METHOD AND FLOW CYTOMETER FOR EXAMINING A HUMAN OR ANIMAL CELL SPECIMEN, AND COMPUTER PROGRAM PRODUCT

Abstract

The invention relates to a method for examining a human or animal cell specimen, comprising the steps: a) providing at least one combinatorial cluster (CMP) which is characteristic of a disease, the CMP characterizing the colocalization and/or anticolocalization of multiple biological features in a voxel; b) determining N tags which are sufficient to determine CMP; c) bringing the cell specimen into contact with the N tags; d) measuring the cell specimen by means of flow cytometry to obtain flow cytometric data; and e) using the flow-cytometric data to check whether the CMP occurs in the cell. The invention further relates to a flow cytometer (10).

Claims

1-15. (canceled)

16. A method for examining a human or animal cell specimen, comprising the steps of: a) providing at least one combinatorial cluster (CMP) characteristic of a disease, wherein the CMP characterizes the colocalization and/or anticolocalization of multiple biological features in a voxel; b) determining N tags, which are sufficient for determining the CMP; c) contacting the cell specimen with the N tags; d) measuring the cell specimen by means of flow cytometry while obtaining flow-cytometric data; and e) based on the flow-cytometric data, checking if the CMP occurs in the cell specimen.

17. The method according to claim 16, wherein a body fluid blood and/or blood plasma, and/or mononuclear cells of the blood and/or a tissue specimen are provided as the cell specimen.

18. The method according to claim 16, wherein the at least one CMP is determined based on a database and/or by means of a multi-epitope ligand cartography (MELK) and/or ICM (imaging cycler microscopy) method and/or by means of a MELK robot system.

19. The method according to claim 16, wherein the disease is a tumor disease and/or an inflammatory disease and/or an autoimmune disease and/or a disease resembling AI phenomena and/or a disease, which includes pathological homing processes.

20. The method according to claim 1, wherein the N tags and the cell specimen are each divided into at least two groups and measured.

21. The method according to claim 16, wherein the cell specimen is divided into at least one first partial specimen, which comprises the at least one CMP, and a second partial specimen, which does not comprise the at least one CMP, after measurement.

22. The method according to claim 16, wherein the disease is amyotrophic lateral sclerosis (ALS) if the cell specimen is a blood specimen and the CMP occurring in the cell specimen includes one or more from the group of CD16, CD8, NeuN, Bax, Bcl2, CD11 b, CD138, CD16A, CD29, CD2, CD45RA, CD49d, CD54, CD56, CD57, CD58, CD62L, CD3, HLADR, immunoglobulin G, MHCII, MHCI, SIRT1, RAC1, BMX, GAK, JNK2, MAPKK6, OTUB2, PRKAR2A, SMAD2, SMAD4 and STAP2.

23. The method according to claim 16, wherein the disease is prostate cancer if the cell specimen is prostate tissue and the at least one CMP includes one or more from the group of CD26 and CD29.

24. The method according to claim 16, wherein the disease is a cutaneous lymphoma if the cell specimen is a skin specimen and the at least one CMP includes one or more from the group of HLA-DQ, CD2, CD3, CD4, CD7, CD8, CD10, CD13, CD18, CD18, CD26, CD29, CD36, CD44, CD45, CD49f, CD54, CD56, CD57, CD58, CD62L, CD71, CD80 and HLA-DR.

25. The method according to claim 16, wherein before examining the flow-cytometric data, at least one gate is first defined, by means of which a subset of the cell specimen and/or of the data for the check is selected.

26. The method according to claim 16, wherein the measurement of the cell specimen is effected in high throughput with at least 5000 cells per second.

27. The method according to claim 16, wherein constituents of the cell specimen, which comprise the at least one CMP, are classified as pathogenic and/or redundant.

28. The method according to claim 27, wherein constituents of the cell specimen classified as pathogenic and/or redundant are extracorporeally removed, by means of an apheresis method and/or by means of an apheresis device.

29. A flow cytometer, comprising: a control device, which can be coupled to a database, to determine at least one combinatorial cluster (CMP) characteristic of a disease, wherein the CMP characterizes the colocalization and/or anticolocalization of multiple biological features in a voxel; a determination device for determining N tags, which are sufficient for determining the CMP; a dosing device, by means of which the N tags can be contacted with a cell specimen; a measurement device for measuring the cell specimen by means of flow cytometry while obtaining flow-cytometric data; and a checking device, by means of which it can be checked if the CMP occurs in the cell specimen, based on the flow-cytometric data.

30. A computer program product, which can be loaded into a memory of a control device of a flow cytometer according to claim 29, wherein the computer program product comprises program means to execute the steps of the method when the program is executed by a processor device of the control device.

Description

[0024] FIG. 1 an ICM documentation of an extracorporeal photopheresis treatment of a patient with the effect thereof on an ALS-specific cell;

[0025] FIG. 2 a diagram of a toponome fingerprint; and

[0026] FIG. 3 a schematic representation of a flow cytometer according to the invention.

[0027] The imaging cycler microscopy (ICM) is the basic technology for the detection of large molecular networks in intact cells or tissues with virtually unlimited combinatorial molecular discriminability per data point (“power of combinatorial molecular discrimination per date point”, PCMD). High PCMD was shown for up to 100 proteins or more (with k=100 different proteins and 65536 values per pixel output of a 16 bit CCD camera in an ICM, for example 65536 k values per voxel result). Such and similar ICM data is specific to disease and can be lifesaving, but ICM methods currently cannot yet be employed as high-throughput methods for the clinical care. The method according to the invention avoids this disadvantage by a multi-stage method flow. In a first step, CMPs specific to disease are provided, which for example have been detected on cells specific to disease by means of ICM and the spatial PCMD code of which is expressed in the form of combinatorial clusters (CMPs). These CMPs, which define the colocalization and/or anticolocalization of a plurality of proteins/molecules/target structures in a voxel, can be expressed as a sum code per cell. In a 1/0 or true/false notation, such a code can for example be expressed as a binary number 0000001001, wherein each digit of the binary number corresponds to a predefined protein/molecule and a predefined target structure, respectively. Of course, other notations and data encoding types (e. g. decimal notation, hexadecimal notation, xml, json etc.) can generally also be used.

[0028] In that one plots all of the possible CMPs for a given tag number (e. g. for 10 tags 0000000000, 0000000001, 0000000010, 0000000011, 0000000100, 0000000101, 0000000110, 0000000111, 0000001000, 0000001001, 0000001010 etc. up to 111111111 or in decimal notation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc. up to 1024) on an abscissa axis (x-axis) and the frequency H of the concerned CMP found in the cell specimen on the ordinate axis (y-axis), diagrams can be created, which are characteristic of the toponome of the measured cell specimen and can be used for “fingerprinting” of diseases. As already mentioned, each binary digit stands for the presence (1) or non-presence (0) of a predetermined target structure (e. g. a CD) in a predetermined voxel. Similarly, such “toponome fingerprints” can be used for identifying potential medical problems by comparison to a “toponome fingerprint” of a cell specimen classified as “healthy”. FIG. 2 exemplarily shows such a diagram of a “toponome fingerprint”, from which the CMPs characteristic of a certain disease and their respective frequency are apparent. The creation and/or representation and/or use of such toponome fingerprint diagrams represent a separate inventive aspect.

[0029] This conversion and representation has proven to be helpful to fast and accurately identify cells specific to disease in blood to therapeutically deplete them as a result. The highly dimensional ICM-PCMD code can then be transformed in a pan-cellular I/O code and be used for the analysis of a cell specimen within the scope of a high-throughput measurement in a flow cytometer. This allows to measure the cellular toponome specific to disease of many patients per day and laboratory and to transfer it into a specific depletion therapy. Thereby, a toponome therapy specific to disease of for example cellular autoimmune processes specific to organ is allowed for the first time.

[0030] The imaging cycler microscopy (ICM) allows to spatially resolve large molecular networks—so-called toponomes—in the morphologically intact tissue. Therein, a virtually arbitrarily high combinatorial molecular resolution per data point or voxel (power of combinatorial molecular discrimination per data point, PCMD) is achieved.

[0031] For example, in a basal lamina (BL) of the human skin, ICM can both structurally and functionally discriminate 6 layers within the BL with a diameter of 125 nm (Schubert, W. (2018): A Platform for Parameter Unlimited Molecular Geometry Imaging Obviously Enabling Life Saving Measures in ALS. Advances in Pure Mathematics, 8, 321-334. https://doi.org/10.4236/apm.2018.83017). Therefore, ICM allows high structural resolution and functional resolution at the same time at one and the same location. Remarkably, these ICM layers are delimited from each other by sharp separating lines. These separating lines, which are here referred to as Riemann-Zeta-like function (RZLF), obviously serve to the fact that the different functions of the 6 layers can be kept biologically separated from each other. Based on the correlations identified by means of ICM, mononuclear cells of the blood were identified with ICM in the sporadic form of the ALS, which immigrate into the pyramidal tract (1st motoneuron), where they compress motoric axons (so-called axotomy-competent cells (ACC)). With the aid of the ICM, thus, the mechanisms of the disease ALS could be directly visualized in the affected tissue. This data implies that the detection of ACC in the blood indicates an active and finally lethal axotomy process such that an absolute indication for immediate therapeutic depletion of these cells is given.

[0032] FIG. 1 shows an ACC measured by ICM (post-mortem) in image a), an ACC of an ALS patient measured by ICM in image b) and an ACC cell of the patient measured by ICM after he was treated with extracorporeal photopheresis (ECP), in image c). The table below FIG. 1a)-c) shows the definition of different CMPs, wherein each CMP has been color-coded. Therein, CMPs, in which CD8 and CD16 are colocalized, are specific to disease for the ALS.

[0033] As one sees in FIG. 1, the ACC were subcellularly severely damaged by the therapeutic treatment with photopheresis. This damage could be evidenced by ICM over a longer period of time in multiple examinations. Parallel in time, the clinical disease signs of the patient regressed. These observations are consistent with collected data, which shows that the number of the ACC per liter of blood correlates with the progression speed of the disease.

[0034] The above described facts imply that the depletion therapy of the ACC is an effective lifesaving measure in case of ALS and should be available for all ALS patients, in particular since there is no alternative as a lifesaving therapy. However, a problem is in that the ALS often could only be diagnosed in an advanced stage heretofore.

[0035] In order to reach many patients with above mentioned indication and other similar indications in case of organ-specific autoimmune processes or autoimmune-similar processes, e.g. Hashimoto Thyroiditis or multiple sclerosis, analogously to the procedure in case of ALS, the following method is performed in case of these and similar indications or suspected cases:

[0036] 1. If CMPs specific to disease are not yet known, ICM diagnostics is performed on mononuclear cells (MNZ) of the blood: [0037] 1) MNZ are isolated from whole blood; [0038] 2) Isolated MNZ are shortly air-dried and then deep-frozen via isopentane in liquid nitrogen and stored deep-frozen until use; [0039] 3) an ICM measurement is performed as follows: [0040] The MNZ specimen is rehydrated with PBS at room temperature and then placed on the object table for measurements in the ICM and cyclically marked with a marker library, which includes N tags. Therein, N can be up to 100 or more. The ICM marker combinations are determined in that the tag combinations present in the MNZ are determined for each tag. The tag combinations can then be expressed as CMP in the form of an I/O encoding. All of the markers can then be plotted in a diagram for illustration, wherein the colocalization and anticolocalization code (I/O) is plotted on the x-axis of a diagram and the respective frequency is plotted on the y-axis. Such a diagram is exemplarily shown in FIG. 2 as already mentioned. [0041] 4) As soon as the finding under (3) is present, the search for the MNZ specific to disease with the same marker library is transferred to a flow cytometer, which for example comprises 6 lasers and 21 fluorescence channels, for the same patient: Here, each individual MNZ is regarded as a point, which expresses a given 1/0 code specific to disease. This MNZ form can then be quantified in high throughput per patient before, during and after the ECP therapy and be subcellularly exactly measured by intermitting ICM. [0042] 5. The advantage of this approach is in that the ECP allows an exact toponome measurement in a two-step method in high throughput by coupling of ICM/flow cytometry for the first time. Thereby, it is possible to toponomically exactly measure all of the relevant patient groups.

[0043] If the CMP data is already known, it can for example be provided from a database, optionally transformed and used for the flow-cytometric measurement.

[0044] FIG. 3 shows a schematic representation of a flow cytometer 10 according to the invention. The flow cytometer 10 includes a control device 12, which is wirelessly or wired coupled to a presently external database 14 for data exchange to determine at least one combinatorial cluster (CMP) characteristic of a disease, wherein the CMP characterizes the colocalization and/or anticolocalization of multiple biological features in a voxel. Among other things, the control device 12 usually comprises a memory and a processor device to execute software. Basically, the database 14 can also be a part of the flow cytometer 10. Furthermore, the flow cytometer 10 includes a determination device 16 for determining N tags, which are sufficient for determining the CMP. Generally, the determination device 16 can be omitted if the required information about the required N tags can be otherwise provided. Furthermore, the flow cytometer 10 comprises a dosing device 18, by means of which the N tags can be contacted with a cell specimen (not shown), as well as a measurement device 20 for measuring the cell specimen by means of flow cytometry while obtaining flow-cytometric data. Generally, the dosing device 18 can also be omitted if the cell specimen has already been contacted with the N tags outside of the flow cytometer 10. Finally, the flow cytometer 10 includes a checking device 22, by means of which it is checked if the CMP occurs in the cell specimen, based on the flow-cytometric data. If the CMP occurs in the cell specimen, it is to be assumed that the patient suffers a disease, of which the found CMP is characteristic. This finding allows an early diagnosis and therapy derivable therefrom.

[0045] The parameter values specified in the documents for definition of process and measurement conditions for characterizing specific characteristics of the inventive subject matter are to be regarded as encompassed by the scope of the invention also within the scope of deviations—for example due to measurement errors, system errors, weighing errors, DIN tolerances and the like.