Flow cytometer measurement method and kit for implementing the measurement method

Abstract

In a flow cytometric measurement method, an analytical medium is provided, which contains a fluid and a biological cell contained therein. A labeling molecule is provided and brought into contact with the analytical medium so that if the cell has a target structure located on the surface of the cell, the labeling molecule can specifically bind to the target structure. Flow cytometric measurements of a single cell are captured as first and second physical parameters. The first parameter is the fluorescent radiation emitted by the labeling molecule when the labeling molecule is excited. The cells are classified based on the flow cytometric measurements. A first calibrator and a second calibrator are provided, which have solid particles that match in shape, size and material. The target structure that matches the target structure of the cell is fixed on the surface of the first calibrator. The second calibrator does not have the target structure. Before capturing the flow cytometric measurements, the calibrator is mixed with the analytical medium. The corresponding first and second flow cytometric measurements of the calibrator and the cell are captured. The normalized first flow cytometric measurement of the cell is formed by the first flow cytometric measurement of the first calibrator, the first flow cytometric measurement of the second calibrator and the first flow cytometric measurement of the cell.

Description

[0001] The present invention relates to a flow cytometric measurement method, wherein an analysis medium is provided, the medium comprising a fluid and biological cells to be classified contained therein, wherein at least one marker molecule is provided and brought into contact with the analysis medium so that if the cell has at least one target structure located on the cell surface, the marker molecule can specifically bind to this target structure, a fluid flow of the analysis medium is generated, wherein the cells individually enter the measurement range of the energy beam and / or the electric field, and for individual cells located in the measurement range, at least a first flow cytometric measurement value as a first physical parameter and a second flow cytometric measurement value as a second physical parameter are recorded in each case, wherein at least the first parameter is the fluorescence radiation emitted by the at least one marker molecule under excitation by the energy beam or the electric field, and the cells are classified according to the flow cytometric measurement values. The present invention also relates to a kit for implementing the method.

[0002] The term flow cytometry (cytometry: cell measurement) describes a measurement method used in biology and medicine. It enables the high-speed analysis of biological cells that pass individually through an electric field or a light beam. Different flow cytometric measurements are recorded, from which the properties of the individual cells can be derived and thus the cells can be classified, depending on their shape, structure and / or color.

[0003] In one form of flow cytometry, fluorescently labeled cells are sorted into different reagent containers according to their staining. The corresponding equipment is called a flow sorter or FACS (Fluorescence Activated Cell Sorting). Sometimes the term FACS (=Fluorescence Activated Cell Sorting) is used for equipment that does not sort the cells, but only analyzes or classifies their properties.

[0004] The principle of the examination is based on the light signal emitted by the cells when they pass through a laser beam. The sample is surrounded by an envelope current and passes into a microchannel of a high-precision cuvette made of glass or quartz in such a way that each cell is guided one by one through the measuring range of the laser beam. The resulting scattered light or fluorescence signal is recorded by a detector. The result is quantitative information about each individual cell. By analyzing a large number of cells in a very short time interval (>1000 cells / s), representative information about the cell population contained in the analysis medium can be quickly obtained.

[0005] In parallel with scattered light, fluorescent colors can also be measured in a flow cytometer. Only a few cells fluoresce on their own. Therefore, marker molecules such as dyes are used, which bind to certain target structures of the cell. For example, if the dyes DAPI (4',6-diamidino-2'-phenylindole dihydrochloride) and propidium iodide are used, they bind to the deoxyribonucleic acid (DNA) of the cell (DAPI) or are intercalated therein (propidium iodide) - i.e. intercalated between the bases. The brightness of the fluorescent radiation emitted by at least one marker molecule fixed to the cell when it passes through the laser beam can be used to determine how much DNA the cell contains.

[0006] Antibodies labeled with fluorescent dyes can also serve as marker molecules. Antibodies are usually directed against certain surface proteins, such as CD-classified proteins (CD: Cluster of Differentiation). After the labeling has been completed, the cells can be classified according to these characteristics if necessary. The number of dyes that can be used and the information density can be increased by using lasers of different colors and, most importantly, using filters. Antibodies are the most frequently used molecules for labeling surface proteins of cells. A marker molecule is understood to be an antibody labeled with at least one optical marker and / or other molecule, suitable for specifically binding to a target structure located on the surface of a cell (e.g. a cancer cell) contained in the analysis medium and optically marking the cell.

[0007] Although in the case of fluorescently labeled cells, flow cytometry can both measure the number of labeled cells and obtain measurements of the fluorescence signal emitted by individual cells, quantitative assessment of the fluorescence signal is difficult due to the differences in the flow cytometers used for the measurements, and therefore the comparability of flow cytometric measurements cannot be guaranteed when performed with different flow cytometers. In particular, flow cytometric readings are affected by variations in the intensity of the laser radiation used to illuminate the measurement area, tolerances of the optical filters, the design of the flow cell (fluidics), the reagents used, the detectors, and the samples containing the labeled molecules.

[0008] Although it is possible to quantify the fluorescence readout of a cell by comparing the first flow cytometric readout to a threshold, this is often set arbitrarily. Accurate quantification of binding events for each cell, and thus universal comparability of fluorescence readouts independent of the platform, is currently not possible.

[0009] It is also known to calibrate a flow cytometer using fluorescent particles placed in a liquid that pass through the measuring range of an energy beam or electric field. However, it has been found that such calibration is not sufficient to accurately compare fluorescence readings measured by different flow cytometers.

[0010] Therefore, the object of the present invention is to provide a flow cytometric measurement method mentioned at the outset, which method enables very accurate quantitative detection of target structures on the cell surface. In addition, the quantitative measurement values ​​obtained by fluorescence measurement should be accurately compared with each other (reference / normalization) when measured with different flow cytometers. Another task of the present invention is to provide a test kit for implementing this method.

[0011] This object is achieved with respect to a flow cytometric measurement method having the features of claim 1. According to the invention, a flow cytometric measurement method is provided, wherein an analysis medium is provided, which contains a fluid and biological cells to be classified contained therein, wherein at least one marker molecule is provided and brought into contact with the analysis medium in such a way that, if the cells have at least one target structure located on the cell surface, the marker molecule can specifically bind to this target structure, and a fluid flow of the analysis medium is generated. Here, the cells individually enter the measurement region of the energy beam and / or the electric field, wherein for individual cells located in the measurement region, the energy beam and / or the electric field is applied to each cell. Therein, a flow of the analysis medium is generated, the cells individually enter the measurement range of the energy beam and / or the electric field, and for individual cells located in the measurement range, at least a first flow cytometric measurement value as a first physical parameter and a second flow cytometric measurement value as a second physical parameter are recorded in each case, wherein at least the first parameter is fluorescence radiation, and the at least one marker molecule fluoresces under excitation by the energy beam or the electric field. Therein, the cells are classified according to the flow cytometric readings. Wherein at least one first calibrant and at least one second calibrant are provided, each calibrant comprising solid particles of inorganic and / or polymeric material that is not water soluble, the solid particles of the at least one first calibrant and the at least one second calibrant matching in shape, size and material. Wherein at least one first calibrant has at least one target structure on its surface, which corresponds to at least one target structure of the cell and is fixed on the solid particles of the first calibrant, wherein at least one second calibrant does not have the target structure. Wherein before the flow cytometric measurement is performed, the at least one first calibrant and the at least one second calibrant are mixed with an analysis medium so that at least one marker molecule present in the analysis medium binds to at least one target structure of the first calibrant, wherein the calibrants are introduced individually one by one into a measurement region in a fluid stream. Wherein first and second flow cytometric measurements corresponding to the cell are recorded for the at least one first calibrant and the at least one second calibrant, wherein a parameter related to the second flow cytometric measurement of the cell and the second flow cytometric measurement of the calibrant is selected so as to distinguish the calibrant from the cell based on the second measurement. wherein the normalized first flow cytometer reading of at least one cell is formed by at least one first flow cytometer reading of at least one first calibrator, at least one first flow cytometer reading of at least one second calibrator, and a first flow cytometer reading of at least one cell.

[0012] The present invention is based on the recognition that the tolerance of the reagents used to detect the binding of the marker molecules to the target structures of the cells, the variability of the marker molecules and the composition of the analytical medium have a great influence on the flow cytometric measurement values ​​of the cells to be classified. These tolerances are particularly affected by the following factors: the age of the marker molecules, the storage method and conditions of the marker molecules before the flow cytometric measurement process, and substances contained in the analytical medium in addition to the cells, such as inhibitors, which make it more difficult for the marker molecules to bind to the target structures or replace the marker molecules in binding to the target structures. In addition, environmental conditions such as temperature can also affect the flow cytometric readings of cells.

[0013] In the method according to the invention, these influences are compensated by providing standardized first and second calibrators, carrying out corresponding flow cytometric readings of these calibrators and cells, and normalizing the measured cell flow cytometric readings by means of the flow cytometric readings of the calibrators. This allows accurate quantification of the binding events occurring between each cell and the marker molecule or its antibody. The normalized flow cytometric readings also allow accurate comparison of flow cytometric measurements performed using different flow cytometers and / or using reagents or marker molecules with different properties. This makes it possible, in particular, to determine cell subpopulations belonging to a class by cluster analysis. Since the calibrators are not living cells, but solid particles as carriers of at least one target structure, the calibrators are stable over a long period of time, i.e. they can be stored for a long time without significant changes in their properties related to the detection of the target structure.

[0014] The parameter space for each analysis depends on the number of maximal binding events per cell and the measurement parameters. This means that, for example, when three parameters are determined, they generate a three-dimensional space whose axes are not necessarily at right angles to each other. The type of labeling and the labeling density (number of fluorophores per antibody) as well as the binding strength and specificity of the antibodies (or labeling molecules) used also play a role.

[0015] In the method according to the invention, normalized flow cytometric values ​​are provided for the cells, which are independent of the physical properties of the flow cytometer used to implement the method, independent of the tolerances of the reagents and labeling molecules used, and independent of substances and active agents that may be contained in the analysis medium besides the cells.

[0016] In an advantageous embodiment of the invention, the difference between the first flow cytometer reading of the first calibrator and the first flow cytometer reading of the second calibrator is proportional to the difference between the first flow cytometer reading of the cell and the first flow cytometer reading of the second calibrator, forming a normalized first flow cytometer reading. When the first flow cytometer reading of the cell is equal to the first flow cytometer reading of the first calibrator, the normalized flow cytometer reading of the cell is preferably 100%. When the first flow cytometer reading of the cell is consistent with the first flow cytometer reading of the second calibrator, the value of the normalized flow cytometer reading of the cell is zero. The first calibrator is preferably designed so that the first flow cytometer measurement value of at least one first calibrator is equal to or slightly higher than the expected maximum value of the first flow cytometer measurement value of the cell, in particular up to 3%, up to 5%, up to 10% or up to 25% higher than the expected first flow cytometer measurement value of the first calibrator. The corresponding expected value can be determined experimentally.

[0017] In a preferred embodiment of the invention, a plurality of identical first calibrators and a plurality of identical second calibrators are provided and mixed with the analytical medium before recording the flow cytometer reading of each calibrator, wherein the average first flow cytometer reading of the first calibrator is formed from the first flow cytometer reading of the first calibrator and the average first flow cytometer reading of the second calibrator is formed from the first flow cytometer reading of the second calibrator; and

[0018] - wherein the difference between the average first flow cytometer reading of the first calibrator and the average first flow cytometer reading of the second calibrator is proportional to the difference between the first flow cytometer reading of the cell and the average first flow cytometer reading of the second calibrator to form a normalized first reading of the cell.

[0019] or

[0020] wherein, in order to form a normalized first measured value for a cell population consisting of a plurality of cells, first flow cytometric measured values ​​of a single cell are recorded in each case, wherein an average first flow cytometric measured value of the cell population is formed from these flow cytometric measured values, and wherein in order to form a normalized first measured value for a plurality of cells,

[0021] a) the difference between the mean first flow cytometer reading of the first calibrator and the mean first flow cytometer reading of the second calibrator is proportional to the difference between the first mean flow cytometer reading of the population of cells and the mean first flow cytometer reading of the second calibrator, or

[0022] b) For a signal having a predetermined signal intensity, in particular a signal of 100%, forming a quotient between the average first flow cytometric measurement value and the average first flow cytometric measurement value of the calibrator.

[0023] The average value can be determined using known statistical methods, in particular using the arithmetic mean. If necessary, the normalized first measured value can be additionally scaled, whereby the scaling factor can correspond to the quotient of the average first flow cytometer measured value of the first calibrator and the number 100, for example.

[0024] In another embodiment of the invention, a first calibration factor is provided for at least one first calibrator, the factor corresponding to the ratio between the intensity of the measured signal of the first calibrator and the intensity of the measured signal of a first reference calibrator having a target structure, the first measured signal is detected as the first physical parameter of the first calibrator, and a first flow cytometric measurement value of the first calibrator is formed by the first measured signal and the first calibration factor, wherein a second calibration factor is provided for the second calibrator, the factor corresponding to the ratio between the intensity of the measured signal of the second calibrator and the intensity of the measured signal of a second reference calibrator not having a target structure, and wherein the second measured signal is acquired as the first physical parameter of the second calibrator, and the first flow cytometric measurement value of the second calibrator is formed by the second measured signal and the second calibration factor.

[0025] The calibration factor makes it possible to compare flow cytometer measurements obtained with different batches of the first calibrator and / or the second calibrator, even more precisely to one another. The calibration factor is preferably measured experimentally under precisely defined conditions.

[0026] First, an expected value of a first flow cytometric measurement is determined for each of the first and second reference calibrators of the first batch production. Instead of the analytical medium, a buffer with defined, constant properties is used, in which the first and second reference calibrators are placed. The buffer contains no biological cells. In this buffer, corresponding first flow cytometric values ​​of a number of identical or substantially identical first or second reference calibrators are measured. From the reference flow cytometric measurements obtained in this way, an expected value for each of the first and second reference calibrators is determined using known statistical methods.

[0027] In a further step, expected values ​​for the first and second calibrants of the second batch are determined in a corresponding manner.

[0028] The first calibration factor is then determined by forming a quotient from the expected value of the first flow cytometer measurement of the second batch of first calibrators and the expected value of the first flow cytometer measurement of the first reference calibrator. Similarly, the second calibration factor is determined by taking the quotient of the expected value of the first flow cytometer reading of the second calibrator and the expected value of the first flow cytometer reading of the second reference calibrator.

[0029] In an advantageous embodiment of the invention, at least two types of first calibrants are provided and mixed with the analytical medium, wherein the different types of calibrants differ from one another, in particular in terms of the surface occupancy density of the solid particle surface and / or the arrangement of at least one target structure thereof, so that the signal intensity of the first flow cytometric measurement of the first calibrant type is greater than the signal intensity of the first flow cytometric measurement of the second calibrant type and less than the signal intensity of the first flow cytometric measurement of the second calibrant type. This makes it possible to compensate for the influence of nonlinear signals during the acquisition of the flow cytometric measurement values.

[0030] Advantageously, the fluid contains at least two different cell populations, which differ in that the first flow cytometric measurement value of the first population of cells is within a first signal intensity range, the first flow cytometric measurement value of the second population of cells is within a second signal intensity range outside the first signal intensity range, and the signal intensity of the first flow cytometric measurement value of the first calibrator type is selected in such a way that it is between the first and second signal intensity ranges. The calibrator of the first calibrator type generates a signal intensity that allows the reference ranges of the two populations to be distinguished from each other, i.e. cells having a first flow cytometric value that is less than the first flow cytometric value of the first calibrator type belong to the first population, and cells having a higher flow cytometric value belong to the second population.

[0031] In a preferred embodiment of the invention, the largest dimension of the solid particles is less than 20 μm, in particular less than 15 μm, preferably less than 10 μm, and / or the smallest dimension of the solid particles is greater than 4 μm, in particular greater than 5 μm, preferably greater than 6 μm. Suitable solid particles are, for example, microspheres with a diameter of 6-10 μm. The solid particles preferably have a size similar to that of the cells in the analysis medium.

[0032] The solid particles are preferably synthetic particles. The solid particles may include polystyrene, melamine, latex and / or silicate or consist of any of these materials.

[0033] The labeling molecule may be a molecule that binds to the target antigen on the surface of a cell or calibrant and can subsequently be used to detect these target antigens. Such molecules are, for example, antibodies and their fragments, lectins, other binding proteins (e.g., protein A).

[0034] The target antigen on the first calibrant is preferably covalently fixed. Methods for producing such covalent bonds are known in the prior art, including, for example, binding to amines (via crosslinkers), thiophenes, epoxides, aldehydes, maleimides and other groups. Suitable methods and chemical conversions thereof are described in the literature (Hermanson, Greg T., "Bioconju-gate Techniques, Third Edition" (2013). However, non-covalent methods, such as His-tag, biotin-streptavidin, protein G, protein A, pre-fixed antibodies, can also be used.

[0035] The target antigen or target structure on the first calibrator can be, for example, a protein, a peptide, a receptor, an allergen, a glycosylated protein, a liposaccharide, an oligosaccharide and a polysaccharide, a nucleic acid, and fragments and derivatives thereof.

[0036] In a preferred embodiment of the invention, at least one second flow cytometer measurement value comprises a forward scatter measurement value of the energy beam occurring within the measurement range and / or a side scatter measurement value of the energy beam occurring within the measurement range. These measurements can be obtained in a simple manner by suitable sensors. The forward scatter measurement value is a measurement of size, while the side scatter measurement value is a measurement of the granularity of the cell or calibrant. On the basis of these measured variables, cells can be distinguished from calibrants. Therefore, the measured flow cytometer values ​​can be clearly assigned to cells and calibrants based on the forward and side scatter values.

[0037] However, it is also possible that the at least one second flow cytometric measurement is a fluorescence measurement of a fluorescence radiation emitted by a label molecule which is different from the fluorescence radiation of the first flow cytometric measurement.Preferably, the wavelength of the fluorescence radiation of the first and second flow cytometric readings is different.

[0038] Advantageously, at least one first flow cytometric measurement is recorded for the cells and the first and second calibrators, if in each case at least two, in particular at least several, different marker molecules corresponding to the number of measurement channels are provided and contacted with the analysis medium, and if at least one first calibrator has at least several target structures corresponding to the number of measurement channels, wherein each structure is able to bind specifically to one of the different marker molecules when contacted with the associated marker molecule.

[0039] However, it is also conceivable that, for the cells and the first and second calibrators, in each case at least one first flow cytometer measurement value is recorded on several measurement channels, at least different marker molecules corresponding to the number of measurement channels are provided and contacted with the analysis medium, at least two first calibrators of the calibrator type are provided, at least one first calibrator of the first calibrator type has at least one first target structure on its surface that corresponds to the at least one first target structure of the cell and is adjusted on solid particles of the calibrator, at least one first calibrator of the second calibrator type has at least one second target structure on its surface that corresponds to the at least one second target structure of the cell and is immobilized on solid particles of the calibrator, and at least one first calibrator of the first calibrator type has a second target structure on its surface and at least one first calibrator of the second calibrator type has no first target structure on its surface. In this case, at least several different first calibrators corresponding to the number of measurement channels can be provided and mixed with the analysis medium, and these first calibrators can have different target structures, each having a binding specificity for a different one of the marker molecules.

[0040] The use of several measurement parameters and their calibrants is only limited by the number of measurement channels of the measuring instrument and the available fluorophores. Six to eight parameters can be routinely recorded. For the analysis of AML cells, 30 relevant parameters have been recorded to date, but they had to be recorded in several measurements. With modern equipment, measurements can also be performed in the near-infrared range, and significantly more than eight parameters can be recorded. If subclasses of cells are to be recorded in such a parameter space, the resolution of the measurement is important. The number of measurements that can be recorded per measurement channel can be increased by using calibrants from another particle class, whose solid particles differ in size from those of the calibrants from the first particle class.

[0041] Since the first calibrant (relative signal intensity 100%) allows the expected maximum signal to be recorded in a device-specific manner, the resolution can be increased (e.g. by adjusting the laser power or the amplifier power of the detector in order to obtain the maximum resolution from the measurement). With such a recorded and processed signal, cell populations can now be detected with the help of mathematical procedures (e.g. cluster analysis - a standard mathematical procedure described at https: / / de.wikipedia.org / wiki / Clusteranalyse). This means that very variable diseases like leukemia can be better typed and thus also better treated, since corresponding markers of treatment response can be recorded and evaluated. In the special case of lymphomas and leukemias, there are markers that are known to have a strong influence on the prognosis of the disease. For example, current studies have shown that, in addition to known markers such as CD38, the expression of CD49d has a very strong influence on the course of chronic lymphocytic leukemia (CLL) (DOI: 10.1111 / j.1365-2141.2011.08725.x).

[0042] In a preferred embodiment of the present invention, the target structure on the first calibrator comprises at least one antigen, preferably at least one protein and / or at least one peptide and / or at least one receptor and / or at least one allergen and / or at least one glycosylated protein and / or at least one liposaccharide and / or at least one oligosaccharide and / or at least one polysaccharide and / or at least one nucleic acid and / or at least one fragment and / or derivative of the above substances.

[0043] In an advantageous embodiment of the invention, the target structure of at least one first calibrant is immobilized on solid particles of the first calibrant via at least one activated carboxyl group and / or at least one activated NH2 group. This allows the target structure, such as an antigen, to be stably coupled and immobilized on the solid particles. Corresponding microparticles are commercially available.

[0044] Advantageously, at least one target structure immobilized on the solid particles of the first calibrant corresponds to one of the typical target structures of at least one blood cancer cell. The method according to the invention can then be used to classify leukemia cells. In particular, fast-growing M-type cells can be distinguished from less aggressive D-type cells.

[0045] In a preferred embodiment of the invention, at least one target structure immobilized on the solid particles of the first calibrant corresponds to one of the target structures found within the B cell receptor (BCR) on the surface of chronic lymphocytic leukemia (CLL) cells. These leukemia cells are particularly aggressive. Due to its high affinity and specificity, this target structure is very suitable for flow cytometry diagnosis (FACS).

[0046] The calibrant is added to the analytical medium, such as blood, during the measurement. A suitable concentration of the calibrant is preferably 1x10 5 pcs / 1x10 6 During the measurement, the calibrants are identified and separated from the target cells by forward scatter (size) and side scatter (granularity) parameters.

[0047] With regard to the test kit, the above-mentioned object is achieved by the features of claim 14.

[0048] The present invention, the kit comprises:

[0049] - at least one first calibrant, comprising a first solid particle of a water-insoluble inorganic and / or polymeric material, the surface of the first calibrant having at least one target structure fixed on the first solid particle,

[0050] - at least one second calibrant comprising second solid particles matching the first solid particles in shape, size and material, the surface of the second calibrant being free of target structures, and

[0051] - At least one labeled molecule that specifically binds to the target structure.

[0052] With such a kit, it is possible to very precisely and quantitatively record target structures on the surface of biological cells contained in the fluid to be classified during flow cytometric measurements. The corresponding measured values ​​can be precisely compared with one another - even if they were measured by different flow cytometers. At least one target structure is preferably an antigen. If necessary, the kit can additionally contain a buffer.

[0053] In a preferred embodiment of the kit, several different target structures are arranged on the surface of at least one first calibrant, which are immobilized on first solid particles. For each of these target structures, the kit has at least one binding-specific label molecule for the respective target structure. With such a kit, parallel measurements can be performed. The parallel loading of the calibrants and the target structures is only limited by the occupancy density of the solid particles and therefore by the maximum achievable signal.

[0054] However, it is also conceivable that the kit for parallel measurements has several different first calibrators, which have different target structures on their surface. Each first calibrator can only have one target structure. It is also possible that at least one first calibrator has several regions with target structures, which have binding specificity for the same marker molecule.

[0055] The labeling molecule preferably carries a fluorescent dye. Such a dye is usually covalently bound to the labeling molecule. A large number of such dyes are commercially available, for example Cy3, Cy5, Cy7, FITC, rhodamine, phycoerythrin, lysamine; a list of various dyes can be found at the following website: https: / / en.wikipedia.org / wiki / Fluorophore.

[0056] In a preferred embodiment of the invention, the kit comprises at least one data carrier on which are stored at least one first calibration factor of a first calibrator and / or at least one second calibration factor of a second calibrator. With such a kit, tolerances of the calibrators can be compensated. This is particularly advantageous if the measurement results of flow cytometric measurement processes are to be compared with one another, and these measurement processes were carried out with different batches of the first or second calibrators. The data carrier may be a machine-readable data carrier, for example, there may be a carrier provided with a barcode or a QR code. However, it is also possible that the calibration factors are printed in digital form on a document belonging to the kit, such as an instruction manual or a packaging belonging to the kit.

[0057] Advantageously, the kit comprises at least two types of first calibrants, and if the different types of calibrants differ from each other, in particular in the surface occupancy density on the surface of the solid particles and / or the arrangement of at least one of their target structures, such that they generate measurement signals with different signal intensities during flow cytometric measurements when their target structures are labeled with labeling molecules, such a kit can be used for regression in flow cytometric measurements where the fluorescence signal has a nonlinear response.

[0058] Further details, features and advantages of the invention will become apparent from the following description of embodiments with reference to the accompanying drawings, which show:

[0059] Figure 1 is a graphical representation of the measurements of the second calibrator population (zero calibrator) obtained by means of flow cytometry, the measured intensity PE being plotted on the horizontal axis and the number of binding events n being plotted on the horizontal axis.

[0060] Figure 2 is similar to Figure 1 , but showing the measured values ​​of a first calibrant population of a first calibrant type.

[0061] Figure 3 is similar to Figure 1 , but showing the measured values ​​of a population of first calibrants of a second calibrant type.

[0062] Figure 4 is a standard curve, where every 2.1·10 6The amount of CD23 protein per solid particle is plotted on the horizontal axis, and the MFI value (mean fluorescence reading) is plotted on the vertical axis.

[0063] Figure 5 Graph of the measured signals for the parameters size and granularity, wherein the forward scatter (FSC) assigned to the parameter size is plotted on the horizontal axis and the side scatter (SSC) assigned to the parameter granularity is plotted on the vertical axis of the fluorescence radiation of cells and calibration particles.

[0064] Figure 6 is a graphical representation of the measured intensity values ​​of IgM and CD19 of cells and calibration particles, wherein the intensity of IgM is plotted on the horizontal axis and the intensity of CD19 is plotted on the vertical axis.

[0065] Figure 7 is a graphical representation of the intensity values ​​of IgD and CD19 measured in cells and calibration particles, wherein the intensity of IgD is plotted on the horizontal axis and the intensity of CD19 is plotted on the vertical axis.

[0066] Figure 8 is a graphical representation of the intensity values ​​of IgD and IgM measured in cells and calibration particles, with the intensity of IgD being plotted on the horizontal axis and the intensity of IgM being plotted on the vertical axis.

[0067] Fig. 9 is based on Figures 6 to 8 A three-dimensional representation of the measurement space, and

[0068] Fig.10 Yes Figures 6 to 9 Illustration of the cluster analysis performed on the measurement space shown.

[0069] In a first embodiment of the present invention, a disease-associated antigen is provided as a target structure. IgM (immunoglobulin M) is provided as a first target structure, and IgD (immunoglobulin D) is provided as a second target structure.

[0070] In addition, a plurality of solid particles, namely microspheres made of polystyrene and having a diameter of 6 μm, are provided. The solid particles are matched in shape, size and material.

[0071] The surface of the solid particles is coated with carboxyl groups (-COOH) to which they are bound. NH2 groups may be present on the surface of the solid particles to replace or supplement the carboxyl groups. The first and second target structures may be bound to the surface of the solid particles via the carboxyl groups and / or the NH2 groups.

[0072] In addition, a first and a second marker molecule are provided. The first marker molecule is an antigen having binding specificity for the first target structure IgM. The second marker molecule is an antigen having binding specificity for the second target structure IgD. The marker molecules each have at least one optical label that emits fluorescent radiation when excited with appropriate excitation radiation.

[0073] 1.1 Coupling of disease-related antigens to solid particle surfaces

[0074] Thirteen solid particle groups are provided, each particle group comprising the same number of solid particles having carboxyl groups on the surface.

[0075] The first target structure is coupled to the solid particle surfaces of six solid particle groups, and the second target structure is coupled to the solid particle surfaces of another six solid particle groups. In addition, the following substances are added to the solid particles:

[0076] - 2.00 μg IgM of the first solid particle population,

[0077] - Second solid particle population 1.00 μg IgM,

[0078] - The third solid particle population 0.40 μg IgM,

[0079] - The fourth solid particle group 0.20 μg IgM,

[0080] - The fifth solid particle group 0.08 μg IgM,

[0081] - The sixth solid particle group 0.04 μg IgM,

[0082] - The seventh solid particle group 2.00 μg IgD,

[0083] - The eighth solid particle group 1.00 μg IgD,

[0084] - Ninth solid particle group 0.40 μg IgD,

[0085] - The tenth solid particle group 0.20 μg IgD,

[0086] - 0.08 μg IgD of the eleventh solid particle group, and

[0087] - 12th solid particle group 0.04 μg IgD,

[0088] Every 1.2·10 6Each of the solid particles was added to the respective solid particle group and incubated for 60 minutes with constant stirring in binding buffer (50 mM MES, pH 5.2; 0.05% Proclin). Coupling was performed according to the standard protocol and activated with EDAC (carbodiimide). The solid particles were washed and activated in EDAC solution (20 mg / ml).

[0089] After centrifugation at 800 g to separate the single solid particle population, the supernatant was removed and kept as a control measurement of the remaining antigen concentration. The solid particles were taken into the wash buffer and resuspended. After repeated centrifugation, the supernatant was discarded and the particles were added to the wash and storage buffer (10 mM Tris, pH 8.0; 0.05% BSA; 0.05% Proclin) to saturate the unoccupied reactive groups.

[0090] Neither the first target structure nor the second target structure is coupled to the surface of the thirteenth solid particle. Instead, the reactive COOH group contacts the blocking protein, allowing the blocking protein to non-specifically bind to the COOH group. For example, bovine serum albumin or hydrolyzed casein can be used as the blocking protein.

[0091] 1.2 Determine the concentrations of IgM and IgD for conjugation.

[0092] Each of the thirteen solid particle groups was measured using a flow cytometer (FACS). To this end, the solid particles of the first to sixth solid particle groups and the first marker molecule (about 0.5 μg per measurement) were suspended in a liquid and incubated in the dark for 10 minutes. The sample was centrifuged and the supernatant was discarded. Wash once with 2 ml of PBS buffer (phosphate buffered saline) and then centrifuged again. After discarding the supernatant, the solid particles were introduced into 100-200 μl PBS. The suspension thus obtained passed through the nozzle of a flow cytometer, which limited the measurement range so that the solid particles in PBS entered the measurement range of the laser beam alone. The laser beam excites the first marker molecule that specifically binds to the first target structure, causing it to emit fluorescent radiation. Fluorescence measurement is obtained from this radiation with the help of an optical detector. A large number of fluorescence values ​​(intensity values) are measured for each solid particle group. An average value (MFI value or median fluorescence intensity) is formed by the fluorescence values ​​recorded for individual solid particles of each solid particle group.

[0093] In the same manner, the solid particles of the seventh to twelfth solid particle groups are measured with the help of a flow cytometer. The laser beam excites the second labeling molecule specifically bound to the second target structure, causing it to emit fluorescent radiation.

[0094] Although human IgM (immunoglobulin M) can be easily obtained from serum, it has been found that human recombinant monomeric IgM is more suitable for coupling to solid particles, especially for stability after coupling. In serum, IgM usually exists in the form of pentamers. Studies have shown that the binding of IgM pentamers does not usually result in complete binding. Some IgM molecules of the pentamer are not covalently bound to the particles. Uncontrolled degradation can cause the flow cytometer to lose resolution in subsequent uses. Therefore, recombinantly produced monomeric IgM and IgD are used for both IgM and IgD (immunoglobulin D).

[0095] exist Figures 1 to 3 In the figure, the fluorescence measurement values ​​and average values ​​of the first, second and seventh solid particle groups are shown in a graph. The fluorescence radiation intensity PE of each solid particle group measured by flow cytometry is plotted on the horizontal axis, and the number of binding events n is plotted on the horizontal axis.

[0096] from Figure 4 As can be seen, in each case, the average of the individual fluorescence values ​​was 2.1·10 6 The amount of CD23 protein in each solid particle PM was plotted as a coupling curve. A corresponding coupling curve was generated for the second target structure.

[0097] From the respective coupling curves, the amount of the first or second target structure required for the desired fluorescence intensity or signal intensity can be read. Based on the coupling curves, the maximum average intensity I of each target structure is determined and defined. max Then, the 75% value (P 75 ) and 25% value (P 25 ) is defined as follows:

[0098] P 75 =I max 0.75

[0099] P 25 =P 75 / 10

[0100] In the first example, the coupling results in the following values:

[0101] I max =21700

[0102] P 75 =21700 0.75 = 16275

[0103] P 25 =16275 / 10=1627

[0104] These values ​​are set as reference. The appropriate protein amount can now be read from the coupling curve. 75 For example, this is every 2.1·106 1.3μg per solid particle.

[0105] 1.3 Preparation of calibrants for two CLL characteristic parameters: IgM and IgD

[0106] The aim was to prepare several calibrator pools for each of the first and second targets (IgM and IgD).

[0107] The first calibrant population comprises a plurality of first calibrants of a first calibrant type that are matched, each of which has a solid particle on the surface of which IgM is immobilized as a first target structure, in such a way that in a flow cytometric analysis, the fluorescence signals of the individual first calibrants of the first calibrant type each reach a relative intensity of 25%.

[0108] The second calibrant group comprises a plurality of matched first calibrants of the second calibrant type. Each of these calibrants has the same solid particles as the first calibrant of the first calibrant type. The same target structure as the surface of the first calibrant of the first calibrant type is immobilized on the surface of the solid particles of the first calibrant of the second calibrant type. However, the surface occupancy density of the first calibrant of the second calibrant type and the arrangement of the target structure are selected in such a way that the fluorescence signal of the first calibrant of the second calibrant type reaches 75% of the relative intensity in the flow cytometric analysis.

[0109] The third calibrator group comprises a plurality of first calibrators of a matched third calibrator type. Each of these calibrators has the same solid particles as the first calibrators of the first and second calibrator types. On the first calibrator of the third calibrator type, a second target structure that specifically binds to IgD is immobilized, so that the fluorescence signal of the first calibrator of the third calibrator type reaches 25% of the relative intensity in flow cytometer analysis.

[0110] The fourth calibrant population comprises a plurality of matched first calibrants of a fourth calibrant type. Each of these calibrants has the same solid particles as the first calibrants of the first, second and third calibrant types. The target structures immobilized on the surface of the solid particles of the first calibrants of the fourth calibrant type are the same as the target structures immobilized on the surface of the first calibrants of the third calibrant type. However, the surface density and the arrangement of the target structures of the first calibrants of the fourth calibrant type are selected in such a way that the fluorescence signal of the first calibrants of the fourth calibrant type reaches 75% of the relative intensity in the flow cytometer analysis.

[0111] A fifth calibrator group is also created, comprising a plurality of matched first calibrators of the fifth calibrator type. Each of these calibrators has the same solid particles as the first calibrators of the first, second, third and fourth calibrator types. Two different target structures are immobilized on the surface of the solid particles of the first calibrator of the fifth calibrator type. One of the target structures is the same as the first target structure (binding specificity for IgM) and the other target structure is the same as the second target structure (specific antigen binding for IgD). The area density and arrangement of the antigens IgM and IgD of the first calibrator of the fifth calibrator type are selected so that the fluorescence signal of the first calibrator of the fifth calibrator type reaches a relative intensity of 75% for IgM and IgD in the flow cytometric analysis.

[0112] In addition, a negative group is created, which contains a large number of matched second calibrants. Each of them is composed of a solid particle on which neither the first target structure (IgM) nor the second target structure (IgD) is immobilized. In the negative group, the carboxyl (-COOH) or NH2 groups on the surface of the solid particles non-specifically bind to the blocking protein. The solid particles of the second calibrant are the same as those of the first calibrant.

[0113] To couple the antigen to the solid particles, the required amount of solid particles for each population was transferred to a reaction vessel. The solid particles were centrifuged at 800 g for 5 minutes and the supernatant was discarded. The solid particles were then brought into the coupling buffer. Coupling was performed according to the previously described protocol, using sufficient amounts of IgM for the first, second and fifth calibrator populations and sufficient amounts of IgD for the third, fourth and fifth calibrator populations.

[0114] The calibrant groups provided in this manner are listed below:

[0115] First Calibrator:

[0116] a) IgM calibrator: PD 25 -IgM concentration Int 25

[0117] PD 75 -IgM concentration Int 75

[0118] b) IgD calibrator: PD 25 -IgD concentration Int 25

[0119] PD 75 -IgD concentration Int 75

[0120] c) IgM and IgD calibrators: P md -IgM and IgD concentrations Int75

[0121] Second calibrant: P o -Neither IgM nor IgD

[0122] The individual calibrators are measured by FACS (fluorescence activated cell sorting) using commercially available labeled antibodies. The calibrators are combined into a kit for later use in diagnostics. In this way, the measurement space is defined, standardized and normalized for later FACS measurements for diagnostic purposes.

[0123] 1.4 FACS analysis of blood samples using calibrators.

[0124] A kit is provided, comprising:

[0125] a) a first group of first calibrants of a first calibrant type, comprising a plurality of first calibrants PD described in Section 1.3 25 , on the surface of which IgM is immobilized as the first target structure.

[0126] b) a second group of first calibrants of the first calibrant type, comprising a plurality of first calibrants PD described in Section 1.3 75 , on the surface of which IgM is immobilized as the first target structure.

[0127] c) a first group of first calibrants of a second calibrant type, comprising a plurality of first calibrants PD described in Section 1.3 25 , on the surface of which IgD is immobilized as the first target structure.

[0128] d) a second group of first calibrants of a second calibrant type, comprising a plurality of first calibrants PD described in Section 1.3 75 , on the surface of which IgD is immobilized as the first target structure.

[0129] e) A second calibrant group, comprising a plurality of second calibrants Po described in Section 1.3.

[0130] f) A first population of marker molecules, each having a first antigen and having binding specificity for a first target structure IgM. The first marker molecules each have a first optical label coupled to the first antigen and emit a first fluorescent radiation when excited with an appropriate first excitation radiation.

[0131] g) a second population of marker molecules, each comprising a second antigen, having binding specificity for a second target structure IgD, each of which has a second optical label coupled to the second antigen and emits a second fluorescent radiation when excited with an appropriate second excitation radiation.

[0132] h) Phosphate buffered saline (PBS buffer).

[0133] In FACS-based blood cell analysis, the number of cells to be classified (lymphoma cells) is determined in the analysis medium blood after blood sampling, and the required number of cells for each FACS analysis is added to the reaction vessel (e.g. 1x10 cells per measurement). 6 Then the labeled molecule (about 0.5 μg per measurement) and the calibrant P o , PD 25 , PD 75 and incubate in the dark for 10 minutes. After the incubation time is over, fill the volume with 2 ml of lysis-fixation buffer and incubate again for 10 minutes. In this step, the cells are not only fixed, but also most of the erythrocytes are ruptured. These are not critical for the measurement, but may interfere with it.

[0134] Centrifuge the sample and discard the supernatant. Wash once with 2 ml of PBS buffer (phosphate buffered saline) and centrifuge again. After discarding the supernatant, place the cells in 100-200 μl PBS.

[0135] The resulting analysis medium is then analyzed in a FACS flow cytometer. In a FACS flow cytometer, the analysis medium passes through a nozzle opening with a defined measuring range, so that the cells labeled with the marker molecule and the different calibration objects each enter the measuring range of the laser beam.

[0136] Using a FACS flow cytometer, a first and a second flow cytometer reading are recorded for each calibrator that enters the measurement range and each cell that enters the measurement range. The first flow cytometer reading depends on the fluorescence emitted by at least one labeled molecule bound to the relevant calibrator or cell in the measurement area due to the excitation of the laser beam. The two second flow cytometer readings include a forward scatter reading and a side scatter reading, which are used to measure the scattered radiation generated when the laser beam is irradiated on the relevant calibrator located in the measurement range or the relevant cell located in the measurement range.

[0137] The value of the second flow cytometer is compared with the reference value. Based on the result of the comparison, it can be determined that the second flow cytometer reading is caused by the scattering of the laser beam on the cell or the calibrator, because the scattering properties of the cell and the calibrator are different. Different calibrators can be distinguished by their signal levels of the first flow cytometer reading. Therefore, based on the result of the comparison, the first flow cytometer reading is assigned to the cell or the calibrator.

[0138] In the case of a calibrator, the first flow cytometer reading is compared with the comparison intervals assigned to different calibrator particle populations. Thus, the first flow cytometer reading can be assigned to a calibrator of a specific calibrator particle population. This work can be automated, for example by appropriate software.

[0139] For each individual target structure (IgM or IgD), a single calibrator P was determined. o , PD 25 and PD 75 The respective average values ​​of the first flow cytometric measurement values ​​(fluorescence intensity) of . These form the basis for the reference measurement space for the measurement. o The first flow cytometric measurement of is used to determine the background of the channel. In addition, the calibrant P o The first flow cytometric readings allow an indication of the quality of coupling of the antigens of the labeled molecules to their assigned target structures in each case (negative control).

[0140] In addition, the average of the first flow cytometric readings was determined for cells of each individual target structure.

[0141] In the embodiment, the following average values ​​of the first flow cytometric measurements are determined for IgM:

[0142] First calibrant PD of first calibrant type 25 :1600

[0143] First calibrant PD of first calibrant type 75 ::16000

[0144] The second calibrant P o :25

[0145] Cells of the first target structure: 5600

[0146] First, background noise is removed from the measured values. Background noise is caused by non-specific binding of the antibodies used to label the molecules to solid particles or cells of the calibrant. To this end, a first calibrant PD of the first calibrant type is obtained. 25 and PD 75 , the second calibration object P o The first flow cytometric measurement of the first calibrator type was subtracted from the first flow cytometric measurement of the first calibrator type, respectively. This resulted in the following adjusted first flow cytometric reading of IgM:

[0147] First calibrant PD of first calibrant type 25 :1600–25=1575

[0148] First calibrant PD of first calibrant type 75 :16000–25=15975

[0149] The second calibrant P o :25–25=0

[0150] Cells of the first target structure: 5600–25=5575.

[0151] The reference unit is the calibrant PD 25 and PD 75 Defined. Calibrator PD of the first calibrator type 75 The first accurate (reinigten) flow cytometer reading of is assigned 100 reference units. Therefore, one reference unit corresponds to the adjusted first flow cytometer reading 15975 / 100=159.75. This number is also called the first proportionality factor. The first calibrator PD 25 The adjusted first flow cytometer reading corresponds to 1575 / 159.75=9.86 reference units. A first normalized measurement is formed by the adjusted first flow cytometer measurement of the first target structure of the cell and the first proportionality factor: 5575 / 159.75=34.90 reference units.

[0152] In the example, the following average values ​​of IgD were determined for the first flow cytometer measurement values.

[0153] First calibrant PD of second calibrator type 25 :3103

[0154] First calibrant PD of second calibrator type 75 ::31030

[0155] The second calibrant P o :144

[0156] Second target structure cells: 4450

[0157] This results in the adjusted first flow cytometry values ​​of IgD as follows:

[0158] First calibrant PD of second calibrant type 25 :3103–144=2959

[0159] First calibrant PD of second calibrant type 75 :31030–144=30886

[0160] The second calibrant P o :144–144=0

[0161] Cells of the second target structure: 4450-144=4306.

[0162] Again, the calibration object PD 25 and PD 75 The reference unit is defined. Calibrator PD of the second calibrator type 75The adjusted first flow cytometer reading of is assigned 100 reference units. Therefore, one reference unit corresponds to the adjusted first flow cytometer reading 30886 / 100=308.86. This number is also called the second scaling factor. 25 The adjusted first flow cytometer reading corresponds to 2959 / 308.86=9.58 reference units. A second normalized reading is formed by the adjusted first flow cytometer reading of the second target cell structure and the second proportionality factor: 4306 / 308.86=13.94 reference units.

[0163] By specifying reference units, the adjusted first flow cytometer reading of the cells becomes independent of the characteristics and settings of the flow cytometer used and can be compared with a corresponding adjusted first flow cytometer reading having different settings than the first flow cytometer or having different characteristics.

[0164] In a later step, if necessary, the normalized measurements of the cell relative to the reference units can be correlated with the measurements of the coupling curve ( Figure 4 ). The coupling curve corresponds to a logistic function (Gauss-Lorentz distribution). With the help of the "best fit" curve, it is possible to determine from the values ​​given in reference units the absolute values ​​which correspond to the amount of the respective target structure located on the cell.

[0165] 2.1 Preparation of calibrants using three CLL characteristic parameters IgM, IgD and CD19

[0166] In the second embodiment, compared with the first embodiment, the number and combination of individual groups are expanded to use additional parameters. As described for IgM and IgD, a standard curve of effective intensity is first established for humans and recombinant B lymphocyte antigen CD19. With reference to this, the corresponding quantity is used for coupling.

[0167] First Calibrator:

[0168] a) IgM calibrator: PD 25 -IgM concentration Int 25

[0169] PD 75 -IgM concentration Int 75

[0170] b) IgD calibrator: PD 25 -IgD concentration Int 25

[0171] PD 75 -IgD concentration Int 75

[0172] c) CD19 calibrant: PD 25 -CD19 concentration Int 25

[0173] PD 75 -CD19 concentration Int 75

[0174] d) IgM and IgD calibrators: P md -IgM and IgD concentrations Int 75

[0175] e) CD19 and IgM calibrators: P CD19 / m -CD19 and IgM concentrations

[0176] Int 75

[0177] f) CD19 and IgD calibrators: P CD19 / d -CD19 and IgD concentrations

[0178] Int 75

[0179] Second calibrant: P o -Neither IgM, IgD, nor CD19

[0180] This process can be extended and varied to any number of parameters.

[0181] 2.2 Schematic diagram of immunoassay using calibration particles

[0182] The cells to be sorted and the calibrant particles are measured and analyzed simultaneously in a flow cytometer. Figure 5 As shown, the first step is to separate the measurement signal of the cells and the measurement signal of the calibrant particles according to the parameters size and granularity. Size is represented by the forward scatter value (FSC) of the energy beam and granularity is represented by the side scatter value (SSC) of the energy beam. At this stage, the cells and calibrant particles to be analyzed are selected (gating). Figure 5 In FIG. 1 , the measured values ​​of the selected cell (cell population) are delimited by a first ellipse 1 and the measured values ​​of the selected calibrator (calibrator population) are delimited by a second ellipse 2 .

[0183] The different parameters (color channels) of these populations can now be analyzed together. The calibrant particles form the reference grid for each channel. Figures 6 to 9 In FIG. 1 , an exemplary analysis of three parameters (IgM, IgD and CD19) is shown in the form of a graph, wherein

[0184] -l(lgM) is the average intensity of IgM measured,

[0185] -l(lgD) is the mean intensity of IgD measured, and

[0186] - I(CD19) is the average intensity of the fluorescence radiation of the antigen CD19 measured. A first calibrant PD of the first type 25 - The measured values ​​of the IgM population are marked with reference number 3, the first calibrator PD of the first type 75 The measured values ​​of the second population of IgM are marked with reference number 4. The first calibrator population PD of the second calibrator type 25 - The measured value of IgD is designated by reference number 5, the first calibrator group PD of the second calibrator type 75 - The measured value of IgD is designated by reference number 6, the second calibrator group P o The measured value of is designated by reference number 7. Calibrator PD 25 - The measured values ​​of CD19 are designated by reference number 8, calibrant PD 75 - The measured value of CD19 is designated by reference number 9, the calibrant P md - The measured values ​​of IgM and IgD are designated by reference number 10, calibrator P CD19 / m - The measured values ​​for CD19 and IgM are designated by reference number 11, while the calibrator P CD19 / d - Measurements of CD19 and IgD are designated by reference number 12.

[0187] The calibrant particles have several purposes:

[0188] 1. Use positive and negative controls that detect the staining properties of the antibody, compared to the cells under the same conditions.

[0189] 2. Definition and standardization of the measurement space, so that

[0190] i) Independence from specific devices and / or equipment is achieved.

[0191] ii) A profile of the cells to be analyzed can be established with respect to the parameters to be analyzed (e.g.

[0192] e.g., IgM, IgD, CD19), and

[0193] iii) It laid the foundation for automatic analysis.

[0194] Regarding the multi-parameter measurement space defined and standardized in this way ( Fig. 9 ), the values ​​of each cell are normalized and can be combined into groups or clusters according to their existing profiles. This clustering is the basis for creating the antigenic profile of the cells to be analyzed.

[0195] Fig.10The results of the cluster analysis are shown graphically. Each circle represents a subgroup of a cluster, where the diameter of the circle is a measure of the measurements belonging to the relevant subgroup relative to the total number of measurements. The data refer to the mean fluorescence intensity (MFI) of the initial group marked by the circle with the largest diameter. The MFI values ​​for CD19, IgM and IgD for this initial group are X, Y and Z. The MFI values ​​for the other subgroups also change accordingly. Therefore, if the value of the initial group is X=1000, then the MFI of the group X+200 for the relevant parameter is 1200.

Claims

1. A flow cytometric measurement method for recording flow cytometric measurement values ​​using a plurality of measurement channels, the method providing an analysis medium, the analysis medium comprising a fluid and biological cells to be classified contained therein, wherein at least one different marker molecule corresponding to the number of measurement channels is provided and brought into contact with the analysis medium so that each marker molecule can specifically bind to at least one target structure located on the cell surface that specifically binds to the marker molecule; if the cell has such a target structure, a fluid flow of the analysis medium is generated, where the cell individually enters the measurement range of an energy beam and / or an electric field, wherein for a single cell located in the measurement range, at least a first flow cytometric measurement value and a second flow cytometric measurement value are recorded in each case; wherein at least a first flow cytometric measurement value of the fluorescence radiation emitted by the marker molecule when excited by the energy beam or the electric field is recorded, wherein the cells are classified according to the flow cytometric measurement values, wherein at least a first calibrant and at least a second calibrant are provided, each having solid particles of a water-insoluble inorganic and / or polymeric material, wherein the solid particles of at least one first calibrant and at least one second calibrant match in shape, size and material; wherein at least one first calibrator has a number of target structures on its surface corresponding to the number of measurement channels, and when the target structures are in contact with the label molecules, they specifically bind to the different label molecules, respectively, wherein the target structures correspond to the structures of cells and are fixed on solid particles of the first calibrator, and wherein at least one second calibrator does not have the target structure; wherein before recording the flow cytometer measurement value, at least one first calibrator and at least one second calibrator are mixed with an analysis medium so that at least one label molecule located in the analysis medium binds to at least one target structure of the first calibrator, wherein the calibrants are introduced into the measurement range one by one in the fluid flow; Wherein at least one first flow cytometric measurement value of cells and first and second calibrants of multiple measurement channels are recorded respectively, wherein at least one second flow cytometric measurement value of the calibrant distinguished from the cells includes a forward diffusion measurement value of the forward scattering of the energy beam occurring within the measurement range and a side scattering measurement value of the side scattering of the energy beam occurring within the test range, and wherein for each measurement channel, in order to form a normalized first flow cytometric measurement value of at least one cell, the difference between the first flow cytometric measurement value of the first calibrant and the first flow cytometric measurement value of the second calibrant is proportional to the difference between the first flow cytometric measurement value of the cell and the first flow cytometric measurement value of the second calibrant.

2. A flow cytometer measurement method using multiple measurement channels to record flow cytometer measurement values, the method provides an analysis medium, the analysis medium contains a fluid and biological cells to be classified contained therein, wherein at least one different labeling molecule corresponding to the number of measurement channels is provided and contacted with the analysis medium, so that each labeling molecule can specifically bind to at least one target structure of the specific binding labeling molecule located on the cell surface; if the cell has such a target structure, a fluid flow of the analysis medium is generated, where the cell individually enters the measurement range of the energy beam and / or the electric field, wherein for a single cell located within the measurement range , in each case at least a first flow cytometric measurement value and a second flow cytometric measurement value are recorded; wherein at least a first flow cytometric measurement value of the fluorescence radiation emitted by the marker molecule when excited by the energy beam or the electric field is recorded, wherein the cells are sorted according to the flow cytometric measurement value, wherein a plurality of identical first calibrants and a plurality of identical second calibrants are provided, each having solid particles of water-insoluble inorganic and / or polymeric material, wherein the solid particles of at least one first calibrant and at least one second calibrant match in shape, size and material; wherein the first calibrants have on their surface particles corresponding to the number of measurement channels a plurality of target structures that specifically bind to the different marker molecules when the target structures are in contact with the marker molecules, wherein the target structures correspond to the structures of the cells and are fixed to the solid particles of the first calibrator, wherein at least one second calibrator does not have the target structure; wherein before recording the flow cytometric measurement values, the first calibrator and the second calibrator are mixed with an analytical medium so that at least one marker molecule located in the analytical medium binds to at least one target structure of the first calibrator, wherein the calibrants are introduced individually one by one into a measurement range in a fluid stream; wherein first flow cytometric measurements of the cells and the first and second calibrators of a plurality of measurement channels are recorded respectively, wherein the second flow cytometric measurements of the calibrators distinguished from the cells respectively include forward diffusion measurements of the forward scatter of the energy beam occurring within the measurement range and side scatter measurements of the side scatter of the energy beam occurring within the measurement range, and wherein in order to form a normalized first measurement value of the cells, the difference between the average first flow cytometric measurement value of the first calibrator and the average first flow cytometric measurement value of the second calibrator is proportional to the difference between the first flow cytometric measurement value of the cells and the average first flow cytometric measurement value of the second calibrator.

3. A flow cytometric measurement method for recording flow cytometric measurement values ​​using a plurality of measurement channels, the method providing an analysis medium comprising a fluid and biological cells to be classified contained therein, wherein at least one different marker molecule in a number corresponding to the number of measurement channels is provided and brought into contact with the analysis medium so that each marker molecule can specifically bind to at least one target structure located on the cell surface that specifically binds to the marker molecule; if the cell has such a target structure, a fluid flow of the analysis medium is generated, where the cell individually enters the measurement range of an energy beam and / or an electric field, wherein for a single cell located in the measurement range, at least a first flow cytometric measurement value and a second flow cytometric measurement value are recorded in each case; wherein at least a first flow cytometric measurement value of the fluorescence radiation emitted by the marker molecule when excited by the energy beam or the electric field is recorded, wherein the cells are classified according to the flow cytometric measurement values, wherein a first calibrant and at least one second calibrant of at least two calibrant types are provided, each having solid particles of a water-insoluble inorganic and / or polymeric material, wherein the solid particles of the first calibrant and at least one second calibrant match each other in terms of shape, size and material; wherein at least one first calibrator of a first calibrator type has at least one first target structure on its surface, which corresponds to at least one first target structure of a cell and is immobilized on solid particles of the calibrator, wherein at least one first calibrator of a second calibrator type has at least one second target structure on its surface, which corresponds to at least one second target structure of a cell and is immobilized on solid particles of the calibrator, wherein at least one first calibrator of the first calibrator type does not have a second target structure on its surface and at least one first calibrator of the second calibrator type does not have a first target structure on its surface, wherein at least one second calibrator does not have the at least one first target structure and the at least one second target structure; wherein before recording the flow cytometric measurement, the at least two calibrator types of the first calibrator and the at least one second calibrator are mixed with an analysis medium so that at least one marker molecule located in the analysis medium binds to the at least one target structure of the first calibrator, wherein the target structures of the at least two calibrator types of the first calibrator are specifically bound to one of the different marker molecules, wherein the calibrants are introduced individually one after the other into the measurement range in the fluid stream;wherein at least one first flow cytometric measurement value of the cell and the first and second calibrators of the plurality of measurement channels are recorded respectively, wherein at least one second flow cytometric measurement value of the calibrator distinguished from the cell comprises a forward diffusion measurement value of the forward scatter of the energy beam occurring within the measurement range and a side scatter measurement value of the side scatter of the energy beam occurring within the test range, and wherein for each measurement channel respectively to form a normalized first flow cytometric measurement value of at least one cell, the difference between the first flow cytometric measurement value of the first calibrator and the first flow cytometric measurement value of the second calibrator is proportional to the difference between the first flow cytometric measurement value of the cell and the first flow cytometric measurement value of the second calibrator.; 4. A flow cytometer measurement method using multiple measurement channels to record flow cytometer measurement values, the method provides an analysis medium, the analysis medium contains a fluid and biological cells to be classified contained therein, wherein at least one different labeling molecule corresponding to the number of measurement channels is provided and contacted with the analysis medium, so that each labeling molecule can specifically bind to at least one target structure of the specific binding labeling molecule located on the cell surface; if the cell has such a target structure, a fluid flow of the analysis medium is generated, where the cell individually enters the measurement range of the energy beam and / or the electric field, wherein for a single cell located within the measurement range, In one embodiment, at least a first flow cytometric measurement and a second flow cytometric measurement are recorded; wherein at least a first flow cytometric measurement of the fluorescence radiation emitted by the marker molecule when excited by the energy beam or the electric field is recorded, wherein the cells are sorted according to the flow cytometric measurement, wherein a plurality of identical first calibrants of a first calibrant type, a plurality of identical first calibrants of a second calibrant type and a plurality of identical second calibrants are provided, each having solid particles of water-insoluble inorganic and / or polymeric material, wherein the solid particles of the first calibrant and the second calibrant match in shape, size and material; wherein a plurality of first calibrants of a first calibrant type respectively have at least one first target structure on their surface, which corresponds to at least one first target structure of a cell and is immobilized on solid particles of the respective calibrant, wherein a plurality of first calibrants of a second calibrant type respectively have at least one second target structure on their surface, which corresponds to at least one second target structure of a cell and is immobilized on solid particles of the respective calibrant, wherein the first calibrants of the first calibrant type do not have a second target structure on their surface and the first calibrants of the second calibrant type do not have a first target structure on their surface, wherein the second calibrant does not have the at least one first target structure and the at least one second target structure; wherein before recording the flow cytometric measurement, the first calibrants and the second calibrants are mixed with an analysis medium so that at least one label molecule located in the analysis medium binds to the at least one target structure of the first calibrants, wherein the calibrants are introduced individually one after the other into the measuring range in a fluid stream; Wherein at least one first flow cytometric measurement value of cells and first and second calibrants of multiple measurement channels are recorded respectively, wherein second flow cytometric measurement values ​​of the calibrants distinguished from the cells respectively include forward diffusion measurement values ​​of forward scattering of the energy beam occurring within the measurement range and side scatter measurement values ​​of side scattering of the energy beam occurring within the test range, and wherein in order to form a normalized first flow cytometric measurement value of the cells, the difference between the average first flow cytometric measurement value of the first calibrant and the average first flow cytometric measurement value of the second calibrant is proportional to the difference between the first flow cytometric measurement value of the cells and the average first flow cytometric measurement value of the second calibrant.

5. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: A first calibration factor is provided for at least one first calibrator, which corresponds to the ratio between the measurement signal intensity of the first calibrator and the measurement signal intensity of a first reference calibrator indicating a target structure, relative to the measurement signal of the first flow cytometer measurement value; wherein the first measurement signal is recorded as a first flow cytometer measurement value of the first calibrator, the first flow cytometer measurement value of the first calibrator being formed by the first measurement signal and the first calibration factor; wherein a second calibration factor is provided for the second calibrator, which corresponds to the ratio between the measurement signal intensity of the second calibrator and the measurement signal intensity of a second reference calibrator not having the target structure, relative to the measurement signal of the first flow cytometer measurement value, and the second measurement signal is recorded as the first flow cytometer measurement value of the second calibrator, the first flow cytometer measurement value of the second calibrator being formed by the second measurement signal and the second calibration factor.

6. The flow cytometer measurement method according to claim 1 or 2, characterized in that: A first calibrant of at least two calibrant types is provided and mixed with an analytical medium in such a way that the calibrants of the different calibrant types differ from each other in terms of surface occupancy density at the surface of the solid particles and / or arrangement of at least one target structure thereof; wherein the signal intensity of a first flow cytometric measurement of the first calibrant type is greater than the signal intensity of a first flow cytometric measurement of the second calibrant type and is less than the signal intensity of the first flow cytometric measurement of the second calibrant type.

7. The flow cytometer measurement method according to claim 6, characterized in that: The fluid contains at least two different cell populations that are different from each other, such that a first flow cytometer measurement value of cells of a first cell population is within a first signal intensity range, a first flow cytometer measurement value of cells of a second cell population is within a second signal intensity range outside the first signal intensity range, and the signal intensity of the first flow cytometer measurement value of a first calibrant type is selected to be between the first and second signal intensity ranges.

8. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The maximum size of the solid particles is less than 20 μm.

9. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The minimum size of the solid particles is greater than 4 μm.

10. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The material of the solid particles is polystyrene, melamine, latex or silicate.

11. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The target structure on the first calibrator comprises at least one antigen comprising at least one allergen and / or at least one fragment and / or derivative thereof.

12. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The target structure on the first calibrator comprises at least one antigen, which comprises at least one protein and / or at least one liposaccharide and / or at least one oligosaccharide and / or at least one nucleic acid and / or at least one fragment and / or derivative thereof.

13. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The target structure on the first calibrator comprises at least one antigen comprising at least one peptide and / or at least one glycosylated protein and / or at least one polysaccharide and / or at least one fragment and / or derivative thereof.

14. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The target structure on the first calibrator comprises at least one antigen comprising at least one receptor and / or at least one fragment and / or derivative thereof.

15. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The target structure of at least one first calibrant is immobilized on the solid particles of the first calibrant via at least one activated carboxyl group and / or at least one activated NH2 group.

16. The flow cytometer measurement method according to any one of claims 1 to 4, characterized in that: The at least one second flow cytometric measurement is a fluorescence measurement of fluorescent radiation emitted by the label molecule, the fluorescent radiation having a different wavelength than the fluorescent radiation of the first flow cytometric measurement.

17. A kit for implementing the method according to claim 1 or 2, comprising - at least one first calibrant having first solid particles made of a water-insoluble inorganic and / or polymeric material, wherein the first calibrant has at least two target structures immobilized on its surface, - at least one second calibrant having second solid particles matching the first solid particles in shape, size and material, wherein the surface of the second calibrant is free of target structures, and - at least one labeling molecule whose number corresponds to the number of target structures immobilized on the first solid particle and which specifically binds to one of the different target structures respectively when in contact with the corresponding target structure.

18. A kit for implementing the method according to claim 3 or 4, comprising - at least one first calibrant of a first calibrant type and at least one first calibrant of a second calibrant type, each having first solid particles consisting of a water-insoluble inorganic and / or polymeric material, wherein the first calibrant of the first calibrant type has a first target structure on its surface, the first target structure being immobilized on the solid particles of the first calibrant of the first calibrant type, wherein the first calibrant of the second calibrant type has a second target structure on its surface, the second target structure being immobilized on the solid particles of the first calibrant of the second calibrant type, wherein the first calibrant of the first calibrant type does not have the second target structure on its surface and the first calibrant of the second calibrant type does not have the first target structure on its surface, - at least one second calibrant having second solid particles matching the first solid particles in shape, size and material, wherein a surface of the second calibrant is free of the at least one first target structure and the at least one second target structure, and - at least one different marker molecule in a number corresponding to the number of calibrator types of said first calibrator, wherein different target structures of said at least two calibrator types of said first calibrator are respectively specifically bound to one of said different marker molecules.

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