EP4326440A1 - Procédé de détection de cellules contenant un noyau dans un échantillon liquide d'un patient au moyen d'un dispositif microfluidique et dispositif microfluidique - Google Patents

Procédé de détection de cellules contenant un noyau dans un échantillon liquide d'un patient au moyen d'un dispositif microfluidique et dispositif microfluidique

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Publication number
EP4326440A1
EP4326440A1 EP22723643.7A EP22723643A EP4326440A1 EP 4326440 A1 EP4326440 A1 EP 4326440A1 EP 22723643 A EP22723643 A EP 22723643A EP 4326440 A1 EP4326440 A1 EP 4326440A1
Authority
EP
European Patent Office
Prior art keywords
microfluidic device
lysate
sample liquid
mixing
cell
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.)
Pending
Application number
EP22723643.7A
Other languages
German (de)
English (en)
Inventor
Samir KADIC
Tianxing Du
Franz Laermer
Jochen Hoffmann
Anne SEROUT
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.)
Robert Bosch GmbH
Original Assignee
Robert Bosch GmbH
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 Robert Bosch GmbH filed Critical Robert Bosch GmbH
Publication of EP4326440A1 publication Critical patent/EP4326440A1/fr
Pending legal-status Critical Current

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    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0642—Filling fluids into wells by specific techniques
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • 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
    • G01N15/06—Investigating concentration of particle suspensions
    • 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
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/075—Investigating concentration of particle suspensions by optical means
    • 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
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • 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
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N2015/0687—Investigating concentration of particle suspensions in solutions, e.g. non volatile residue

Definitions

  • the invention is based on a method for detecting nucleated cells in a sample liquid of a patient and a microfluidic device according to the species of the independent claims.
  • the subject matter of the present invention is also a computer program.
  • Circulating tumor cells have emerged as a promising and clinically relevant biomarker for the study of malignant tumors in recent years.
  • Known as liquid biopsy it is therefore one of the main research areas of modern oncology.
  • a time-resolved quantification of CTCs per normalized volume of blood from a cancer patient allows the course of the disease to be tracked precisely and in real time (real-time monitoring), to make therapy decisions that are tailored to the individual disease situation, or even to be able to make predictions about progression-free survival .
  • WO 2012/138882 A2 describes a microfluidic device with microcavities for detecting biological cells using magnetic elements. Disclosure of Invention
  • the approach presented here can advantageously be used to carry out an automated medical analysis of a patient sample, which can advantageously be connected to time-critical examinations.
  • the presented approach can therefore save time.
  • a method for detecting nucleated cells in a sample liquid of a patient using a microfluidic device comprising a providing step, a dispensing step and an identifying step.
  • a mixed signal is provided at an interface to a mixing device, the mixed signal causing the sample liquid to be mixed with a lysis buffer in a mixing chamber of the microfluidic device in order to obtain a lysate.
  • an application signal is output, which causes the lysate to be applied to a carrier substrate of the microfluidic device in order to obtain a cell sediment and a cell suspension of the lysate.
  • the nucleated cells are identified from the cell sediment.
  • nucleated cells can be understood to mean cells that have a cell nucleus.
  • these cells contain genetic information in the cell nucleus that allows reproduction of the cell from the genetic information in this cell nucleus allows.
  • Cells without a nucleus can be, for example, red blood cells (erythrocytes) or blood platelets (thrombocytes).
  • the sample liquid can be a blood sample from the patient, for example.
  • the microfluidic device can be formed, for example, as a lab-on-chip cartridge or at least include one.
  • the sample liquid can be pumped back and forth in or before the step of providing, for example in the mixing chamber, so that the concentration and distribution of the lysate is uniform.
  • the lysis buffer can be in the form of a chemical fluid, for example, which has properties such that erythrocytes, for example, can be separated from leukocytes and additionally or alternatively from tumor cells.
  • the lysis buffer can be in the form of an ammonium chloride lysis buffer (ACK lysis buffer), in order to produce a density difference in the cells, for example.
  • the lysate can be agitated within the mixing chamber for a predetermined period of time and in particular at least five minutes in order to achieve a homogeneous concentration of the components of the lysate.
  • the carrier substrate can be formed as a chip, for example.
  • the lysate is applied to the support substrate where it is allowed to rest for a predetermined period of time. Sedimentation can advantageously take place during this period of time, so that the cell sediment, ie cells that are still intact, are deposited on the carrier substrate and can finally be identified in the identification step.
  • the method may comprise a step of washing the lysate prior to the identifying step using a washing buffer to render the lysate optically transparent.
  • a washing buffer to render the lysate optically transparent.
  • the lysate can thereby be cleaned in such a way that it becomes clear and accordingly a more precise identification of the nucleated cells can take place.
  • sources of error can be reduced.
  • the washing buffer used for this can advantageously be in the form of a phosphate-buffered saline solution (PBS).
  • PBS phosphate-buffered saline solution
  • the lysate in the washing step, can be washed using the washing buffer which is isotonic and additionally or alternatively pH-neutral.
  • the washing buffer which is isotonic and additionally or alternatively pH-neutral.
  • the mixing signal can be provided in the providing step, which causes a mixing of a quantity of the lysis buffer that is dependent on a quantity of the sample liquid.
  • a corresponding mixing ratio of the sample liquid and the lysis buffer can advantageously be specified.
  • the quantities of the two liquids can advantageously be made available automatically.
  • the nucleated cells from the cell sediment can be optically detected and additionally or alternatively quantified.
  • an illness and additionally or alternatively a severity of the illness can advantageously be determined.
  • the mixed signal can be provided in the providing step in order to mix the sample liquid with the lysis buffer.
  • the lysis buffer can have a fluorescent dye for determining a cell type of the nucleated cells.
  • the dye can act on the nucleated cells of the lysate, so that the cell sediment differs in color from the cell suspension and it is therefore advantageously easier to see whether and in what quantity, for example, tumor cells are in the lysate.
  • the method may comprise a step of introducing the fluorescent dye into the lysis buffer prior to the providing step.
  • this can result in time savings.
  • the method can also include a step of introducing the sample liquid into the microfluidic device.
  • the sample liquid can be introduced manually by a user or alternatively by machine.
  • An embodiment of the approach presented here is also advantageous as a method for detecting nucleated cells in a sample liquid of a patient using a variant of a microfluidic device presented here, the method including a step of mixing the sample liquid with a lysis buffer in a mixing chamber of the microfluidic Device having to obtain a lysate.
  • the method also includes a step of applying the lysate to a carrier substrate of the microfluidic device in order to obtain a cell sediment and a cell suspension of the lysate.
  • the method includes a step of identifying the nucleated cells from the cell sediment.
  • the method comprises a step of washing the lysate before the identification step using a washing buffer in order to make the lysate optically transparent.
  • the wash buffer is, for example, isotonic and/or pH-neutral. This results in the advantages already mentioned above.
  • nucleated cells from the cell sediment are optically detected and/or quantified in the identification step. This results in the advantages already mentioned above.
  • the lysis buffer has a fluorescent stain for determining a cell type of the nucleated cells, in particular a step of supplying the fluorescent stain to the lysis buffer being provided before the mixing step.
  • the method includes a step of introducing the sample liquid (105) into the microfluidic device. This results in the advantages already mentioned above.
  • a microfluidic device for detecting nucleated cells in a sample liquid of a patient
  • the microfluidic device can be formed as a lab-on-chip cartridge.
  • the microfluidic device has a mixing chamber for receiving the sample liquid and a lysis buffer to obtain a lysate.
  • the microfluidic device has a carrier substrate for receiving the lysate, in order to obtain a cell sediment and a cell suspension of the lysate, and a detection chamber.
  • the carrier substrate is and/or can be arranged in the detection chamber.
  • the microfluidic device can be used in connection with rapid tests, since it enables a method for detecting nucleated cells in a patient sample in one of the variants mentioned above to be carried out in a timely advantageous manner.
  • the microfluidic device can advantageously be designed to analyze a blood sample, for example.
  • the carrier substrate can be shaped like a chip, for example.
  • the detection chamber can have a height that is less than a width and a length of the detection chamber.
  • the detection chamber can, for example, have a height of 320 micrometers and, for example, a square base area with, for example, edge lengths of 12.5 mm ⁇ 12.5 mm. Additionally or optionally, dimensions of the mixing chamber can be 13 mm x 13 mm x 10 mm.
  • the carrier substrate can have a plurality of microcavities.
  • the cell sediment can be deposited in the individual microcavities.
  • the individual microcavities can be arranged in a honeycomb pattern.
  • the microfluidic device can have a buffer storage chamber which is designed to store the lysis buffer and release it into the mixing chamber.
  • the buffer storage chamber can advantageously have a predetermined capacity, which can correspond to the required amount of lysis buffer.
  • an evaluation device for evaluating a cell sediment in a microfluidic device having a mixing device and an identification unit.
  • the mixing device is designed to mix the sample liquid with the lysis buffer in the mixing chamber in order to obtain a lysate.
  • the identification unit is designed to identify the nucleated cells from the cell sediment.
  • the evaluation device can have a control unit or a control device for activating and/or executing the steps of the method in one of the aforementioned variants.
  • the mixing device can have a pump unit or be designed as such, for example.
  • the sample liquid and the lysis buffer can advantageously be mixed to form a homogeneous lysate.
  • the identification unit can advantageously include a microscope unit and at least one light source or be formed as such.
  • the aforementioned method can be implemented, for example, in software or hardware or in a mixed form of software and hardware, for example in a control device or a control unit.
  • control device can have at least one computing unit for processing signals or data, at least one memory unit for storing signals or data, at least one interface to a sensor or an actuator for reading in sensor signals from the sensor or for outputting control signals to the actuator and/or or have at least one communication interface for reading in or outputting data that are embedded in a communication protocol.
  • the arithmetic unit can be, for example, a signal processor, a microcontroller or the like, with the memory unit being able to be a flash memory, an EEPROM or a magnetic memory unit.
  • the communication interface can be designed to read in or output data wirelessly and/or by wire, wherein a communication interface that can read in or output wire-bound data can, for example, read this data electrically or optically from a corresponding data transmission line or output it to a corresponding data transmission line.
  • a control device can be understood to mean an electrical device that processes sensor signals and outputs control and/or data signals as a function thereof.
  • the control unit can have an interface that can be designed in terms of hardware and/or software.
  • the interfaces can be part of what is known as a system ASIC, for example, which contains a wide variety of functions of the control device.
  • the interfaces can be separate integrated circuits or to consist at least partially of discrete components.
  • the interfaces can be software modules which are present, for example, on a microcontroller alongside other software modules.
  • a computer program product or computer program with program code which can be stored on a machine-readable carrier or storage medium such as a semiconductor memory, a hard disk memory or an optical memory and for carrying out, implementing and/or controlling the steps of the method according to one of the embodiments described above, is also advantageous is used, especially if that Program product or program running on a computer or device.
  • FIG. 1 shows a schematic representation of a microfluidic device according to an embodiment
  • Fig. 2 is a flow chart of a method according to a
  • Embodiment for detecting nucleated cells in a sample liquid of a patient Embodiment for detecting nucleated cells in a sample liquid of a patient
  • FIG. 3 shows a flow chart of a method according to an embodiment for detecting nucleated cells in a
  • FIG. 4 shows a schematic exemplary embodiment of a cross section of a detection chamber of a microfluidic device
  • FIG. 5 shows a schematic exemplary embodiment of a cross section of a detection chamber of a microfluidic device
  • FIG. 6 shows an evaluation device according to an embodiment and a microfluidic device
  • FIG. 7 shows a schematic exemplary embodiment of a cross section of a detection chamber of a microfluidic device
  • Fig. 8 shows a schematic embodiment of a cross section of a
  • FIG. 9 shows a schematic representation of a flow profile in a microcavity according to an embodiment.
  • the same or similar reference symbols are used for the elements which are shown in the various figures and have a similar effect, with a repeated description of these elements being dispensed with.
  • FIG. 1 shows a schematic representation of a microfluidic device 100 according to an embodiment.
  • the microfluidic device 100 is designed to identify nucleated cells in a sample liquid 105 of a patient.
  • the microfluidic device 100 is formed in particular as a lab-on-chip cartridge.
  • the microfluidic device 100 has a mixing chamber 110 for receiving the sample liquid 105 and a lysis buffer, which together form a lysate.
  • the elements shown in FIG. 1 essentially only provide an overview of components that can be implemented on the microfluidic device 100; a more precise depiction or description of the location or position of these elements or components cannot be inferred from FIG. 1 .
  • the microfluidic device 100 has a carrier substrate 115 for receiving the lysate in order to obtain a cell sediment and a cell suspension of the lysate, and a detection chamber 120 .
  • the carrier substrate 115 is and/or can be arranged in the detection chamber 120 .
  • the microfluidic device 100 is designed, for example, to shorten an analysis time of the sample liquid 105 since it can be used as a cartridge in connection with, for example, rapid tests.
  • the sample liquid 105 is implemented as blood from the patient, for example, which is examined for tumor cells (CTCs), for example.
  • CTCs tumor cells
  • the microfluidic device 100 has an inlet interface 125, which is designed to admit the sample liquid 105 into an interior of the device 100, where it ultimately flows by means of a method for detecting nucleated cells is examined, as explained in more detail in one of the following figures.
  • the sample liquid 105 is introduced into the microfluidic device 100, for example manually or alternatively automatically using a filling device 130, for example by means of a pipette.
  • the mixing chamber 110 is flatter than the detection chamber 120 according to this exemplary embodiment.
  • the carrier substrate 115 is arranged in the detection chamber 120 and is implemented or can be implemented, for example, as a removable or insertable microchip.
  • the carrier substrate 115 is formed as a permanently installed chip, for example.
  • the carrier substrate 115 only optionally has a plurality of microcavities 135 which are formed, for example, as indentations on a surface of the carrier substrate 115 .
  • the microcavities 135 are arranged, for example, in a honeycomb pattern on the carrier substrate 115 .
  • the microfluidic device 100 Only optionally does the microfluidic device 100 have a buffer storage chamber 140 which is designed to store the lysis buffer and deliver it into the mixing chamber 110 .
  • the approach presented here enables a fully automated and isolation-free quantification of circulating tumor cells from whole blood in a microfluidic environment. More specifically, a microfluidic counterpart to an isolation-free and previously only manual method is provided, which includes preparation of the blood sample up to CTC quantification.
  • the use is of particular interest for automated microfluidic systems such as the microfluidic device 100 described here, which offer analyzes at a so-called point-of-care (PoC), i.e. are subject in particular to time-critical boundary conditions and only provide limited space for reagents and sample material .
  • PoC point-of-care
  • sample liquid 105 The method described in the following figure and the microfluidic device 100 also generally allow detection of all nucleated cells from whole blood, which is referred to here as sample liquid 105 . This concerns in particular leukocytes, but also endothelial cells and/or stem cells, so that alternative blood analyzes can also be carried out.
  • the core elements of the presented approach include a chronological composition of individual steps and/or components.
  • a selective lysis of the sample liquid 105 is performed using, for example, an ACK lysis buffer to maximize the density differences between lysed erythrocytes and non-lysed, still intact nucleated cells.
  • the dyes required for later detection and still to be incubated are added to the lysis buffer at this point, so that an "AII-in-One-Buffer" is created.
  • the core information does not necessarily have to be obtained via bright-field transmitted-light microscopy, in particular not in the event that this possibility does not exist in the microfluidic device 100 or the associated optical path cannot be made transparent enough.
  • the cell nucleus could be fluorescently stained with a standard DNA dye in order to demonstrate “nucleus present or not?”.
  • the resulting and already partially stained lysate which is also referred to as blood lysate, is transferred to the relatively flat and large-area detection chamber 120, with the natural and homogeneously distributed sedimentation of all intact nucleated cells under lysed erythrocytes being distributed over the detection area a structure of microcavities 135 or microwells located at the bottom of the chamber 120 is awaited.
  • a negative selection is designed "quasi isolation-free" and thus in the most ideal form possible.
  • the sedimentation time is used as the remaining incubation time for the dyes used, for example in the form of parallelization.
  • CTC detection using fluorescence microscopy is possible, for example, after gently washing away the erythrocyte membrane, “EH” for short, and hemoglobin, “Hb” for short, which act as an optical barrier, and which are mostly not yet at the bottom of the chamber Sedimented nucleated cells remain in the microcavities 135 and/or are protected by them from being washed away. Is the path of light on the other hand, transparent enough, which means the microfluidic system is transparent, and if the system is still not inclined, both the chip with microcavities 135 and the washing process are optional. In this case, the cells can sediment directly onto a planar and transparent carrier substrate 115 .
  • All information, including the core information, can then be extracted by fluorescent staining and optical detection on the underside of the substrate, since the optical barrier is virtually non-existent in this case. If the optical barrier due to the lysate is thin overall and therefore transparent enough, the analysis described is carried out without a chip with microcavities 135 and without a washing process via fluorescence channels, for example via reflected light microscopy.
  • the approach presented reduces cell losses and/or cell damage, since CTC detection takes place (quasi) without isolation, which means that only the smallest sample volumes are processed and the CTC quantification can be standardized.
  • microfluidic integrability automation is possible, since the need for classic laboratory equipment that cannot or only with difficulty be combined with microfluidic environments, such as centrifuges, containers, vessels, etc., as well as manual processing steps with dead times between different process stations, is reduced. Furthermore, optionally, a volume of necessary reagents and sample liquid 105 is reduced, since effective washing and the subsequent quantification of stained cells, for example by a conventional reflected-light microscope or optical detection system after a reflected-light setup with a reduced working distance is possible. Microcavities 135 offer the advantage of being able to operate the microfluidic device 100 against an inclination.
  • FIG. 2 shows a flow chart of a method 200 according to an embodiment for detecting nucleated cells in a sample liquid of a patient.
  • the method 200 is controlled or performed in connection with a microfluidic device, for example, as was described in FIG. 1 .
  • the nucleated cells to be recognized are, for example, tumor cells, leukocytes, endothelial cells or stem cells.
  • the method 200 comprises a step 205 of providing, a step 210 of outputting and a step 215 of identifying.
  • a mixed signal is provided at an interface to a mixing device, the mixed signal causing the sample liquid to be mixed with a lysis buffer in the mixing chamber of the microfluidic device in order to obtain a lysate.
  • the sample liquid is mixed with the lysis buffer for a predetermined period of time, which is at least five minutes according to this exemplary embodiment.
  • an application signal is output which causes the lysate to be applied to the carrier substrate of the microfluidic device in order to obtain a cell sediment and a cell suspension of the lysate, for example after a minimum sedimentation time has elapsed.
  • the nucleated cells are identified from the cell sediment in order to recognize colored cells, for example.
  • the method 200 include a step 220 of introducing the sample liquid into the microfluidic device, as a result of which the method 200 is initiated, for example.
  • a quantity of sample liquid comprises, for example, 500 ⁇ l, of which, for example, 100 ml are mixed with the lysis buffer in step 205 of providing.
  • the lysis buffer includes a fluorescent stain for determining a cell type of the nucleated cells.
  • the fluorescent colorant is optionally already contained in the lysis buffer or is supplied to the lysis buffer in an optional step 225 of supply before step 205 of providing. This ensures that the nucleated cells absorb the dye and thus become optically recognizable.
  • the mixing signal is provided in step 205 of providing, which causes a mixing of a quantity of the lysis buffer that is dependent on a quantity of the sample liquid.
  • the quantities of the lysis buffer and the sample liquid ideally have a predetermined mixing ratio in order to obtain a meaningful result in step 215 of identification.
  • the method 200 includes a step 230 of washing the lysate before Step 215 of identifying using a wash buffer to make the lysate optically transparent.
  • the wash buffer is, for example, isotonic and/or pH-neutral. Consequently, it is easier to detect the nucleated cells in step 215 of identifying.
  • the nucleated cells are optically detected and/or quantified in step 215 of identification from the cell sediment, for example using a microscope device.
  • the sample liquid also referred to as a blood sample
  • the microfluidic device In other words, according to this exemplary embodiment, the sample liquid, also referred to as a blood sample, is placed in the microfluidic device. All subsequent steps continue to be fully automated on-chip. In particular, microfluidic unit operations such as sample transport, mixing, washing, etc. take place according to a programmed sequence and without human intervention.
  • step 205 of providing whole blood is mixed with the ACK lysis buffer in a suitable working ratio within the relatively high mixing chamber and continuously mixed for a necessary lysis time for a homogeneous concentration.
  • the fluorescence dyes required for optical detection or classification of CTCs from leukocytes are already added to the lysis buffer in the respective working concentration. Since it is a closed microfluidic system, the dyes are incubated in parallel, or in step 225 of the feeding. No additional separate waiting time is therefore required for the incubation.
  • Hb hemoglobin
  • the membrane becomes impermeable to further flow of Hb and other proteins of comparable size.
  • the resulting lysate is pumped into the flat, large-area detection chamber in step 210 of dispensing.
  • the height of the chamber is selected in such a way that the sedimentation time of the smallest intact cells containing a nucleus is limited to a predetermined maximum value, which is also referred to as PoC suitability.
  • erythrocytes through the selective lysis have experienced a diffusive exchange of media between the cell interior and the cell exterior, a net density difference between them and the surrounding medium is effectively only composed of the cell membrane and is thus virtually negligible.
  • lysed cells have a sedimentation rate of almost zero and "swim around in the medium".
  • unlysed cells ie in particular cells containing a nucleus, which still have an intact membrane function, retain a relatively large difference in density to the surrounding medium and sediment at high speeds compared to erythrocyte membranes.
  • the carrier substrate which is structured with microcavities, is optionally located at the bottom of the chamber.
  • the effective detection area of the chip as well as the dimensions and thus also the total number of cavities on the carrier substrate within this effective area are selected in such a way that the cells on the floor can easily be isolated in a monolayer as soon as the intact nucleated cells have settled into the cavities are and form the cell sediment. Loading can be assisted by gently "pumping" the cell suspension back and forth, which also brings cells into the cavities that were previously deposited on ridges, for example. Ideally, all CTCs within the wells are easily identified and distinguished from leukocytes in step 215 of identifying. Furthermore, a scanning time for a microscope, for example, is limited to a predetermined maximum value by the effective detection area of the chip.
  • a gentle washing process with washing buffer is also carried out.
  • the washing procedure should be designed in such a way that captured cells are not swirled out of the microwells. It is important to ensure that a flow velocity is selected to be small enough. Nevertheless, the washing process takes place quickly enough to keep the overall process time short. Furthermore, sufficient optical transparency is achieved to observe the fluorescently stained cells within the microcavities, which are also referred to as wells, and to reliably distinguish them from the background. This achieves, for example, a negative selection that is as robust as possible and a reduction in interfacial scattering due to the homogeneous distribution of the Hb. Consequently a reduction in intensity when the light passes through the optical barrier due to scattering effects is also drastically reduced and any remaining traces of the lysate due to a non-ideal washing process are acceptable.
  • FIG. 3 shows a flow chart of a method 200 according to an embodiment for detecting nucleated cells in a sample liquid of a patient.
  • the method 200 shown here corresponds to the method 200 described in FIG. 2. Only the method 200 according to this exemplary embodiment is shown in a time profile. This means that step 205 of providing according to this exemplary embodiment is carried out after step 220 of introducing the sample liquid, which is also referred to as preparation. According to this exemplary embodiment, a duration of the method 200 is measured from the step 205 of providing. According to the flowchart shown here, step 210 of outputting, which can also be referred to as “chip loading”, takes place after the predetermined period of time has elapsed, which according to this exemplary embodiment is five minutes.
  • the washing step 230 is carried out after a further 10 minutes, which are referred to as the minimum sedimentation time.
  • step 215 of identifying is carried out after at least 20 minutes have elapsed after the start of the time measurement.
  • an initially "generous" blood volume of, for example, > 500 ml is entered into the microfluidic system. For example, this is the only manually executable processing step in the entire method 200, together with the taking of blood Dimensions ⁇ 13mm x ⁇ 13mm x ⁇ 10mm converted. In this case, for example, the minimum volume of ACK buffer required for a selective lysis is 400 ⁇ l.
  • the "AII-in-One-Buffer" contains all colors necessary for fluorescent detection in the respective working concentrations for tumor cell specificity, propidium iodide (PI) for live-dead staining and/or a stain useful for cell nucleus detection.
  • the cell suspension In order to counteract sedimentation and thus uneven cell concentrations, the cell suspension is continuously mixed until lysis is complete, with a minimum lysis time of approx. 5 minutes.
  • the necessary hydrodynamic pressure is to be designed in such a way that no damage to the cells can be induced, for example by generating a reduced shearing force.
  • the flat detection chamber with the same capacity.
  • the volume is selected in such a way that after the sedimentation of all intact nucleated cells, 10 ml of blood can be analyzed on the chamber floor for a final quantification.
  • the dimensions of such an exemplary chamber are 12.5 mm ⁇ 12.5 mm for an effective detection area of the carrier substrate, so that the detection area is approximately 156 mm 2 .
  • a chamber height is, for example, 320 ⁇ m.
  • step 210 of outputting can be carried out in ⁇ 10 minutes. If the diameter of the largest expected CTCs from solid tumors in an analysis is 30 pm, then, for example, cavities with a diameter of 40 pm and a depth of 28 pm are advantageous.
  • Cavities are to be arranged, for example, in a classic matrix or hexagonally packed as circles, hexagons or squares. In all configurations, it is important to maximize the base area of the cavities relative to the total area of the chip for the most efficient possible loading of the carrier substrate, i.e. the webs, which are also referred to as partitions, between adjacent cavities to be selected as small as possible, preferably ⁇ 10 ⁇ m, such as 3 pm.
  • a structured chip with microcavities includes, for example, silicon that has been etched at the appropriate locations.
  • a photosensitive lacquer with the appropriate thickness can also be used, which is either laminated to a suitable substrate as a finished dry resist or spun onto it and cured as a lacquer and, in a final processing step, photolithographically using a photomask and UV light opened in the desired places.
  • the cavities can also be formed from a polymer film, which has been applied to a substrate in the appropriate thickness, by means of a laser, for example by means of a USP laser.
  • a carrier substrate is obtained with a total of approx. 97600 cavities.
  • 10 pl of blood contains between 40,000 and 110,000 leukocytes, which corresponds to a target blood equivalent in the final optical detection in step 215 of identification.
  • an average of between 0.41 and 1.13 cells can be expected per tray, which corresponds to relatively good isolation. Ideally, one cell per well can be found.
  • the washing buffer used is clear, which means it is optically transparent, isotonic and pH-neutral to ensure biological compatibility with the cells.
  • a phosphate-buffered saline solution PBS
  • PBS phosphate-buffered saline solution
  • the 50 pl chamber volume is to be exchanged 3 times within 5 minutes to ensure sufficient optical transparency, i.e. rinsed out with the washing buffer, this is the result a volume flow of 0.5 ml/s, for example, to be set constant on average.
  • a volume flow of 0.5 ml/s for example, to be set constant on average.
  • the previously considered tumor cell is not whirled out of a cavity with a width of 40 ⁇ m, a depth of 28 ⁇ m and ridges that are 3 ⁇ m wide once it has completely settled to the bottom. Such a washing process is therefore practicable.
  • the quantification ie the step 215 of identification, is carried out using a reflected-light fluorescence microscope, for example.
  • the cells relevant for the statistics are living tumor cells with a nucleus.
  • the step 215 of identification is started, for example, after about 20 minutes, which corresponds to a time saving by a factor of ⁇ 3 compared to manual processing.
  • a selective erythrocyte lysis (RBC) is conceivable, which hydrodynamically and biologically considered converts RBCs into a form that is favorable for the detection process.
  • RBC erythrocyte lysis
  • the rigidity and effective cell diameter of RBCs are significantly reduced and lysed RBCs can no longer sustain the expression of antigens, thereby avoiding clumping.
  • the cell property which is used as a distinguishing criterion for the elimination of RBCs from leukocytes (WBC) and CTCs is the cell density.
  • Intact RBCs have a mean density of approximately 1110 kg/m 3 , slightly heavier than nucleated cells (WBCs and CTCs) with densities between 1070 kg/m 3 and 1090 kg/m 3 .
  • WBCs and CTCs nucleated cells
  • both cell types i.e. anucleate RBCs and nucleated WBCs and CTCs, have a significant density difference to the surrounding medium, which is similar to water.
  • the medium usually has ⁇ 1000 kg/m 3 .
  • both cell types sediment to the ground at about the same rate and a specificity that can be used for spatial separation does not yet exist.
  • the RBCs once the RBCs are lysed, they only have about 1 to 3% of the density of an intact erythrocyte, which is why they assume a density difference of almost zero after diffusive media equalization between the cell interior and cell exterior during lysis in suspension. The actually remaining difference in density is effectively only made up by the thin cell membrane. Therefore, while nucleated cells remain unaffected by lysis and maintain their density difference to the medium (sedimentation rate constant), RBCs have a near-zero sedimentation rate and float in suspension. WBCs and CTCs sediment through these floating RBC membranes to the bottom.
  • FIG. 4 shows a schematic embodiment of a cross section of a detection chamber 120 of a microfluidic device.
  • a carrier substrate 115 and a lysate 400 are arranged in the detection chamber 120, as was described in FIG. 1, for example.
  • the lysate 400 has a fluorescent dye for recognizing and/or identifying nucleated cells 402 .
  • a cell type of the nucleated cells 402 can be identified, e.g. tumor cells 403, since they absorb the color contained in the stain better than other nucleated cells 402, e.g. leukocytes 404.
  • the detection chamber 120 has a height 405 of 320 ⁇ m, for example, in which the lysate 400 is arranged. According to this exemplary embodiment, a state is thus shown which is achieved by the outputting step, as was described, for example, in one of FIGS.
  • the carrier substrate 115 has the plurality of microcavities 135 which, according to this exemplary embodiment, are arranged in a uniformly distributed manner on the carrier substrate 115 .
  • the microcavities 135 are thereby separated from adjacent microcavities 135 by partition walls 410 .
  • the microcavities 135 are formed as part of the carrier substrate 115 . They have a depth 415 of 28 ⁇ m, for example, and are designed to receive the nucleated cells 402 as cell sediment after a sedimentation time has elapsed, as is shown in the following figure.
  • FIG. 5 shows a schematic embodiment of a cross section of a detection chamber 120 of a microfluidic device.
  • the carrier substrate 115 shown here corresponds, for example, to the carrier substrate 115 described in FIG. 4.
  • FIG. 5 shows a state inside the detection chamber 120 that is reached according to this exemplary embodiment in the dispensing step after the minimum sedimentation time.
  • FIG. 6 shows an evaluation device 600 according to an embodiment and a microfluidic device 100.
  • the evaluation device 600 is designed to evaluate a cell sediment in the microfluidic device 100.
  • FIG. The microfluidic device 100 shown here corresponds, for example, to the microfluidic device 100 described in Fig. 1.
  • the evaluation device 600 has a mixing device 605 for mixing the sample liquid with the lysis buffer in the mixing chamber, as described for example in FIG. 1, in order to obtain a lysate. Furthermore, the evaluation device 600 has an identification unit 610 which is designed to identify the nucleated cells from the cell sediment. Furthermore, the evaluation device 600 according to this exemplary embodiment has a control unit 615, which is designed to control and/or execute the steps of a method for detecting nucleated cells in a sample liquid of a patient, as described in one of FIGS.
  • the mixing device 605 is in the form of a pump device, for example, which is designed to pump the sample liquid with it to mix with the lysis buffer.
  • the identification unit 610 is designed, for example, as a microscopy unit, which includes a light source, for example. This advantageously quantifies the lysate after it has been placed on the carrier substrate 115 .
  • the carrier substrate 115 is quadrangular, more precisely square, so that a width 620 and a length 625 of the carrier substrate 115 have the same dimensions.
  • a depth 630 of the carrier substrate 115 is smaller than the length 625 and/or the width 620.
  • the carrier substrate 115 is shown enlarged in an enlarged section 635, so that the plurality of microcavities 135 is illustrated.
  • the control unit 615 is designed to provide a mixed signal 640 to an interface to the mixing device 605 .
  • the mixing signal 640 causes the sample liquid to be mixed with the lysis buffer in the mixing chamber of the microfluidic device 100 in order to obtain the lysate.
  • the control unit 615 is designed to output an application signal 645, for example to an interface to an application unit 650 external to the device, in order to apply the lysate to the carrier substrate 115 of the microfluidic device 100 in order to bring about the cell sediment and a cell suspension of the lysate to obtain, for example, after a minimum sedimentation time.
  • the control unit 615 is designed to identify the nucleated cells from the cell sediment, for example using an identification signal 655 and the identification unit 610 .
  • FIG. 7 shows a schematic embodiment of a cross section of a detection chamber 120 of a microfluidic device.
  • the detection chamber 120 shown here corresponds, for example, to the detection chamber 120 described in one of FIGS. 1, 4 or 5.
  • a snapshot during the optional step of washing is shown.
  • a washing buffer 700 is introduced into the detection chamber 120 which has a flow 705 for washing the cell suspension 500 .
  • the microcavities 135 and their partition walls 410 prevent the cell sediment 505 from being washed away.
  • FIG. 8 shows a schematic embodiment of a cross section of a detection chamber 120 of a microfluidic device.
  • the detection chamber 120 shown here corresponds, for example, to the detection chamber 120 described in FIG. 7.
  • a snapshot of the identification step is shown.
  • the washing phase of the lysate 400 shown in FIG. 7 has been completed.
  • the light source 800 shown here represents the identification device described in FIG. 6 , which is designed to identify and/or quantify the cell sediment 505 .
  • a further enlarged view 805 of the plurality of microcavities 135 is also shown.
  • the microcavities 135 are shown from above.
  • the microcavities 135 are also round in shape according to this exemplary embodiment and have a spacing 810 from one another that is smaller than a diameter 815 of a microcavity 135.
  • the spacing 810 corresponds to a thickness of at least one of the partition walls 410.
  • the nucleated cells 402 present in the cell sediment 505 are only optionally colored differently, whereby the cell type is recognizable.
  • FIG. 9 shows a schematic representation of a flow pattern 900 of a liquid for a microfluidic device according to an embodiment.
  • the flow pattern 900 occurs, for example, in a microfluidic device that has a plurality of microcavities, as was described in one of FIGS. 1 or 4 to 8.
  • a cavity section 905 and a chamber section 910 of the detection chamber are shown.
  • the liquid flows linearly in the chamber section 910 while it is swirled in the cavity section 905 .
  • the cavity section 905 represents a flow behavior within a microcavity that is surrounded by partition walls. This prevents the cell sediment from being entrained and/or damaged by the flow.
  • an embodiment includes an "and/or" link between a first feature and a second feature, this should be read in such a way that the embodiment according to one embodiment includes both the first feature and the second feature and according to a further embodiment either only that having the first feature or only the second feature.

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Abstract

L'invention concerne un procédé de détection de cellules contenant un noyau dans un échantillon liquide (105) d'un patient au moyen d'un dispositif microfluidique (100), le procédé comprenant une étape de fourniture, une étape de délivrance et une étape d'identification. L'étape de fourniture implique la fourniture d'un signal de mélange à un moyen de mélange, le signal de mélange provoquant le mélange du liquide échantillon (105) avec un tampon de lyse dans une chambre de mélange (110) du dispositif microfluidique (100) pour obtenir un lysat. L'étape de sortie implique l'émission d'un signal d'application qui effectue l'application du lysat sur un substrat de support (115) du dispositif microfluidique (100) pour obtenir un sédiment cellulaire et une suspension cellulaire du lysat. L'étape d'identification implique l'identification des cellules contenant un noyau à partir du sédiment cellulaire.
EP22723643.7A 2021-04-20 2022-04-20 Procédé de détection de cellules contenant un noyau dans un échantillon liquide d'un patient au moyen d'un dispositif microfluidique et dispositif microfluidique Pending EP4326440A1 (fr)

Applications Claiming Priority (2)

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DE102021203897.2A DE102021203897A1 (de) 2021-04-20 2021-04-20 Verfahren zum Erkennen von kernhaltigen Zellen in einer Probenflüssigkeit eines Patienten unter Verwendung einer mikrofluidischen Vorrichtung und mikrofluidische Vorrichtung
PCT/EP2022/060378 WO2022223593A1 (fr) 2021-04-20 2022-04-20 Procédé de détection de cellules contenant un noyau dans un échantillon liquide d'un patient au moyen d'un dispositif microfluidique et dispositif microfluidique

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WO (1) WO2022223593A1 (fr)

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IT1391619B1 (it) 2008-11-04 2012-01-11 Silicon Biosystems Spa Metodo per l'individuazione, selezione e analisi di cellule tumorali
EP3193170B1 (fr) 2011-04-05 2020-09-16 Purdue Research Foundation Système micro-fluidique utilisant des micro-ouvertures pour une détection de haut débit de unitees
CN106660058B (zh) * 2014-05-16 2019-09-17 克维拉公司 用于执行自动化离心分离的设备、系统和方法
AU2018323449B2 (en) * 2017-08-29 2020-09-03 Bio-Rad Laboratories, Inc. System and method for isolating and analyzing cells
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