Device and method for detecting flourescent marked biological components

FIELD: medicine.

SUBSTANCE: device comprises a measuring cavity for receiving and introducing a fluid sample. The measuring cavity has a set fixed thickness not exceeding 170 micrometres. The measuring cavity has a section fit for acquisition of its image. Within the measuring cavity, there is a dry reagent. The reagent contains as a component, a molecule conjugate with phosphor used for binding with biological components and with all other reacting components. The reacting components are soluble and/or suspended in the fluid sample. The method involves mixing of the reagent with the liquid sample to be introduced in the measuring cavity. A section of the sample in the measuring cavity is exposed to electromagnetic radiation of wavelength corresponding to wavelength of phosphor excitation. Phosphor marked biological components are detected through-thickness of the measuring cavity. Further, numerical analysis of the digital image follows to identify the biological components showing phosphor and to determine amounts of the biological components showing phosphor in the sample. The biological components are discernible on the digital image as fluorescing points emitting electromagnetic radiation of wavelength corresponding wavelength of phosphor emission.

EFFECT: device and method allow for higher effectiveness of numerical volume concentration of fluorescent marked biological components of the fluid sample.

24 cl, 4 dwg

The technical field

The present invention relates to a device for sampling, to method and system for detection and determination of numerical concentration of fluorescently labeled biological components in a liquid sample.

The level of technology

When analyzed biological sample, such as sample cells, it is desirable to be able to identify the various components of the sample, such as various types of cells present. These various components demonstrate appropriate molecular patterns, such as cell surface markers by which components may vary. Through the use of molecules, conjugated with a fluorophore, adapted to associate with these molecular structures, biological components can be fluorescently labeled. In this area there are several known techniques for detection and analysis of fluorescently labeled components, mainly of cells, primarily techniques of flow cytometry and fluorescence microscopy.

During flow cytometry suspended fluorescently labeled cells pass one after another through a flow channel in front of the laser beam, and can be measured fluorescence at multiple wavelengths and scattering of light forward and perpendiculares direction. Thus can be decomposed tagging several different fluorophores, as well as the size and heterogeneity of the cells. Methods in flow cytometry are described, for example, in U.S. patent No. 3826364, 4248412 and 5047321.

Fluorescence microscopy, as a rule, is carried out by irradiating a fluorescent sample, which should be analysed, usually smeared on subject glass microscope, using electromagnetic radiation of a specific shorter wavelength, which causes the fluorophores in the sample to absorb a specified radiation, and subsequently to emit electromagnetic radiation of a specific higher wavelength. The emitted radiation is detected using a microscope equipped with a chromatic filter or equivalent monochromator, which makes possible the passage of essentially only the emitted radiation with a longer wavelength.

In U.S. patent No. 4125828 and 2006/0017001 describes fluorescent microscopes and methods of detection of fluorescence of the sample smeared on the subject the microscope.

In U.S. patent No. 5932428 describes the mixture of the sample and components for analysis to quantify fluorescently dyed target components of the blood sample with a tool for image analysis. In one aspect of atanta U.S. No. 5932428 whole blood is mixed with dried fluorescently labeled antibody and zwitterionic detergent, then mix with the blood introduced into the capillary for scanning. The filled capillary is scanned using a laser beam, which tapers in the form of a Gaussian banners, which crosses the capillary. Thus, the laser illuminates the area in the form of a column in a capillary tube whose volume is equal to the square of the Gaussian banners, multiplied by the depth of the lumen of the capillary, and excites any fluorescent material in this area. The fluorescence of this area is measured by the light detector. Then, the laser illuminates a different region of the capillary, and the fluorescence is also detected, and the like. Thus, a given volume of the sample is scanned for fluorescence.

In the document U.S. No. 2006/0024756 describes a device, method and algorithm for the quantitative determination of fluorescent and magnetic labeled cells. In accordance with the described method, all cells are marked by means of fluorescently, but only the target cells are marked by means of magneto. The sample with the labeled cells are placed in a chamber or cuvette between the two wedge-shaped magnets for selective movement of magnetically labeled cells to the surface of the observation cell. LED lights cells, and CCD camera (digital camera charge-coupled) captures the image of fluorescent light emitted by the target cells. Tagging clearmode take place in a cell or chamber, used for analysis, or the sample is moved in such a cuvette or chamber after a sufficient time to allow for tagging cells. The volume of the cell is known and is used to determine the absolute concentration of the target cells in the blood sample. However, this requires waiting until the target cells will not move under the action of magnets on the surface of the observations before they can be detected and counted.

European patent EP 0422708 describes a device for use in procedures chemical tests. The device includes a cavity defined by two flat and parallel walls, one of which holds covalently immobilized antibodies, and this or another wall holds dried, but not covalently immobilized fluorescently labeled antibodies. The aim is to use the device for sandwich assays of antigen that can bind with the immobilized and labeled antibodies. A liquid sample containing the antigen, which is to be analyzed is drawn under the action of capillary forces in the device, dissolving labeled antibodies. The antigen is bound by the immobilized antibody and labeled antibody binds to the antigen, with a fluorescently labeled antibody concentrate on the article is the NCA, holding covalently immobilized antibodies. This wall is made of glass or other light transmissive material and has the ability to carry out light like optical fiber or waveguide. The presence of antigen in a liquid sample is detected by measuring the intensity of light at the end of the wall, the light is conducted by the wall, which is emitted fluorescent antibodies that are present on the wall.

The invention

The present invention is the creation of a simple analysis for the detection of fluorescently labeled biological components in a liquid sample. In accordance with one aspect of the present invention the task is to create a simple analysis for the determination of numerical concentration of fluorescently labeled biological components in a liquid sample. An additional object of the present invention is to provide a quick analysis without the need for complicated devices or excessive sample preparation.

These tasks are partially or completely solved by using a device for sampling, method and system in accordance with the independent claims. Preferred embodiments of clear from the dependent claims.

In accordance with one of the speakers who projects the present invention relates, thus, the device for sampling for detection of biological components in a liquid sample, with the specified device for sampling includes a measurement cavity for receiving a liquid sample, where the measurement cavity has a predetermined fixed thickness. Device for sampling also contains a reagent, which is located in the dry form inside the measurement cavity, specified reagent contains a molecule conjugate with the fluorophore.

In accordance with another aspect of the present invention relates to a method for detecting biological components labeled with a fluorophore, in a liquid sample. The method includes the mixing of the reagent containing molecule, conjugate with the fluorophore, with the liquid sample, so that the molecule conjugated with a fluorophore that binds to a specific molecular structure of the biological component in a liquid sample, the introduction of liquid sample into a measurement cavity of the device for sampling, while the measurement cavity has a predetermined fixed thickness; the irradiation area of the sample in the measurement cavity electromagnetic radiation with a wavelength corresponding to the wavelength of excitation of the fluorophore; and detecting the labeled fluorophore biological components throughout the thickness measuring floor is STI, the specified detection includes receiving a digital image of the irradiated area in the measuring cavity.

Device for sampling provides the ability to directly obtain a sample of whole blood in the measuring cavity and shipping it for analysis. There is no need for sample preparation. In fact, the blood sample can passivates in the measuring cavity directly from the punctured finger of the patient. Supply device for sampling reagent makes it possible reaction in the device for sampling, which makes the sample is ready for analysis. The reaction is initiated when blood comes into contact with the reagent. Thus, there is no need for sample preparation by hand, which makes the analysis particularly suitable for direct implementation in the exam room while the patient waits.

Because the reagent is provided in a dry form, a device for sampling can be transported and stored for extended periods of time without affecting consumer device properties for sampling. Thus, the device for sampling with the reagent can be produced and prepared long before the implementation of the analysis of the blood sample.

Device for sampling according to the present izaberete the Oia may thus, easily and with good reproducibility even be used untrained person and, optionally, in the usual standardized laboratory environment, as the device for sampling can be a part of the ready set, where the input sample unit for sampling should only be brought into contact with the sample to obtain a sample in a form ready for analysis.

In addition, a fixed thickness of the measurement cavity provides the ability to determine the number of biological components per unit volume of liquid sample. Because the method is adapted to detect labeled with fluorophore biological components throughout the depth of the measuring cavity, it is possible to carry out rapid analysis of liquid sample. There is no need to wait until the biological components of interest, will accumulate in the measuring cavity or fall on the surface of the observation.

Biological components of the liquid sample can represent, for example, eukaryotic cells such as mammalian cells (e.g., leukocytes and platelets); bacteria; viruses and macromolecules, such as DNA.

The liquid sample may represent, for example, a bodily fluid, such as undiluted whole blood, urine or spinnomozgove the I fluid; or culture of cells, such as mammalian cell culture or bacterial culture. The liquid sample can be a undiluted biological fluid, which is not subjected to any pretreatment. Pre-processing a biological sample, such as dilution, centrifugation, lizirovania, leads to a decrease of accuracy when mapping a quantified target cells and analyzed volume. The more stages of pre-treatment, the less the accuracy of quantitative determination. By eliminating the need to use any type of pre-treatment before the introduction of the sample in ready-to-use device for sampling the method further simplified.

Thus, it is possible detection of the presence or quantity, for example, a particular type of cells in the blood sample.

Device for sampling may contain an element body having two flat (planar) surface to determine the specified measuring cavity. The planar surface may be located at a specified distance from each other to determine the thickness of the sample with the objective optical measurements. This means that the device for sampling provides a certain thickness for optical measurements, which is th can be used to accurately determine the number of labeled fluorophore biological components per unit volume of liquid sample. The volume of the analyzed liquid sample will be determined by the thickness of the measurement cavity and the area of the sample image which get. Thus, a certain amount could be used to correlate the number of labeled fluorophore biological components to the sample volume, which is determined by the number of labeled fluorophore biological components per unit volume.

Measuring the cavity preferably has a uniform thickness is 50-170 micrometers. The thickness equal to at least 50 micrometers, provides that the measuring cavity does not give a liquid sample, such as sample cells, blurred in the form of a monolayer, allowing for analysis of a larger volume of liquid sample on a small cross-sectional area. Thus, a sufficiently large volume of liquid sample to obtain reliable values for the number of labeled fluorophore biological components can be analyzed using a relatively small image of a sample with cells. Thickness, more preferably, is at least 100 micrometers, which allows for the analysis of a smaller cross-sectional area or for analysis of a larger sample volume. In addition, the thickness of at least 50 micrometers, and more preferably 100 micrometers, also simplifies the fabrication and the measurement cavity, having a certain thickness, between two planar surfaces.

For most samples, such as blood sample, located in the cavity, having a thickness of not more than 170 μm, the number of labeled fluorophore biological components such as cells in a blood sample is so low that there will be only small deviations due to components that are overlapping each other. However, the effect of such variance will be associated with the number of labeled fluorophore biologically components and can thus, at least to some extent, processed through statistical adjustments, at least for large values of the number of labeled fluorophore biologically components. This statistical adjustment may be based on the calibrations of the measuring device. The difference would be even smaller for the measurement cavity, having a thickness not more than 150 μm, you can use more simple calibration. This thickness may not even require any calibration for overlapping biological components.

In addition, the thickness of the measurement cavity is sufficiently low to provide a measuring device for obtaining a digital image so that the entire depth of the measuring cavity can the be analyzed simultaneously. If the measuring device must be used in the system increase, it will be difficult to get greater depth of field. For this reason, the thickness of the measurement cavity preferably should not exceed 150 microns, so that the whole thickness simultaneously analyzed in a digital image. Depth of field can be set to work with the thickness of the measurement cavity 170 micrometers.

A digital image can be obtained with a depth at least corresponding to the thickness of the measurement cavity. This means that there will be sufficient focus throughout the thickness of the sample, so that the whole thickness of the measurement cavity can be analyzed on the digital image of the sample. Thus, there is no need to expect that, for example, the cells will settle in the measuring cavity, at the same time, the analysis is reduced. Choosing not very sharp focus on specific part of the sample, it is sufficient to focus on the entire thickness of the sample, in order to make possible the identification number of labeled fluorophore biologically components in the sample. This ensures that the fluorescent component can sometimes be diluted and still be regarded as being in focus depth of field.

A fixed thickness of the measurement cavity is eleet possible the analysis of a specific sample volume. In particular, the area of the measuring cavity is adapted to the image to ensure that the analysis of a particular sample volume, can be obtained numerical determination of the volume concentration of the biological component in the sample. The area of the image which is obtained together with the thickness of the measurement cavity, provides a certain amount of the sample. By counting the number of labeled biological components inside the static displacement can be easily obtained, the number of biological components in a single sample volume. Volume numerical concentration can be obtained by analyzing the digital image volume. Thus, three-dimensional numerical concentration can be obtained without the necessity of passing the sample prior to the analyzer that is carried out in accordance with the principle of flow cytometry.

Device for sampling can be supplied with the reagent, which can be applied to the surface dissolved in a volatile liquid, which evaporates, leaving the reagent in dry form.

Is the preferential dissolution of the reagent in the volatile liquid before introduction into the measuring cavity. This ensures that the liquid can effectively evaporate from the narrow space of the measuring cavity during production and on the cooking device for sampling.

The reagent preferably can be dissolved in an organic solvent, more preferably, dissolved in methanol. These solvents are volatile and can accordingly be used for drying of the reagent on the surface of the measuring cavity.

The reagent, including all its components, according to the present invention preferably is soluble and/or suspendiruemye in a liquid sample, which must be assessed, and preferably is designed to stay in solution/suspension for analysis. Because, as stated above, the method is adapted to detect labeled with fluorophore biological components throughout the thickness of the measurement cavity, therefore, there is no need to move or immobilization of biological components of interest on the surface of the observations, there is no need to immobilization or preventing other by dissolution/suspension of the reagent or any component of the reagent. On the contrary, the use of soluble/suspendiruemye reagent, preferably readily soluble/suspendiruemye reagent facilitates the mixing of the reagent with the liquid sample and accelerates any reaction between the reagent and the liquid sample containing the biological component, which should be measured.

Reagenta the present invention contains a molecule conjugate with the fluorophore. The fluorophore or fluorochrome is defined here as the rest of the molecule which causes a molecule to be fluorescent. The molecule is fluorescent, if it emits electromagnetic radiation of a specific wavelength in response to what she is exposed to radiation of another wavelength.

Used in most cases, the fluorophores or fluorochromes include, for example, the fluorescein isothiocyanate (FITC), phycoerythrin (PE), protein pyridiniomethyl (PerCP), allophycocyanin (APC) and cyanin-5.5 (Cy5.5).

Molecule conjugated with a fluorophore, preferably adapted to bind to a specific molecular structure of the biological component. Examples of such molecules include, but are not limited to, ligands, receptors, antigens, antibodies and antibody fragments. Examples of fragments of antibodies are, for example, the fragment binding to the antigen (Fab) and single-chain variable fragment (scFv). Antibodies and antibody fragments are preferred because they are relatively easy to work with relationship to all types of molecular structures, and there are many schemes of conjugating them with different types of fluorophores.

The molecular structure can be any specific molecular structure of biologicheskikh the component, for example, a cell surface marker, such as CD4 or CD8, or intracellular structure, such as DNA. The cell surface marker is defined here as any molecular characterization of the plasma membrane of cells, which is accessible from the outside of cells, such as antigen or epitope. This ensures that all types of cells can be detected for any purpose, such as the detection and quantification of CD4+ for the purpose of monitoring HIV infection.

The number of molecules conjugated with a fluorophore, preferably is selected so that there is enough to associate with biological components. To ensure that essentially all of the target biological molecules adequately marked by means of using molecules conjugated with a fluorophore, within a reasonable time, molecules, conjugated with the fluorophore must be present in excess. However, still remain unbound molecules conjugated with a fluorophore, in a mixed sample, and it is desirable that the unbound concentration is maintained low enough to reduce the background fluorescence when the sample is analyzed. Thus, molecules, conjugated with a fluorophore, must not be present in too great excess. The ratio of bound and unbound maul is cool, conjugated with a fluorophore depends on the affinity between the molecules, conjugated with a fluorophore, and the biological component and the time allotted for mixing molecules conjugated with a fluorophore, with a biological component.

Device for sampling may further comprise the input for sample connecting the measuring cavity with the external space of the device for sampling a particular input is adapted for sampling a liquid sample. The input sample may be placed for receiving the liquid sample by capillary forces, and the measuring cavity can optionally extract the fluid from the inlet into the cavity. As a result, the liquid sample can easily be selected in the measuring cavity simply by managing the input sample in contact with the liquid. Then the capillary force of the input sample and the measuring cavity will extract into a measurement cavity of a well-defined amount of fluid. Alternatively, the liquid sample can passivate or injected into the measuring cavity through the application of an external force to the pumping device for sampling. In accordance with another alternative liquid sample can be drawn into the pipette, and then introduced into the measuring cavity by means of a pipette.

Device for sampling which may be disposable, that is, it is intended to be used only once. Device for sampling forms part of a set for the implementation of the account labeled with fluorophore biological components, as the device for sampling capable of receiving a liquid sample and hold all reagents required to provide a sample for counting. It is possible, in particular, because the device for sampling adapted for a single use only and can be formed without taking into account the capabilities of the device for sampling and re-application of the reagent. Also the device for sampling may be formed in the plastic material and for this reason be produced at low cost. Thus, it may be economically efficient use of disposable devices for sampling.

In accordance with one variant of the method of detecting labeled with fluorophore biological components in a liquid sample, the device for sampling contains a reagent, which is located in the dry form inside the measurement cavity, where the reagent contains a molecule conjugate with the fluorophore. Then the mixing is carried out by introducing the liquid sample into a measurement cavity for contact with the reagent. This lake is achet, there is no need for sample preparation. The reaction can be initiated when blood comes into contact with the reagent. Thus, there is no need for sample preparation by hand, which makes the analysis particularly suitable for direct implementation in the exam room, while the patient waits.

However, in accordance with an alternative embodiment of the mixing of the reagent with the liquid sample can be carried out before the liquid sample is introduced into the measuring cavity. In accordance with another alternative, the mixing may be carried out at least in two stages where the first stage is performed before the sample is introduced into the measuring cavity, and the second stage is carried out in the measuring cavity. This ensures that the sample preparation, at least partly carried out device for sampling, and that he gets in a device for sampling. However, the advantage of using the device for sampling with measurement cavity with a fixed thickness, remains. Thus, the method provides the possibility of determining the number of biological components per unit volume of liquid sample.

In addition, there is no need to wait biological components, represents the General interest, will settle within the measuring cavity or fall on the surface of the observation.

For irradiation of the sample, which should be studied, it is preferable to use a radiation source adapted in order not to give radiation with wavelengths that correspond to the wavelengths that are emitted by the fluorophores of the sample or close to them, to reach the sample, because it would prevent the detection of the emitted radiation. To obtain such a radiation limited to wavelengths, preferably uses a radiation source in combination with a chromatic filter. Alternatively, it may use a laser, which emits a specific wavelength, which is absorbed by the fluorophore. Prism or a raster (grid) can also be used to route only certain wavelengths of radiation from the radiation source to the sample. The radiation source preferably is a light emitting diode (LED), but may use any source of radiation, such as laser or conventional incandescent bulb. LED is preferred because it is relatively cheap and reliable.

Detection of labeled fluorophore biological components preferably includes obtaining a digital image of the irradiated sample in the measurement cavity, where the biological is the cue components, demonstrating the fluorophore, stand out as fluorescent dots that emit electromagnetic radiation with a wavelength corresponding to the wavelength of emission of the fluorophore. Digital image conveniently be obtained by using a digital CCD camera, built-in fluorescent microscope, convenient to the microscope preferably adapted through the use of chromatic filter to give only electromagnetic radiation with a wavelength of emission of the fluorophore able to reach the camera.

Detection of labeled fluorophore biological components, preferably, further includes a numerical analysis of the digital image to identify biological components, demonstrating the fluorophore, and the determination of the number of biological components, demonstrating the fluorophore in the sample. This ensures that the detection and/or by biological components can easily be obtained using image analysis by computer. Thus, it can be obtained in a reliable and reproducible results.

The liquid sample is preferably introduced into a measurement cavity of the device for sampling through the capillary input sample by capillary forces.

A digital image can be obtained when ispolzovaniem increase 3-50×, more preferably, 3-10×. In these ranges increase most biological components, such as mammalian cells, which is the target for this method, it is enough to increase detection, while the depth of field must adjust for the overlap sample thickness. Low magnification provides what can be obtained a large depth of field. However, if you use low magnification, the detection of biological components can be complex. Lower magnification can be used by increasing the number of pixels in the output image, that is, by increasing the resolution of digital images. Thus, it is possible using the zoom 3-4×, still preserving the possibility of detecting biological components such as mammalian cells.

The analysis may include identification of areas of strong emission of electromagnetic radiation of a particular wavelength corresponding to the wavelength of emission of the fluorophore, which is labeled biological component, on the digital image. In addition, the analysis may include identification of the light points on the digital image resulting from the specific emission of electromagnetic radiation. Because molecules is, conjugated with a fluorophore, can accumulate around the target biological components specific emission of fluorescence can have peaks at certain points. These points will form a light point on a digital image that can be detected as the corresponding target biological component.

The analysis can also include e-the increase of the received digital image. While the sample is increased to enlarge a digital image of the sample obtained digital image itself, you can use electronics to increase to simplify finding the difference between objects that are depicted very close to each other on the received digital image.

If two or more different molecules, conjugated with fluorophore conjugated, respectively, with different fluorophores that emit electromagnetic radiation, respectively, with different wavelengths, and adapted to associate, respectively, with different molecular structures are contained in the reagent can be obtained an image of each emitted wavelength of radiation to the sample. Then these images may overlap each other, while the analysis may show some biological components, pre is maintained by a single molecular structure, some other representing the other, and some representing them both. If you use more than two different molecules, conjugated with a fluorophore, the reasoning is similar.

In another embodiment, the present invention relates to a system for determining the numerical concentration-labeled fluorophore biological components in a liquid sample, said system contains a device for sampling, as defined above; and to the measuring device containing a holder device for sampling, adapted to receive a device for sampling, which contains the liquid sample in the measurement cavity, a source adapted for irradiating a liquid sample with electromagnetic radiation of a given wavelength, the system receiving the image containing means for receiving a digital image of the irradiated sample in the measurement cavity, where conjugated with fluorophore biological components differ digital image by obtaining selective images using different wavelengths of electromagnetic radiation, and an image analyzer adapted to analyze the received digital image to identify conjugated with fluorophore biological components and to determine the number of conjugated with fluorophore biological components in a liquid sample.

The measuring device can use the device properties for sampling, as described above, for analysis of a liquid sample, which is drawn directly into the measuring cavity. The measuring device can obtain an image of a certain volume of model for the determination of numerical concentration of the biological component in the sample.

Brief description of drawings

The present invention will now be described in more detail by way of examples with reference to the accompanying drawings.

Figure 1 is a schematic view of the device for sampling in accordance with the embodiment of the present invention.

Figure 2 is a schematic view of the device for sampling in accordance with another embodiment of the present invention.

Figure 3 is a schematic view of the measuring system in accordance with the embodiment of the present invention.

Figure 4 is a block diagram of a method in accordance with the embodiment of the present invention.

A detailed description of the preferred option exercise

Referring now to Figure 1, will be described device 10 for sampling in accordance with the embodiment of us who Otsego invention. The device 10 for sampling is disposable and should be discarded after use for analysis. This means that the device 10 for sampling does not require complex treatment. Device for sampling molded into the plastic material and is produced by injection molding. This makes manufacture of the device 10 for sampling is simple and cheap, while the cost of the device for sampling is reduced.

The device 10 for sampling contains the element 12 of the housing that has a base 14, which may relate to the operator, without causing any interference in the assay results. The base 14 also has projections 16, which are connected with the holder in the device for analysis. The protrusions 16 are arranged in such a way that the device for sampling 10 will be properly positioned in the device for analysis.

The device 10 for sampling further comprises an inlet 18 for the sample. The inlet 18 for the sample is defined between opposite walls in a device for sampling, the walls are so close to each other that at the inlet 18 for sample creates capillary forces. Input 18 for sample communicating with the external space of the device for sampling in order to make possible the flow of blood in the device 10 for selection of exemplary is. The device 10 for sampling further comprises a camera for the account labeled with fluorophore biologically components, such as cells, in the form of a measurement cavity 20 located between the opposite walls inside the device 10 for sampling. The measurement cavity 20 has a capability message to the input 18 for the sample. The walls defining the measurement cavity 20, are located closer to each other than the walls of the entrance 18 to the sample, so that capillary forces can extract the blood from the inlet 18 to the sample in the measurement cavity 20.

Wall of the measuring cavity 20 are located at a distance of 140 micrometers from each other. The distance is the same throughout the measurement cavity 20. The thickness of the measurement cavity 20 determines the amount of blood that is investigated. Since the analysis result should be compared with the volume of the blood sample that is investigated, the thickness of the measurement cavity 20 should be maintained very accurately, i.e. only a very small variation in thickness is permissible within the measuring cavity 20 and between the measuring cavities 20 different devices 10 for selection of sample. The thickness makes it possible to analyze a relatively large sample volume over a small area of the cavity. The thickness of theoretically allows the cells to be placed one on top of another in izmeritelnoi cavity 20. However, the number of cells in the sample, such as a blood sample, is so low that the likelihood of this is very low.

The device 10 for sampling adapted for measuring quantities of cells labeled with fluorophore, over 0.5·10⁹cells per liter of blood. When smaller quantities of cells in the sample volume is too small to calculate a statistically significant quantities of cells. In addition, when the number of labeled fluorophore cells exceeds 12·10⁹cells per liter of blood, the influence of the cells located to overlap each other, will become important when measuring the number of cells. With the high number of cells labeled with fluorophore-labeled cells will cover approximately 8% of the cross-section of the sample that is irradiated, when the thickness of the measurement cavity 140 micrometers. Thus, to obtain the correct number of cells labeled with a fluorophore, this effect should be taken into account. For this reason, can be used for statistical adjustment of values of the number of labeled cells over 12·10⁹labeled cells per liter of blood. This statistical adjustment will increase with the increase in the number of cells labeled with fluorophore, as the impact of overlapping labeled cells will be great for larger amounts, the tion of cells. Statistical adjustment can be determined by calibration of the measuring instrument. Alternatively, the statistical correction can be determined at a General level to configure the measuring device to be used in conjunction with the device 10 for sampling. It is assumed that the device 10 for sampling could be used for analysis of the number of cells labeled with fluorophore, reaching 50·10⁹Mechernich cells per liter of blood.

In accordance with an alternative embodiment of the detection-labeled fluorophore biological components is used to determine whether a particular biological component in the sample. In this embodiment, it is not necessary in the implementation of the counting volume numerical concentration and, thus, the presence of a biological component can be detected even for very small quantities of the component in the sample.

The surface of the wall of the measuring cavity 20 at least partially covered by the reagent 22. The reagent 22 may be dried by freezing, dried thermally or in a vacuum and applied to the surface of the measurement cavity 20. When the sample is taken into a measurement cavity 20, the sample comes into contact with the dried reagent 22 and in Ziarul the binding reaction between the reagent 22 and the components of the sample.

The reagent 22 is applied by placing the reagent 22 in the measurement cavity 20 using a pipette or switchgear. The reagent 22 is dissolved in methanol, when fed into a measurement cavity 20. The solvent with the reagent 22 fills the measurement cavity 20. Then carry out the drying, so that the solvent evaporates, and the reagent 22 is attached to the surface of the measurement cavity 20.

Because the reagent should be dry on the surface of the narrow space, the liquid will have a very small surface in contact with the surrounding atmosphere, for this reason, the evaporation of the liquid becomes difficult. Thus, it is preferable to use a volatile liquid, such as methanol, which makes possible an efficient evaporation of the liquid from the narrow space of the measuring cavity.

In accordance with an alternative method of manufacturing the device 10 for sampling is formed by connecting the two parts together, with one part forms a lower wall of the measuring cavity 20, and the other part forms the upper wall of the measuring cavity 20. This makes it possible drying of the reagent 22 on the outside surface before the two parts are connected to each other. Thus, the reactant 22 can be dissolved in water as the solvent does not have life is volatile.

The reagent 22 may contain one or more antibodies conjugated to fluorophores. Antibodies are adapted to bind to a specific molecular structure, characteristic for the target biological component, such as a cell. The structure may be a cell surface marker, such as CD4 or CD8. When a blood sample comes into contact with the reagent 22, antibodies will act by binding to specific molecular structure of the target blood cells, thus, accumulates in the cells. The reagent 22 should preferably contain a sufficient number of antibodies to clearly mark the part of the target cells, essentially covering all cells. This means that essentially all of the labeled cells are fluorescent and thus can easily be detected on the digital image of the sample. In addition, often will be present an excess of antibodies conjugated with a fluorophore, which will be blended in the blood plasma. Excess antibody will give a homogeneous low background level of fluorescence in the blood plasma. Accumulated antibody on target cells will be visible above the background level of fluorescence.

The reagent 22 may also contain other components that may be active, i.e. participating in the chemical linking, for example, with cells obraztcova, or inactive, i.e. not participating in the binding. Active components can, for example, to adapt to facilitate binding of the antibodies with their corresponding target molecular structures. Inactive components can, for example, to adapt to improve the attachment of the reagent 22 to the surface of the wall of the measuring cavity 20.

Within a few minutes the blood sample reacts with the reagent 22, so that an antibody labelled with a fluorophore will bind to the target cells.

Referring to Figure 2, will be described in another variant implementation of the device for sampling. Device 110 for sampling includes a camera 120, forming the measuring cavity. Device 110 for sampling has an input 118 into the chamber 120 to transfer the blood into the chamber 120. The camera 120 is connected with a pump (not shown) through a pipe 121 to the suction. The pump can exert a suction force in the chamber 120 through the tube 121 to the suction, so that the sample is sucked into the chamber 120 through the inlet 118. Device 110 for sampling can be disconnected from the pump before being measured. Like the measurement cavity 20 of the device 10 for sampling in accordance with the first embodiment, the camera 120 has a thickness that defines the thickness of the sample, which should be explored. In addition to t the th, reagent 122 is applied to the chamber wall 120 to interact with the sample.

Referring now to Figure 3, will be described a system for the detection and determination of numerical concentration-labeled fluorophore biological components. The system 30 includes a holder 32 for the sample reception device 10 for sampling a blood sample. The holder 32 of the sample is adapted to receive the device 10 for sampling, so that the measuring cavity 20 of the device for sampling is properly positioned in the system 30. The system 30 includes a light source 34 to illuminate the sample in the device 10 for sampling. The light source 34 may be a LED, which in combination with the chromatic filter 48 emits light corresponding to the wavelength of excitation of the fluorophore used together with the sample. In addition, the wavelength should be chosen so that the absorption of the fluorescent compounds of the sample, other than the components labelled with a fluorophore, was relatively low. In addition, the walls of the device 10 for sampling should be essentially transparent to this wavelength. After passing through the chromatic filter light is directed to the sample through the use of a dichroic mirror 35. The fluorophores, which are accumulated around (or inside) labeled biological to the components of the sample, such as the cells will absorb this light 48 specific wavelength and emit light 50 more specific wavelength. This emitted light 50 higher wavelength gets the opportunity to pass through the dichroic mirror and entering the system image creation 36, so that the components will appear in the digital image of the sample as highlights or points. If you get a color image, labeled cells will appear as dots of a particular color. If you get a black and white image, labeled cells appear as bright spots on a dark background.

Alternatively, the light source 34 may be a light from the incandescent lamp in combination with chromatic filter or a laser.

Alternatively, the light 48 may be routed directly to the sample at an angle, without the use of dichroic mirrors.

In addition, the system 30 includes a system for obtaining image 36, which is located above the holder 32 of the sample. Thus, the receiving system image 36 is located to receive radiation 50, which is emitted by the blood sample. System 36 receiving the image may contain system 38 optical zoom and funds 40 image acquisition. To prevent light emitted by the fluorophores of the sample, the means 40 to the floor of the treatment image may be used chromatic filter. The system 38 of the increase can be adjusted to provide increasing 3-50×, more preferably 3-100×, and most preferably, 3-4×. In these ranges of magnification is possible to distinguish the labeled cells. The image can be obtained with a higher resolution to make it possible to use a smaller increase. In addition, the depth of field 38 system magnification can be adjusted so that it at least corresponds to the thickness of the measurement cavity 20.

System 38 increase may contain a lens or system of lenses 42 of the lens, which is located near the holder 32 of the sample and the lens or system of lenses 44 of the eyepiece, which is located at some distance from the lens 42 of the lens. The lens 42 of the lens provides the first increase of the sample which is further increased by using lenses 44 eyepiece. The lens 42 of the lens can be placed between the dichroic mirror 35 and the holder 32 of the sample. System 38 increase may contain additional lenses for the implementation of the respective increase and create an image of the sample. System 38 increase is positioned so that the sample in the measurement cavity 20 when placed in the holder 32 of the sample will focus on the image plane means 40 receiving the image.

Means 40 receiving the picture from the Oia provided for obtaining a digital image of the sample. Means 40 receiving the image can be any kind of digital cameras, such as CCD camera. The pixel size of a digital camera imposes restrictions on the system 36 image acquisition, so that the circle scattering in the image plane could not exceed the size of a pixel within the depth of field. However, the labeled cells can still be detected, even if they are somewhat blurred, and for this reason the circle scattering can be such that it exceeds the size of the pixel, the same being considered within the depth of field. Digital camera 40 will receive a digital image of the sample in the measurement cavity 20, where the entire thickness of the sample is essentially focused in the digital image for the account labeled blood cells. System 36 receiving the image will determine the area of the measurement cavity 20, which will be depicted in the digital image. The area that is depicted, together with the thickness of the measurement cavity 20 defines a sample volume that is displayed. System 36 image acquisition is configured to obtain images of blood samples in the device 10 for sampling. There is no need to change system settings 36 image acquisition. Preferably, the system 36 image acquisition is located inside the housing, so that the settings of the e change randomly.

Alternatively, the system 30 may contain more than one system 36 image acquisition, while the emitted fluorescence of different wavelengths can be directed to the appropriate different systems produce images. Direction different wavelengths of light in different systems 36 image acquisition can be achieved by using, for example, one or more dichronic mirrors or gratings.

Also can be used multiple light sources 34, the sample can be irradiated with light of several different wavelengths simultaneously or sequentially. This can be achieved through the use of multiple LED in combination, at least one chromatic filter for each. Convenient to all chromatic filters used in the system 30 could be placed on the carousel, on which all the most frequently needed chromatic filters can be placed, so that the particular filter that is required for the specific detection, can easily switch to the active position. The filter is in the active position when it crosses the light from the LED before it reaches the sample, if the filter is used for excitation light or when he crosses the light of the fluorescence from the sample before it reaches the means 40 image acquisition, if f is ltr is used for the light emitted.

The system 30 further comprises an analyzer 46 images. The analyzer 46 images connected with digital camera 40 for receiving digital images from a digital camera 40. The analyzer 46 images provided to identify structures on the digital image that correspond to the labeled cells for counting the number of labeled cells, which are present in the digital image. Thus, the analyzer 46 images can be used to identify bright spots on a dark background. The analyzer 46 images can be adapted for electronic zoom digital images first, before analyzing the digital image. This ensures that the analyzer 46 images may be able to more easily distinguish the labeled cells, which are represented close to each other, even if the electronic zoom digital images will make the digital image is somewhat blurred.

The analyzer 46 images can calculate the number of labeled blood cells per volume of blood by dividing the number of labeled blood cells that are identified on the digital image volume of the blood sample, which is well defined, as described above. Volume numerical concentration of labeled blood cells may be represented by the and the display device 30.

The analyzer 46 images can be realized as the host processor, which contains codes for analysis of the image.

Referring to Figure 4, will be described by way of fluorescently labeled biological components. The method will be described specifically with reference to the method of detection and determination of numerical concentration-labeled T lymphocytes. However, the person skilled in the art will understand that the method can be modified for detection and determination of numerical concentration of other biological components. The appropriate reagent for labeling biological components of interest should be used, and the irradiation and detection must be adapted to the wavelengths of excitation and emission of selected fluorophores, as will be clear to experts in this field.

The method of detection and determination of numerical concentration of T lymphocytes involves taking a blood sample in a device for sampling, the stage 102 having a fixed thickness of 140 μm. Undiluted sample of whole human blood is collected in a device for sampling. The sample may be selected from capillary blood or venous blood. A sample of capillary blood can flow into the measuring cavity directly from the punctured finger of the patient. Education is the EC's blood comes into contact with the reagent 22 in the device for sampling, initiating a reaction of binding. The reagent contains one labeled with Fitc antibody anti-CD4 and one APC labeled antibody anti-CD8. Within a few minutes the blood sample reacts with the reagent 22, so labeled by the fluorophore antibodies will bind with markers CD4 helper T lymphocytes with markers CD8 T lymphocyte killer cells of the blood sample, respectively, and now the sample is ready for analysis. Device for sampling is placed in the device for analysis, the stage 104. The analysis can be initiated by pressing the button of the device for analysis. Alternatively, the analysis is automatically initiated by the device, detecting the presence of devices for sampling.

The sample is irradiated with LED in combination with chromatic filter adapted to allow the passage only of light of about 450 nm, the stage 106. This allowed the light goes directly to the sample at a small angle relative to the upper surface of the device for sampling. LED light is absorbed by the fluorophores Fitc, aiming lymphocytes CD4+, while Fitc emits light of about 500 nm. CCD camera is used to obtain the fluorescent image of the sample without any optical zoom, the stage 108. The camera is in connection with chromatic filter adapted to pass only the light on the wavelength of about 500 nm in the camera. This ensures that the digital image will contain bright spots/areas in positions labeled helper T lymphocytes.

The sample was then again, in the same manner as described above is irradiated with light, now in combination with chromatic filter, which allows passage of only light about 590 nm through the sample, thus exciting the fluorophores APC, aiming T killer lymphocytes CD8+. By analogy with detection of cells labeled with Fitc, use a chromatic filter, enabling only the light about 640 nm to pass in the CCD camera, thus receive the second digital image, this time containing bright spots/areas in the provisions of labeled T lymphocytes, killer cells present in the sample.

The obtained digital image is transferred to an image analyzer, performing the analysis with the electronic image magnification, the stage 110, for counting the number of bright points on the corresponding digital image. The image analyzer is thus capable of determining concentrations of helper T lymphocytes and T lymphocytes, killer cells, respectively, in the blood sample. The image analyzer is also able to combine the two images to determine whether there are any cells that, against expectations, demonstrate both CD4 and CD8.

Alternatively, LM is the cue pattern whole blood, in this case, can react or partially react with the reagent 22 (antibodies) outside the device 10 for sampling, after which unreacted or partially reacted sample can be selected in the device for sampling.

In one embodiment, the implementation of the reagent 22 contains labeled with fluorophore secondary antibody having affinity for the primary antibody, a secondary antibody is present in the measurement cavity 20 of the device 10 for sampling in the dry form. The liquid sample, thus, the first out of the device for sampling, processed by a primary antibody that binds to a specific desired molecular structure of the target biological components of the sample. The sample, including primary antibodies, and then is collected in a device for sampling, holding secondary antibody. Thus, the secondary antibodies are mixed with the sample and bind with the primary antibodies, which, in turn, bind to the target biological components, and the target biological components are marked by means of a fluorophore. This implementation ensures that the antibody conjugated with a dry fluorophore, can be used for marking many different kinds of biological components, insofar as those whom onanti pre-processed primary antibody having an affinity for him. Thus, pretreatment of the sample makes it possible to use the device 10 for sampling for many different applications, and there is no need to adapt the device for selection of samples for use in the detection of only one biological component.

It must be emphasized that the preferred embodiments of described herein are not limiting, and many alternative embodiments are possible within the scope of protection defined by the attached claims.

1. Device for sampling for the detection of biological components in a liquid sample, containing:
measurement cavity for receiving the liquid sample, and measuring the cavity has a predetermined fixed thickness not exceeding 170 μm, while the measurement cavity has a portion which is adapted to receive his image, to support the analysis of a particular sample volume, whereby can be obtained numerical determination of the volume concentration of the biological component in the sample; and
the reagent, which is available in dry form inside the measurement cavity, and the reagent contains, as its components, molecule, conjugate with the fluorophore, which is adapted for binding the deposits with biological components, and with all the other reactive components, if any, within the measuring cavity are adapted to bind with biological components,
where all reactive components within the measuring cavity are adapted to bind with biological components that are soluble and/or suspendiruemye in a liquid sample.

2. Device for sampling according to claim 1, where the molecule conjugated with a fluorophore, is adapted to bind to a specific molecular structure of the biological component.

3. Device for sampling according to claim 1 or 2, where the device for sampling includes a case element having two planar surfaces that define the specified measuring cavity.

4. Device for sampling according to claim 3, where the planar surface is located at a given distance from each other to determine the thickness of the sample for optical measurements.

5. Device for sampling according to claim 1, where measuring the cavity has a uniform thickness of from 50 to 170 μm.

6. Device for sampling according to claim 5, where the measurement cavity has a uniform thickness of at least 100 microns.

7. Device for sampling according to claim 5 or 6, where the measurement cavity has a uniform thickness of not more than 150 μm.

8. Device for selection of treatment the samples according to claim 1, optionally containing input for sample connecting the measuring cavity with an external device space for sampling, and the entrance is made with the possibility of selection of the liquid sample.

9. A device for sampling of claim 8, where the input is adapted to filter a liquid sample by capillary forces.

10. Device for sampling according to claim 1, where the reagent is deposited on the surface dissolved in a volatile liquid, which evaporates, leaving the reagent in dry form.

11. Device for sampling according to claim 1, where the molecule conjugated with a fluorophore, is an antibody or antibody fragment.

12. Device for sampling according to claim 1, where the device for sampling is disposable.

13. Method of detecting labeled with fluorophore biological components in a liquid sample, comprising:
the mixing of the reagent containing molecule, conjugate with the fluorophore, with the liquid sample, so that the molecule conjugated with a fluorophore that binds to a specific molecular structure of the biological component in the liquid sample;
the introduction of liquid sample into a measurement cavity of the device for sampling and measuring the cavity has a predetermined fixed thickness not exceeding 170 microns;
the irradiated area of the sample in the measurement cavity is electromagnitism radiation wavelength, the appropriate wavelength of excitation of the fluorophore;
detection of labeled fluorophores biological components throughout the thickness of the measurement cavity, and the detecting includes receiving a digital image of the irradiated area in the measuring cavity; and
numerical analysis of digital images to identify biological components, demonstrating the fluorophore, and the determination of the number of biological components, demonstrating the fluorophore in the sample;
where biological components, demonstrating the fluorophore, differ on the digital image as fluorescent dots that emit electromagnetic radiation of a wavelength corresponding to the wavelength of emission of the fluorophore.

14. The method according to item 13, where said device for sampling contains a reagent, placed in dry form inside the measurement cavity, and the reagent contains a molecule conjugate with a fluorophore, and a specified mixing receive through the introduction of a liquid sample into a measurement cavity for coming in contact with the reagent.

15. The method according to item 13 or 14, where the digital image is obtained using the optical zoom 3-50x, more preferably 3-10x.

16. The method according to item 13 or 14, where the specified analysis includes the identification of areas of digital depicts the I, the resulting emission of electromagnetic radiation.

17. The method according to item 13 or 14, where the specified analysis involves identifying points in a digital image, resulting from the emission of electromagnetic radiation.

18. The method according to item 13 or 14, where the specified analysis includes overlaying two or more images, each image shows the corresponding specific emitted wavelengths.

19. The method according to item 13 or 14, where the specified analysis includes an electronic zoom of the received digital image.

20. The method according to item 13 or 14, where the liquid sample is introduced into the measuring cavity of the device for sampling through the capillary input sample by capillary forces.

21. The method according to item 13 or 14, where the specified digital image to be obtained with a depth at least corresponding to the thickness of the measurement cavity.

22. The method according to item 13 or 14, where the volume of the analyzed liquid sample is defined by the thickness of the measurement cavity and the sample area, for which you get the picture.

23. The method according to item 13 or 14, where the specified irradiation is performed using a light source containing the led.

24. The method according to item 13 or 14, where the specified wavelength corresponding to the wavelength of the excitation gain is through the use of LEDs in combination with chromatic filter.