Method for analyzing platelets in a blood sample

Abstract

The present invention relates to a method for analyzing platelets present in a blood sample, the method comprising the steps of: (a) adding a ligand that binds to MCH I coupled to a fluorescent pigment to the sample, (b) measuring the mean fluorescence intensity (MFI) of the platelets by flow cytometry. 血小板 ), and c) measuring the mean "forward scatter" parameter (FSC) using flow cytometry and determining the MFI 血小板 / FSC ratio. The present invention also relates to an in vitro method for diagnosing peripheral thrombocytopenia or central thrombocytopenia in a subject.

Description

Technical Field

[0001] The present invention relates to the field of in vitro blood analysis methods, in particular to the field of methods for analyzing platelets present in a blood sample. More specifically, the method of the present invention can be used for in vitro diagnosis of peripheral thrombocytopenia or central thrombocytopenia in a subject. Background Art

[0002] Platelets are blood cells that lack a nucleus and are produced by the division of megakaryocytes in the bone marrow. They are normally present in human blood at an average concentration of 150,000 to 400,000 per μL and play an important role in hemostasis, a biological process that encompasses all phenomena that prevent and stop bleeding.

[0003] Many conditions are associated with abnormalities in the number of platelets in the blood. These may be either an increased concentration of platelets in the blood (known as thrombocytosis), or a decreased concentration of platelets in the blood (known as thrombocytopenia).

[0004] Most thrombocythaemias are caused by severe bone marrow damage (particularly myeloproliferative syndromes). Detection of these types of thrombocythaemia is necessary so that appropriate medical treatment can be instituted. Initial analysis of the percentage of platelets and young platelets is a simple process that can easily guide exploratory steps that may include analysis of a bone marrow biopsy, a rather invasive procedure.

[0005] There are two types of thrombocytopenia:

[0006] - Central or essential thrombocytopenia, which is caused by defects in the quality and / or quantity of megakaryocyte formation and manifests itself in a decrease in platelet production, resulting in a low platelet concentration in the blood. Central thrombocytopenia can have different causes, which may be genetic or acquired, such as vitamin B12 deficiency, certain cancers, bone marrow dysplasia, liver damage or certain treatments, such as chemotherapy or radiotherapy. In some cases, the cause is unknown. Some diseases are known to be associated with central thrombocytopenia, such as giant platelet syndrome (Bernard-Soulier syndrome) or May-Hegglin anomaly (MYH9).

[0007] - Peripheral thrombocytopenia is characterized by a decrease in the number of circulating platelets and a compensatory increase in megakaryopoiesis and, therefore, an increase in platelet production. Peripheral thrombocytopenia is usually caused by the destruction of circulating platelets, for example in the presence of certain drugs (e.g. heparin, quinine), by the action of autoantibodies or antibodies that bind to platelets. Thus, peripheral thrombocytopenia is characterized by a higher proportion of young platelets (sometimes called immature platelets). Idiopathic thrombocytopenic purpura (ITP) of autoimmune origin is the most common cause of peripheral thrombocytopenia. Peripheral thrombocytopenia can also be explained by the patient's inflammatory condition, which can lead to the consumption of circulating platelets or have an allergic origin.

[0008] Thrombocytopenia may lead to bleeding, which may take the form of petechiae or purpura, abnormal bleeding from the mouth or nose, menorrhagia, and hematomas.

[0009] The treatment of thrombocytopenia depends on its cause. Therefore, it is important to be able to distinguish between central thrombocytopenia and peripheral thrombocytopenia.

[0010] Thrombocytopenia is usually identified by a blood test called a complete blood count or CBC or hemogram, which measures the number of platelets in the blood. If the platelet count is less than 150 x 10 9 If the platelet count is less than 1 / L of blood, thrombocytopenia is diagnosed. However, measuring the platelet count alone cannot distinguish between central thrombocytopenia and peripheral thrombocytopenia.

[0011] Therefore, determining the number of young platelets (i.e., platelets that have just been produced by the bone marrow) in the blood is particularly interesting for distinguishing central thrombocytopenia from peripheral thrombocytopenia. Young platelets are distinguished from other circulating platelets by a higher content of large RNA (messenger RNA and ribosomal RNA) and a slightly larger size. Thiazole orange (TO) or its analogs (such as acridine orange) are dyes that increase fluorescence emission 1000-fold after binding to RNA and are used to identify young platelets by flow cytometry. However, the analysis of young platelets using TO and its analogs is tricky, especially in humans where the young platelet population usually only accounts for 1% to 2% of total platelets. In fact, part of the fluorescence of TO (or its analogs) in platelets is insensitive to treatment with RNases that target cytosolic RNA, which can be explained by the fluorescence after binding to mitochondrial nucleic acids or nucleotides present in dense granules. Therefore, labeling platelets with TO (or its analogs) revealed two populations: the first, called "TO" and the second, called "TO". 亮 ”, whose strong fluorescence was significantly reduced by RNase treatment, so it has a higher RNA content and effectively corresponds to young platelets; the second one is called “TO 暗”, whose TO (or its analogs) have weak fluorescence and are insensitive to RNase treatment, thus they contain little or no cytosolic RNA and do not correspond to young platelets.

[0012] Despite this limitation, the procedure used to date to identify the cause of thrombocytopenia (i.e., central or peripheral) is based on the use of TO analogs to determine the proportion of young platelets in a whole blood sample, known as the “immature platelet fraction” or “IPF.” For example, it can be called process. The process uses an algorithm that integrates platelet size and fluorescence in the presence of an acridine orange derivative, which is a molecule with properties similar to those of TO in terms of RNA. This process is used to obtain a percentage of IPF, which can be used to make a clinical diagnosis in the face of isolated thrombocytopenia. It is believed that high IPF levels reflect peripheral thrombocytopenia, while conventional IPF levels (less than 15%) measured by the Sysmex method can reflect central thrombocytopenia. However, as mentioned above, some acridine orange binds to mitochondrial nucleic acids or nucleotides in dense granules, which are insensitive to RNase treatment and may produce false positives.

[0013] Therefore, there is a real need for an easy-to-use method which dispenses with the use of thiazole orange (TO) or one of its analogs, such as acridine orange, so as to be able to analyse the presence of platelets in a blood sample in a reliable and reproducible manner, in particular for the in vitro diagnosis of peripheral thrombocytopenia or central thrombocytopenia. Summary of the invention

[0014] The inventors have demonstrated that flow cytometric measurements of expression levels of major histocompatibility complex class I (MHC I) molecules in the plasma membrane of platelets correlate with platelet age, with higher expression levels corresponding to "young platelets". Therefore, the inventors have developed a method for analyzing platelets in a blood sample by flow cytometry using an anti-MHC I alpha chain ligand coupled to a fluorochrome. This analytical method is particularly suitable for distinguishing peripheral thrombocytopenia from central thrombocytopenia.

[0015] A first object of the present invention relates to a method for analyzing the presence of platelets in a blood sample, said method comprising the following steps:

[0016] a) adding a ligand that binds to MHC I coupled to a fluorescent pigment to the sample;

[0017] b) Flow cytometry was used to measure the mean fluorescence intensity (MFI) of platelets 血小板 );as well as

[0018] c) Measure the mean forward scatter (FSC) parameter using the flow cytometer and determine the MFI 血小板 / FSC ratio.

[0019] A second object of the present invention relates to a method for identifying a population of platelets present in a blood sample, said method comprising the following steps:

[0020] a) analyzing the presence of platelets in a blood sample by implementing the method of the present invention; and

[0021] b) using the results of step a) to identify a population of platelets.

[0022] A third object of the present invention relates to a method for in vitro diagnosis of peripheral thrombocytopenia or central thrombocytopenia in a subject, the method comprising the following steps:

[0023] a) analyzing platelets present in a blood sample of a subject by implementing the analysis method of the present invention, or identifying platelet populations present in a blood sample of a subject by implementing the identification method of the present invention; and

[0024] b) using the result of step a) to diagnose peripheral thrombocytopenia or central thrombocytopenia.

[0025] A fourth object of the present invention relates to an in vitro method for monitoring the therapeutic efficacy of central thrombocytopenia or peripheral thrombocytopenia, the method comprising the following steps:

[0026] a) analyzing the platelets present in a blood sample of a patient who has undergone treatment for central thrombocytopenia or peripheral thrombocytopenia by implementing the analysis method of the present invention, or identifying the platelet population present in a blood sample of a patient who has undergone treatment for central thrombocytopenia or peripheral thrombocytopenia by implementing the identification method of the present invention;

[0027] b) using the result of step a) to determine the therapeutic efficacy of peripheral thrombocytopenia or central thrombocytopenia.

[0028] A fifth object of the present invention relates to an in vitro method for predicting the development of an inflammatory disease or a cardiovascular disease, said method comprising the following steps:

[0029] a) analyzing platelets present in a blood sample of a subject by implementing the analysis method of the present invention, or identifying platelet populations present in a blood sample of a subject by implementing the identification method of the present invention;

[0030] b) using the result of step a) to predict the development of inflammatory disease or cardiovascular disease.

[0031] The sixth object of the present invention relates to a kit for implementing the method for diagnosing, monitoring the therapeutic efficacy or predicting the development of inflammatory diseases or cardiovascular diseases according to the present invention, the kit comprising:

[0032] - a ligand that binds to MHC I coupled to a fluorochrome;

[0033] - internal standard; and

[0034] -manual.

[0035] Surface expression of MHC I molecules is higher on platelets from young mice. A) Representative FACS analysis of TO and MHC I expression on mouse platelets. Platelets were labeled with PE or Alexa-647-conjugated anti-GPIbβ antibody and thiazole orange (TO). GPIbβ+ platelets were selected and analyzed for MHC I expression by co-labeling with anti-β2m antibody or PE-conjugated anti-MHC I antibody revealed by Alexa-647-conjugated goat anti-mouse secondary antibody. B) MFI of MHC I or MFI of GPIbβ was calculated by comparing the MFI of MHC I with TO. 暗 or TO 亮 The ratio of the FSC parameter to the MFI on platelets was used to assess the surface expression of MHC I and GP1bβ molecules.

[0036] Surface expression of MHC I molecules in vivo decreases during platelet aging. Platelets from iDTR PF4 cre mice treated with diphtheria toxin (DT) were isolated, biotinylated and transfused into WT mice. Flow cytometric analysis of: A) MHC I expression, B) GPIbβ expression and C) biotinylated transfused platelets and endogenous TO at 5 min, 24 h, 48 h and 72 h after transfusion. 暗 Platelets or TO 亮 TO labeling on platelets. Results are expressed as the ratio between the MFI of the labeling and the MFI of the FSC parameter.

[0037] In response to thiazole orange, HLA I molecules are preferentially expressed by highly fluorescent human platelets. A) Representative FACS analysis of TO and MHC I expression on human platelets. Platelets were labeled with PE-conjugated anti-GPIb and TO. GPIbβ+ platelets were selected and stained with A647-conjugated anti-MHC I and PE-CY7-conjugated anti-β2m antibodies to analyze MHC I expression. B) The expression of HLA I molecules was analyzed by counting the expression of HLA I molecules in TO. 暗 Platelets or TO 亮The cell surface expression of MHC I and GP1bβ molecules was assessed by the ratio between MHC I MFI (anti-β2m or anti-HLA I marker) or GP1bβ MFI on platelets and the FSC parameter MFI.

[0038] Human platelets expressing a high density of MHC I molecules exhibit characteristics of young platelets. Representative FACS analysis of ribosomal protein expression, TO, and MHC I molecule expression on human platelets. Human platelets were fixed, permeabilized, co-labeled with PE-conjugated anti-GPIb, mAb ribosomal P protein antibody, A647-conjugated anti-MHC I, PE-CY7-conjugated anti-β2m antibody, and TO, and then analyzed by FACS. A) Selection of GPIbβ+ platelets. TO 亮 Platelets were used to analyze ribosomal protein expression. The expression of MHCI molecules was determined by TO 亮 B) Analysis of ribosomal P protein or TO in populations expressing HLA I molecules strongly or weakly. 亮 The distribution of markers was analyzed.

[0039] Cell surface expression of MHC I is a reliable criterion for determining the proportion of young platelets in patients. A) Detection of TO in blood samples from healthy donors by FACS analysis 亮 and HLA I 高 Platelet percentage and use Systematic MFI percentage (n=6). B) Blood samples from healthy donors or MyH9-deficient patients were analyzed and then the MFI of TO and HLA I on platelets was determined by FACS analysis. Results are expressed as the MFI of HLA I (W6 / 32 or β2m) or the ratio of TO labeling to FSCMFI. C) FACS analysis of TO labeling of different blood samples before and after RNase treatment shows the TO in each case. 亮 The percentage of platelets. D) Citrated blood samples from healthy donors were activated with 0.1 μM, 0.5 μM, 1 μM, 10 μM, 50 μM or 100 μM TRAP before estimation of IPF percentage or analysis of HLA I / FSC or β2m / FSC ratios by flow cytometry (n=3-5). DETAILED DESCRIPTION

[0040] The Applicant has developed a method for analyzing platelets in a blood sample in an accurate and reproducible manner. In particular, the use of this method enables the origin (central or peripheral) of thrombocytopenia to be determined in vitro.

[0041] definition

[0042] "Platelets" are blood cells produced in the bone marrow by the division of megakaryocytes. Mammalian platelets lack a nucleus, and human platelets have a lifespan of about 7 to 10 days before being cleared by macrophages in the spleen or liver. Platelets are an important component of initial hemostasis. In humans, platelets have a diameter of 2 to 4 microns, and their normal concentration in the blood is 150 to 400 × 10 per liter of blood. 9 Platelets. Their number decreases with age. Platelets express major histocompatibility complex (MHC) class I molecules on their surface.

[0043] The term "young platelets" refers to platelets newly produced by the bone marrow, ie, platelets that are less than 24 hours old, such as less than 12 hours old.

[0044] When the platelet concentration is less than 150 × 10 9 When the platelet level is less than 100,000, the platelet level is considered low, a condition called thrombopenia (thrombocytopenia), and the risk of bleeding is increased.

[0045] When the platelet concentration is greater than 400×10 9 When the number of platelets exceeds 50, the platelet level is considered high, called "thrombocytosis," and is associated with the risk of blood clots.

[0046] There are two types of thrombocytopenia:

[0047] - Central or essential thrombocytopenia, which is caused by defects in the quality and / or quantity of megakaryocyte formation and leads to decreased platelet production (blood platelet concentration less than 150 × 10 per liter of blood). 9 Central thrombocytopenia can have different causes, which may be genetic or acquired, such as vitamin B12 deficiency, certain cancers, bone marrow dysplasia, liver damage, or certain treatments, such as chemotherapy or radiation therapy. In some cases, the cause is unknown. Some diseases are known to be associated with central thrombocytopenia, such as giant platelet syndrome or May-Hegglin anomaly (MYH9).

[0048] - Peripheral thrombocytopenia is characterized by a decrease in the number of circulating platelets, a compensatory increase in megakaryopoiesis and, therefore, an increase in platelet production. Peripheral thrombocytopenia is usually caused by the destruction of platelets, for example in the presence of certain drugs (e.g. heparin, quinine), by the action of autoantibodies or antibodies that bind to platelets. Thus, peripheral thrombocytopenia is characterized by a higher proportion of young platelets (sometimes called immature platelets). Idiopathic thrombocytopenic purpura (ITP) of autoimmune origin is the most common cause of peripheral thrombocytopenia. Peripheral thrombocytopenia can also be explained by the patient's inflammatory condition, which can lead to the consumption of circulating platelets or have an allergic origin.

[0049] Major histocompatibility complex class I (MHC I) molecules constitute the self-recognition system and present peptide antigens to T lymphocytes. In humans, MHC I is called human leukocyte antigen (HLA) class I. These molecules are expressed on the plasma membrane of almost all nucleated cells and platelets. They consist of a non-covalent association of α chains (including 3 immunoglobulin-like domains: α1, α2 and α3) and β2 microglobulin (β2m). MHC I expressed on the plasma membrane is always associated with peptides that stabilize MHC I. MHC I is particularly involved in the presentation of antigens to CD8 T lymphocytes, enabling the discrimination between self and non-self.

[0050] As used herein, the term "blood sample" includes any sample containing platelets. For example, the blood sample is obtained from the blood of a subject. Preferably, it is a whole blood sample collected in the presence of an anticoagulant according to the best practices of those skilled in the art. The anticoagulant can be EDTA or citrate.

[0051] The term "ligand" in the meaning of the present invention refers to a molecule that specifically binds to a target. For example, in the context of the present invention, a ligand that binds to MHC I thus refers to a ligand that specifically binds to MHC I.

[0052] In a specific embodiment of the invention, the ligand is unable to bind to the β2m chain that is not associated with the α chain, ie, is unable to bind to the soluble β2m chain.

[0053] In another specific embodiment of the invention, the ligand is capable of binding to the β2m chain that is not associated with the α chain. In this specific embodiment, it is advantageous to use a sufficiently high concentration of the ligand so that when performing the method described in the invention, the soluble β2m chain present in the blood sample does not interfere with the binding to MHC I.

[0054] In a particular embodiment of the invention, the ligand is capable of binding to all isotypes of MHC I, ie isotype A (HLA A), isotype B (HLA B) and isotype C (HLA C).

[0055] The ligand can be an antibody, an antibody fragment, an aptamer, a protein, a peptide or a small molecule. The ligand that can be used in the context of the present invention can be easily obtained by those skilled in the art. For example, the Selex process of aptamers or the immunization and cloning process of antibodies can be mentioned. In a preferred embodiment, the ligand is an antibody or an antibody fragment. Several antibodies that can be used in the present invention are commercially available, such as the antibody W6 / 32 produced by the hybridoma ATCC HB-95, such as the antibody W6 / 32 that can be obtained from Biolegend and has been coupled to APC, or the anti-human β2m antibody (item number: 316318) that can be obtained from Biolegend and has been coupled to the fluorophore PE / Cy7.

[0056] The term "antibody" is used herein in its broadest sense and includes various antibody structures, particularly monoclonal antibodies, having the desired antigen-binding activity. These may be human, humanized, chimeric or non-human species antibodies, such as murine antibodies.

[0057] In the context of the present invention, the ligand is coupled to a means allowing its detection in flow cytometry, preferably a fluorochrome (or fluorophore). The term "fluorochrome" refers to a molecule capable of emitting fluorescence after excitation. The fluorescence intensity (or MFI) emitted by the fluorochrome can be measured by any known method, for example using a flow cytometer capable of detecting and measuring the fluorescence intensity of the fluorochrome.

[0058] "Flow cytometry" is a widely used technique that allows particles or cells to be counted and characterized as they move at high speed in a laser beam. Flow cytometry is a particularly advantageous technique because it allows the fluorescence intensity of individual particles, molecules or cells to be measured by passing them one by one in front of a laser beam.

[0059] An "internal standard" within the meaning of the present invention allows the normalization of the fluorescence intensity (MFI) measured in flow cytometry. In the context of the present invention, the internal standard is preferably a component that binds to the ligand. In particular, the internal standard is a polystyrene microsphere to which the ligand can be adsorbed. Many internal standards suitable for different types of ligands are commercially available, such as the "BD Compbead No. 552843" standard, which can be used when the ligand is an antibody comprising a kappa chain of a mouse antibody.

[0060] In the meaning of the present invention, "subject" refers to a mammal, such as a dog, a cat, a sheep, a cow or a human, preferably, the subject is a human. In the meaning of the present invention, "healthy subject" refers to a subject who does not have any disease causing abnormal platelet count and does not take any drug.

[0061] The term "diagnosis" is used herein in its broadest sense and is commonly and fully understood by those skilled in the art. For example, this term means determining the likelihood, risk or probability that a subject has a condition.

[0062] The terms "treatment" or "treatment" or "responsive to treatment" within the meaning of the present invention refer to the improvement, attenuation, reversal or inhibition of the progression of a disorder. In particular, treatment of a disorder may include restoring normal platelet production in the bone marrow.

[0063] Methods for analyzing platelets or identifying platelet populations

[0064] The present invention relates to a method for analyzing platelets present in a blood sample, the method comprising the following steps:

[0065] a) adding a ligand that binds to MHC I coupled to a fluorescent pigment to the sample;

[0066] b) Flow cytometry was used to measure the mean fluorescence intensity (MFI) of platelets 血小板 ).

[0067] The present invention relates to a method for analyzing platelets present in a blood sample, the method comprising the following steps:

[0068] a) adding a ligand that binds to MHC I coupled to a fluorescent pigment to the sample;

[0069] b) Flow cytometry was used to measure the mean fluorescence intensity (MFI) of platelets 血小板 ); and, advantageously

[0070] c) Measure the mean forward scatter (FSC) parameter using the flow cytometer and determine the MFI 血小板 / FSC ratio.

[0071] The applicants have indeed found that the level of MHC I expression on the surface of platelets is correlated with platelet age. Thus, younger platelets express more MHC I on their plasma membrane than older platelets.

[0072] In particular, the applicant has found that the level of MHC I expression on the surface of platelets normalized to the FSC parameter is correlated with their age. Thus, younger platelets have a higher density of MHC I molecules on their plasma membrane than older platelets.

[0073] Step a)

[0074] Step a) comprises adding a ligand that binds to MHC I coupled to a fluorescent pigment to the sample. This step is performed, for example, by adding the fluorescent pigment-coupled ligand to the blood sample under shaking. Shaking is used to homogenize the mixture to promote contact between the labeled ligand and the platelets.

[0075] The incubation time should be sufficient to contact the labeled part with the platelets. Typically, for antibodies, the incubation time used by technicians is 20 min to 30 min, but can be shortened according to the part used, and then can be less than 5 min, such as less than 4 min, less than 3 min, less than 2 min, less than 1 min, less than 30 seconds. The incubation time can also be longer than 5 min, longer than 10 min, or even longer than 15 min.

[0076] The analytical method of the present invention is particularly easy to implement because it can be implemented using a whole blood sample. However, it is not limited to the use of a specific blood sample. For example, it can be any part of whole blood containing platelets, such as platelet-rich plasma (PRP) or platelets separated from whole blood. Preferably, the blood sample is a whole blood sample.

[0077] In certain embodiments, the blood sample may contain components other than those naturally present in the blood sample, such as a buffer for adjusting the pH, or a suitable anticoagulant, such as EDTA or citrate.

[0078] In a particular embodiment, the ligand may be selected from an antibody, an antibody fragment, an aptamer, a protein, a peptide, or a small molecule. The appropriate amount of ligand can be easily determined by the experimenter. For example, if the ligand is an antibody, it may be added in a concentration range of 0.1 μg / mL to 10 μg / mL. The ligand is added to the sample under saturation conditions to ensure proper platelet labeling.

[0079] In a particular embodiment, the ligand is an antibody or antibody fragment, such as the W6 / 32 antibody produced by the hybridoma ATCC HB-95 or a fragment thereof that retains its ability to bind to MHC I. It may also be an antibody that binds β2m, in which case the antibody should be used at a sufficiently high concentration so that soluble β2m present in the blood does not interfere with the practice of the method.

[0080] The ligand is coupled to a means allowing its detection by flow cytometry, preferably a fluorochrome (or fluorophore). The ligand may be coupled directly or indirectly, preferably directly, to the fluorochrome. Methods for coupling ligands to fluorochromes are widely described in the literature. Therefore, the coupling does not present particular technical difficulties.

[0081] The method is not limited to a specific fluorochrome, as long as it can be detected by flow cytometry. Advantageously, the fluorochrome is one whose excitation and emission wavelengths do not interfere with the reagents used to detect RNA. Non-limiting examples of fluorophores are cited below and in the Examples: FITC (fluorescein isothiocyanate), PE (R-phycoerythrin), APC (allophycocyanin), PerCP (peridinin chlorophyll protein), PE-CF594, PerCP-Cy5.5, PE-Texas Red, GFP (green fluorescent protein), YFP (yellow fluorescent protein) or CFP (cyan fluorescent protein).

[0082] Step b)

[0083] Step b) comprises measuring the mean fluorescence intensity (MFI) of platelets by flow cytometry. 血小板 ). Of course, flow cytometry is able to detect and measure the fluorescence of fluorochromes. The measurement is performed automatically by the flow cytometer. The parameterization of flow cytometry does not present any particular technical difficulties. In particular, the skilled person will know how to set up the flow cytometer according to the selected fluorochrome.

[0084] The measurement performed in step b) allows the identification of platelets bound by the labeled ligand and allows the identification of one or more platelet populations, in particular young platelet populations.

[0085] Step c)

[0086] Step c) comprises measuring the mean forward scatter (FSC) parameter using the flow cytometer and determining the MFI 血小板 / FSC ratio. In addition to fluorescence intensity, flow cytometry can also measure other parameters that can indicate particle size, such as platelet size. The parameter most commonly measured to indicate platelet size is forward scatter (FSC), hereinafter also referred to as "FSC 血小板 This can be FSC-A, FSC-H, or FCS-W. FSC-A is usually used.

[0087] In a specific embodiment, the MFI 血小板 The measured values ​​were normalized to the mean fluorescence intensity (MFI) of the internal standard. 标准 ) to obtain the normalized mean fluorescence intensity (MFI) of platelets 归一化的血小板 In step a), preferably, the internal standard is added to the sample before or after the ligand, or simultaneously with the ligand (preferably before the ligand). The experimenter can easily determine the amount of the appropriate internal standard, preferably setting it to an amount of the same order of magnitude as the number of platelets.

[0088] MFI 血小板 Normalization of measurements to an internal standard is particularly advantageous because it overcomes inter-experimental, inter-laboratory and / or inter-experimenter variability.

[0089] In a specific embodiment, the internal standard is bound to a fluorochrome-coupled ligand, for example, the fluorochrome-coupled ligand is adsorbed onto the internal standard. Thus, in this specific embodiment, MFI 标准 It is the average fluorescence intensity emitted by the fluorochrome bound to the standard and is measured using a flow cytometer capable of detecting and measuring the fluorescence of the fluorochrome. The flow cytometer should be adjusted to distinguish the labeled platelets from the labeled internal standard. Generally, the distinction is made by size. Therefore, when distinguishing by size, it is very important to choose the size of the labeled standard so that the standard can be easily distinguished from the platelets.

[0090] Advantageously, the internal standard is a polystyrene microbead on which a fluorochrome-coupled ligand is adsorbed. For example, it can be a polystyrene microbead coated with an antibody that binds to the fluorochrome-coupled ligand. For example, when the ligand is an antibody (e.g., a chimeric antibody) comprising a kappa chain of a mouse antibody, the commercial product "BD Compbead No. 552843" that can be used can be mentioned.

[0091] In a preferred embodiment, the method of the present invention further comprises the step c) measuring the mean FSC diffusion parameter (FSC) by flow cytometry and determining the MFI 血小板 / FSC or MFI 归一化的血小板 / FSC ratio. Determine MFI 血小板 / FSC or MFI 归一化的血小板 The MFI / FSC ratio is particularly advantageous because it allows a more detailed analysis of the platelets. This enables a more detailed differentiation of one or more platelet populations, especially young platelet populations. Simultaneously with step b), step c) is automatically performed by flow cytometry. In this particular embodiment, the ratio can be automatically determined using the values ​​measured by flow cytometry. For example, the ratio can be determined by calculating (MFI 血小板 / FSC 血小板 ) / (MFI 标准 / FSC 标准 ) ratio to obtain MFI 归一化的血小板 / FSC ratio. Compared with FSC, which indicates platelet size 血小板 Same, FSC 标准 The parameter indicates the standard particle size.

[0092] The present invention also relates to a method for identifying a population of platelets, preferably a population of young platelets, present in a blood sample, the method comprising the following steps:

[0093] a) analyzing the presence of platelets in a blood sample by implementing the method of the present invention; and

[0094] b) using the results of step a) to identify a population of platelets, preferably a population of young platelets.

[0095] Based on the above parameters, identification of platelet populations was performed automatically by flow cytometry.

[0096] Diagnostic Methods

[0097] The present invention relates to a method for in vitro diagnosis of peripheral thrombocytopenia or central thrombocytopenia in a subject, the method comprising the following steps:

[0098] a) analyzing platelets present in a blood sample of a subject by implementing the method for analyzing platelets of the present invention; and

[0099] b) using the result of step a) to diagnose peripheral thrombocytopenia or central thrombocytopenia.

[0100] The results obtained by analyzing platelets according to the invention allow the diagnosis of peripheral thrombocytopenia in a subject. In particular, the MFI parameter in said subject is compared with the same parameter measured in a healthy subject. 血小板 、MFI 归一化的血小板 、MFI 血小板 / FSC ratio and / or MFI 归一化的血小板 An increase in the FSC / FSC ratio gives a positive diagnosis of peripheral thrombocytopenia.

[0101] The increase of the above parameters in the subject under consideration compared to the same parameters measured in healthy subjects reflects an increase in the number of young platelets in the subject under consideration and demonstrates an increased bone marrow activity, thus allowing the diagnosis of peripheral thrombocytopenia.

[0102] In a preferred embodiment, the magnitude of increase in one or more of the above parameters is greater than 1.5, such as greater than 2, greater than 2.5, greater than 3 or greater than 3.5.

[0103] The results obtained by analyzing platelets according to the invention allow diagnosis of central thrombocytopenia in a subject. In particular, by the parameter MFI in said subject 血小板 、MFI 归一化的血小板 、MFI 血小板 / FSC ratio and / or MFI 归一化的血小板 A FSC / FSC ratio that is similar or identical to the same parameter measured in healthy subjects gives a positive diagnosis of central thrombocytopenia.

[0104] In a particular embodiment, the subject has previously been diagnosed as suffering from thrombocytopenia or as being at risk of suffering from thrombocytopenia.The diagnostic method according to the invention will then make it possible to know the origin (central or peripheral) of the thrombocytopenia.

[0105] Methods for monitoring treatment effectiveness

[0106] The present invention also relates to an in vitro method for monitoring the therapeutic efficacy of central thrombocytopenia or peripheral thrombocytopenia, the method comprising the following steps:

[0107] a) analyzing the platelets present in a blood sample of a patient who has undergone treatment for central thrombocytopenia or peripheral thrombocytopenia by implementing the analytical method of the present invention;

[0108] b) using the result of step a) to determine the therapeutic efficacy of central thrombocytopenia or peripheral thrombocytopenia.

[0109] Prognostic methods

[0110] The present invention also relates to an in vitro method for predicting the development of an inflammatory disease or a cardiovascular disease, said method comprising the following steps:

[0111] a) analyzing platelets present in a blood sample of a subject by implementing the analysis method of the present invention;

[0112] b) using the result of step a) to predict the development of inflammatory disease or cardiovascular disease.

[0113] In a specific embodiment, the inflammatory disease is sepsis. In another specific embodiment, the cardiovascular disease is atherosclerosis.

[0114] Diagnostic kits

[0115] The present invention also relates to a kit for diagnosing peripheral thrombocytopenia or central thrombocytopenia in a subject, for monitoring the efficacy of treatment, or for predicting the development of inflammatory diseases or cardiovascular diseases, the kit comprising:

[0116] - a ligand that binds to MHC I coupled to a fluorochrome;

[0117] - internal standard; and

[0118] - Instructions for carrying out the in vitro diagnostic method according to the invention.

[0119] Treatment

[0120] The present invention also relates to a method for treating peripheral thrombocytopenia or central thrombocytopenia in a subject, the method comprising:

[0121] - carrying out the in vitro diagnostic method according to the invention; and

[0122] -When the diagnosis is positive, start appropriate treatment for the subject.

[0123] Suitable treatments are particularly described in reference [8], which is incorporated into the present application by reference, in particular the sections "First-line therapy", "Second-line therapy", "Third-line therapy", "Other new agents" and Table 2 on pages 18 and 19.

[0124] Example

[0125] Materials and methods

[0126] Reagents

[0127] Reagents are commercially available. Thiazole orange (TO) was from Sigma-Aldrich. Xylazine and ketamine (Imalgene ) were from Bayer and Merial, respectively. Biotin was provided by ThermoFischer, and diphtheria toxin (DT) was provided by Santa Cruz.

[0128] Antibodies and flow cytometry

[0129] Rat monoclonal antibodies against the extracellular domain of platelet glycoprotein GPIbβ (Ram1, [7]) were prepared in our laboratory and conjugated to Alexa 647 or 488. Anti-HLAI mouse antibody (clone W6 / 32) conjugated to allophycocyanin (APC) (item number: 311410), human anti-β2m conjugated to PE-CY7 (item number: 316318), and human anti-CD45 conjugated to APC-cy7 (clone 2D1) (item number: 368516) were from Biolegend. Mouse anti-H2 (clone M1 / 42) conjugated to phycoerythrin (PE) (item number: 125506) and streptavidin conjugated to APC-CY7 (item number: 405208) were purchased from Biolegend. Mouse (clone S19.8) anti-b2 microglobulin antibody (β2m) (item number: 555299) was from BD Pharmingen. Mouse anti-CD45 antibody conjugated to PE-cy7 (clone 30-F11) (item number: 25-0451) was provided by Invitrogen. Human anti-ribosomal P protein antibody was kindly provided by Professor Sun from Yang-Ming University, Taiwan, China [1].

[0130] Whole blood was diluted in 10 volumes of PBS containing 1% BSA and 6mM EDTA. Before labeling with one or more antibodies, Fcγ receptors were blocked with a 1 / 200 dilution of "FcR blocking" reagent (Milteny Biotec). After a washing step, cells were reverse labeled with 1 μg / ml TO in PBS for 15 min. The samples were then diluted in 20 volumes of 1% paraformaldehyde within 45 minutes and analyzed by flow cytometry (LSR Fortessa TM Cell analyzer, BD Biosciences) was used for analysis. Data were analyzed using BD FACSDiva software.

[0131] Mouse

[0132] Experiments were performed on 8-10 week old C57Bl6 / J mice purchased from Charles River Laboratories. Mice positive for both the diphtheria toxin receptor transgene and PF4 (designated herein as PF4-cre / iDTR) were generated as described previously [2].

[0133] Platelet transfusion

[0134] PF4-cre / iDTR mice were induced to develop severe thrombocytopenia by injecting diphtheria toxin (DT) (100 ng / day, ip) for 4 days. Eight days after the last injection, blood was collected from the abdominal aorta of anesthetized mice using citrate (3.8%) as an anticoagulant, and platelets were isolated as described previously [3]. Washed cross-linked platelets were biotinylated and platelets were isolated at 1.2·10 6 The cells were resuspended at a concentration of 1:1 / μL and 150 μL of the suspension was injected into the retro-orbital sinus of WT mice. Blood samples were collected 5 minutes after infusion (time 0) and 30 min, 2 h, 24 h, 48 h and 72 h later.

[0135] Ethics statement

[0136] Human blood samples were obtained from Etablissement Volunteers and unpaid blood donors were recruited by the Facility du Sang-Grand Est (EFS-Alsace), where the study was conducted. At each blood draw, donors signed an informed consent form indicating that the sample could be used for research purposes. Ethical approval for animal experiments was in accordance with EU Directive 2010 / 63 / EU. This study was approved by the Comitérégional d'éthique pour l'expérimentation animale de Strasbourg (CREMEAS (CEEA 35)).

[0137] Statistical analysis

[0138] Statistical analysis was performed using GraphPad software (Prism 5.02).

[0139] result

[0140] Platelets from young mice express more MHC I molecules on their surface

[0141] First, we analyzed whether the expression of class I molecules on the surface of mouse platelets depends on their age. In mice, thiazole orange (TO) labeling allows two populations of platelets to be easily distinguished, of which about 10% show strong TO fluorescence (TO 亮 ) and were younger than 12 hours old [2]. The expression of MHC I molecules on the platelet surface was assessed by flow cytometry after labeling with anti-β2m or anti-MHC I (H2) antibodies and subsequent TO. 亮 The expression of class I molecules on platelets appears to be higher than that on TO 暗 To exclude the fact that younger platelets might express more MHC I molecules due to their larger size, we calculated TO 暗 andTO 亮 The ratio of the mean fluorescence intensity (MFI) of the β2m or heavy chain (H2) marker in the population to the mean FSC parameter (a parameter related to cell size). 亮 The ratio of the group is significantly higher than that of TO 暗 Platelet population (H2 / FSC ratio, TO 亮 14.55+ / -0.46, TO 暗 was 8.35+ / -0.14, p<0.001; and β2m / FSC ratio, TO 亮 is 44.78+ / -16.50, TO 暗 In contrast, when we analyzed the expression of the major platelet-specific glycoprotein GPIbβ, the ratios of the two platelet populations were similar (GPIb / FSC ratio, TO 亮 is 247.07+ / -3.57, TO 暗 is 254.70+ / -8.15, n=3, ns).

[0142] Collectively, these data suggest that under steady-state conditions, platelets from young mice express more MHC I molecules on their surface.

[0143] Surface expression of class I molecules decreases in vivo as platelets age

[0144] We then determined whether the surface expression of MHC I on platelets is affected by their aging in vivo. We utilized iDTR PF4 cre mice to obtain a young, synchronous population of platelets [2]. Daily administration of diphtheria toxin (DT) for 4 days induced ablation of mature megakaryocytes, blocked platelet production and resulted in progressive thrombocytopenia. Four days after cessation of treatment (day 8), megakaryocyte production and platelet production were significantly improved, resulting in the transient presence of the vast majority of young platelets (data not shown and [2]). Therefore, platelets were isolated from DT-treated iDTR PF4 cre mice on day 8, biotinylated and transfused into WT mice. MHC I expression on biotinylated transfused platelets was analyzed by flow cytometry at different times after transfusion and compared with endogenous platelet MHC I expression. The results showed that 5 min after transfusion, the (TO mean fluorescence) / (mean FSC) ratio of biotinylated platelets was higher, in contrast to endogenous TO. 亮 After 24 hours, the ratio was comparable to endogenous TO 暗 The same was true for platelets, confirming the in vivo aging of transfused platelets. Notably, the expression of MHC I on biotinylated transfused platelets decreased gradually over time. Five minutes after transfusion, the expression of MHC I on biotinylated platelets was similar to that of endogenous TO. 亮 The expression level on platelets was similar. After 24 hours, the expression level was significantly reduced to the same level as endogenous TO. 暗 The present study showed that in vivo, the expression levels of MHC I molecules on the surface of platelets are related to their age. In contrast, GPIbβ expression on transfused platelets was stable over 72 hours.

[0145] Collectively, these data highlight the relevance of MHC I expression parameters in identifying young platelets, less than 24 h old.

[0146] TO 亮 Human platelets preferentially express HLA I molecules

[0147] In humans, TO 亮 Platelets make up 1% to 2% of the total platelet population. To characterize the expression of HLA I molecules on the surface of human platelets, the latter were co-labeled with anti-β2m antibody, pan anti-HLA class I antibody W6 / 32[4], and then counter-labeled with TO. Flow cytometric analysis showed that, as observed in mice, TO 亮 Platelets express higher levels of HLA I molecules.

[0148] To analyze the distribution of HLA I molecules in the platelet population independent of platelet size, we used the same approach as for mouse platelets, namely the ratio of the HLA I MFI / FSC parameter. Again, we found that TO 亮 This ratio of platelets is higher than TO 暗 Platelets (β2m / FSC ratio, TO 亮 11.58±1.25, TO 暗 was 6.59±0.762, n=7, p<0.05; and W632 / FSC ratio, TO 亮 is 16.02±1.99, TO 暗 8.46±0.93, n=7, p<0.001). 暗 andTO 亮 The GPIbβ MFI / FSC ratios were similar in young platelets, highlighting the specificity of HLA I molecule expression on young platelets.

[0149] Human platelets that strongly express HLA I molecules have higher ribosomal protein content, a characteristic of young platelets

[0150] The ribosomal P protein antigen is present in the C-terminal part of the three ribosomal subunits (RLP0, 2, and 3) and is required for mRNA translation [5]. Since mRNA translation occurs mainly in the first few hours of platelet life [2], it is expected that mRNA translation will be activated at least in the first few hours of platelet life. 亮 Ribosomal P protein antigen can be detected in platelets.

[0151] Human platelets were fixed, permeabilized, and co-labeled with antibodies against ribosomal P protein antigen, anti-β2m antibody, pan anti-HLA I antibody, W6 / 32, and TO before analysis by flow cytometry.

[0152] Labeling with antibodies against ribosomal P protein showed that 13.3±% (n=4) of human platelets expressed this protein, while in TO 亮 In platelet subsets, it was 83±% (n=4), indicating that the antigen was more expressed by younger platelets. In addition, analysis of MHC I molecule expression showed that in the dark gray TO 亮 We then analyzed the distribution of ribosomal P protein in the populations with high or low expression of HLA I molecules (labeled with W6 / 32 or anti-β2m). 高 Expressed in platelets, but only in a minority of HLA I 低 The expression of mitochondria and prostate in platelets was 82.77±10.63% and 10.47±3.59%, respectively, n=4.

[0153] The expression level of HLA I molecules on platelets can be used as a standard to evaluate the proportion of young platelets in patients

[0154] To investigate the relevance of these observations in clinical applications, we evaluated the expression of HLA I molecules on the surface of human platelets from healthy blood donors or patients with conditions associated with an increased proportion of young platelets in the circulation. We first compared the expression of HLA I molecules in healthy volunteers with those in patients with healthy volunteers. 高 ” and TO 亮 Platelet percentage and Percentage of donated IPF (n=6). In healthy volunteers, HLA I 高 andTO 亮 Platelet percentages were similar (2.25% ± 0.26 vs 2.17% ± 0.19, n = 6), however IPF percentages were more heterogeneous but still below 15% (6.7% ± 1.6, n = 6).

[0155] Finally, we analyzed the expression of HLA I molecules on the platelet surface of two patients with myosin 9 deficiency, whose The analysis showed a higher percentage of IPF (64.9% and 42.7% IPF, respectively).

[0156] Citrated blood from each patient was co-labeled with antibodies against β2m, pan anti-HLA I antibody, W6 / 32, and TO before analysis by flow cytometry. Of note, although both patients (P1 and P2) had higher TO / FSC ratios compared with healthy volunteers, only one patient (P1) had higher β2m / FSC and W6 / 32 / FSC ratios compared with controls. Considering the fact that TO can bind not only to RNA but also to particulate matter, RNaseA treatment was performed before TO labeling. Flow cytometric analysis showed that RNaseA treatment significantly reduced TO in patient 1. 亮 Platelet ratio (TO after RNaseA treatment) 亮 The TO before RNase A treatment was 4.9%. 亮 15.9%), and this treatment had an effect on the TO of patient 2. 亮 Platelet population was not affected (TO after RNaseA treatment) 亮 The TO before RNase A treatment was 26.1%. 亮 is 29.4%).

[0157] Since it is known that estimates of IPF may be biased by platelet activation, particularly due to the presence of aggregates [6], we have examined whether surface expression of HLA I molecules could serve as a more reliable criterion for assessing the proportion of young platelets.

[0158] Therefore, in the presence of Prior to technical analysis, citrated blood was incubated with different concentrations of the platelet aggregation reagent TRAP (0.1 μM, 0.5 μM, 1 μM, 10 μM, 50 μM, or 100 μM). Although TRAP was not significantly increased starting with 0.5 μM, the platelet aggregation was not significantly increased with TRAP. The IPF of the technique increased significantly (IPF in the 0.5 μM TRAP condition was 10.7 ± 8% and in the non-activated condition was 4.2 ± 0.8%; n = 3), and the β2m / FSC or W632 / FSC ratios remained stable (1.3 ± 0.4 in the 0.5 μM TRAP condition and 1.5 ± 0.3 in the non-activated condition, n = 3). It is noteworthy that with a higher dose of TRAP (100 μM), the HLA I / FSC ratio increased, which could be due to the exposure of the HLA I complexes present in the particles to the plasma membrane. However, it is interesting to emphasize that this increase is still within the reference values ​​found in healthy volunteers.

[0159] These data suggest that for samples from patients with a high proportion of young platelets, the expression level of HLA I molecules is higher than that of TO markers or The technology is more reliable.

[0160] Interest in FSC (FSC-A) normalization

[0161] a) Blood samples from 7 healthy blood donors and 1 patient with giant platelet syndrome were analyzed using the method described in the present invention, optionally calculating the ratio to FSC (ie with and without FSC normalization).

[0162] For the results obtained without normalization, BSS patients may suffer from peripheral thrombocytopenia. In fact, the unnormalized data seem to indicate that the platelets of BSS patients express more HLA I molecules on their surface. Therefore, this patient will have more young platelets than healthy blood donors.

[0163] Calculation of the ratio to FSC showed that BSS patient platelets did not express more HLA I molecules on their surface. Therefore, BSS patients did not have more young platelets than healthy donors. Therefore, normalization avoided false positives.

[0164] b) Blood samples from 21 healthy blood donors, 1 patient with myelofibrosis (MF), 2 patients with May-Hegglin abnormality (MYH9) and 2 patients with thrombotic thrombocytopenic purpura (ITP) were analyzed using the method described in the present invention, optionally calculating the ratio to FSC (i.e. with and without FSC normalization).

[0165] Regardless of platelet size, all patients would be suspected of having more young platelets and therefore be labeled as having “peripheral thrombocytopenia.” This finding is consistent with the condition in patients with MF and ITP (enhanced bone marrow activity), whereas MYH9 patients may not have enhanced bone marrow activity.

[0166] The ratio to platelet size showed that MYH92 patients were actually false positives and did not have more young platelets than healthy blood donors.

[0167] discuss

[0168] The results suggest that measuring the level of MHC I expression in relation to platelet size in flow cytometry is useful for platelet analysis, particularly for identifying young platelets. It was indeed shown that young platelets express more MHC I on their surface, a level that decreases as platelets age. All of these results emphasize the importance of the MHC I / FSC ratio in assessing the proportion of young platelets in a subject.

[0169] Until now, clinicians have typically used TO to visualize RNA markers to analyze the presence of young platelets in a sample (TO 亮 ). However, this technique has limitations because the reagents used to bind cytosolic RNA (TO) can also bind nucleotides contained in the particles. Measurement of MHC I expression avoids nonspecific labeling of RNA.

[0170] Furthermore, measurement of MHC I expression avoids another disadvantage, namely the presence of aggregates in the samples analyzed. False positives are generated in the method.

[0171] By measuring the parameter MFI 血小板 The results obtained by the inventors are particularly accurate when the measurements of MHC I expression levels are normalized to the FSC parameter, which enables in particular an indication of particle size (e.g. platelet size), and then normalized to the fluorescence intensity of the internal standard (MFI 标准 ). This normalization makes it possible in particular to compensate for inter-experimental, inter-laboratory and / or inter-experimenter variability.

[0172] When the inventors determined (MFI 血小板 / FSC 血小板 ) / (MFI 标准 / FSC 标准 ) ratio (i.e., MFI 归一化的血小板When platelets are analyzed using flow cytometry (FSC / FSC), even more accurate results are obtained. This ratio allows platelets to be analyzed with great precision. Ultimately, it allows for a more refined distinction between one or more platelet populations, particularly young platelet populations. In summary, the expression level of MHC I measured in flow cytometry is a good marker for analyzing platelets present in a blood sample, particularly for determining the proportion of young platelets. Therefore, the expression density of MHC I molecules has become a preferred marker for diagnosing peripheral thrombocytopenia or central thrombocytopenia in a subject and for monitoring the therapeutic efficacy of central thrombocytopenia or peripheral thrombocytopenia.

[0173] References cited in this application as "[reference number]":

[0174] 1. Sun, KH, et al., Monoclonal antibodies against human ribosomal Pproteins penetrate into living cells and cause apoptosis of jurkat T cells in culture. Rheumatology (Oxford), 2001.40(7): p.750-6.

[0175] 2. Angenieux, C., et al., Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity. PLoS One, 2016.11(1): p.e0148064.

[0176] 3.Hechler, B., et al., Platelets are dispensable for antibody-mediatedtransfusion-related acute lung injury in the mouse, J Thromb Haemost, 2016.14(6): p.1255-67.

[0177] 4.Tran,T.M.,et al.,The epitope recognized by pan-HLA class I-reactivemonoclonal antibody W6 / 32 and its relationship to unusual stability of theHLA-B27 / beta2-microglobulin complex.Immunogenetics,2001.53(6):p.440-6.

[0178] 5.Derylo,K.,et al.,The uL10 protein,a component of the ribosomal P-stalk,is released from the ribosome in nudeolar stress.Biochim Biophys Acta,2018.1865(1):p.34-47.

[0179] 6.Miyazaki,K.,et al.,Immature platelet fraction measurement isinfluenced by platelet size and is a useful parameter for discrimination ofmacrothrombocytopenia.Hematology,2015.20(10):p.587-92.

[0180] 7.Perrault,C.et al.,Novel Monoclonal Antibody against theExtracellular Domain of GPIbModulates vWF Mediated Platelet Adhesion,ThrombHaemost,2001.86:p.1238-48.

[0181] 8.Nomura,S.,Advances in Diagnosis and Treatments for ImmuneThrombocytopenia,Clinical Medicine Insights:Blood Disorders,2016.9:p.15-22.

Claims

1. A method for analyzing platelets present in a blood sample, the method comprising the following steps: a) adding a ligand that binds to MHCI coupled to a fluorescent dye to the sample; b) Measuring the mean fluorescence intensity (MFI) of the platelets using flow cytometry 血小板 ; as well as c) Measure the mean forward scatter (FSC) parameter using the flow cytometer and determine the MFI 血小板 / FSC ratio.

2. The method of claim 1, wherein the blood sample is a whole blood sample or a portion of whole blood containing platelets.

3. The method of any one of claims 1 or 2, wherein the ligand is an antibody or an antibody fragment.

4. The method of claim 3, wherein the antibody is the W6 / 32 antibody produced by hybridoma ATCC HB-95 or a derivative thereof that retains the ability to bind to MHC I.

5. The method according to any one of claims 1 or 2, wherein the fluorescent dye is a fluorescent dye whose excitation wavelength and emission wavelength do not interfere with a reagent for detecting RNA.

6. The method according to any one of claims 1 or 2, wherein the MFI 血小板 The measured values ​​were normalized to the mean fluorescence intensity (MFI) of the internal standard. 标准 , to obtain the normalized platelet mean fluorescence intensity (MFI) 归一化的血小板 , and step c) comprises measuring the mean forward scatter FSC parameter using said flow cytometry to determine the MFI 归一化的血小板 / FSC ratio.

7. The method of claim 6, wherein the internal standard is bound to the ligand coupled to the fluorescent pigment.

8. The method according to claim 7, wherein the internal standard is polystyrene microbeads bound to a ligand coupled to the fluorescent pigment.

9. The method of any one of claims 1 or 2, wherein the ligand at least partially binds to the MHC I alpha chain.

10. A method for identifying a population of platelets present in a blood sample, the method comprising the steps of: a) analyzing platelets present in a blood sample by implementing the method according to any one of claims 1 to 9; as well as b) using the results of step a) to identify a population of platelets.

11. The method of claim 10, wherein: The platelet population is a young platelet population.

12. Use of a ligand that binds to MHC I coupled to a fluorescent pigment in the preparation of a reagent for in vitro diagnosis of peripheral thrombocytopenia or central thrombocytopenia in a subject, wherein: The use is the use of the ligand bound to MHCI coupled to a fluorescent dye in the preparation of a reagent for analyzing platelets present in a subject's blood sample by implementing the method described in any one of claims 1 to 9, or identifying platelet populations present in a subject's blood sample by implementing the method described in any one of claims 10 or 11, thereby diagnosing peripheral thrombocytopenia or central thrombocytopenia.

13. The use according to claim 12, wherein The use is that the ligand bound to MHC I coupled to a fluorescent pigment is used in the preparation of a ligand for detecting MHC I in the subject through MFI 血小板 / FSC ratio and / or MFI 归一化的血小板 The invention relates to a reagent for giving a positive diagnosis of peripheral thrombocytopenia by comparing the FSC / FSC ratio with an increase in the same parameter measured in healthy subjects.

14. The use according to claim 12, wherein The use is for preparing the ligand bound to MHC I coupled to a fluorescent pigment for measuring the MFI parameter in the subject 血小板 / FSC ratio and / or MFI 归一化的血小板 The invention relates to a reagent for making a positive diagnosis of central thrombocytopenia by showing that the FSC / FSC ratio is similar or identical to the same parameter measured in healthy subjects.

15. Use of a ligand that binds to MHC I coupled to a fluorescent pigment in the preparation of a reagent for in vitro prediction of the development of an inflammatory disease or a cardiovascular disease, wherein: The use is the use of the ligand bound to MHCI coupled to a fluorescent dye in the preparation of a reagent for analyzing platelets present in a blood sample from a subject by implementing the analytical method described in any one of claims 1 to 9, or identifying a platelet population present in a blood sample from a subject by implementing the method described in any one of claims 10 or 11 to predict the development of an inflammatory disease or a cardiovascular disease.

16. Use of a ligand that binds to MHC I coupled to a fluorescent pigment in the preparation of a reagent for in vitro monitoring of the therapeutic efficacy of central thrombocytopenia or peripheral thrombocytopenia, wherein: The use is the use of the ligand bound to MHC I coupled to a fluorescent dye in the preparation of a reagent for analyzing platelets present in a blood sample of a patient who has undergone treatment for central thrombocytopenia or peripheral thrombocytopenia by implementing the analytical method described in any one of claims 1 to 9, or for identifying platelet populations present in a blood sample of a patient who has undergone treatment for central thrombocytopenia or peripheral thrombocytopenia by implementing the analytical method described in any one of claims 10 or 11, thereby determining the therapeutic efficacy of central thrombocytopenia or peripheral thrombocytopenia.

17. A kit for implementing the method according to any one of claims 7 to 11, the kit comprising: - a ligand that binds to MHC I coupled to a fluorochrome; - internal standard; and -manual.