PROCESS FOR CARRYING OUT AN ALLERGY TEST, PROCESS FOR DETERMINING DEGRANULATION IN CELLS, DEVICE FOR CARRYING OUT AN ALLERGY TEST AND MICROFLUIDIC CHIP

Abstract

A process for carrying out an allergy test is based on a blood sample being taken. The blood sample is contacted in vitro with at least one allergen. At least one allergic reaction or the absence of the at least one allergic reaction is observed via a microscope (11) directly and/or optically. To make it possible to carry out an allergy test with a higher validity and/or better accuracy, a position of granules is observed via the microscope (11). The granules are observed in different planes or horizontal planes.

Claims

1. A process for carrying out an allergy test, the process comprising: taking a blood sample; contacting the blood sample with at least one allergen in vitro; and observing in vitro at least one allergic reaction or an absence of the at least one allergic reaction by means of a microscope directly and/or optically comprising observing a position of granules is by means of the microscope, wherein the granules are observed in different planes or horizontal planes.

2. A process in accordance with claim 1, wherein degranulation of immunocytes, granulocytes, basophilic cells, basophilic granulocytes and/or mast cells, of the blood sample is observed with the microscope in case of an allergic reaction, and an absence of degranulation is classified as a non-allergic reaction and a presence of degranulation is classified as an allergic reaction on the basis of the direct and/or optical observation of the immunocytes on contacting with the at least one allergen.

3. A process in accordance with claim 1, wherein after taking the blood sample and before contacting the blood sample with the at least one allergen, the blood sample is processed to increase a percentage of immunocytes, wherein the percentage of immunocytes in the blood sample is increased to more than 5%, or higher, and a percentage of red blood cells and/or of white blood cells different from immunocytes, granulocytes, basophilic cells, basophilic granulocytes and/or mast cells is reduced.

4. A process in accordance with claim 1, wherein a microfluidic chip is used for contacting the blood sample with the at least one allergen, wherein immunocytes of the blood sample are arranged in at least one reaction chamber of the microfluidic chip, and the at least one reaction chamber and/or at least one reference chamber of the microfluidic chip has an at least partially transparent configuration.

5. A process in accordance with claim 4, wherein at least one allergen is guided into the at least one reaction chamber, and the at least one allergen is especially guided both into the at least one reaction chamber containing the immunocytes and into a reference chamber without immunocytes, and a respective reference chamber is associated with each reaction chamber.

6. A process in accordance with claim 4, wherein immunocytes of the blood sample are arranged on or in the microfluidic chip by means of a microfluidic cell trap, electrophoresis and/or dielectrophoresis, immunocytes are positioned and/or held by means of electrophoresis and/or dielectrophoresis in at least one reaction chamber, and at least one reference chamber is provided as a chamber free from immunocytes by means of electrophoresis and/or dielectrophoresis.

7. A process in accordance with claim 1, wherein the position and/or a change in the position of granules of immunocytes are observed directly and/or optically by means of the microscope, and an especially digital, holographic microscopy, shearography and/or a quantitative phase contrast microscopy are preferably carried out with the microscope.

8. A process in accordance with claim 1, wherein live immunocytes of the blood sample are identified by means of the microscope, the identification of live immunocytes being carried out in an automated manner, and only immunocytes identified as live immunocytes are taken into account during the observation and/or the analysis of a reaction of the immunocytes on contacting the immunocytes with the at least one allergen.

9. A process in accordance with claim 1, wherein a position of granules in immunocytes, a motion of the granules and/or degranulation are observed over an observation time of up to 10 minutes or over an observation time of 60 sec to 300 sec, the observation time being started with the contacting of the blood sample with the at least one allergen, and a plurality of reaction chambers of a microfluidic chip are observed several times consecutively at predefined time intervals during the observation time.

10. A process in accordance with claim 1, wherein the observation and/or analysis are carried out in an automated manner, comprising using a digital image recording to observe a reaction of the blood sample upon contacting the blood sample with the at least one allergen and/or using an image processing software to provide and/or analyze recorded images.

11. A process for determining degranulation in cells, especially with a process in accordance with claim 1, in which a blood sample is taken, and in which the blood sample is contacted in vitro with at least one reaction partner, wherein degranulation or the absence of degranulation is observed directly and/or optically by means of a microscope, the determination of the degranulation being carried out especially on the basis of an observation and/or measurement of a quantitative phase contrast.

12. A device for carrying out an allergy test with a process comprising taking a blood sample, contacting the blood sample with at least one allergen in vitro, and observing in vitro at least one allergic reaction or an absence of the at least one allergic reaction directly and/or optically comprising observing a position of granules, wherein the granules are observed in different planes or horizontal planes, the device comprising: a microscope; and an at least partially transparent microfluidic chip for the direct and/or optical observation of an allergic reaction or for observing the absence of an allergic reaction, wherein the microscope is configured to generate a holographic image.

13. A device in accordance with claim 12, wherein the microfluidic chip comprises at least one reaction chamber, and the microfluidic chip comprises transparent window surfaces in an area of the at least one reaction chamber on two sides facing away from one another, and a reference chamber, with transparent window surfaces associated with the at least one reaction chamber on two sides facing away from one another.

14. A device in accordance with claim 13, wherein the at least one reaction chamber and/or a reference chamber have a base or a respective transparent window surface of 100 m100 m on the two sides facing away from one another, and the at least one reaction chamber and/or the reference chamber have a height of less than 1 mm.

15. A device in accordance with claim 12, further comprising a computer, wherein the microscope comprises a digital, holographic microscope, a shearography microscope and/or a phase contrast microscope, the microfluidic chip is arranged between a light source and an objective lens device, and the microscope is connected to the computer for viewing, recording and/or analyzing images and/or data.

16. A microfluidic chip comprising: at least one reaction chamber; a reference chamber; electrodes for an electrophoretic and/or dielectrophoretic positioning of immunocytes in the at least one reaction chamber, wherein the reference chamber is formed adjacent to the reaction chamber based on a shape and/or orientation of at least one of the electrodes, the at least one of the electrodes comprising a strand electrode having a deflection and/or an arch to form the reference chamber.

17. A process in accordance with claim 1, further comprising providing a device comprising a microscope and an at least partially transparent microfluidic chip for the direct and/or optical observation of an allergic reaction or for observing the absence of an allergic reaction, wherein the microscope is configured to generate a holographic image, wherein the microfluidic chip is used for the step of contacting the blood sample and the microscope is used for the step of observing.

18. A process in accordance with claim 17, wherein the microfluidic chip comprises at least one reaction chamber and the microfluidic chip comprises transparent window surfaces in an area of the at least one reaction chamber on two sides facing away from one another, and a reference chamber, with transparent window surfaces associated with the at least one reaction chamber on two sides facing away from one another.

19. A process in accordance with claim 18, wherein the at least one reaction chamber and/or a reference chamber have a base or a respective transparent window surface of 100 m100 m on the two sides facing away from one another, and the at least one reaction chamber and/or the reference chamber have a height of less than 1 mm.

20. A process in accordance with claim 19, wherein: the microscope comprises a digital, holographic microscope, a shearography microscope and/or a phase contrast microscope; the microfluidic chip is arranged between a light source and an objective lens device; and the microscope is connected to a computer for viewing, recording and/or analyzing images and/or data.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In the drawings:

[0032] FIG. 1 is a schematic lateral view of a device according to the present invention;

[0033] FIG. 2 is a schematic detail top view of a microfluidic chip according to the present invention; and

[0034] FIG. 3 is a schematic flow chart for a process according to the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0035] Referring to the drawings, FIG. 1 shows a schematic lateral view of a device 10 according to the present invention. The device 10 has a microscope 11. The microscope 11 is configured in this exemplary embodiment as a digital holographic microscope. The microscope 11 may be configured, in particular, to generate a holographic image. The microscope 11 has, furthermore, an objective lens device 12 and a sensor device 13 in this exemplary embodiment. The sensor device 13 may be configured as a detector and/or as an image sensor, especially a CCD sensor.

[0036] The microscope 11 is connected to a computer 14. As a result, data of the microscope 11 or of the sensor device 13 can be transmitted to the computer 14 by means of a data line 15. Furthermore, the microscope 11 can be controlled by means of the computer 14.

[0037] The device 10 has a light source 16. The light source 16 is configured as an LED in this exemplary embodiment. As an alternative, the light source 16 may be a laser. The light source 16 is arranged in this schematic view such that an optical axis 17 is oriented in the direction of the microscope 11. The optical axis 17 is shown here as a broken line. Furthermore, a spatial modulator, especially a so-called SLM (Spatial Light Modulator), not shown more specifically here, may be present for the light of the light source 16.

[0038] The device 10 has a lens device 18. The lens device is configured as a lens-filter device in this exemplary embodiment. The lens device 18 is configured to focus and/or filter a light beam 19. The light beam 19 is indicated here schematically by means of dotted lines. The lens device 18 may have one or more lenses. Furthermore, the lens device 18 is arranged between the light source 16 and the microscope 11 on the optical axis 17. The lens device 18 can be controlled in this exemplary embodiment to set or change the focus. The lens device 18 may be controlled, for example, by means of the computer 14.

[0039] Finally, the device 10 has a microfluidic chip 20. The microfluidic chip 20 may be positioned and/or held by means of a carrying device, which is not shown in more detail here. The microfluidic chip 20 is arranged between the light source 16 and the microscope 11. The microfluidic chip 20 is positioned here on the optical axis 17 between the lens device 18 and the objective lens device 12. The microfluidic chip 20 has an at least partially transparent configuration. As a result, the light beam 19 can be guided, starting from the light source 16, through the microfluidic chip 20 to the microscope 11. As an alternative, the microfluidic chip 20 may be configured such that it is transparent on one side only, in which case the irradiation and the observation or measurement are performed from the same side.

[0040] The microfluidic chip 20 has at least one reaction chamber 21. The microfluidic chip 20 has transparent window surfaces 22, 23 at least in the area of the at least one reaction chamber 21. The window surfaces 22, 23 are arranged on two sides of the microfluidic chip 20, which face away from one another. The plane of the microfluidic chip 20 or of the window surfaces 22, 23 is oriented obliquely and in this exemplary embodiment essentially at right angles to the optical axis 17.

[0041] A focus 24 of the light beam 19 is positioned within the reaction chamber 21. The position of the focus 24 within the at least one reaction chamber 21 can be changed by means of a suitable control, especially the computer 14. For example, the focus 24 can be shifted essentially in the longitudinal direction of the optical axis 17. As a result, different planes or horizontal planes can be observed within the at least one reaction chamber 21.

[0042] FIG. 2 shows a schematic detail of a microfluidic chip 20 according to the present invention. The schematic detail is shown here as a top view. The microfluidic chip 20 has a plurality of reaction chambers 21, but only a single reaction chamber 21 is shown here. The microfluidic chip 20 has a plurality of separating elements 25. The position and/or size of the at least one reaction chamber 21 can be defined by means of the separating elements 25. The separating elements 25 are used, in particular, to separate a plurality of reaction chambers 21. The separating elements 25 are configured as partitions in this exemplary embodiment.

[0043] The reaction chamber 21 has an access opening 26. Cells or immunocytes of a blood sample, which are not shown here in more detail, can enter the reaction chamber 21 by means of the access opening 26. The term blood sample may be used for a direct blood sample or for a serum from a direct blood sample, which serum was mixed or brought into contact with cells of special cell lines.

[0044] Furthermore, the microfluidic chip 20 has at least one microchannel 27. Each reaction chamber 21 is connected, in particular, with at least one microchannel 27. At least one allergen, not shown here in more detail, can be guided by means of the microchannel 27 into the reaction chamber 21. The access openings 26 and the microchannel 27 are arranged in this exemplary embodiment on sides of the reaction chamber 21 that face away from one another. Furthermore, both the access opening 26 and the microchannel 27 are configured in this exemplary embodiment by means of two separating elements 25 arranged parallel to one another and mirror-symmetrically to one another.

[0045] The microfluidic chip 20 has a dielectrophoretic positioning device 28. The dielectrophoretic positioning device 28 has a plurality of electrodes 29, 30, 31. The electrodes 29, 30, 31 have an essentially strand-like configuration. Furthermore, the electrodes 29, 30, 31 are oriented essentially parallel to one another. The dielectrophoretic positioning device 28 or the electrodes 29, 30, 31 are arranged or configured such that cells or immunocytes can be positioned dielectrophoretically in the at least one reaction chamber 21. The electrode 31 located closest to the reaction chamber 21 has a deflection 32 in the area of the reaction chamber 21 or of the access opening 26. The deflection 32 is formed in the direction of the reaction chamber 21 or of the access opening 26. The deflection 32 is embodied in this exemplary embodiment as a type of bulge of the electrode 31. As an alternative, the deflection 32 may have an essentially C-, U- or V-shaped configuration. The deflection 32 partially protrudes in this exemplary embodiment into the area of the access opening 26. In case of a plurality of reaction chambers 21 arranged next to one another, the electrode arranged closest to the reaction chambers 21 may have a meandering configuration. An arch is formed in this case in the area of the reaction chambers 21 in the direction of the reaction chamber 21 and a respective arch pointing away from the separating elements 25 is formed in areas of the separating elements 25.

[0046] Due to the deflection 32, a reference chamber 33 is formed between the electrode 31 having the deflection 32 and the electrode 30 located closest hereto. Due to the dielectrophoretic action of the positioning device 28, it can be achieved that no cells or immunocytes can be positioned within the reference chamber 33 and they can only be positioned in the reaction chamber 21. At the same time, at least one allergen can be guided by means of the microchannel 27 both into the reaction chamber 21 and the reference chamber 33. As an alternative, the at least one allergen can only be guided into the reaction chamber 21 and not into the reference chamber 33.

[0047] The reaction chamber 21 and the reference chamber 33 can be observed directly and/or optically by means of window surfaces 22, 23, as is shown in FIG. 1. The reference chamber 33 makes possible the observation of superimposed light, which passes through the reaction chamber 21 and through the reference chamber 33. A first part of the superimposed light can pass through the reaction chamber 21 and another part of the superimposed light can pass through the reference chamber 33. A quantitative phase contrast microscopy is made possible hereby.

[0048] FIG. 3 shows a schematic flow chart for a process according to the present invention. The process will be explained below in more detail taking into account the device 10 and the microfluidic chip 20 according to FIGS. 1 and 2.

[0049] After a start of the process according to step S10, a blood sample is taken with step S11. For example, a blood sample may be taken from a person or a patient in the usual manner by means of a needle or syringe. However, relatively small quantities of blood are sufficient for the process according to the present invention. In particular, a blood sample quantity of less than 50 mL, less than 20 mL or less than 1 mL is sufficient.

[0050] The blood sample is subsequently processed according to step S12. An additive is added to the blood sample within the framework of the processing of the blood sample in this exemplary embodiment in order to prevent clotting of the blood. Furthermore, the percentage of the immunocytes to be tested is increased in the blood sample within the framework of the processing of the blood sample. Red blood cells are removed for this purpose from the blood sample in this exemplary embodiment by means of processes that are known per se. Furthermore, nonrelevant white blood cells may likewise be removed according to processes that are known per se. The basophilic cells are isolated or their percentage is increased in the blood sample in this exemplary embodiment. It is sufficient in this exemplary embodiment to increase the percentage of basophilic cells in the blood sample in a range of 10% to 20%. The blood sample is then suspended or the cells are then suspended in a suitable medium in order to keep the cells alive for a predefined time, especially for up to 24 hours.

[0051] The blood sample is subsequently applied to or introduced into a microfluidic chip 20 according to step S13. The microfluidic chip 20 may be configured here such that the blood sample or the immunocytes to be tested are guided into the microfluidic chip 20 by means of capillary forces. As an alternative, the blood sample or the immunocytes may be pumped into the microfluidic chip 20 by means of a suitable device.

[0052] Immunocytes are then positioned in at least one reaction chamber 21 according to step S14. The positioning may be carried out here by means of suitably configured cell traps, microchannels or an electrophoretic or dielectrophoretic positioning device 28. A plurality of immunocytes are arranged each time in a reaction chamber 21 by means of the dielectrophoretic positioning device 28 in this exemplary embodiment. Furthermore, immunocytes are guided into a plurality of reaction chambers 21 and are held there by means of the dielectrophoretic positioning device 28.

[0053] At least one allergen is subsequently introduced according to step S15. At least one allergen is guided, especially in the liquid form, into the reaction chamber 21 and into the corresponding reference chamber 33 by means of the microchannel 27 in this exemplary embodiment.

[0054] An optical monitoring of the immunocytes is carried out according to step S16. The optical monitoring may already be started prior to the introduction of the at least one allergen, together with the introduction of the at least one allergen or immediately after the introduction of the at least one allergen. Optical monitoring is carried out in this exemplary embodiment by means of the microscope 11. The optical monitoring is embodied in this exemplary embodiment as a quantitative phase contrast microscopy. Granules of the immunocytes are observed here directly and/or optically by means of the microscope 11. The observation is carried out, in particular, over a predefined time of 60 sec to 300 sec or longer in order to determine whether degranulation takes place after contacting the immunocytes with the at least one allergen. The microscope 11 may make possible for this a digital image recording or a digital image acquisition. A plurality of immunocytes are observed within the framework of the observation or monitoring performed in different planes or horizontal planes of the at least one reaction chamber 21. The microscope 11 may be configured for this especially for generating a holographic image.

[0055] An analysis of the optical observation or monitoring is then carried out according to step S17. If degranulation is observed after contacting the immunocytes with the at least one allergen, this is classified as an allergic reaction. If, by contrast, no observable degranulation takes place, this is considered to be a non-allergic reaction.

[0056] The process then ends according to step S18.

[0057] While specific embodiments of the invention have been shown and described in detail to illustrate the application of the principles of the invention, it will be understood that the invention may be embodied otherwise without departing from such principles.