Method, kit and computer program product for the diagnosis of arthritis and / or prognostic assessment of disease progression in arthritis

Abstract

The invention relates to a method and a kit for the diagnosis of arthritis and / or prognostic assessment of disease progression in arthritis, to a method for monitoring the disease progression in arthritis following a diagnosis of arthritis using the method, and to a computer program product comprising instructions for carrying out the method.

Description

[0001] Method, kit and computer program product for diagnosing arthritis and / or prognostic assessment of the disease course in arthritis

[0002] The invention relates to a method and a kit for diagnosing arthritis and / or prognostic assessment of the course of arthritis, a method for monitoring the course of arthritis after a diagnosis of arthritis using the method, and a computer program product comprising commands for executing the method.

[0003] Rheumatoid arthritis (RA) is a chronic autoimmune disease of the joints. In addition to affecting the joints, internal organs and the cardiovascular system can also be affected. The causes of the disease are still largely unknown.

[0004] Currently, the presence of autoantibodies in a patient's serum is one of the diagnostic methods used. In addition to detecting rheumatoid factor (autoantibodies directed against the body's own IgG molecules), the detection of anti-CCP antibodies, which target citrullinated side chains of proteins, can lead to an earlier diagnosis of rheumatoid arthritis (Schellekens et al., 1998). Comparably high sensitivity is achieved by detecting antibodies against mutated citrullinated vimentin (MCV) (Poulsom and Charles, 2008). Commercial test kits are already available for both anti-CCP and anti-MCV antibodies, enabling simple and rapid detection and thus providing a diagnosis of rheumatoid arthritis.However, the detection of systemic autoantibodies in the blood only allows for a very limited assessment of the stage of the disease, especially the status of joint destruction.

[0005] EP 2 965 084 B1 relates to a method for the prognostic assessment of the disease course in rheumatoid arthritis and / or the diagnosis of rheumatoid arthritis by cytokine analysis of a human whole blood sample, wherein, after incubation with a stimulating agent, the concentration of at least two cytokines selected from TNF-α, IL-8, IFNγ, IL-10, IL-1β, and IL-6 is determined in the cell-free supernatant. Cytokine analysis allows for an estimation of the course of therapy but has low sensitivity and specificity for the diagnosis of rheumatoid arthritis.

[0006] Jäger et al. describe a close relationship in rheumatoid arthritis between calcium ions released from bone lesions, fetuin-based and calcium phosphate-containing calciprotein particles (CPP), and the pro-inflammatory response of monocytes and macrophages to ionized calcium and CPP (Jäger et al., 2020). This pro-inflammatory response has been shown to be particularly pronounced in patients with untreated disease or those with very high disease activity. This pro-inflammatory response is triggered by the uptake and digestion of CPP in the lysosomes of monocytes, which are dependent on a calcium ion-induced signal from the calcium-sensing receptor (CaSR).It is therefore desirable to use a diagnostic procedure for rheumatoid arthritis in which the concentration of pro-inflammatory CPP and other pro-inflammatory calcium phosphate particles in the blood serum of patients can be determined.

[0007] One method for detecting CPP relies on the sedimentation of calciprotein particles by high-speed centrifugation. By determining the total fetuin A content in the serum before and after centrifugation, an indirect calculation of the concentration of the calcium phosphate particles that can be sedimented by centrifugation is performed. A disadvantage of this indirect determination is its high degree of inaccuracy.

[0008] JP 6566697 B2 discloses a method for detecting CPP in human serum, whereby the particles are specifically stained with a fluorescent dye (OsteoSense™) and excess free dye is removed by gel filtration. The concentration of CPP in the sample is determined by quantifying the fluorescence signal using a fluorescence scanner in cell culture plate format. Smith et al. describe the measurement of CPP using a flow cytometer (Smith et al., 2017). A disadvantage of the former method is that it provides no information about the type, size, and composition of the fluorescently labeled particles, and furthermore, it cannot detect potential non-specific binding of the dye, which could distort the results.Due to the limited spatial resolution of the method, this applies almost equally to flow cytometer-based determination, even though individual particles can at least be differentiated there.

[0009] The object of the invention is therefore to provide an improved detection of calcium phosphate particles in serum for a method and / or a kit for the diagnosis of arthritis and / or for the prognostic assessment of the disease course in arthritis.

[0010] According to the invention, the problem is solved by the method, the kit, and the computer program product according to the independent claims. Advantageous embodiments of the invention are specified in the dependent claims. A first aspect of the invention relates to a method for diagnosing arthritis and / or for prognostic assessment of the disease course in arthritis, comprising the steps: a. providing an isolated serum sample, wherein the serum sample is diluted in the range of 1:5 to 1:100; b. staining particles in the isolated serum sample with at least one calcium phosphate-specifically binding fluorescent dye and with at least one lipid membrane-specifically binding fluorescent dye, wherein at least the emission wavelength of the at least one calcium phosphate-specifically binding fluorescent dye and the at least one lipid membrane-specifically binding fluorescent dye differs; c.Separation of unbound fluorescent dye, i.e., fluorescence flow cytometry (time-resolved measurement) with at least the excitation and emission wavelength of the fluorescent dye specifically binding at least one calcium phosphate and the fluorescent dye specifically binding at least one lipid membrane, and e. computer-implemented evaluation of the fluorescence flow cytometry based on the fluorescence intensity of the fluorescent dye specifically binding at least one calcium phosphate and the fluorescent dye specifically binding at least one lipid membrane.

[0011] Advantageously, the method according to the invention is simple and reproducible and enables early diagnosis of arthritis and / or prognostic assessment of the disease course. Advantageously, the method according to the invention allows for prognostic assessment of the disease course, selection of treatment options, monitoring of treatment success, and thus also further treatment decisions, in particular regarding the continuation, change, or discontinuation of therapy.

[0012] In embodiments, the method according to the invention is carried out in the sequence of steps a), b), c), d) and e).

[0013] In some embodiments, the method is used for the diagnosis and / or prognostic assessment of the disease course in rheumatoid arthritis or psoriatic arthritis. In other embodiments, the method is used for monitoring the success of therapy and / or for further treatment decisions, particularly in the case of therapy using TNF inhibitors. The term "TNF inhibitors" (also TNF blockers) refers to drugs that inhibit tumor necrosis factor (TNF) and thereby inflammatory processes.

[0014] In embodiments, the isolated serum sample is a sample from a mammal. In embodiments, the isolated serum sample is a human serum sample or a serum sample from a bovine, pig, horse, goat, sheep, dog, cat, mouse, guinea pig, or rabbit. In preferred embodiments, the isolated serum sample is a human serum sample.

[0015] It is advisable to provide the isolated serum sample as supernatant after centrifugation of a blood sample, for example at about 3,000 g for about 10 minutes.

[0016] In embodiments, the isolated serum sample is provided in step a) by incubation at a temperature in the range of 10°C to 25°C, preferably in the range of 15°C to 25°C, or by incubation at a temperature in the range of 30°C to 40°C, preferably in the range of 35°C to 39°C.

[0017] In embodiments, after incubation at a temperature in the range of 10°C to 25°C, preferably in the range of 15°C to 25°C, or at a temperature in the range of 30°C to 40°C, preferably in the range of 35°C to 39°C, the serum sample is mixed, preferably vortexed.

[0018] In embodiments, when providing the isolated serum sample in step a), a dilution to a concentration in the range of 1 :5 to 1 :100 takes place, preferably after incubation at a temperature in the range of 10°C to 25°C, preferably in the range of 15°C to 25°C, or at a temperature in the range of 30°C to 40°C, preferably in the range of 35°C to 39°C.

[0019] In embodiments, the isolated serum sample is prepared in step a) by incubating it at a temperature in the range of 15°C to 25°C, or by incubating it at a temperature in the range of 35°C to 39°C; and by dilution to a concentration in the range of 1:5 to 1:100. In embodiments, the serum sample is prepared in step a) by dilution in the range of 1:5 to 1:100, preferably in the range of 1:5 to 1:40, and particularly preferably in the range of 1:15 to 1:25.

[0020] In embodiments, the isolated serum sample, the calcium phosphate-specific binding fluorescent dye, and the lipid membrane-specific binding fluorescent dye are mixed, wherein calcium phosphate-containing particles are bound by the calcium phosphate-specific binding fluorescent dye and lipid membrane-containing particles are bound by the lipid membrane-specific binding fluorescent dye.

[0021] The term "calcium phosphate particles" refers to particles in serum samples containing calcium ions (Ca). 2+ ) and phosphate ions (PO4) 3 ") understood. Calcium phosphate particles also conveniently include calciprotein particles. The term "calciprotein particles" refers to particles containing calcium ions (Ca 2+ ), Phosphation ions (PO4 3 ") and fetuin A understood. Calciprotein particles represent an inherent mineral buffer system responsible for trapping excess Ca 2+ - and PC>4 3"-ions are responsible for preventing calcification outside the skeleton.

[0022] The term "calcium phosphate specifically binding fluorescent dye" refers to a fluorescent dye that is conjugated, i.e., covalently bonded, to a molecule that specifically binds calcium phosphate.

[0023] In embodiments, the calcium phosphate-specifically binding fluorescent dye is a bisphosphonate-conjugated fluorescent dye.

[0024] Advantageously, a bisphosphonate-conjugated fluorescent dye binds specifically to amorphous or crystalline calcium phosphate. In embodiments, the calcium phosphate-specifically binding fluorescent dye is an OsteoSense™ dye (Perkin Eimer Co.). Under OsteoSense TMThe term "dye" refers to a fluorescent dye to which the bisphosphonate pamidronate is covalently bound via amine coupling. In some embodiments, the calcium phosphate-specifically binding fluorescent dye is IVISense Osteo 680 Fluorescent Probe™ (Perkin Eimer Co.).

[0025] In embodiments, the fluorescent dye is selected from Rhodamine, FITC (fluorescein isothiocyanate), IRDye®, or Alexa Fluora. In embodiments, the calcium phosphate-specifically binding fluorescent dye is a bisphosphonate-conjugated near-infrared fluorescent dye, in particular IRDye™ (excitation 668 nm, emission 687 nm).

[0026] In embodiments, the calcium phosphate-specifically binding fluorescent dye is a calcein dye, in particular Fluorexon™.

[0027] The term "lipid membrane-specific binding fluorescent dye" refers to a fluorescent dye that is conjugated, i.e., covalently bound, to a molecule that specifically binds to lipid membranes.

[0028] In embodiments, the lipid membrane-specifically binds the fluorescent dye phosphatidylserine in the lipid membrane.

[0029] In some embodiments, the phosphatidylserine-specifically binding fluorescent dye is a lactadherine-conjugated fluorescent dye. In other embodiments, the lipid membrane-specifically binding fluorescent dye is lactadherine-FITC. In other embodiments, the lipid membrane-specifically binding fluorescent dye is bovine FITC-labeled lactadherine, Prolytix (Haematologic Techn.). Advantageously, staining with a lipid membrane-specifically binding fluorescent dye allows differentiation from so-called extracellular vesicles (EVs) in peripheral blood, which are physically and physicochemically similar to calcium phosphate particles.

[0030] Advantageously, at least the emission wavelength of the fluorescent dye that binds at least one calcium phosphate specifically and the fluorescent dye that binds at least one lipid membrane specifically differs.

[0031] In embodiments, the coloring of particles in the isolated serum sample is achieved by adding 0.5 pl to 5 pl of serum sample to 10 pl to 100 pl of a calcium phosphate-specifically binding fluorescent dye solution with a concentration in the range of 5 pM to 50 pM.

[0032] In embodiments, the coloring of particles in the isolated serum sample is carried out by adding 0.5 pl to 5 pl serum sample to 10 pl to 100 pl of a lipid membrane-specific binding fluorescent dye solution with a concentration in the range of 5 pM to 50 pM.

[0033] In this embodiment, step b. additionally involves staining with at least one fluorescent dye used for cell tracking, particularly for determining cell proliferation, especially in the cytoplasm (cell division tracking dye). Such dyes are known to those skilled in the art (see Tempany et al. 2017, Rieger 2022, and Lyons et al. 2013). Advantageously, staining with this fluorescent dye (cell tracking dye or cell division tracking dye) has been observed to have low cytotoxicity and only minimal effects on cell proliferation and biology.

[0034] In some embodiments, the fluorescent dye (“cell tracking dye” or “cell division tracking dye”) is selected from the group comprising CellTrace™ Blue, CellTrace™ Violet, CellTrace™ CFSE, CellTrace™ Yellow (CTY), and CellTrace™ Far Red (ThermoFisher Scientific). In other embodiments, the fluorescent dye is CellTrace™ Yellow (CTY).

[0035] In embodiments, the at least one fluorescent dye used for labeling cells, particularly for determining cell proliferation, especially in the cytoplasm, is a succinimidyl ester-based fluorescent dye, in particular a carboxyfluorescein succinimidyl ester (CFSE). With these succinimidyl ester-based fluorescent dyes, especially carboxyfluorescein succinimidyl esters (CFSE), cleavage occurs through esterases located in the cytoplasm, releasing a fluorescent dye. Advantageously, intact vesicles containing cytoplasm can be stained by additional staining with this fluorescent dye.

[0036] Advantageously, at least the emission wavelength of the at least one fluorescent dye used for labeling cells, in particular for determining cell proliferation, especially in the cytoplasm, in particular carboxyfluorescein obtained after cleavage of the carboxyfluorescein succinimidyl ester by esterases located in the cytoplasm, differs from the emission wavelength of the at least one calcium phosphate-specifically binding fluorescent dye and the at least one lipid membrane-specifically binding fluorescent dye.

[0037] In some embodiments, the staining of particles in the isolated serum sample is achieved by adding 0.5 pl to 5 pl of serum sample to 10 pl to 100 pl of a solution of the fluorescent dye ("cell tracking dye," "cell division tracking dye") with a concentration in the range of 5 pM to 50 pM. In some embodiments, the staining of the particles in the serum sample takes place at room temperature, particularly at a temperature in the range of 20°C to 26°C. In other embodiments, the staining of the particles in the serum sample is carried out with gentle shaking.

[0038] In embodiments, the staining of the particles in the serum sample takes place for a period of time ranging from 5 min to 10 h, preferably for a period of time ranging from 30 min to 2 h.

[0039] In some embodiments, the separation of unbound fluorescent dye is carried out by means of gel filtration.

[0040] In step d), the fluorescence measurement is appropriately carried out at least with the excitation and emission wavelength of the fluorescent dye that binds at least one calcium phosphate specifically and the fluorescent dye that binds at least one lipid membrane specifically.

[0041] In some embodiments, a multispectral measurement is performed in step d). In some embodiments, a time-resolved multispectral measurement is performed in the fluorescence and visible ranges.

[0042] In embodiments, in step d) time-resolved microscopic multispectral images are taken in visible light and in the corresponding fluorescence channels using a high-resolution digital camera.

[0043] In preferred embodiments, fluorescence flow cytometry is video-based fluorescence flow cytometry. The term "video-based fluorescence flow cytometry" (also called imaging fluorescence flow cytometry) refers to a flow cytometer with an attached microscope. Advantageously, video-based fluorescence flow cytometry determines the fluorescence intensity and the spatial position of the fluorescent object. Advantageously, video-based fluorescence flow cytometry enables the detection of nanoparticles in the size range between 30 nm and 100 nm, which are not detectable by light microscopy. The term "nanoparticles" refers to particles with a diameter in the range of 1 nm to 100 nm. Furthermore, the high event rate of flow cytometry is advantageously combined with the benefits of single-cell imaging in conjunction with microscopy.In some embodiments, the ImageStream™ (Luminex™) is used for video-based fluorescence flow cytometry. In other embodiments, the ImageStream Mark II™ (Luminex™) is used for video-based fluorescence flow cytometry.

[0044] In preferred embodiments, fluorescence flow cytometry is a high-resolution flow cytometry. Advantageously, high-resolution flow cytometry allows for the differentiation of individual particles in the serum sample.

[0045] In embodiments, fluorescence flow cytometry is performed using a multispectral, video-based or imaging flow cytometer with 1 to 12 channels, preferably with 8 to 12 channels.

[0046] In some embodiments, bright-field microscopy is performed in step d). Bright-field microscopy refers to a microscopy technique in which objects are observed in a bright field, with the light passing through the object (transmitted light microscopy) or the reflected light (reflected light microscopy) entering the microscope objective (microscope illumination). In some embodiments, the particle size is determined.

[0047] In embodiments, step e) involves a computer-implemented evaluation of the flow fluorescence measurement using the following steps:

[0048] • Removal of results that do not show fluorescence of the calcium phosphate-specifically binding fluorescent dye,

[0049] • Evaluation of the fluorescence of the calcium phosphate specifically binding

[0050] Fluorescent dye and the fluorescence of the lipid membrane-specifically binding fluorescent dye by grouping the particles according to the

[0051] Fluorescence intensity,

[0052] • Determination of the concentration and / or total fluorescence of the

[0053] Calcium phosphate-positive particles of varying fluorescence intensities,

[0054] • Comparison of the concentration and / or total fluorescence of calcium phosphate-positive particles of different fluorescence intensities with a cutoff value determined using serum samples from healthy patients.

[0055] Advantageously, various particles are measured using the method according to the invention: Calcium phosphate-containing particles: exhibit fluorescence of the calcium phosphate-specifically binding fluorescent dye (“calcium phosphate-positive”) and may exhibit fluorescence of the lipid membrane-specifically binding fluorescent dye (“lipid membrane-positive” or “lipid membrane-negative”), Extracellular vesicles (EVs): exhibit fluorescence of the lipid membrane-specifically binding fluorescent dye (“lipid membrane-positive”) and do not exhibit fluorescence of the calcium phosphate-specifically binding fluorescent dye (“calcium phosphate-negative”).

[0056] The calcium phosphate-containing particles also include calciprotein particles (CPPs), which contain calcium ions (Ca). 2+ ), Phosphation ions (PO4 3Calciprotein particles (CPPs) contain fetuin A. They do not exhibit fluorescence of the fluorescent dye that binds specifically to lipid membranes ("lipid membrane negative"). CPPs are not detectable by light microscopy.

[0057] Other calcium phosphate-containing particles exhibit both fluorescence of the calcium phosphate-specific fluorescent dye ("calcium phosphate-positive") and fluorescence of the lipid membrane-specific fluorescent dye ("lipid membrane-positive"). Some of these calcium phosphate-containing particles are detectable by light microscopy, while others are below this detection limit.

[0058] In embodiments, step e) involves removing results that do not exhibit fluorescence of the calcium phosphate-specific binding fluorescent dye and the lipid membrane-specific binding fluorescent dye.

[0059] In embodiments, results that do not exhibit fluorescence of the calcium phosphate-specifically binding fluorescent dye are removed by removing results with fluorescence below a threshold value of the fluorescence signal area (fluorescence intensity), in particular less than 100 AU (arbitrary units of fluorescence intensity). Advantageously, separating results with fluorescence below a threshold value of the fluorescence signal area removes non-specific background signals.

[0060] Advantageously, by separating results that show fluorescence of the lipid membrane-specifically binding fluorescent dye and no fluorescence of the calcium phosphate-specifically binding fluorescent dye, results from extracellular vesicles (EVs) that are irrelevant for the evaluation are removed. In embodiments, step e. involves a computer-implemented evaluation of the fluorescence flow cytometry based on the fluorescence intensity of the at least one calcium phosphate-specifically binding fluorescent dye, the at least one lipid membrane-specifically binding fluorescent dye, and the fluorescence of the at least one fluorescent dye used for labeling cells, in particular for determining cell proliferation, especially in the cytoplasm.

[0061] In some embodiments, the evaluation involves classifying the fluorescence intensity of the calcium phosphate-specifically binding fluorescent dye.

[0062] It is advisable to perform at least one calibration and / or measurement of samples from healthy subjects and representative patients to classify the fluorescence intensity, in particular for the fluorescence intensity of the calcium phosphate-specific binding fluorescent dye and the lipid membrane-specific binding fluorescent dye.

[0063] In embodiments, the fluorescence intensity is classified into the following ranges: 1) between 100 arbitrary units of fluorescence intensity (AU) and 200 AU, 2) between 200 AU and 300 AU, 3) between 300 AU and 500 AU, 4) between 500 AU and 1000 AU, 5) between 1000 AU and 2000 AU, and 6) above 2000 AU. Advantageously, the particles with different fluorescence intensities have different particle sizes and maturation states and therefore differ in their calcium phosphate complex content.

[0064] In some embodiments, the evaluation distinguishes between results exhibiting fluorescence of the lipid membrane-specifically binding fluorescent dye and results lacking such fluorescence, based on a threshold value of the fluorescence signal area (fluorescence intensity), particularly less than 5000 AU (arbitrary units of fluorescence intensity). Advantageously, the additional differentiation of individual calcium phosphate-positive particle populations with respect to lipid membrane staining enables a more precise distinction between healthy individuals and patients with rheumatoid arthritis.

[0065] In embodiments, results exhibiting fluorescence of the lipid membrane-specifically binding fluorescent dye above the threshold are classified into the following ranges according to the fluorescence intensity of the calcium phosphate-specifically binding fluorescent dye: 7) between 300 AU and 500 AU, 8) between 500 AU and 1000 AU; 9) between 1000 AU and 2000 AU, and 10) above 2000 AU.

[0066] In embodiments, the concentration of calcium phosphate-positive particles of different fluorescence intensities per microliter of serum sample is determined.

[0067] In embodiments, the total fluorescence per group is calculated from the fluorescence intensity multiplied by the concentration of calcium phosphate-positive particles.

[0068] In some embodiments, the total fluorescence of calcium phosphate-positive particles of varying fluorescence intensities is compared to a threshold value determined from serum samples of healthy patients. In other embodiments, the threshold value for a pathological increase in calcium phosphate particle concentration is the median plus two standard deviations of the examined cohort of healthy controls.

[0069] In preferred embodiments, the total fluorescence of the calcium phosphate-positive particles is determined with a fluorescence intensity in the range of 500 AU to over 2000 AU, particularly preferably in the range over 2000 AU, for the diagnosis of rheumatoid arthritis. Advantageously, healthy patients exhibit a very low concentration of calcium phosphate-positive particles with this fluorescence intensity.

[0070] In preferred embodiments, the overall fluorescence of the lipid membrane-positive and calcium phosphate-positive particles is determined with respect to the fluorescence intensity of the calcium phosphate-specifically binding fluorescent dye in the range of 500 AU to over 2000 AU, particularly preferably in the range over 1000 AU, for the diagnosis of rheumatoid arthritis. Advantageously, a particularly good association between the diagnosis of rheumatoid arthritis and an increase in fluorescence in this group is observed.

[0071] In embodiments, the total fluorescence of the calcium phosphate-positive particles with a fluorescence intensity in the range of 100 AU to 200 AU and in the range of 200 AU to 300 AU is determined for the assessment of the course of therapy. Advantageously, the calcium phosphate-positive particles with a fluorescence intensity in the range of 100 AU to 200 AU and in the range of 200 AU to 300 AU are calciprotein particles that do not exhibit fluorescence from the fluorescent dye that specifically binds to lipid membranes. In embodiments, after a diagnosis of arthritis, in particular rheumatoid arthritis, using the method according to the invention, monitoring of the disease course in patients is necessary.

[0072] In embodiments, after a diagnosis of arthritis, in particular rheumatoid arthritis, the course of the disease is monitored using the method according to the invention, comprising the following steps:

[0073] (1) Provision of an isolated serum sample wherein the serum sample is diluted in the range of 1:5 to 1:100,

[0074] (2) Staining of particles in the serum sample with at least one calcium phosphate-specifically binding fluorescent dye,

[0075] (3) Separation of unbound fluorescent dye,

[0076] (4) Dissolving the particles and releasing the fluorescent dye,

[0077] (5) Fluorescence measurement and

[0078] (6) Evaluation of the fluorescence intensity of the fluorescent dye that binds at least one calcium phosphate specifically.

[0079] In embodiments, to monitor the course of arthritis after a diagnosis of arthritis, in particular rheumatoid arthritis, the method according to the invention comprises the following steps:

[0080] (1) Provision of an isolated serum sample wherein the serum sample is diluted in the range of 1:5 to 1:100,

[0081] (2) Staining of particles in the serum sample with at least one calcium phosphate-specifically binding fluorescent dye,

[0082] (3) Separation of unbound fluorescent dye,

[0083] (4) Dissolving the particles and releasing the fluorescent dye,

[0084] (5) Fluorescence measurement and

[0085] (6) Evaluation of the fluorescence intensity of the fluorescent dye that binds at least one calcium phosphate specifically.

[0086] Advantageously, the method for monitoring disease progression allows disease activity to be easily determined by ascertaining the total fluorescence intensity of the fluorescent dye that specifically binds at least one calcium phosphate. A further aspect of the invention relates to a method for monitoring the course of arthritis after a diagnosis of arthritis using the method according to the invention, comprising the steps

[0087] (1) Provision of an isolated serum sample wherein the serum sample is diluted in the range of 1:5 to 1:100,

[0088] (2) Staining of particles in the serum sample with at least one calcium phosphate-specifically binding fluorescent dye,

[0089] (3) Separation of unbound fluorescent dye,

[0090] (4) Dissolving the particles and releasing the fluorescent dye,

[0091] (5) Fluorescence measurement and

[0092] (6) Evaluation of the fluorescence intensity of the fluorescent dye that binds at least one calcium phosphate specifically.

[0093] In embodiments, the particles are dissolved in step (4) using ethylenediaminetetraacetic acid (EDTA).

[0094] In step (5) it is appropriate to perform a fluorescence measurement at least with the excitation and emission wavelength of the fluorescent dye that binds at least one calcium phosphate specifically.

[0095] In embodiments, the measurement in step (5) is carried out using a fluorescence microtiter plate reader.

[0096] In embodiments, a multispectral measurement is performed in step (5).

[0097] A high fluorescence intensity is advantageously obtained at a high concentration of the calcium phosphate-specifically binding fluorescent dye and thus at a high concentration of calcium phosphate particles.

[0098] In some embodiments, the threshold for a pathological increase in calcium phosphate particle concentration is defined as the median plus twice the standard deviation of the examined cohort (group) of healthy controls. In other embodiments, the threshold for a pathological increase in calcium phosphate particle concentration is defined as the median of the examined cohort (group) of rheumatoid arthritis patients. The median is the value that lies exactly in the middle of a data distribution. In some embodiments, the evaluation is performed by calibration with hydroxyapatite. Advantageously, the fluorescence intensity of the calcium phosphate-specifically binding fluorescent dye bound to the hydroxyapatite is measured in the same way as the serum samples, and a calibration curve is generated.The calibration curve can be used to calculate the amount of fluorescent dye that binds to hydroxyapatite and to determine the calcium phosphate concentration in the blood (serum sample).

[0099] In some embodiments, following an arthritis diagnosis, the procedure for monitoring the course of arthritis and / or the success of therapy is performed, whereby the fluorescence intensity of the fluorescent dye specifically binding at least one calcium phosphate in the serum sample is determined. In other embodiments, following an arthritis diagnosis, the procedure for monitoring the course of the disease is performed after a period of 6 to 12 months and / or when the therapy is changed. In other embodiments, following an arthritis diagnosis, the procedure for monitoring the course of the disease is performed after a period of approximately 3 months during uncontrolled activity phases.

[0100] In some embodiments, after staining the particles in the serum sample, centrifugation takes place at 16,000 g to 24,000 g for a duration of 1.5 h to 2 h. In other embodiments, the fluorescence measurement is performed directly from the resulting precipitate.

[0101] In some embodiments, after staining the particles in the serum sample, filtration with a pore size filter takes place. Advantageously, filtration using a pore size filter separates particles of different sizes.

[0102] Another aspect of the invention relates to a kit for diagnosing arthritis and / or prognostic assessment of the course of arthritis comprising i. at least one calcium phosphate-specific binding fluorescent dye and ii. at least one lipid membrane-specific binding fluorescent dye.

[0103] In embodiments, the kit further comprises at least one fluorescent dye used for labeling cells, particularly for determining cell proliferation, especially in the cytoplasm. In embodiments, the at least one fluorescent dye used for labeling cells, particularly for determining cell proliferation, is selected from the group comprising CellTrace™ Blue, CellTrace™ Violet, CellTrace™ CFSE, CellTrace™ Yellow (CTY), and CellTrace™ Far Red (ThermoFisher Scientific). In embodiments, the at least one fluorescent dye used for labeling cells, particularly for determining cell proliferation, is CellTrace™ Yellow (CTY).

[0104] In embodiments, the kit further comprises at least one buffer for diluting the isolated serum sample. In embodiments, the buffer is an isotonic buffer, preferably a cell culture medium, most preferably Dulbecco's Modified Eagle Medium (DMEM).

[0105] In embodiments, the kit further comprises a computer program product comprising instructions which, when the method is executed by a computer, cause the computer to execute step e) of the method according to the invention.

[0106] In some embodiments, the kit also includes instructions for carrying out the method according to the invention.

[0107] In some embodiments, the kit is used for the diagnosis of arthritis and / or the prognostic assessment of the disease course in arthritis.

[0108] Another aspect of the invention relates to a data processing device comprising means for carrying out step e) of the method according to the invention.

[0109] In embodiments, the data processing device comprises means for performing the following steps:

[0110] • Removal of results that do not show fluorescence of the calcium phosphate-specifically binding fluorescent dye,

[0111] • Evaluation of the fluorescence of the calcium phosphate specifically binding

[0112] Fluorescent dye and the fluorescence of the lipid membrane-specifically binding fluorescent dye by grouping the particles according to the

[0113] Fluorescence intensity,

[0114] • Determination of the concentration and / or total fluorescence of the

[0115] Calcium phosphate-positive particles of varying fluorescence intensities,

[0116] • Comparison of the concentration and / or total fluorescence of calcium phosphate-positive particles of different fluorescence intensities with a threshold value determined from serum samples of healthy patients. In embodiments, the fluorescence of the calcium phosphate-specifically binding fluorescent dye, the fluorescence of the lipid membrane-specifically binding fluorescent dye, and the fluorescence of the at least one fluorescent dye used for labeling cells, in particular for determining cell proliferation, are evaluated by grouping the particles according to their fluorescence intensity.

[0117] Another aspect of the invention relates to a computer program product comprising instructions which, when the method is executed by a computer, cause the computer to execute step e) of the method according to the invention.

[0118] In embodiments, the computer program product includes instructions which, when executed by a computer, cause the computer to perform the following steps:

[0119] • Removal of results that do not show fluorescence of the calcium phosphate-specifically binding fluorescent dye,

[0120] • Evaluation of the fluorescence of the calcium phosphate-specific binding fluorescent dye and the fluorescence of the lipid membrane-specific binding fluorescent dye by grouping the particles according to their fluorescence intensity,

[0121] • Determination of the concentration and / or total fluorescence of calcium phosphate-positive particles of different fluorescence intensities,

[0122] • Comparison of the concentration and / or total fluorescence of calcium phosphate-positive particles of different fluorescence intensities with a cutoff value determined using serum samples from healthy patients.

[0123] In embodiments, the fluorescence of the calcium phosphate-specific binding fluorescent dye, the fluorescence of the lipid membrane-specific binding fluorescent dye, and the fluorescence of the at least one fluorescent dye used for labeling cells, in particular for determining cell proliferation, are evaluated by grouping the particles according to their fluorescence intensity.

[0124] Another aspect of the invention relates to a computer-readable medium on which the computer program product is stored. For the realization of the invention, it is also advantageous to combine the aforementioned inventive configurations, embodiments, and features of the claims.

[0125] Examples of implementation

[0126] The invention will now be explained in more detail using an exemplary embodiment. This embodiment relates to a cohort of patients with rheumatoid arthritis and a smaller group of patients with psoriatic arthritis, and is intended to describe the invention without limiting it.

[0127] The invention is explained in more detail with the aid of drawings. These drawings show

[0128] Fig. 1 shows an image of the detection of individual particles and their fluorescence microscopic representation. A) In the dot plot shown, the fluorescence intensity of the OsteoSense stain (x-axis) is plotted against that of the lactadherine stain (y-axis). B) Schematic representation of the particle distribution in the dot plot and the evaluation gates 1-10 used. C) Representative microscopic images for gates 1-10 are shown individually, with Channel 2 (Ch02) showing the fluorescence signal of the lipid membrane-specifically binding fluorescent dye, Channel 5 (Ch05) showing the fluorescence signal of the calcium phosphate-specifically binding fluorescent dye, Channel 4 (Ch04) showing the light microscopic image, and Channel 6 (Ch06) showing the side-scattered light.

[0129] Fig. 2 shows the relationship between calcium phosphate particle concentrations and rheumatoid arthritis in healthy patients (HD, control) and patients with rheumatoid arthritis (RA). A) Calcium phosphate particle concentration in gate 10 (lactadherin-positive and an OsteoSense fluorescence intensity above 2000 AU). B) Calcium phosphate particle concentration in gates 4 to 6 (lactadherin-negative and an OsteoSense fluorescence intensity in the range of 500 AU to over 2000 AU) and in gates 8 to 10 (lactadherin-positive and an OsteoSense fluorescence intensity in the range of 500 AU to over 2000 AU). C) Comparison of calcium phosphate particle concentrations in healthy patients (HD), patients with rheumatoid arthritis (RA) with low disease activity (DAS28 < 3.2), and patients with high disease activity. (DAS28>3,2), (ns - no significant difference, p<0,05, ** p<0.01 , *** p<0.005).

[0130] Fig. 3 shows the relationship between calcium phosphate particles and disease activity, autoantibody production and bone destruction in rheumatoid arthritis. A) Total fluorescence of calcium phosphate particles in gate 10 (lactadherin-positive and an OsteoSense fluorescence intensity above 2000 AU) in healthy patients (HD) and patients with rheumatoid arthritis (RA), B) Correlation of the total fluorescence of calcium phosphate particles in gate 10 with disease activity (DAS 28), C) Correlation of the concentration of anti-citrullinated peptide antibodies (ACPA) measured in patients with rheumatoid arthritis with the calcium phosphate particle concentrations in gate 10 (lactadherin-positive and an OsteoSense fluorescence intensity above 2000 AU), D) Calcium phosphate particle concentrations in gates 1-5 in patients with RA and erosive disease (ED) and in patients without erosions (non-ED).

[0131] Fig. 4 shows the course of the calcium phosphate particle concentrations measured in gates 1 and 2 in rheumatoid arthritis under TNF inhibition, especially before TNF inhibition and after 12 weeks of therapy with TNF inhibitors.

[0132] Fig. 5 Comparison of the OsteoSense fluorescence results in Gate 10 with the total fluorescence in the entire sample without prior grouping of the fluorescence results: A) Correlation of OsteoSense fluorescence in Gate 10 with the total fluorescence in the entire sample, B) Correlation of OsteoSense fluorescence with disease activity (DAS 28).

[0133] Fig. 6 shows an image of the detection of individual particles and their fluorescence microscopic representation. The dot plot shows the fluorescence intensity of the OsteoSense (OS) stain (x-axis) plotted against that of the lactadherin stain (LA, y-axis, top image) and against the CTY stain (y-axis, bottom image) in serum samples (A) from healthy subjects and (B) from patients with rheumatoid arthritis, compared to bright-field measurement (BF).

[0134] Implementation of the inventive method

[0135] The embodiment includes preparing the serum sample by diluting it 1:20 and labeling it with the dye OsteoSense™ (IVISense Osteo 680 Fluorescent Probe™, Perkin Eimer) as a calcium phosphate-specific binding fluorescent dye and lactadherene (bovine FITC-labeled lactadherene, Prolytix™, Haematologic Techn.) as a lipid membrane-specific binding fluorescent dye. The excess, unbound fluorescent dye is removed by gel filtration.

[0136] Samples are measured using video flow cytometry with the ImageStream Mark II™ to detect fluorescence-labeled calcium phosphate particles in human serum. Microscopic multispectral images are acquired in visible light and in the corresponding fluorescence channels using a high-resolution digital camera, stored in an analyzable database, and quantified using specialized evaluation mechanisms. The calcium phosphate particles are analyzed using the fluorescence of the two dyes OsteoSense and Lactadherin.

[0137] The evaluation of the measured video flow cytometry files is performed in the following steps: Events that are simultaneously OsteoSense and lactadherin-negative are excluded from the analysis. The separation of a fluorescence-negative from a fluorescence-positive population is clearly possible in channel 2 (lactadherin) (see Fig. 1). The intensity of the OsteoSense fluorescence (channel 5) is initially evaluated such that all events below a threshold value of the fluorescence signal area (100 arbitrary units of fluorescence intensity) are excluded, as these cannot be attributed to particles but represent nonspecific background staining.

[0138] Lactadherene-positive, OsteoSense-negative events are excluded from the analysis. These events contain no OsteoSense-positive calcium phosphate particles and are most likely attributable to calcium-free extracellular vesicles (EVs).

[0139] OsteoSense-positive events are classified into different intensity levels, defined by the following cut-off values: OsteoSense fluorescence between 100 AU and 200 AU, 200 AU and 300 AU, 300 AU and 500 AU, between 500 AU and 1000 AU, between 1000 AU and 2000 AU, and above 2000 AU. These different populations of calcium phosphate particles represent different sizes or different stages of maturation, which differ in their calcium phosphate content and, consequently, in their fluorescence intensity. As a result of this analysis, the concentration of each individual calcium phosphate particle species per microliter of serum can be calculated and reported.

[0140] OsteoSense-positive events are subdivided into lactadherin-positive and lactadherin-negative events (cut-off fluorescence intensity of over 5000 AU).

[0141] The events included in the analysis will subsequently be used as parameters for diagnostic classification.

[0142] Diagnosis of rheumatoid arthritis based on the concentration of OsteoSense and lactadherin-positive calcium phosphate particles

[0143] For the diagnosis of rheumatoid arthritis, the measured concentration of calcium phosphate particles in gate 10, which are simultaneously OsteoSense- and lactadherin-positive, can be used. The measured concentration of such simultaneously OsteoSense- and lactadherin-positive particles is elevated in rheumatoid arthritis patients (n = 74) (Fig. 2) and allows for the confirmation of a diagnosis of rheumatoid arthritis in patients with joint symptoms.

[0144] The median plus two standard deviations of the healthy control cohort was identified as the threshold for a pathological increase in calcium phosphate particle concentration. An alternative threshold for determining disease activity is the median of the study cohort of rheumatoid arthritis patients, which was also higher than that of the healthy controls.

[0145] Patients with rheumatoid arthritis exhibit significantly elevated concentrations of calcium phosphate particles, which are also lactadherine-positive. This applies both to particles in gate 10 (Fig. 2A) and to the combined total concentration in gates 8 to 10 (Fig. 2B).

[0146] The diagnosis of rheumatoid arthritis is particularly reliable in patients with high disease activity (DAS28 > 3.2) because the concentration of simultaneously osteosense- and lactadherene-positive calcium phosphate particles measured at gate 10 in this patient cohort is especially high. This diagnostic value of high concentrations is reflected in the significantly increased concentration of such simultaneously osteosense- and lactadherene-positive calcium phosphate particles shown in Fig. 2C in patients with disease activity greater than DAS 3.2 compared to age-matched healthy controls.

[0147] The DAS28 (Disease Activity Score 28: score = score / evaluation; disease activity = disease activity) is a system for the quantitative assessment of the disease status and progression of rheumatoid arthritis, which is determined from the following parameters: number of tender joints, number of swollen joints, erythrocyte sedimentation rate and an assessment of the disease status / activity by the patient, and has values ​​in the range of 2.0 to 10.0.

[0148] A definitive diagnosis of rheumatoid arthritis is particularly reliable in patients with an erosive, joint-destructive course of the disease. In addition to confirming the diagnosis, assessing the likely prognosis of these patients, who often experience severe joint destruction and resulting impairments, is also important. Rheumatoid arthritis activity can be determined by measuring the concentration of OsteoSense- and Lactadherin-positive calcium phosphate particles.

[0149] In addition to diagnosing rheumatoid arthritis, the method according to the invention can also be used to determine the activity of the disease.

[0150] Currently, disease activity is determined using the DAS28 system (see above) and by measuring the production of anti-citrullinated peptide antibodies (ACPA). This activity assessment is clinically important for monitoring the success of the therapy, for future treatment decisions, and for estimating prognosis.

[0151] The total OsteoSense fluorescence (total fluorescence) is used as a diagnostic parameter, calculated as a measure of the calcium phosphate particles present. This is derived from the number of events in one of the three relevant gates multiplied by the mean fluorescence intensity in the respective gate. Total fluorescence is significantly increased in patients with rheumatoid arthritis compared to healthy controls (Fig. 3A). Within the RA patient group, increased total OsteoSense fluorescence in gate 10 correlates significantly with disease activity (DAS28) (Fig. 3B) and with the level of ACPA (antibodies against citrullinated peptides) measurable in the patients (Fig. 3C).

[0152] Figure 3D shows calcium phosphate particle concentrations in gates 1-5 in patients with RA and erosive joint destruction (ED) and in patients without erosions (non-ED). The particle count is significantly increased in patients with rheumatoid arthritis and erosive joint destruction compared to patients without erosions.

[0153] The total fluorescence at gate 10 therefore allows both the confirmation of a diagnosis of rheumatoid arthritis in patients with high disease activity and the determination of disease activity. Assessment of the therapeutic response to TNF inhibitors is based on the concentration profile of OsteoSense-positive, lactadherene-negative calcium phosphate particles.

[0154] The method according to the invention can also be used to assess the therapeutic response in rheumatoid arthritis, thereby facilitating the decision on whether to continue therapy in unclear clinical situations. For this purpose, the osteosense fluorescence in gates 1 and 2 is determined before and after therapy and compared.

[0155] The particles in gates 1 and 2 are characterized by very low osteosense fluorescence (osteosense fluorescence in the range of 100 to 300 AU) and the complete absence of lactadherine fluorescence. However, they are clearly distinguishable from osteosense- and lactadherine-negative particles in the high-resolution analysis.

[0156] In a longitudinal treatment study, biologic-naïve patients with a disease duration of less than three years and high disease activity despite methotrexate therapy were started on TNF inhibitor therapy. TNF inhibitor therapy can significantly influence disease activity and, in particular, its erosiveness. After twelve weeks of therapy, the median of the study cohort showed a comparatively good clinical response with a marked and significant decrease in DAS28.

[0157] The therapeutic response was demonstrated by a significant decrease in the concentration of calcium phosphate particles in both gate 1 and gate 2 (Fig. 4). The concentrations of calcium phosphate particles in gates 1 and 2 decrease significantly after 12 weeks of TNF inhibitor therapy.

[0158] To date, no comparable diagnostic parameter exists that also allows conclusions to be drawn about the underlying pathogenesis process of the disease.

[0159] Activity assessment of rheumatoid arthritis based on the determination of total OsteoSense fluorescence in serum samples

[0160] Following a diagnosis of rheumatoid arthritis using the method according to the invention, disease activity can be assessed using a simplified procedure comprising the determination of total OsteoSense fluorescence in serum samples using a plate reader (scanner). The serum samples are treated with the fluorescent dye OsteoSense as described above. The excess fluorescent dye not bound to calcium phosphate is removed by gel filtration. For this purpose, diluted serum is passed through a commercially available gel filtration column after staining. This column retains free dye molecules due to its specificity, allowing stained calcium phosphate particles to pass through and be collected in the eluate. The particles are dissolved using ethylenediaminetetraacetic acid (EDTA). The calcium phosphate particle-bound OsteoSense fluorescence is then determined using a suitable plate reader with a fluorescence scanner.The values ​​determined after OsteoSense staining are a measure of the concentration of all calcium phosphate particles in the sample. This is evident in the correlation between the content of OsteoSense-positive calcium phosphate measured photometrically in the plate reader with the OsteoSense fluorescence of the calcium phosphate particles in Gate 10 (lactadherin-positive), measured according to the embodiment described above (Fig. 5A), and with the disease activity (DAS28) (Fig. 5B).

[0161] This simplified procedure allows for a routine assessment of the disease and therapy progress after a diagnosis has been made.

[0162] Diagnosis of rheumatoid arthritis based on the concentration of OsteoSense- and lactadherin-positive calcium phosphate particles in combination with CFSE staining. Analysis of human serum revealed the presence of OsteoSense-positive, CaP-loaded particles that were simultaneously lactadherin-positive and therefore represent vesicles with phosphatidylserine incorporated into lipid membranes (within the frame, shown in Fig. 6A and B, top images). In addition, OsteoSense-negative, lactadherin-positive extracellular vesicles and membrane-free, OsteoSense-positive particles (to the left of the frame), which likely represent CPPs (below the frame), were also detected. To confirm that lactadherin-positive events represent membrane-bound, CaP-loaded extracellular vesicles, the fluorescent dye CellTrace™ Yellow (CTY), which stains intact vesicles containing cytoplasm, was used.Co-staining with CTY showed that the majority of lactadherene-positive CaP particles are indeed intact, membrane-bound, CaP-containing extracellular vesicles exposing phosphatidylserine (within the frame shown in Fig. 6A and B, lower figures). Image analysis of CTY-positive vesicles showed that most are visible in the bright-field channel. The vesicle size can be estimated from the area detected in the bright-field channel, since an area of ​​7.8 pixels corresponds to a diameter of 1 pm. Based on this ratio, a size range of approximately 500 nm to over 1 pm could be derived for visible phosphatidylserine+CaP particles. Non-patent literature cited.

[0163] Jäger, E., Murthy, S., Schmidt, C., Hahn, M., Strobel, S., Peters, A., Stäubert, C., Sungur, P., Venus, T., Geisler, M., et al. (2020) Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis. Nat. Commun. 11.

[0164] Lyons AB, Blake SJ, Doherty KV (2013) Flow cytometric analysis of cell division by dilution of CFSE and related dyes. Curr Protoc Cytom. Chapter 9:9.11.1-9.11.12. doi: 10.1002 / 0471142956. cy0911s64. PM ID: 23546777.

[0165] Poulsom, H., and Charles, P.J. (2008) Antibodies to citrullinated vimentin are a specific and sensitive marker for the diagnosis of rheumatoid arthritis. Clin. Rev. Allergy Immunol. 34, 4-10.

[0166] Rieger AM (2022) Flow Cytometry and Cell Cycle Analysis: An Overview. Methods Mol Biol. 2579:47-57. doi: 10.1007 / 978-1 -0716-2736-5_4. PMID: 36045197.

[0167] Schellekens, G.A., De Jong, B.A.W., Van Den Hoogen, F.H.J., Van De Putte, L.B.A., and Van Venrooij, W.J. (1998) Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J. Clin. Invest. 101, 273.

[0168] Smith, E.R., Hewitson, T.D., Cai, M.M.X., Aghagolzadeh, P., Bachtier, M., Pasch, A., and Holt, S.G. (2017) A novel fluorescent probe-based flow cytometric assay for mineral-containing nanoparticles in serum. Sei. Rep. 7.

[0169] Tempany JC, Zhou JH, Hodgkin PD, Bryant VL (2018) Superior properties of CellTrace Yellow™ as a division tracking dye for human and murine lymphocytes. Immunol Cell Biol. 96(2):149-159. doi: 10.1111 / imcb.1020. Epub 2017 Dec 15. PMID: 29363164; PMCID: PMC6446909.

Claims

Patent claims 1. A method for diagnosing arthritis and / or prognostically assessing the course of arthritis, comprising the steps of: a. providing an isolated serum sample, the serum sample being diluted in the range of 1:5 to 1:100; b. staining particles in the isolated serum sample with at least one calcium phosphate-specific fluorescent dye and with at least one lipid membrane-specific fluorescent dye, wherein at least the emission wavelength of the at least one calcium phosphate-specific fluorescent dye and the at least one lipid membrane-specific fluorescent dye differs; c. separating unbound fluorescent dye; d. fluorescence flow cytometry using at least the excitation and emission wavelengths of the at least one calcium phosphate-specific fluorescent dye and the at least one lipid membrane-specific fluorescent dye; and e.Computer-implemented evaluation of fluorescence flow cytometry based on the fluorescence intensity of at least one calcium phosphate-specifically binding fluorescent dye and at least one lipid membrane-specifically binding fluorescent dye.

2. Method according to claim 1, characterized in that a diagnosis and / or prognostic assessment of the course of the disease is carried out in rheumatoid arthritis or psoriatic arthritis.

3. Method according to claim 1 or 2, characterized in that, during the provision of the isolated serum sample • incubation at a temperature in the range of 15°C to 25°C, or • incubation at a temperature in the range of 35°C to 39°C, and dilution to a concentration in the range of 1:5 to 1:100 is carried out.

4. Method according to one of claims 1 to 3, characterized in that the calcium phosphate-specifically binding fluorescent dye is a bisphosphonate-conjugated fluorescent dye.

5. Method according to one of claims 1 to 4, characterized in that the lipid membrane-specifically binding fluorescent dye phosphatidylserine binds specifically in the lipid membrane and is a lactadherene-conjugated fluorescent dye.

6. Method according to one of claims 1 to 5, characterized in that in step b. additionally a staining with at least one fluorescent dye, which is used for marking cells, in particular for determining cell proliferation, is carried out.

7. Method according to one of claims 1 to 6, characterized in that the separation of unbound fluorescent dye is carried out by gel filtration.

8. Method according to one of claims 1 to 7, characterized in that in step d) a time-resolved microscopic multispectral measurement is performed in the fluorescence range and in the visible range.

9. Method according to any one of claims 1 to 8, characterized in that the computer-implemented evaluation of the fluorescence flow cytometry in step e) is carried out by means of the following steps: • Removal of results that do not show fluorescence of the calcium phosphate-specifically binding fluorescent dye, • Evaluation of the fluorescence of the calcium phosphate specifically binding Fluorescent dye and the fluorescence of the lipid membrane-specifically binding fluorescent dye by grouping the particles according to the Fluorescence intensity, • Determination of the concentration and / or total fluorescence of the Calcium phosphate-positive particles of varying fluorescence intensities, • Comparison of the concentration and / or total fluorescence of calcium phosphate-positive particles of different fluorescence intensities with a cutoff value determined using serum samples from healthy patients.

10. Method for controlling the course of arthritis after a diagnosis of arthritis using the method according to any one of claims 1 to 9, comprising the steps (1) Provision of an isolated serum sample wherein the serum sample is diluted in the range of 1:5 to 1:100, (2) Staining of particles in the serum sample with at least one calcium phosphate-specifically binding fluorescent dye, (3) Separation of unbound fluorescent dye, (4) Dissolving the particles and releasing the fluorescent dye, (5) Fluorescence measurement and (6) Evaluation of the fluorescence intensity of the fluorescent dye that binds at least one calcium phosphate specifically.

11. Kit for the diagnosis of arthritis and / or prognostic assessment of the course of arthritis comprising i. at least one calcium phosphate-specific binding fluorescent dye and ii. at least one lipid membrane-specific binding fluorescent dye.

12. The kit according to claim 11 further comprising at least one fluorescent dye which is used for labeling cells, in particular for determining cell proliferation.

13. Use of a kit according to claim 11 or 12 for the diagnosis of arthritis and / or prognostic assessment of the course of arthritis.

14. Computer program product comprising instructions which, when executed by a computer, cause the computer to perform step e. according to claim 1.

15. Computer program product according to claim 14, comprising instructions which, when the method is executed by a computer, cause the computer to perform the steps according to claim 9.

16. Computer-readable medium on which the computer program product according to claim 14 or 15 is stored.

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