the distance between the closest sensor elements 11 of the two pairs 12, 13 35 ym.
  • Figure 2 shows an aggregate of a monocyte 21 and some platelets 22 at two positions, once above the first pair 12 and once above the second pair 13.
  • the unit generates a signal sequence, as it is also shown in Figure 2.
  • the characteristic signal A is generated.
  • Signal A is essentially characterized by a temporally narrowly limited amplitude of high amplitude.
  • the characteristic signal B When repainting the second pair 13, the characteristic signal B is generated.
  • Signal B is characterized by a Langge ⁇ solid waveform with two similar peaks of a mean amplitude of the tude to as Standardampli- is used 24th
  • the two peaks of the signal B over ⁇ overlap the close spacing of the sensor elements 11 of the first pair 12 and so form the signal A.
  • the larger From ⁇ stand of the sensor elements 11 of the second pair 13 ensures that there do not overlap with these peaks.
  • the signals described are due to the flow rate and thus the time required for the cell aggregate from the first pair 12 to the second pair 13, separated in time by the time interval tl. Other types of cells and cell aggregates used in this
  • FIG. 3 shows which signal sequence is produced when a single marked thrombocyte cell 22 is swept over the sensor elements 11. This results in a sweep of the f ⁇ th pair 12 again, the characteristic signal sequence B, since the ratio of sizes of cell and the first pair 12 in about the ratio of the sizes of aggregate of a monocyte 21 and some platelets 22 and the second pair 13 ent - speaks.
  • a charac ⁇ C signals are available in the form of two clearly separate rashes.
  • the time interval t2 between the two signals is in This case is significantly greater than the time interval tl.
  • Figure 4 shows the signal sequence through a medium-sized conglomerate 41 from some labeled platelet cells 22, in this example exactly eleven cells produced during sweeping the Sen ⁇ sorieri. 11 This results in a sweep of the first pair 12 again this time the characteristic signal sequence ⁇ A having a peak of large amplitude, since the sensor elements 11 of the first pair 12 due to their small distance not dissolve the individual components of the conglomerate 41 Kgs ⁇ NEN. With a time interval of the magnitude of approximately t1, a signal of the type of the characteristic signal B arises, but this time with a significantly increased amplitude.
  • Figure 5 shows the signal sequence through a large conglomerate 51 from a GroE ßeren labeled platelet cells 22, in this example, more than thirty cells produced during sweeping the Sen ⁇ sorimplantation. 11 So the characteristic Sig ⁇ nal façade A produced during a sweep of the first pair 12 again this time with a peak of large amplitude since the Sensorele ⁇ elements 11 of the first pair 12 can not resolu ⁇ sen due to their small distance, the individual portions of the large conglomerate 51st Since the large conglomerate 51 is greater than the Ab ⁇ stand of the pairs 12, 13 to each other, no zeitli ⁇ cher distance creates more between the first and second signal but the signals are overlapped in part.
  • the second pair 13 a characteristic signal D high Ampli tude ⁇ arises because the large conglomerate 51 is greater than the distance of the sensor elements 11 of the second pair. 13 Also, the signal sequence that was developed for the large conglomerate 51 is distinguishable from the other types of cells and aggregates.
  • the different occurring cells and aggregates can be distinguished by the following table.
  • different sizes and cell / cell aggregates can be measured by analyzing the different signal forms.
  • M / T an aggregate of monocyte 21 and platelets 22
  • T is a single platelet cell 22
  • the sensor geometry is adapted to the expected geometry or size of the analyte to be measured, and on the other hand, the distance between two sensor strips is set to produce immune cell / platelet aggregates (diameter: 15-25) of individual platelets (2 -5ym) in the same sample.
  • the spacing of the pairs 12, 13 to each other aggregates allows the additional exclusion of cell ⁇ which are greater than the target structure, that is in present example greater than about 25 ym.
  • the resulting signal combinations can be inferred to the currently measured cell or cell combination.
  • a) adaptation of the sensor geometry to the size of the analyte magnetic particles such as metal particles or magnetically marked biochemical particles such as proteins or liposomes as well as magnetically labeled biological particles such as animal cells, microorganisms and Viruses.
  • a time-of-flight measurement allows a statement about the size of the analyte.
  • the arrangement of two sensors with different geometries allows the differentiation of particles of different sizes and their composition by an exclusion method. The form of the individual signal and the time sequence of two signals is a specific criterion.
  • the amplitude of the signal allows the differentiation of particle agglomerates of different composition depending on their magnetization.
  • the agglomerate is magnetically labeled (platelet 22), while the other component remains unlabeled (monocyte 21).
  • the un ⁇ marked component affects the magnetization and size of the entire agglomerate.
  • the measurement of an analyte may be performed (in complex fluids, inter alia, blood, urine, or secretions) without purification or Ver ⁇ Phymbiariae.
  • An optical Transpa ⁇ ence is not necessary.
  • the cells used are (for example, primary phagocytes of the immune system) size Zvi ⁇ rule 15 and 30ym.
  • the platelets however, have a size between 2 and 5ym. This results in an area for the Distances.
  • the distance between the sensor elements 11 of the first pair 12 may be between 1 and 4 ym
  • the distance between the sensor elements 11 of the second pair 13 may be between 20 and 30 ym and the distance between the closest sensor elements 11 of the two pairs 12, 13 between 30 and 40 yards.
  • the optimal geometry can be specified experimentally.
  • Exemplary embodiment 2 Marking of platelets 22 within cell aggregates together with microorganisms (bacteria, viruses or fungi / yeasts)
  • Platelets 22 are becoming increasingly important during the process of primary immune defense, where they interact with immune cells or, in the case of ITP, directly interact with foreign organisms such as bacteria, viruses or fungi and yeasts. In general, a differentiation of these two causes of thrombocytopenia (ITP or infection) is crucial for a subsequent selection of a drug treatment.
  • ITP thrombocytopenia
  • platelets 22 are also able to include these on phagocytosis and neutrali ⁇ Sieren. During this process, platelets 22 are also able to present MHC-I antigens (found mainly on immune cells but also on platelets 22) on their surface to alert the immune system. Labeling MHC-1 in the blood and counting the cells may indicate immunothrombocytopenia. Large cells can be identified as immune cells and small cells as platelets 22.
  • Example 3 Marking of endogenous Fresszel ⁇ len of the immune system within aggregates with large Zel- len (circulating tumor cells, own immune cells)
  • the body's own scavenger cells are capable of circulating Tu ⁇ morzellen that identifies the immune system as foreign bodies were rendered harmless by phagocytosis (ingestion) and subsequent digestion.
  • the diameter of a phagocyte is the one hand, significantly RESIZE ⁇ SSER, on the other hand, these cells also present specific antigens (MHC-1) on their surface during and after completion of this operation.
  • MHC-1 specific antigens
  • Embodiment 4 Measurement of fibrin based stei ⁇ gender viscosity during coagulation:
  • the viscosity of the blood increases due to the formation of fibrin from fibrinogen.
  • fibrin the final step during the Bloody ⁇ rinsted
  • the viscosity of the blood increases continuously until it finally comes to a halt.
  • the speed of blood-borne particles also decreases. The slowdown in the
  • Particles in the coagulating blood can be used as a measure of its increasing viscosity and placed in direct correlation with the increasing proportion of insoluble fibrin.
  • the time-of-flight measurement uses, for example, the distance between the two pairs 12, 13 and the signals generated by the pairs when an analyte moves past.
  • Embodiment 5 Magnetic beads can be used as an internal standard for the flow rate Since the flow rate of blood Spen ⁇ which may vary due to different viscosities of different output, an internal standard should be introduced into the sample, which allows the flow rate at the beginning to determine each measurement. Such a standard can consist of magnetic particles which should clearly differ from the analyte (much smaller or much larger) so that a confusion with the analyte, ie the actual cells or cell conglomerates, can be excluded.
  • the sensor signals of the Sensorelemen ⁇ te 11 are not inverted in time but follow without inverting successive , At a time overlap of the signals arise thereby also characteristic Sig- nalformen depending on the size of the respective Analy ⁇ th compared with the spacing of the sensor elements 11 from one another.

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Abstract

The invention relates to an arrangement for quantifying cells while differentiating between at least two different sizes of cell types and/or cell conglomerate types in a cell suspension, comprising a magnetic field-sensitive sensor which has at least one first and second pair of sensor elements. The sensor elements of the first pair are connected as part of a Wheatstone bridge and have a first spacing of between half and double a first average size of a first cell or cell conglomerate type to be measured. The sensor elements of the second pair are connected as part of a Wheatstone bridge and have a second spacing of between half and double a second average size of a second cell or cell conglomerate type to be measured. A third spacing of the two closest sensor elements of the pairs is greater than the larger of the two average sizes. The arrangement also comprises a channel for conducting the cell suspension past the sensor elements.

Description

Beschreibung description
Verfahren und Anordnung zur Detektion von Zellen in einer ZellSuspension Method and device for detecting cells in a cell suspension
Die Erfindung betrifft ein Verfahren sowie eine Anordnung zur Detektion und insbesondere Zählung von Zellen in einer Zellsuspension . Die Detektion von Zellen und Zellinteraktionen innerhalb ein und derselben Blutprobe mit Hilfe von magnetoresistiven Methoden ist bislang ein ungelöstes Problem. Derartige Wechsel¬ wirkungen sind jedoch wichtig für die medizinische Diagnos¬ tik, um möglichst schnell auf ein bestimmtes Krankheitsbild zu schließen. The invention relates to a method and an arrangement for detecting and in particular counting cells in a cell suspension. The detection of cells and cell interactions within one and the same blood sample using magnetoresistive methods has been an unsolved problem. However, such changes ¬ effects are important for medical Diagnos ¬ policy to close as quickly as possible to a particular clinical picture.
Eines dieser Krankheitsbilder ist die Thrombozytopenie, also die zu geringe Anzahl an Thrombozyten bzw. Blutplättchen im Blut. Thrombozytopenie kann aufgrund einer Blutgerinnungsstö- rung oder einer erhöhten Aktivität des Immunsystems gegen die körpereigenen Thrombozyten ( Immunothrombozytopenie) entste¬ hen. Eine Immunothrombozytopenie kann also Autoimmunerkran¬ kung ( immunthrombozytische Purpura oder idiopathische throm¬ bozytopenische Purpura, ITP) auftreten, bei der das eigene Immunsystem Thrombozyten aufspürt und entfernt. Immunothrombozytopenie kann jedoch auch auftreten, wenn die Anzahl an Thrombozyten während einer Infektionskrankheit drastisch sinkt. Dabei führen Thrombozyten Aufgaben innerhalb des Pro¬ zesses der Immunabwehr aus. Die Thrombozyten treten dabei entweder in direkte Wechselwirkung mit Immunzellen (Monozyten) und bilden dabei Immunzell/Thrombozyten-Aggregate oder in direkte Wechselwirkung mit den eingedrungenen Mikroorganismen (Bakterien, Viren, Hefen/Pilzen) . In beiden Fällen werden die Thrombozyten von Monozyten aufgespürt und ent- fernt. Monozyten sind im Blut zirkulierende Zellen des Immun¬ systems und die Vorläufer der u. a. in den Geweben lokali¬ sierten Makrophagen sowie eines Teils der dendritischen Zellen. Thrombozyten innerhalb solcher Aggregate sind für Aufga- ben während der Blutgerinnung bzw. Hämostase nicht mehr verfügbar. Die resultierende Senkung der Thrombozytenzahl durch akute Immunreaktionen kann mit einer Gerinnungsstörung verwechselt werden. Die rasche Differenzierung dieser beiden Krankheitsbilder (Gerinnungsstörung oder Autoimmunerkrankung) kann die Diagnostik beschleunigen. Die vorliegende Erfindung ermöglicht unter anderem die Zählung von Immun- zell/Thrombozyten-Aggregaten im Vollblut. One of these syndromes is thrombocytopenia, ie the low number of platelets or blood platelets in the blood. Thrombocytopenia may be due to a clotting disorder or increased activity of the immune system against the body's own platelets (immunothrombocytopenia) entste ¬ hen. A Immunothrombozytopenie can Autoimmunerkran ¬ kung (immunthrombozytische purpura or idiopathic thrombogenic ¬ bozytopenische purpura, ITP) occur, then, in the tracking's own immune system platelets and removed. However, immunothrombocytopenia may also occur if the number of platelets decreases dramatically during an infectious disease. Here platelets perform tasks within the Pro ¬ zesses the immune defense. The platelets occur either in direct interaction with immune cells (monocytes) and thereby form immune cell / platelet aggregates or in direct interaction with the invaded microorganisms (bacteria, viruses, yeasts / fungi). In both cases, the platelets are detected by monocytes and removed. Monocytes are circulating in the blood cells of the immune system and the precursor ¬ among other things in the tissues lokali ¬ overbased macrophages as well as a portion of the dendritic cells. Platelets within such aggregates are required for ben during blood clotting or hemostasis is no longer available. The resulting reduction in platelet count by acute immune responses may be confused with a coagulation disorder. The rapid differentiation of these two clinical pictures (coagulation disorder or autoimmune disease) can accelerate the diagnosis. Among other things, the present invention makes it possible to count immune cell / platelet aggregates in whole blood.
Die Detektion von Aggregaten aus Immunzellen mit Thrombozyten ist bisher soweit bekannt nur mittels optischer Durchflusszy- tometrie realisiert. Diese Technologie erfordert die spezifi¬ sche Markierung beider Zelltypen (Immunzellen und Thrombozyten) mit Hilfe von für Fluoreszenz markierten Antikörpern. Weiterhin erfordert die optische Durchflusszytometrie eine komplexe Aufreinigung der zu untersuchenden Zelltypen bzw. eine Entfernung störender Zelltypen wie beispielsweise roter Blutzellen. Ohne diese Aufreinigung wäre die Detektion der verwendeten Fluoreszenzfarbstoffe nicht möglich. The detection of aggregates of immune cells with platelets has so far been known only realized by means of optical flow cytometry. This technology requires the specifi ¬ specific marker of both cell types (immune cells and platelets) with the aid of labeled antibodies for fluorescence. Furthermore, the optical flow cytometry requires a complex purification of the cell types to be investigated or a removal of interfering cell types such as red blood cells. Without this purification, the detection of the fluorescent dyes used would not be possible.
Es ist Aufgabe der vorliegenden Erfindung, ein verbessertes Verfahren sowie eine entsprechende Anordnung zur Detektion und insbesondere Quantifizierung von Zellen in einer Zellsuspension anzugeben, bei denen der eingangs genannte Nachteil vermieden wird. It is an object of the present invention to provide an improved method and a corresponding arrangement for the detection and in particular quantification of cells in a cell suspension, in which the above-mentioned disadvantage is avoided.
Diese Aufgabe wird durch eine Anordnung mit den Merkmalen von Anspruch 1 gelöst. Die Unteransprüche betreffen vorteilhafte Ausgestaltungen der Erfindung. Weiterhin wird die Aufgabe durch ein Verfahren mit den Merkmalen von Anspruch 10 gelöst. This object is achieved by an arrangement having the features of claim 1. The subclaims relate to advantageous embodiments of the invention. Furthermore, the object is achieved by a method having the features of claim 10.
Die erfindungsgemäße Anordnung zur Quantifizierung von Zellen unter Unterscheidung wenigstens zweier verschiedener Größen von Zell-Arten und/oder Zellkonglomerat-Arten in einer Zellsuspension mit einem magnetfeldsensitiven Sensor, der wenigstens ein erstes und zweites Paar von Sensorelementen aufweist, wobei - die Sensorelemente des ersten Paars einen ersten Abstand von zwischen der Hälfte und dem doppelten einer ersten mittleren Größe einer ersten zu vermessenden Art von Zelle oder Zellkonglomerat aufweisen, The arrangement according to the invention for the quantification of cells distinguishing at least two different sizes of cell types and / or cell conglomerate species in a cell suspension with a magnetic field sensitive sensor comprising at least a first and second pair of sensor elements, wherein the sensor elements of the first pair have a first distance of between half and twice a first average size of a first type of cell or cell conglomerate to be measured,
- die Sensorelemente des zweiten Paars einen zweiten Abstand von zwischen der Hälfte und dem doppelten einer zweiten mittleren Größe einer zweiten zu vermessenden Art von Zelle oder Zellkonglomerat aufweisen, the sensor elements of the second pair have a second distance of between half and twice a second average size of a second type of cell or cell conglomerate to be measured,
- ein dritter Abstand der einander nächstliegenden Sensorele- mente der Paare größer ist als die größere beider mittlerer - A third distance of the closest sensor elements of the pairs is greater than the larger of the two middle
Größen, sizes,
sowie mit as well as with
- einem Kanal zur Führung der Zellsuspension an den Sensorelementen vorbei.  - A channel for guiding the cell suspension past the sensor elements.
Für die Erfindung wurde erkannt, dass mittels einer spezifi¬ schen Sensorgeometrie eine Unterscheidung zwischen verschie¬ denen Arten von Zellen und/oder Konglomeraten in einer Zellsuspension möglich ist. Dabei ist vorteilhaft keine Aufreini- gung oder Filterung oder Verdünnung erforderlich, sondern die Zellsuspension kann in ihrem Ausgangszustand belassen werden. Lediglich eine Markierung von wenigstens einem Teil der Zellen mit superparamagnetischen Partikeln ist erforderlich, um am magnetoresistiven Sensor ein Signal zu erzeugen. For the invention, it was recognized that by means of a specifi ¬ rule sensor geometry ¬ which types of cells and / or conglomerates is possible to distinguish between different in a cell suspension. In this case, advantageously no purification or filtration or dilution is necessary, but the cell suspension can be left in its initial state. Only labeling of at least a portion of the cells with superparamagnetic particles is required to generate a signal at the magnetoresistive sensor.
Zweckmäßig umfasst die Anordnung eine Auswerteeinrichtung zur Auswertung eines ersten Signals des ersten und eines zweiten Signals des zweiten Paars, wobei die Auswerteeinrichtung aus¬ gestaltet ist, sowohl den zeitlichen Abstand des ersten und zweiten Signals als auch die Amplitude der beiden Signale aus zuwerten . Conveniently, the assembly includes an evaluation device for evaluating a first signal of the first and a second signal of the second pair, wherein the evaluation device is designed from ¬, zuwerten both the time interval between the first and second signal and the amplitude of the two signals.
Ein bevorzugtes, jedoch keinesfalls einschränkendes Ausfüh¬ rungsbeispiel für die Erfindung wird nunmehr anhand der Figu- ren der Zeichnung näher erläutert. Dabei sind die Merkmale schematisiert dargestellt. Es zeigen A preferred, but by no means limitative exporting ¬ approximately example of the invention will be with reference to the drawing Figu- ren explained in more detail. The features are shown schematically. Show it
Figur 1 ein Messsystem mit Fluidkanal und GMR-Sensor, Figur 2 ein Konglomerat aus Monozyte und Thrombozyten über dem Sensor und das zugehörige Messsignal, Figur 3 eine Thrombozyte über dem Sensor und das zugehörige 1 shows a measuring system with fluid channel and GMR sensor, Figure 2 is a conglomerate of monocyte and platelet over the sensor and the associated measurement signal, Figure 3 shows a platelet over the sensor and the associated
Messsignal ,  Measuring signal,
Figur 4 ein mittelgroßes Konglomerat aus Thrombozyten über dem Sensor und das zugehörige Messsignal, FIG. 4 shows a medium-sized conglomeration of thrombocytes above the sensor and the associated measurement signal,
Figur 5 ein großes Konglomerat aus Thrombozyten über dem Sensor und das zugehörige Messsignal, FIG. 5 shows a large conglomeration of platelets over the sensor and the associated measurement signal,
Figur 6 ein Schema eines GMR-Sensors in paralleler Anordnung in einer Wheatstone-Brücke und Figure 6 is a schematic of a GMR sensor in parallel arrangement in a Wheatstone bridge and
Figur 7 ein Schema eines GMR-Sensors in diagonaler Anordnung in einer Wheatstone-Brücke. Figur 1 zeigt schematisch den prinzipiellen Aufbau eines beispielhaften Sensors 10 gemäß der Erfindung: ein Fluidkanal 20 dient zur Führung und Leitung einer Zellsuspension über Sensorelemente 11 eines GMR-Sensors (Giant Magnetoresistve) . Die Zuführung der Zellsuspension erfolgt durch mikrofluidische Kanalsysteme wie aus der US 20110315635 AI bekannt. Die Sen¬ sorelemente bilden dabei ein erstes Paar 12 und ein zweites Paar 13. Beide Paare 12, 13 sind dabei in für sich genommen bekannter Weise in paralleler Anordnung wie in Figur 6 dargestellt in je einer Wheatstone-Brücke zusammengeschlossen. Das erste Paar 12 erzeugt ein erstes Sensorsignal und das zweite Paar 13 erzeugt ein zweites Sensorsignal. Beide Signale wer¬ den erzeugt, wenn sich magnetisch markierte Zellen oder Konglomerate im Fluidkanal 20 an den Sensorelemente 11 vorbeibe¬ wegen, da die Sensorelemente 11 in der Lage sind, magnetische Felder in ihrer unmittelbaren Nähe zu detektieren. In einer alternativen Ausführungsform können die Sensorelement 11 auch direkt zur Messung herangezogen werden, ohne sie in einer Wheatstone-Brücke zu verschalten. Die Figuren 6 und 7 zeigen die Verbindung zu einer Wheatstone-Brücke in paralleler Anordnung, wie sie in den folgenden Beispielen verwendet wird bzw. in diagonaler Anordnung. Hierbei sind die eigentlichen Sensorelemente 11 elektrisch mittels Leiterbahnen 61 ver- schaltet. Figure 7 is a schematic of a GMR sensor in a diagonal arrangement in a Wheatstone bridge. 1 schematically shows the basic structure of an exemplary sensor 10 according to the invention: a fluid channel 20 serves to guide and guide a cell suspension via sensor elements 11 of a GMR sensor (Giant Magnetoresistve). The delivery of the cell suspension is carried out by microfluidic channel systems as known from US 20110315635 AI. The Sen ¬ sorelemente thereby form a first pair 12 and a second pair 13. Both pairs 12, 13 are made in manner known per se in a parallel arrangement as shown in Figure 6 together in each of a Wheatstone bridge. The first pair 12 generates a first sensor signal and the second pair 13 generates a second sensor signal. Both signals wer ¬ generated when magnetically marked cells or conglomerates in the fluid passage 20 vorbeibe ¬ because of the sensor elements, because the sensor elements 11 are able to detect magnetic fields in their immediate vicinity. In an alternative embodiment, the sensor element 11 can also be used directly for the measurement, without interconnecting it in a Wheatstone bridge. Figures 6 and 7 show the connection to a Wheatstone bridge in parallel arrangement, as used in the following examples or in a diagonal arrangement. In this case, the actual sensor elements 11 are electrically connected by means of conductor tracks 61.
Das erste Ausführungsbeispiel, das anhand der Figuren 2 bis 5 näher erläutert wird, behandelt die spezifische Zählung von Aggregaten von Monozyten 21 und/oder Thrombozyten 22 inner- halb einer Vollblutprobe. Dabei werden die Thrombozyten 22 vorab mit superparamagnetischen Nanopartikeln 23 markiert, die wiederum mit einem spezifischen Antikörper verbunden sind. Wenn die Thrombozyten 22 mit Monozyten 21 in Wechselwirkung treten, präsentieren sie Antigene (beispielsweise CD154) auf ihrer Oberfläche, welche sie während dem Vorgang der Hämostase nicht präsentieren würden. Auf diese Weise kön¬ nen diese Thrombozyten 22 mit Hilfe von spezifisch markierten Nanopartikeln 23 von Thrombozyten 22 unterschieden werden, die an der Blutgerinnung beteiligt sind. Thrombozyten 22, die an der Blutgerinnung beteiligt sind, werden demnach nicht markiert . The first embodiment, which is explained in more detail with reference to FIGS. 2 to 5, deals with the specific counting of aggregates of monocytes 21 and / or platelets 22 within a whole blood sample. Here, the platelets 22 are marked in advance with superparamagnetic nanoparticles 23, which in turn are associated with a specific antibody. When platelets 22 interact with monocytes 21, they present antigens (eg CD154) on their surface which they would not present during the process of hemostasis. Be distinguished in this way Kings ¬ nen this platelet 22 with the help of specifically labeled nanoparticles 23 of platelets 22, which are involved in blood clotting. Platelets 22, which are involved in blood clotting, are therefore not labeled.
Dadurch, dass die Thrombozyten 22 mit superparamagnetischen Nanopartikeln markiert sind, werden die einzelnen Zellen und Aggregate mittels GMR-Sensorik detektierbar . Wenn ein einzelner Thrombozyt 22, ein Monozyten/Thrombozyten-Aggregat oder ein Thrombozytenaggregat 41, 51 über den Sensor geleitet wird, dann entstehen charakteristische Signale. Wenn Thrombo¬ zyten 22 über spezifische Antigen-Antikörper Wechselwirkungen mit Monozyten 21 reagieren, bilden sich Zell/Zellaggregate einer mittleren Größe von etwa 25ym. Because the platelets 22 are labeled with superparamagnetic nanoparticles, the individual cells and aggregates are detectable by means of GMR sensor technology. When a single platelet 22, monocyte / platelet aggregate or platelet aggregate 41, 51 is passed over the sensor, then characteristic signals are produced. If Thrombo cytes ¬ 22 react on specific antigen-antibody interactions with monocytes 21, located cell / cell aggregates of an average size of about 25ym form.
Die Sensorgeometrie des in Figur 1 gezeigten Sensors ist vor¬ teilhaft an die Messaufgabe angepasst. So wird als Abstand der Sensorelemente 11 des ersten Paars 12 2 ym verwendet, weiterhin als Abstand der Sensorelemente 11 des zweiten Paars 13 25 ym und als Abstand der nächstliegenden Sensorelemente 11 der beiden Paare 12, 13 35 ym. Figur 2 zeigt ein Aggregat aus einem Monozyt 21 und einigen Thrombozyten 22 an zwei Positionen, einmal über dem ersten Paar 12 und einmal über dem zweiten Paar 13. Auf dem Weg über die beiden Paare 12, 13 aus Sensorelementen 11 des GMR-Sen- sors erzeugt das Aggregat dabei eine Signalfolge, wie sie ebenfalls in Figur 2 dargestellt ist. Beim Überstreichen des ersten Paars 12 wird das charakteristische Signal A erzeugt. Signal A ist im Wesentlichen durch einen zeitlich eng be- grenzten Ausschlag hoher Amplitude gekennzeichnet. Beim Über¬ streichen des zweiten Paars 13 wird das charakteristische Signal B erzeugt. Signal B zeichnet sich durch einen langge¬ zogenen Signalverlauf mit zwei gleichartigen Peaks einer mittleren Amplitude aus, die im Folgenden als Standardampli- tude 24 verwendet wird. Die beiden Peaks des Signals B über¬ lappen durch den geringen Abstand der Sensorelemente 11 des ersten Paars 12 und bilden so das Signal A. Der größere Ab¬ stand der Sensorelemente 11 des zweiten Paars 13 sorgt dafür, dass dort diese Peaks nicht überlappen. Die beschriebenen Signale sind aufgrund der Fließgeschwindigkeit und damit der Zeit, die das Zellaggregat vom ersten Paar 12 zum zweiten Paar 13 benötigt, zeitlich getrennt durch den zeitlichen Abstand tl. Andere Arten von Zellen und Zellaggregaten, die in diesemThe sensor geometry of the sensor shown in Figure 1 is adapted to the measuring task before ¬ geous. Thus, 2 ym is used as the distance of the sensor elements 11 of the first pair 12, furthermore as the distance of the sensor elements 11 of the second pair 13 25 ym and as the distance between the closest sensor elements 11 of the two pairs 12, 13 35 ym. Figure 2 shows an aggregate of a monocyte 21 and some platelets 22 at two positions, once above the first pair 12 and once above the second pair 13. On the way over the two pairs 12, 13 of sensor elements 11 of the GMR sensor The unit generates a signal sequence, as it is also shown in Figure 2. When sweeping the first pair 12, the characteristic signal A is generated. Signal A is essentially characterized by a temporally narrowly limited amplitude of high amplitude. When repainting the second pair 13, the characteristic signal B is generated. Signal B is characterized by a Langge ¬ solid waveform with two similar peaks of a mean amplitude of the tude to as Standardampli- is used 24th The two peaks of the signal B over ¬ overlap the close spacing of the sensor elements 11 of the first pair 12 and so form the signal A. The larger From ¬ stand of the sensor elements 11 of the second pair 13 ensures that there do not overlap with these peaks. The signals described are due to the flow rate and thus the time required for the cell aggregate from the first pair 12 to the second pair 13, separated in time by the time interval tl. Other types of cells and cell aggregates used in this
Beispiel auftreten können, können anhand ihrer charakteristi¬ schen Signale eindeutig davon und untereinander unterschieden werden. Figur 3 zeigt, welche Signalfolge bei Überstreichen der Sensorelemente 11 durch eine einzelne markierte Thrombo- zyt-Zelle 22 entsteht. So entsteht bei Überstreichen des ers¬ ten Paars 12 wieder die charakteristische Signalfolge B, da das Verhältnis der Größen von Zelle und dem ersten Paar 12 in etwa dem Verhältnis der Größen von Aggregat aus einem Monozyt 21 und einigen Thrombozyten 22 und dem zweiten Paar 13 ent- spricht. Beim Überstreichen des zweiten Paars 13 erzeugt das die einzelne markierte Thrombozyt-Zelle 22 ein charakteristi¬ sches Signal C in Form zweier deutlich getrennter Ausschläge. Der zeitliche Abstand t2 zwischen den beiden Signalen ist in diesem Fall deutlich größer als der zeitliche Abstand tl. So¬ mit ist anhand der Signale eine eindeutige Unterscheidung zwischen einer einzelnen Thrombozyt-Zelle 22 und einem Aggregat aus solchen Zellen und einem Monozyten 21 möglich. For example occur can be clearly distinguished and thereof with each other based on their charac ¬ rule signals. FIG. 3 shows which signal sequence is produced when a single marked thrombocyte cell 22 is swept over the sensor elements 11. This results in a sweep of the f ¬ th pair 12 again, the characteristic signal sequence B, since the ratio of sizes of cell and the first pair 12 in about the ratio of the sizes of aggregate of a monocyte 21 and some platelets 22 and the second pair 13 ent - speaks. When passing over the second pair 13 which generates the single labeled platelet-cell 22 a charac ¬ C signals are available in the form of two clearly separate rashes. The time interval t2 between the two signals is in This case is significantly greater than the time interval tl. Thus ¬ with reference to the signals a clear distinction between a single platelet cell 22 and a unit of such cells and monocytes 21 is possible.
Figur 4 zeigt, welche Signalfolge bei Überstreichen der Sen¬ sorelemente 11 durch ein mittelgroßes Konglomerat 41 aus einigen markierten Thrombozyt-Zellen 22, in diesem Beispiel genau elf Zellen, entsteht. So entsteht bei Überstreichen des ersten Paars 12 wieder diesmal die charakteristische Signal¬ folge A mit einem Peak großer Amplitude, da die Sensorelemente 11 des ersten Paars 12 aufgrund ihres geringen Abstands die einzelnen Anteile des Konglomerats 41 nicht auflösen kön¬ nen. Mit einem zeitlichen Abstand der Größe von etwa tl ent- steht ein Signal der Art des charakteristischen Signals B, allerdings diesmal mit wesentlich vergrößerter Amplitude. So¬ mit ist anhand der Amplitude des Signals des zweiten Paars 13 auch dieses Konglomerat 41 - ohne eine Monozyt-Zelle 21 - von dem Aggregat mit Monozyt 21 unterscheidbar. Noch deutlicher ist der Unterschied zu der Signalfolge eines einzelnen Throm¬ bozyt 22. Figure 4 shows the signal sequence through a medium-sized conglomerate 41 from some labeled platelet cells 22, in this example exactly eleven cells produced during sweeping the Sen ¬ sorelemente. 11 This results in a sweep of the first pair 12 again this time the characteristic signal sequence ¬ A having a peak of large amplitude, since the sensor elements 11 of the first pair 12 due to their small distance not dissolve the individual components of the conglomerate 41 Kgs ¬ NEN. With a time interval of the magnitude of approximately t1, a signal of the type of the characteristic signal B arises, but this time with a significantly increased amplitude. Thus ¬ with reference to the amplitude of the signal of the second pair 13 is even this conglomerate 41 - without a monocyte cell 21 - distinguishable from the aggregate with monocyte 21st Even more significant is the difference to the signal sequence of a single Throm ¬ bozyt 22nd
Figur 5 zeigt, welche Signalfolge bei Überstreichen der Sen¬ sorelemente 11 durch ein großes Konglomerat 51 aus einer grö- ßeren markierten Thrombozyt-Zellen 22, in diesem Beispiel über dreißig Zellen, entsteht. So entsteht bei Überstreichen des ersten Paars 12 wieder diesmal die charakteristische Sig¬ nalfolge A mit einem Peak großer Amplitude, da die Sensorele¬ mente 11 des ersten Paars 12 aufgrund ihres geringen Abstands die einzelnen Anteile des großen Konglomerats 51 nicht auflö¬ sen können. Da das große Konglomerat 51 größer als der Ab¬ stand der Paare 12, 13 zueinander ist, entsteht kein zeitli¬ cher Abstand mehr zwischen dem ersten und zweiten Signal, sondern die Signale überlappen sich in Teilen. Beim zweiten Paar 13 entsteht ein charakteristisches Signal D hoher Ampli¬ tude dadurch, dass das große Konglomerat 51 größer als der Abstand der Sensorelemente 11 des zweiten Paars 13 ist. Auch die Signalfolge, die sich für das große Konglomerat 51 er- gibt, ist von den anderen Arten von Zellen und Aggregaten unterscheidbar . Figure 5 shows the signal sequence through a large conglomerate 51 from a GroE ßeren labeled platelet cells 22, in this example, more than thirty cells produced during sweeping the Sen ¬ sorelemente. 11 So the characteristic Sig ¬ nalfolge A produced during a sweep of the first pair 12 again this time with a peak of large amplitude since the Sensorele ¬ elements 11 of the first pair 12 can not resolu ¬ sen due to their small distance, the individual portions of the large conglomerate 51st Since the large conglomerate 51 is greater than the Ab ¬ stand of the pairs 12, 13 to each other, no zeitli ¬ cher distance creates more between the first and second signal but the signals are overlapped in part. The second pair 13 a characteristic signal D high Ampli tude ¬ arises because the large conglomerate 51 is greater than the distance of the sensor elements 11 of the second pair. 13 Also, the signal sequence that was developed for the large conglomerate 51 is distinguishable from the other types of cells and aggregates.
Somit lassen sich die verschiedenen auftretenden Zellen und Aggregate anhand der folgenden Tabelle unterscheiden. Hier kann man sehen, dass trotz der Markierung nur eines Zelltypus unterschiedliche Größen und Zell/Zell-Aggregate mit Hilfe der Analyse der unterschiedlichen Signalformen gemessen werden können . Thus, the different occurring cells and aggregates can be distinguished by the following table. Here one can see that, despite the labeling of only one cell type, different sizes and cell / cell aggregates can be measured by analyzing the different signal forms.
Dabei bezeichnen: Where:
M/T ein Aggregat aus Monozyt 21 und Thrombozyten 22  M / T an aggregate of monocyte 21 and platelets 22
T eine einzelne Thrombozyten-Zelle 22  T is a single platelet cell 22
TT ein mittelgroßes Aggregat 41 aus Thrombozyten 22  TT a medium aggregate aggregate 41 from platelets 22
TTT ein großes Konglomerat 51 aus Thrombozyten 22.  TTT a large conglomerate 51 from platelets 22.
Vorteilhaft wird hier also einerseits die Sensorgeometrie an die zu erwartende Geometrie bzw. Größe des zu messenden Ana- lyten angepasst und andererseits der Abstand zweier Sensor¬ streifen eingestellt, um Immunzell/Thrombozyten-Aggregate (Durchmesser: 15-25ym) von einzelnen Thrombozyten (2-5ym) in derselben Probe zu unterscheiden. Der Abstand der Paare 12, 13 zueinander erlaubt die zusätzliche Ausschließung von Zell¬ aggregaten, welche größer als die Zielstruktur sind, also im vorliegenden Beispiel größer als etwa 25 ym. Weiterhin lassen die dabei entstehenden Signalkombinationen auf die gerade gemessene Zelle oder Zellkombination rückschließen . Folgende Schritte werden also vorteilhaft vorgenommen bzw. Vorteile ergeben sich: a) Anpassung der Sensorgeometrie an die Größe des Analyten (magnetische Partikel wie Metallpartikel oder magnetisch mar- kierte biochemische Partikel wie Proteine oder Liposome als auch magnetisch markierte biologische Partikel wie tierische Zellen, Mikroorganismen und Viren) . Eine Time-of-Flight- Messung erlaubt eine Aussage über die Größe des Analyten. b) Die Anordnung zweier Sensoren mit unterschiedlichen Geometrien erlaubt die Differenzierung von Partikeln unterschiedlicher Größe und ihrer Zusammensetzung durch ein Ausschlussverfahren. Dabei ist die Form des einzelnen Signals und die zeitliche Reihenfolge zweier Signale ein spezifisches Kriterium. c) Die Amplitude des Signals erlaubt die Differenzierung von Partikelagglomeraten unterschiedlicher Zusammensetzung in Abhängigkeit ihrer Magnetisierung. Dabei ist eine Komponente des Agglomerates magnetisch markiert (Thrombozyt 22), während die andere Komponente unmarkiert bleibt (Monozyt 21) . Die un¬ markierte Komponente beeinflusst die Magnetisierung und Größe des gesamten Agglomerates. d) Die Messung eines Analyten kann in komplexen Flüssigkeiten (u.a. Blut, Urin oder Sekreten) ohne Aufreinigungs- oder Ver¬ dünnungsschritte durchgeführt werden. Eine optische Transpa¬ renz ist nicht notwendig. Im vorliegenden ersten Beispiel haben die verwendeten Zellen (z.B. primäre Fresszellen des Immunsystems) eine Größe zwi¬ schen 15 und 30ym. Die Blutplättchen haben hingegen eine Größe zwischen 2 und 5ym. Daraus ergibt sich ein Bereich für die Abstände. Beispielsweise kann als Abstand der Sensorelemente 11 des ersten Paars 12 zwischen 1 und 4 ym verwendet werden, weiterhin als Abstand der Sensorelemente 11 des zweiten Paars 13 zwischen 20 und 30 ym und als Abstand der nächstliegenden Sensorelemente 11 der beiden Paare 12, 13 zwischen 30 und 40 ym. Die optimale Geometrie kann experimentell konkretisiert werden . Advantageously, on the one hand, the sensor geometry is adapted to the expected geometry or size of the analyte to be measured, and on the other hand, the distance between two sensor strips is set to produce immune cell / platelet aggregates (diameter: 15-25) of individual platelets (2 -5ym) in the same sample. The spacing of the pairs 12, 13 to each other aggregates allows the additional exclusion of cell ¬ which are greater than the target structure, that is in present example greater than about 25 ym. Furthermore, the resulting signal combinations can be inferred to the currently measured cell or cell combination. The following steps are thus advantageously carried out or advantages arise: a) adaptation of the sensor geometry to the size of the analyte (magnetic particles such as metal particles or magnetically marked biochemical particles such as proteins or liposomes as well as magnetically labeled biological particles such as animal cells, microorganisms and Viruses). A time-of-flight measurement allows a statement about the size of the analyte. b) The arrangement of two sensors with different geometries allows the differentiation of particles of different sizes and their composition by an exclusion method. The form of the individual signal and the time sequence of two signals is a specific criterion. c) The amplitude of the signal allows the differentiation of particle agglomerates of different composition depending on their magnetization. One component of the agglomerate is magnetically labeled (platelet 22), while the other component remains unlabeled (monocyte 21). The un ¬ marked component affects the magnetization and size of the entire agglomerate. d) The measurement of an analyte may be performed (in complex fluids, inter alia, blood, urine, or secretions) without purification or Ver ¬ dünnungsschritte. An optical Transpa ¬ ence is not necessary. In the present first example, the cells used are (for example, primary phagocytes of the immune system) size Zvi ¬ rule 15 and 30ym. The platelets, however, have a size between 2 and 5ym. This results in an area for the Distances. For example, the distance between the sensor elements 11 of the first pair 12 may be between 1 and 4 ym, and the distance between the sensor elements 11 of the second pair 13 may be between 20 and 30 ym and the distance between the closest sensor elements 11 of the two pairs 12, 13 between 30 and 40 yards. The optimal geometry can be specified experimentally.
Ausführungsbeispiel 2: Markierung von Thrombozyten 22 inner- halb von Zellaggregaten zusammen mit Mikroorganismen (Bakterien, Viren oder Pilzen/Hefen) Exemplary embodiment 2: Marking of platelets 22 within cell aggregates together with microorganisms (bacteria, viruses or fungi / yeasts)
Den Thrombozyten 22 wird immer mehr Bedeutung während des Prozesses der primären Immunabwehr zugesprochen, wo sie un- terstützend mit Immunzellen oder im Falle von ITP aber auch direkt mit Fremden Organismen wie Bakterien, Viren oder Pilzen und Hefen in Wechselwirkung treten. Generell ist eine Differenzierung dieser beiden Ursachen für eine Thrombozytopenie (ITP oder Infektion) ist ausschlaggebend für eine fol- gende Auswahl einer medikamentösen Behandlung. Platelets 22 are becoming increasingly important during the process of primary immune defense, where they interact with immune cells or, in the case of ITP, directly interact with foreign organisms such as bacteria, viruses or fungi and yeasts. In general, a differentiation of these two causes of thrombocytopenia (ITP or infection) is crucial for a subsequent selection of a drug treatment.
Im Falle einer Viruserkrankung sind Thrombozyten 22 auch in der Lage, diese über Phagozytose aufzunehmen und zu neutrali¬ sieren. Während dieses Vorganges sind Thrombozyten 22 auch in der Lage MHC-I Antigene (vor allem auf Immunzellen aber auch auf Thrombozyten 22 zu finden) auf ihrer Oberfläche zu präsentieren, um das Immunsystem zu alarmieren. Eine Markierung von MHC-1 im Blut und die Zählung der Zellen kann auf eine Immunothrombozytopenie hinwiesen. Dabei können große Zellen als Immunzellen und kleine Zellen als Thrombozyten 22 identifiziert werden. In the case of a viral disease platelets 22 are also able to include these on phagocytosis and neutrali ¬ Sieren. During this process, platelets 22 are also able to present MHC-I antigens (found mainly on immune cells but also on platelets 22) on their surface to alert the immune system. Labeling MHC-1 in the blood and counting the cells may indicate immunothrombocytopenia. Large cells can be identified as immune cells and small cells as platelets 22.
Ausführungsbeispiel 3: Markierung von körpereigenen Fresszel¬ len des Immunsystems innerhalb von Aggregaten mit großen Zel- len (zirkulierende Tumorzellen, eigene Immunzellen) Example 3: Marking of endogenous Fresszel ¬ len of the immune system within aggregates with large Zel- len (circulating tumor cells, own immune cells)
Körpereigene Fresszellen sind in der Lage, zirkulierende Tu¬ morzellen, die vom Immunsystem als Fremdkörper identifiziert wurden, durch Phagozytose (Verschlucken) und anschließendes Verdauen unschädlich zu machen. Während dieses Vorganges wird der Durchmesser einer Fresszelle einerseits signifikant grö¬ ßer, andererseits präsentieren diese Zellen während und nach Abschluss dieses Vorgangs auch spezifische Antigene (MHC-1) auf ihrer Oberfläche. Die magnetische Markierung dieser Anti¬ gene, die Ermittlung der Zellgröße und die anschließende Zäh¬ lung dieser Zellen gibt eine indirekte Auskunft über die nor¬ male oder erhöhte Anzahl an zirkulierenden Tumorzellen. The body's own scavenger cells are capable of circulating Tu ¬ morzellen that identifies the immune system as foreign bodies were rendered harmless by phagocytosis (ingestion) and subsequent digestion. During this operation the diameter of a phagocyte is the one hand, significantly RESIZE ¬ SSER, on the other hand, these cells also present specific antigens (MHC-1) on their surface during and after completion of this operation. The magnetic labeling of these anti ¬ genes, the determination of the cell size and the subsequent Zäh ¬ development of these cells is an indirect information about the male nor ¬ or increased number of circulating tumor cells.
Ausführungsbeispiel 4: Messung der Fibrinbildung anhand stei¬ gender Viskosität während der Blutgerinnung: Embodiment 4: Measurement of fibrin based stei ¬ gender viscosity during coagulation:
Während der Hämostase steigt die Viskosität des Blutes durch die Bildung von Fibrin aus Fibrinogen an. Wenn das Blut durch einen mikrofluidischen Kanal geleitet wird, bewegen sich die Teilchen frei von Reibung mit dem Flüssigkeitsstrom innerhalb dieses Kanals. Wenn Fibrin entsteht (der letzte Schritt während der Blutge¬ rinnung) , steigt die Viskosität des Blutes kontinuierlich an, bis es letztendlich zum Stillstand kommt. Wenn die Viskosität ansteigt und die Fließgeschwindigkeit des Blutes verlangsamt wird, wird auch die Geschwindigkeit der sich im Blut befind- liehen Teilchen zunehmend geringer. Die Verlangsamung derDuring hemostasis, the viscosity of the blood increases due to the formation of fibrin from fibrinogen. As the blood passes through a microfluidic channel, the particles move free of friction with the fluid flow within that channel. When fibrin is formed (the final step during the Bloody ¬ rinnung), the viscosity of the blood increases continuously until it finally comes to a halt. As the viscosity increases and the flow rate of the blood slows, the speed of blood-borne particles also decreases. The slowdown in the
Teilchen im gerinnenden Blut kann als Maß für seine zunehmende Viskosität herangezogen und in direkte Korrelation mit dem steigenden Anteil an unlöslichem Fibrin gestellt werden. Particles in the coagulating blood can be used as a measure of its increasing viscosity and placed in direct correlation with the increasing proportion of insoluble fibrin.
Folglich kann mittels einer Time-of-Flight-Messung auch die Änderung der Viskosität des sich im Kanal befindlichen Blutes gemessen werden. Consequently, by means of a time-of-flight measurement, the change in the viscosity of the blood in the channel can be measured.
Die Time-of-Flight-Messung verwendet dabei beispielsweise den Abstand zwischen den beiden Paaren 12, 13 und die von den Paaren erzeugte Signale bei Vorbeiziehen eines Analyten. The time-of-flight measurement uses, for example, the distance between the two pairs 12, 13 and the signals generated by the pairs when an analyte moves past.
Ausführungsbeispiel 5: Magnetische Beads können als interner Standard für die Fließgeschwindigkeit herangezogen werden Da die Fließgeschwindigkeit von Blut unterschiedlicher Spen¬ der aufgrund unterschiedlicher Ausgangsviskositäten variieren kann, sollte ein interner Standard in die Probe eingebracht werden, welcher erlaubt die Fließgeschwindigkeit am Anfang jeder Messung zu bestimmen. Ein derartiger Standard kann aus magnetischen Partikeln bestehen, die sich eindeutig vom Ana- lyten unterscheiden sollten (sehr viel kleiner oder sehr viel größer) , damit eine Verwechslung mit dem Analyten, also den eigentlichen Zellen oder Zellkonglomeraten, ausgeschlossen werden kann. Embodiment 5: Magnetic beads can be used as an internal standard for the flow rate Since the flow rate of blood Spen ¬ which may vary due to different viscosities of different output, an internal standard should be introduced into the sample, which allows the flow rate at the beginning to determine each measurement. Such a standard can consist of magnetic particles which should clearly differ from the analyte (much smaller or much larger) so that a confusion with the analyte, ie the actual cells or cell conglomerates, can be excluded.
Für die Ausführungsbeispiele 4 und 5 gilt, dass die anfängli¬ che Pumpleistung immer die gleiche ist. It applies to the embodiments 4 and 5 that the anfängli ¬ che pumping power is always the same.
In den Ausführungsbeispielen wurde von einer parallelen Anordnung der Sensorelemente (11) in einer Wheatstone-Brücke ausgegangen. Die einzelnen Sensorelemente 11 eines Paars 12, 13 liefern dabei zeitlich invertierte Signale, die bei einem Überlapp zu den eingangs erläuterten Signalfolgen in Abhängigkeit von den Analyten führt. In the exemplary embodiments, a parallel arrangement of the sensor elements (11) in a Wheatstone bridge was assumed. The individual sensor elements 11 of a pair 12, 13 provide time-inverted signals, which leads to an overlap with the signal sequences described above as a function of the analyte.
Bei Verwendung von Sensorelementen 11, die nicht zu einer Wheatstone-Brücke verschaltet sind oder bei Verwendung einer diagonalen Anordnung der Sensorelemente 11 in der Wheatstone- Brücke gemäß Figur 7 sind die Sensorsignale der Sensorelemen¬ te 11 nicht mehr zeitlich invertiert, sondern folgen ohne Invertierung aufeinander. Bei einer zeitlichen Überlappung der Signale ergeben sich dadurch ebenfalls charakteristische Sig- nalformen in Abhängigkeit von der Größe des jeweiligen Analy¬ ten im Vergleich zum Abstand der Sensorelemente 11 voneinander . With the use of sensor elements 11 which are not connected to form a Wheatstone bridge, or when using a diagonal arrangement of the sensor elements 11 in the Wheatstone bridge as shown in FIG 7, the sensor signals of the Sensorelemen ¬ te 11 are not inverted in time but follow without inverting successive , At a time overlap of the signals arise thereby also characteristic Sig- nalformen depending on the size of the respective Analy ¬ th compared with the spacing of the sensor elements 11 from one another.

Claims

Patentansprüche claims
1. Anordnung (10) zur Quantifizierung von Zellen unter Unterscheidung wenigstens zweier verschiedener Größen von Zell- Arten (21, 22) und/oder Zellkonglomerat-Arten (41, 51) in einer Zellsuspension mit einem magnetfeldsensitiven Sensor, der wenigstens ein erstes und zweites Paar (12, 13) von Sensor¬ elementen (11) aufweist, wobei 1. An arrangement (10) for quantifying cells by distinguishing between at least two different sizes of cell types (21, 22) and / or cell conglomerate types (41, 51) in a cell suspension with a magnetic field-sensitive sensor, comprising at least a first and second Pair (12, 13) of sensor ¬ elements (11), wherein
- die Sensorelemente (11) des ersten Paars (12) einen ersten Abstand (14) von zwischen der Hälfte und dem doppelten einer ersten mittleren Größe einer ersten zu vermessenden Art von Zelle (21, 22) oder Zellkonglomerat (41, 51) aufweisen, the sensor elements (11) of the first pair (12) have a first distance (14) of between half and twice a first average size of a first type of cell (21, 22) or cell conglomerate (41, 51) to be measured
- die Sensorelemente (11) des zweiten Paars (13) einen zwei¬ ten Abstand (16) von zwischen der Hälfte und dem doppelten einer zweiten mittleren Größe einer zweiten zu vermessenden- The sensor elements (11) of the second pair (13) a two ¬ th distance (16) of between half and twice a second average size of a second to be measured
Art von Zelle (21, 22) oder Zellkonglomerat (41, 51) auf¬ weisen, Type of cell (21, 22) or cell conglomeration (41, 51) have ¬,
- ein dritter Abstand (15) der einander nächstliegenden Sensorelemente (11) der Paare größer (12, 13) ist als die grö- ßere beider mittlerer Größen,  a third distance (15) of the closest sensor elements (11) of the pairs larger (12, 13) than the larger of the two middle sizes,
sowie mit as well as with
- einem Kanal (20) zur Führung der Zellsuspension an den Sensorelementen (11) vorbei.  - A channel (20) for guiding the cell suspension past the sensor elements (11).
2. Anordnung (10) gemäß Anspruch 1, bei dem der erste Abstand (14) zwischen 1 und 4 ym, der zweite Abstand (16) zwischen 20 und 30 ym und der dritte Abstand (15) wenigstens 30 ym be¬ trägt . 2. Arrangement (10) according to claim 1, wherein the first distance (14) between 1 and 4 ym, the second distance (16) between 20 and 30 ym and the third distance (15) at least 30 ym be ¬ contributes.
3. Anordnung (10) gemäß Anspruch 1 oder 2 mit einer Auswerteeinrichtung zur Auswertung eines ersten Signals des ersten und eines zweiten Signals des zweiten Paars, derart ausges¬ taltet, dass sowohl der zeitliche Abstand (tl, t2) des ersten und zweiten Signals als auch die Amplitude (24) der beiden Signale ausgewertet werden. 3. Arrangement (10) according to claim 1 or 2 with an evaluation device for evaluating a first signal of the first and a second signal of the second pair, such ausgest ¬ taltet that both the time interval (tl, t2) of the first and second signal as also the amplitude (24) of the two signals are evaluated.
4. Anordnung (10) gemäß Anspruch 3, ausgestaltet zur Erfas¬ sung und Unterscheidung von Thrombozyten (22) mit magneti- scher Markierung, Immunzellen (21) im Verbund mit solchen Thrombozyten (22) und Konglomeraten (41, 51) aus solchen Thrombozyten (22), wobei für das Überstreichen eines der Sensorpaare (12, 13) eine erste Signalamplitude sowie ein erster zeitlicher Abstand zwischen den Signalen gespeichert ist,4. The arrangement (10) according to claim 3, configured for Erfas ¬ measurement and discrimination of platelets (22) with magnetic scher mark, immune cells (21) in conjunction with such platelets (22) and conglomerates (41, 51) from such platelets (22), wherein for sweeping one of the sensor pairs (12, 13) has a first signal amplitude and a first time interval between the signals are stored,
- im Falle, dass die Amplitude des ersten und zweiten Signals größer als die erste Signalamplitude ist, ein Konglomerat (41, 51) aus den Thrombozyten (22) erfasst wird, in the case that the amplitude of the first and second signals is greater than the first signal amplitude, a conglomerate (41, 51) is detected from the platelets (22),
- im Falle, dass die Amplitude des ersten Signals größer als die erste Signalamplitude ist erste und zweite Signal eine ungleiche Amplitude aufweisen, eine Immunzelle (21) im Ver¬ bund mit den Thrombozyten (22) erfasst wird, - in the case that the amplitude of the first signal first and second signals is greater than the first signal amplitude have an unequal amplitude, an immune cell (21) in the Ver ¬ bund with the platelet (22) is detected,
- im Falle, dass der zeitliche Abstand größer als der erste zeitliche Abstand ist, eine einzelne Thrombozyte (22) er¬ fasst wird. - in the event that the time interval is greater than the first time interval, a single platelets (22) being ¬ sums.
5. Anordnung gemäß einem der vorangehenden Ansprüche, bei der die Sensorelemente (11) der Paare (12, 13) jeweils zu 5. Arrangement according to one of the preceding claims, wherein the sensor elements (11) of the pairs (12, 13) respectively
Wheatstone-Brücken verbunden sind. Wheatstone bridges are connected.
EP13726515.3A 2012-06-22 2013-06-03 Method and arrangement for detecting cells in a cell suspension Withdrawn EP2841940A1 (en)

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US6875621B2 (en) * 1999-10-13 2005-04-05 Nve Corporation Magnetizable bead detector
US6736978B1 (en) * 2000-12-13 2004-05-18 Iowa State University Research Foundation, Inc. Method and apparatus for magnetoresistive monitoring of analytes in flow streams
US8133439B2 (en) * 2006-08-01 2012-03-13 Magic Technologies, Inc. GMR biosensor with enhanced sensitivity
DE102007057667A1 (en) * 2007-11-30 2009-09-03 Siemens Ag Device for detecting particles in a fluid
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DE102009047801B4 (en) * 2009-09-30 2014-06-12 Siemens Aktiengesellschaft Flow chamber with cell guide
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