Reagents and methods for diagnosing thrombus events

Abstract

The present disclosure relates to reagents, kits and methods for the detection of anti-PF4 antibodies and the diagnosis of heparin-free thrombotic events, including vaccine-induced thrombocytopenia. The present disclosure provides a method comprising: (i) contacting a whole blood, plasma or serum sample obtained from a subject with an agent comprising: (a) a binding molecule selected from the group consisting of platelet factor 4 protein (PF4), a PF4 fragment capable of binding to an anti-PF4 antibody, and an anti-idiotype antibody of an antiplatelet factor 4 antibody (anti-PF4 antibody); and (b) a solid support wherein the binding molecule (a) is covalently bound to the surface of the solid support (b) and wherein the binding molecule (a) does not comprise heparin or a heparin substitute, and (ii) analysing the sample to detect a complex formed by the reagent and the anti-PF4 antibody wherein detecting the complex indicates that the sample contains the anti-PF4 antibody.

Description

[0001] This application claims the benefit of European patent application No. EP22382635.5 filed on July 1, 2022. Technical Field

[0002] The present invention relates to the field of clinical diagnostics, in particular to the determination of anti-platelet factor 4 (anti-PF4) antibodies, which allows, for example, the diagnosis of non-heparin-related thrombotic thrombocytopenias, such as vaccine-induced thrombotic thrombocytopenia (VITT). Background Art

[0003] Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most important countermeasure in the fight against the current Covid-19 pandemic. One of the first vaccines approved by the European Medicines Agency was a recombinant chimpanzee adenovirus [ChAdOx1-S] vector encoding the spike glycoprotein of SARS-CoV-2, and the COVID-19 vaccine AstraZeneca (also known as ChAdOx1 nCoV-19, AZD1222, Vaxzevria) is an adenovirus vector-based vaccine.

[0004] Unfortunately, several countries have reported cases of cerebral venous sinus thrombosis (CVST) and visceral vein thrombosis, combined with moderate to severe thrombocytopenia, in healthy individuals after vaccination with the ChAdOx1 nCov-19 vaccine. This novel condition, “vaccine-induced immune thrombotic thrombocytopenia (VITT),” is associated with high titers of immunoglobulin G class antibodies against the cationic platelet chemoattractant, platelet factor 4 (PF4; CXCL4). These antibodies potently activate platelets via the platelet FcγIIa receptor, with PF4 significantly enhancing platelet activation. Another adenovirus-based vaccine, the COVID-19 vaccine Janssen (Johnson & Johnson), also appears to be associated with the development of anti-PF4 antibodies.

[0005] PF4 opsonizes the polyanionic surface of pathogens, which promotes the binding of anti-PF4 antibodies. This is likely an ancient evolutionary immune defense mechanism, as anti-PF4-producing B cells can be found in almost all individuals, even in the umbilical cord blood of newborns. However, a strong anti-PF4 antibody response, when misdirected, underlies the thromboembolic diseases, immune heparin-induced thrombocytopenia, and its most severe manifestation, autoimmune heparin-induced thrombocytopenia. Autoimmune heparin-induced thrombocytopenia is characterized by the formation of platelet-activating anti-PF4 antibodies with extremely high affinity, which are reactive even in the absence of heparin. Severe heparin-induced thrombocytopenia is also characterized by "whole-cell" activation (platelets, neutrophils, monocytes, endothelial cells). VITT closely resembles autoimmune heparin-induced thrombocytopenia (HIT) both clinically and serologically (Greinacher et al, "Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination". DOI: 10.1056 / NEJMoa2104840). One of the major risk factors for the development of anti-PF4 antibodies is inflammation and tissue trauma, which greatly increases the risk of developing pathogenic anti-PF4 antibodies.

[0006] In addition to VITT, other thrombotic events unrelated to heparin have been observed (Favaloro et al, 2022 "Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT / HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT / TTS" DOI: 10.3390 / antib1101000). New thrombotic events also include vascular occlusions in the arterial system. The factors that lead to these conditions are largely unknown, but despite this, their diagnosis is as difficult as VITT. Therefore, these conditions are underdiagnosed in current clinical protocols, which mainly include the use of rapid HIT assays or commercial anti-PF4 ELISA assays and functional assays for heparin-dependent antibodies.

[0007] Currently, several commercial immunoassays are available for the diagnosis of HIT, including classic IgG-specific and multispecific ELISA assays (e.g., Asserachrom HPIA IgG, Lifecodes PF4 IgG, Hyphen Biomed Zymutest HIAIgG, AESKULISA HiT II) as well as rapid assays (e.g., AcuStar HIT-lgG (PF4-H) 、 HIT-Ab (PF4-H) , Diamed PaGIA gel, STic Expert). All assays use PF4 protein complexed with heparin or heparin substitutes (such as polyvinyl sulfate) to detect anti-PF4 antibodies (anti-PF4 / H antibodies) triggered by heparin in patients suspected of having HIT. However, the available assays are not suitable for diagnosing VITT. Previous studies have shown that rapid assays have very poor sensitivity for diagnosing VITT, which makes them unsuitable for diagnosing this special condition. On the other hand, the anti-PF4 / heparin or anti-PF4 / PVS ELISA assays of the prior art only detect some VITT cases, and no single ELISA method seems to detect all VITT cases (Platton et al, "Evaluation of laboratory assays for anti-platelet factor 4 antibodies after ChAdOx1 nCOV-19 vaccination". DOI: 10.1111 / jth.15362). Most problematically, even those assays that recognize certain VITT-like antibodies also recognize heparin-dependent HIT-like antibodies, making it impossible for the assays to distinguish between the two antibody specificities. This has led experts to recommend that when an ELISA result is negative, a second ELISA or platelet activation assay should be considered if VITT is strongly suspected clinically, a conventional approach that significantly complicates diagnosis. Even this step can lead to false-negative results if the functional test is performed with heparin enhancement rather than PF4 enhancement. However, the ELISA assays that are more widely available in diagnostic laboratories have a long turnaround time of at least 6 to 8 hours, and only a few laboratories are able to perform platelet activation assays.

[0008] Another problem is that the available immunoassays are designed to measure the presence of anti-PF4 / H antibodies and, therefore, are not specific for the anti-PF4 antibodies found in patients with VITT. Consequently, these immunoassays cannot distinguish between patients with HIT and VITT (if they detect VITT at all). Consequently, in patients receiving heparin, it is impossible to distinguish whether a thrombotic event has been triggered by heparin or by another cause (e.g., vaccination or other unknown cause). This lack of a differential diagnosis of VITT relative to HIT makes it impossible to discern which patients can continue to receive heparin and which patients must interrupt heparin administration, often replacing it with a different, much more expensive antithrombotic drug.

[0009] In addition, an increasing number of patients are thought to have thrombotic diseases that are independent of the presence of heparin or vaccination. In some of these patients, variable proportions of heparin-dependent and heparin-independent anti-PF4 antibodies have been identified. Currently, these antibodies can be distinguished by functional assays. New and widely applicable assays that can distinguish these various anti-PF4 diseases are needed to systematically assess the prevalence of these different patient populations. This is clinically relevant because anti-PF4 heparin-independent antibodies require auxiliary measures (in addition to anticoagulation) to reduce FcγRIIa-dependent cell activation that triggers a large amount of hypercoagulability.

[0010] Therefore, there is an urgent need to provide a sensitive and specific assay for detecting anti-PF4 heparin-independent antibodies for the diagnosis of VITT and other thrombotic events not induced by heparin or vaccination, which is cost-effective, rapid, and easy to implement in clinical practice. Summary of the Invention

[0011] The present inventors have developed a novel method for detecting heparin-independent anti-PF4 antibodies (hereinafter also referred to as "anti-PF4 antibodies") that overcomes the aforementioned disadvantages. The method of the present invention specifically detects anti-PF4 antibodies, but does not detect heparin-dependent HIT-like antibodies. Therefore, the novel method allows for the diagnosis of thrombotic events that are not induced by heparin, such as VITT or other types. The novel method is based on a reagent consisting of a molecule capable of binding to anti-PF4 antibodies and a solid support, wherein the anti-PF4 antibodies are derived from a human patient who is currently suffering from or has previously suffered from a thrombotic event that is not induced by heparin, and the molecule capable of binding to the anti-PF4 antibodies is covalently bound to the surface of the solid support.

[0012] Therefore, a first aspect of the present disclosure provides a reagent comprising: (a) a molecule for binding to an anti-PF4 antibody and (b) a solid support, wherein the molecule is covalently bound to the surface of the solid support.

[0013] Figure 1 An illustrative schematic diagram of a reagent according to the first aspect of the present disclosure is shown. The covalent bond of the molecule for binding anti-PF4 antibodies (from now on also referred to as "binding molecule") to the solid support is important because the inventors found that anti-PF4 antibodies could hardly be detected in a small number of samples from VITT patients by attaching the binding molecule by other means (see Figure 4 and 5 ). It was also found that the binding molecule must not be able to form complexes with heparin or other heparin substitutes (such as PVS). Figure 3 This was confirmed in the results of the present invention, where it can be seen that the plasma of all VITT patients showed a high signal only when tested using the reagent of the present disclosure, which contains only PF4 as the sole binding molecule. Therefore, the binding molecule (a) of the reagent of the first aspect of the present disclosure does not include heparin or any other heparin substitute, that is, the binding molecule (a) does not form a complex with heparin or a heparin substitute.

[0014] In a second aspect, the present disclosure provides a method for preparing a reagent as defined in the first aspect, the method comprising: (i) contacting a solid support with a solution comprising a molecule capable of binding to an anti-PF4 antibody in the presence of a cross-linking agent and, optionally, a catalyst. Alternatively, the method for preparing a reagent as defined in the first aspect may comprise contacting a solid support with a solution comprising a cross-linking agent and, optionally, a catalyst, and (ii) subsequently adding a molecule capable of binding to a human anti-PF4 antibody.

[0015] As outlined above, for reagent purposes it is important that the molecule capable of binding the anti-PF4 antibody (eg, PF4 protein) does not itself form a complex with heparin or a heparin surrogate prior to covalent attachment to the support.

[0016] A third aspect of the present disclosure relates to an agent obtainable by the method defined in the second aspect.

[0017] The fourth aspect of the present disclosure provides a composition comprising the agent defined in the first aspect or the third aspect of the present disclosure.

[0018] The fifth aspect relates to use of the reagent as disclosed in the first or third aspect or the composition as defined in the fourth aspect for detecting anti-PF4 antibodies.

[0019] The sixth aspect relates to the use of the reagent disclosed in the first or third aspect or the composition defined in the fourth aspect for differential detection of anti-PF4 antibodies (without detection of anti-PF4 / heparin antibodies) and / or for diagnosis of thrombotic events not induced by heparin (e.g. VITT).

[0020] The seventh aspect relates to an in vitro method for detecting human anti-PF4 antibodies, comprising: (i) contacting an agent as defined in the first or third aspect or a composition as defined in the fourth aspect with a biological sample obtained from a subject, and (ii) analyzing the sample to detect an immune complex formed by the agent and the anti-PF4 antibody, wherein the formation of the immune complex indicates the presence of the anti-PF4 antibody in the sample.

[0021] Since anti-PF4 antibodies are often present in thrombotic events that are not induced by heparin, the detection of anti-PF4 antibodies allows the diagnosis of these conditions. Therefore, the eighth aspect relates to an in vitro method for diagnosing thrombotic events that are not induced by heparin, comprising: (i) contacting a reagent as defined in the first or third aspect or a composition as defined in the fourth aspect with a biological sample obtained from a subject, and (ii) analyzing the sample to detect immune complexes formed by the reagent and anti-PF4 antibodies, wherein the formation of immune complexes indicates a thrombotic event that is not induced by heparin. The thrombotic events that are not induced by heparin include VITT, as well as other thrombotic events that are not induced by heparin but are not induced by vaccination, such as, for example, spontaneous HIT-like syndrome. It has been demonstrated that anti-PF4 antibodies are present in such thrombotic events that are not induced by heparin or vaccination, for example, in patients with spontaneous HIT-like syndrome, as described in the present disclosure. Figure 7 Anti-PF4 antibodies have also been observed in patients with severe or recurrent thrombotic and / or thrombocytopenic events that were independent of heparin or vaccination.

[0022] The method disclosed herein overcomes many of the shortcomings of current methods for diagnosing thrombotic events not induced by heparin. First, the method has good sensitivity. As observed in the examples below, all samples from VITT patients were correctly identified as positive when tested by the method disclosed herein, while all control samples (from healthy patients) gave negative results ( Figure 2 The relative light unit (RLU) signal values ​​observed for the VITT samples were very high, indicating high sensitivity. Importantly, the methods disclosed herein are also specific for detecting thrombotic events not induced by heparin ( Figure 6 ) and has no cross-reactivity with HIT ( Figure 2). This is also demonstrated by the following examples, in which it can be observed that all samples obtained from HIT patients tested negative with the present method. Therefore, the method disclosed herein allows for the differential diagnosis of VITT or other non-heparin-induced thrombotic events from HIT. Therefore, the method is a very valuable tool for the clinician, who can choose the most appropriate treatment plan based on it. For example, when a patient's sample tests positive with the method disclosed herein, this indicates the presence of VITT or other non-heparin-induced thrombotic events, which can be very serious. This information is valuable to the clinician, who can recommend auxiliary measures to reduce the FcγRIIa-dependent cell activation that triggers a large hypercoagulable state. Typically, this will be achieved by administering intravenous immunoglobulin (IVIG). In addition, when a patient's sample tests positive with the method disclosed herein, but a very specific method for detecting anti-PF4 / heparin antibodies (e.g. Acustar HIT-IgG (PF4-H) Determination and HIT-Ab (PF4-H) ) test negative, the thrombotic event is most likely not induced by heparin, and therefore, the clinician does not need to interrupt heparin therapy. In contrast, when a patient sample tests negative using the methods disclosed herein, but is positive using a very specific method for detecting anti-PF4 / heparin antibodies (e.g., Acustar HIT-IgG (PF4-H) Determination and HIT-Ab (PF4-H) ) test is positive, the thrombotic event was likely induced by heparin, and therefore, clinicians should definitely consider interrupting heparin therapy and possibly replacing it with another anticoagulant. In addition, when a patient's sample tests positive using the methods disclosed herein and is also positive for anti-PF4 / heparin antibodies, clinicians should consider interrupting heparin therapy and possibly replacing it with another anticoagulant, as well as taking auxiliary measures to downgrade FcγRIIa-dependent cellular activation that triggers a large hypercoagulable state, such as IVIG administration.

[0023] Thus, the ninth aspect relates to a method for recommending or initiating a medical regimen for a subject suspected of having a thrombotic event, the method comprising detecting human anti-PF4 antibodies in a biological sample obtained from the subject by the method defined in the seventh aspect, and if human anti-PF4 antibodies are detected, recommending or initiating an appropriate medical regimen. As described above, the appropriate medical regimen includes measures to reduce FcγRIIa-dependent cellular activation that triggers a significant hypercoagulable state, such as administration of IVIG, and may also include heparin, depending on whether anti-PF4 / heparin antibodies are also present.

[0024] The tenth aspect of the present disclosure provides a kit comprising a reagent as defined in the first or third aspect. The eleventh aspect provides a drug box comprising a reagent as defined in the first or third aspect. Finally, the twelfth aspect provides the use of the kit or drug box for detecting anti-PF4 antibodies not induced by heparin or for diagnosing thrombotic events not induced by heparin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of a reagent according to the present disclosure. The figure shows a carboxyl-modified particle covalently coupled to PF4 via an amide bond.

[0026] Figure 2 is a scatter plot showing the results of a chemiluminescent assay using carboxylated particles with covalently attached PF4 (carboxylated PF4 particles, VITTP3) with three different groups of plasma samples: healthy donors (VITT-; n=39); samples from HIT patients (HIT+, n=24) and samples from VITT patients (VITT+; n=69).

[0027] Figure 3 is a scatter plot that compares Acustar HIT IgG (PF4 / H) ( Results of the immunohistochemical assays for HIT (HIT) and carboxylated PF4 particles (VITT P3) in plasma / serum samples from patients with suspected VITT (grey circles) and healthy donor individuals (white circles).

[0028] Figure 4 is a scatter plot comparing the results of the experiments using carboxyl PF4 particles (VITT P3, covalent binding of PF4), magnetic particles with carboxyl functional groups on the surface to which PF4 is adsorbed (VITT P1 and VITT P2), and Acustar HITIgG (PF4 / H) (HemosIL) Results of chemiluminescent assay of plasma samples from patients with suspected VITT (VITT+, grey circles) and healthy donors (white circles).

[0029] Figure 5 is a scatter plot comparing the results of chemiluminescent analysis of plasma samples from VITT+ and VITT- patients using PF4 adsorbed to particles (P1) and carboxy-PF4 particles (P3, covalently coupled).

[0030] Figure 6 is a scatter plot that compares Acustar HIT IgG (PF4 / H)Results of HemosIL HT and carboxy PF4 particles (VITT P3) on two plasma samples from a patient with unexplained thrombotic complications (HIT and VITT negative). Sample 1 (grey circle) was analyzed on three different test days.

[0031] Figure 7 is a scatter plot showing AcuStar HIT-IgG (PF4-H) Results of 6 patients with a VITT-like pattern (thrombocytopenia, thrombosis, strongly positive anti-PF4 / heparin IgG EIA and PIPA) but no previous exposure to heparin or vaccination in the VITT assay (x-axis; cutoff: vertical line) and the new prototype VITT P3 assay (prototype VITT-IgG (PF4)) (y-axis, cutoff: horizontal line).

[0032] Figure 8 is a scatter plot showing AcuStar HIT-IgG (PF4-H) Results of patients with autoimmune HIT (N=70) in the VITT assay (x-axis; cutoff: dashed vertical line) and the new prototype VITT P3 assay (prototype VITT-IgG (PF4)) (y-axis, cutoff: dashed horizontal line). 63% of the samples showed positive results for the new prototype VITT-IgG (PF4) assay. Detailed Description of the Invention

[0034] Unless otherwise specified, all terms used in this application are to be understood according to their ordinary meanings known in the art. Other more specific definitions of certain terms used in this application are set forth below and are intended to apply uniformly throughout the specification and claims, unless otherwise expressly set forth a definition providing a broader definition.

[0035] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Definite articles such as "the" as used herein also include plural nouns unless otherwise stated.

[0036] In a first aspect, the present disclosure relates to a reagent comprising: (a) a binding molecule for binding to an anti-PF4 antibody and (b) a solid support, wherein the binding molecule is covalently bound to a surface of the solid support.

[0037] Platelet factor 4 protein (PF4) is a small cytokine belonging to the CXC chemokine family, also known as chemokine (CXC motif) ligand 4 (CXCL4). This chemokine is released from the α-granules of activated platelets during platelet aggregation and promotes blood coagulation by regulating the action of heparin-like molecules. In one embodiment, the mature human PF4 protein has the following sequence:

[0038]

[0039] For the purposes of this disclosure, "anti-PF4 antibodies" are immunoglobulin G (IgG) antibodies that bind to an epitope on the platelet factor 4 protein that is independent of heparin. "Anti-PF4 / heparin or anti-PF4 / H" are antibodies (IgG, IgA, and IgM) that recognize platelet factor 4 (PF4) associated with heparin or heparin substitutes. The terms "anti-PF4 antibody" (or simply "anti-PF4") and "anti-PF4 heparin-independent antibody" are used interchangeably herein. The terms "anti-PF4 / heparin or anti-PF4 / H antibody" and "anti-PF4 heparin-dependent antibody" are used interchangeably herein. HIT and VITT antibodies bind to different epitopes on PF4. Anti-PF4 / H is present in classic HIT and requires the simultaneous presence of PF4 and pharmacological concentrations of the relevant polyanion to achieve pathogenicity. Antibodies that bind to PF4 alone (heparin-independent antibodies) are present in VITT and other thrombotic events that are not caused by heparin or vaccination. Both pathological types of IgG antibodies activate platelets via FcγRIIa. This method allows the identification of patients who develop thrombocytopenia and thrombosis associated with heparin-independent anti-PF4 antibodies, i.e., even in the absence of any close heparin exposure. As mentioned above, some of these thrombotic events were found not only in the absence of heparin, but also in the absence of vaccination, suggesting that triggers other than pharmacological heparin and Covid-19 vaccines can also trigger prothrombotic anti-PF4 disease.

[0040] A "molecule for binding to an anti-PF4 antibody," also referred to herein as a "binding molecule," is generally understood to be a molecule, typically but not exclusively a polypeptide, that binds to an anti-PF4 antibody in a manner similar to the PF4 protein. For this purpose, binding molecules according to the present disclosure comprise epitopes relevant to PF4. Such epitopes can be linear, comprising contiguous stretches of amino acids, or conformational, consisting of amino acids that are distant in the protein sequence but brought into proximity by protein folding. These conformational epitopes can comprise both contiguous and non-contiguous amino acids throughout the protein.

[0041] The molecule can be a full-length PF4 protein, preferably a human PF4 protein or a highly similar protein. In one embodiment, the binding molecule is a mammalian PF4. In another embodiment, the binding molecule is a primate PF4. In a specific embodiment, the binding protein is human PF4. In one embodiment, PF4 is mature PF4. The PF4 protein can be natural or synthetic. In another embodiment, PF4 is natural, isolated PF4. In another embodiment, PF4 is recombinant PF4. In another embodiment, the binding molecule is a polypeptide that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to human PF4 (SEQ ID NO: 1). In very specific embodiments, the binding molecule is the human PF4 of SEQ ID NO: 1.

[0042] In the present disclosure, the term "identity" refers to the percentage of identical residues in two sequences when the sequences are optimally aligned. If, in the optimal alignment, a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the sequence exhibits identity relative to that position. The identity percentage determines the number of identical residues over a specified length in a given alignment. Therefore, the level of identity between two sequences or ("sequence identity percentage") can be measured as the ratio of the number of identical positions shared by the sequences relative to the number of comparison positions [i.e., sequence identity percentage=(number of identical positions / total number of positions compared) x 100]. Gaps (i.e., positions where residues are present in one sequence but not in the other sequence in the alignment) are considered as positions with different residues and are counted as comparison positions.

[0043] Many mathematical algorithms for quickly obtaining the best comparison and calculating the identity between two or more sequences are known and are integrated into many available software programs.For purposes of the present disclosure, the sequence identity between two amino acid sequences is preferably determined using an algorithm based on a global comparison, such as Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), preferably in the Needle program of EMBOSS bag (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) realizes; Or BLAST global comparison tool (Altschul et al., " Basic local alignment search tool ", 1990, J.Mol.Biol, v.215, 403-410 pages), is carried out using default settings. When the sequence lengths compared are substantially the same, local comparisons can also be used.

[0044] In one embodiment, the binding molecule is a polypeptide capable of binding to an anti-PF4 antibody (e.g., a PF4-reactive antibody) in patients with thrombotic events not triggered by heparin (e.g., VITT). For example, the polypeptide can be a fragment of the PF4 protein, as long as it retains the ability to bind to the anti-PF4 antibody. A conformational epitope within the PF4 protein has been described that binds to the anti-PF4 antibody (Huynh, A. et al.). The conformational epitope is defined by residues R22, H23, E28, K46, N47, K50, K62, and K66 of SEQ ID NO: 1. In one embodiment, the binding molecule is a polypeptide comprising a conformational epitope defined by residues R22, H23, E28, K46, N47, K50, K62, and K66 of SEQ ID NO: 1. The conformational epitope can also be defined as -RH-E-KN-KKK-. In another embodiment, the binding molecule is a PF4 fragment that can bind to the anti-PF4 antibody (hereinafter also referred to as "antibody-binding PF4 fragment"). Assays for binding to anti-PF4 antibodies are known in the art and can be employed by one skilled in the art to determine which PF4 fragments, or in general, which binding molecules, are capable of binding to anti-PF4 antibodies.

[0045] In another embodiment, the binding molecule is an anti-idiotypic antibody to the PF4 antibody. Anti-idiotypic antibodies recognize the antigen-binding site of an antibody and mimic the structure and / or function of its nominal antigen. Methods for obtaining anti-idiotypic antibodies to known antibodies are well known to those skilled in the art. In another embodiment, the binding molecule is an aptamer.

[0046] In a specific embodiment, the reagent according to the first aspect of the present disclosure consists essentially of (a) a molecule capable of binding to an anti-PF4 antibody and (b) a solid support, wherein the binding molecule is covalently bound to the surface of the solid support. Specifically, the binding molecule is selected from PF4, an antibody binding fragment thereof, and an anti-idiotypic antibody to an anti-PF4 antibody. The binding molecule does not contain heparin or any other heparin substitute (i.e., does not form a complex with heparin or another heparin substitute). In other specific embodiments, the reagent according to the first aspect of the present disclosure consists essentially of (a) PF4 and (b) a solid support, wherein PF4 is covalently bound to the surface of the solid support. Again, the PF4 in (a) is not complexed with heparin or any heparin substitute, i.e., the molecule recognized and bound by the anti-PF4 antibody does not include heparin or its substitute.

[0047] A person skilled in the art understands a "heparin substitute" as any anionic substance that binds to the same heparin binding site in the PF4 protein. An example of a heparin substitute is polyvinyl sulfonate (PVS).

[0048] In some embodiments, the solid support of the reagent of first aspect of the present invention is a particle." particle " is generally understood in the art as several physical or chemical properties, and for example shape, volume, density or quality can be attributed to a small localized object therein. They vary greatly in size, shape or quantity, and change from subatomic particles such as electrons to microscopic particles such as atoms and molecules, to macroscopic particles such as powders and other particulate materials. Any material consisting of particles can be referred to as granular. Therefore, it can be believed that the solid support in the reagent of the present disclosure can be a granular solid support.

[0049] In some embodiments, the solid support is a nanoparticle or microparticle. Both nanoparticles and microparticles can have different compositions, such as but not limited to metallic, organic, metal-organic, polymeric, quantum dots or carbon structures. Nanoparticles and microparticles have at least two nanometer-scale dimensions, preferably all three nanometer-scale dimensions, and can have different shapes and sizes, such as balls, rods, disks, tubes and hemispheres. In some embodiments, the particulate solid support has the shape of a ball or bead, rod or tube.

[0050] In some embodiments, the particulate solid support comprises a metal or a metal alloy or consists essentially of a metal or a metal alloy. In a specific embodiment, the particulate solid support comprises a magnetic metal or a metal alloy or consists essentially of a magnetic metal or a metal alloy. As a magnetic material, any magnetic material widely used in the art can be used. For example, one of Fe2O3, Fe3O4 or FePt can be used, but is not limited thereto. Non-limiting metals suitable for solid supports according to the present disclosure can be selected from gold, platinum, silver, titanium, zinc, cerium, iron, copper, thallium and combinations and / or alloys thereof. In other embodiments, the particulate solid support comprises silicon dioxide or consists essentially of silicon dioxide. In other embodiments, the particulate solid support comprises a metal organic framework or consists essentially of a metal organic framework. In other embodiments, the particulate solid support is a quantum dot. In other embodiments, the particulate solid support comprises carbon (e.g., graphite) or consists essentially of carbon (e.g., graphite).

[0051] In some embodiments, the solid support comprises a polymer or is substantially composed of a polymer. For example, nanoparticles based on polymers are known in the art and can be used in reagents of the present disclosure. The suitable polymer of the solid support in the sense of the present disclosure can be selected from latex, polystyrene, alginic acid, gelatin, polylactic acid, chitosan, polylactide-co-glycolide and polycaprolactone. In specific embodiments, the solid support comprises latex or polystyrene or is substantially composed of latex or polystyrene.

[0052] The binding molecule, such as PF4, can be covalently bound to the solid support, for example, via an amide bond, an amine bond, or a thioether bond. In a specific embodiment of the first aspect of the present disclosure, the binding molecule, such as PF4, is bound to the solid support via an amide bond. In some embodiments, the reagent of the first aspect may further comprise a spacer. The "spacer" can be any branched or unbranched carbon chain comprising at least one carbon, one end of which is bound to the solid support and the other end is bound to the binding portion. Preferably, the binding molecule, such as PF4, is bound to the spacer via an amide bond. In a specific embodiment, it can be seen that the reagent of the present disclosure has the following structure:

[0053] SS-(A)-CO-NH-(A)-PF4

[0054] SS-(A)-S-(A)-PF4 SS-(A)-CO-S-(A)-PF4

[0055] Wherein SS is a solid support, A is a spacer that may or may not be present in the reagent, and PF4 is a binding molecule capable of binding to an anti-PF4 antibody.

[0056] The nature of the spacer according to the present disclosure is not limited, as long as it does not negatively affect the function of the reagent, particularly with respect to the binding of the anti-PF4 antibody. Non-limiting spacers for the reagents of the present disclosure are amino acids, peptides, polyethylene glycols, alkyl groups, and the like. In particular embodiments, the reagents of the first aspect of the present disclosure may have the following structure: SS-CO-NR-PF4, wherein R is H or (C1-C4) alkyl. More particularly, the reagent may have the structure SS-CO-NH-PF4.

[0057] Coupling polypeptides and proteins to solid supports (including to different types of particles, which in turn include beads) can be accomplished by several methods well known to those skilled in the art. An overview of some of the main methods is given by Di Marco et al. (Int J Nanomedicine. 2010; 5: 37-49) and Biju (Chem. Soc. Rev., 2014, 43, 744-764) (which are incorporated herein by reference). Some methods involve chemically coupling polypeptides to solid supports using established reagents (e.g., bifunctional cross-linker molecules). These methods can be used to prepare the reagents of the present disclosure.

[0058] A second aspect relates to a method for preparing a reagent as defined above, generally comprising (i) contacting a solid support with a solution comprising a cross-linking agent and an optional catalyst, and (ii) adding a molecule capable of binding to an anti-PF4 antibody. Steps (i) and (ii) can be performed sequentially (step ii after step i) or simultaneously, i.e., contacting the solid support with a solution comprising both a cross-linking agent and a binding molecule and an optional catalyst. All embodiments described above for the binding molecule and the solid support also apply to the method of the second aspect of the present disclosure.

[0059] The solid support may contain functional groups that react with a cross-linking agent. In some embodiments, the solid support contains functional groups selected from carboxyl, hydroxyl, sulfhydryl, tosyl, and amino functional groups. In some embodiments, the solid support is decorated with negatively charged functional groups. In specific embodiments, the solid support is carboxylated.

[0060] Many cross-linking agents are available in the market, and they can be selected for specific requirements (such as chemical specificity, spacer arm length, cleavability). Non-limiting cross-linking agents used in the present disclosure based on their respective functions are shown in Table 1.

[0061] Table 1. Cross-linkers for covalently coupling peptides and proteins to particles according to their respective functions

[0062]

[0063] Abbreviations: SIAB, N-succinimidyl (4-iodoacetyl) aminobenzoate; SMCC, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; SPDP, succinimidyl 3-(2-pyridyldithio) propionate; SPMB, succinimidyl (4-p-maleimidophenyl) butyrate; EDC, 1-ethyl -3-[3-Dimethylaminopropyl]carbodiimide hydrochloride; DCC, dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide; EGS, ethylene glycol bis(succinimidylsuccinate); BS3, bis-(sulfosuccinimidyl) suberate; BMME, bis(maleimidomethyl) ether; DSS, disuccinimidyl suberate; DSP, dithiobis(succinimidylpropionate); -NHS, N-hydroxysuccinimide.

[0064] In certain embodiments, the cross-linking agent is a carbodiimide.In certain embodiments, the methods of the present disclosure are performed in the presence of a catalyst (eg, NHS).

[0065] In other embodiments, binding molecules can be immobilized using molecular or genetic engineering techniques, such as intein-mediated protein ligation, click chemistry, or other similar techniques.

[0066] The method of the second aspect is carried out under the condition that promotes binding molecule to be covalently bound to solid support.Technicians can set up suitable conditions and in one embodiment, can comprise the time that solid support is hatched enough with the solution comprising cross-linking agent, optional catalyst and binding molecule, for example 20 minutes to 5 hours.In a specific embodiment, the incubation time is 1-4 hours.When the method of the present disclosure is carried out in two separate steps, one embodiment considers that the incubation time of step (i) is at least 15 minutes, for example 15 minutes to 1 hour, and the incubation time of step (ii) is 1 to 5 hours.In a specific embodiment, step (i) considers hatching 20 minutes to 45 minutes, and step (ii) considers hatching 1 to 3 hours.In a specific embodiment, hatching is carried out at room temperature.In a specific embodiment, hatching is carried out under continuous mixing.In some embodiments, the method of the present disclosure is carried out in the presence of buffer.In a specific embodiment, steps (i) and (ii) are carried out under slightly acidic pH, for example pH scope is 5 to 6.5.

[0067] In some embodiments, the method further comprises the step of adding a blocking agent after (ii) (step (iii). The blocking agent blocks the PF4-free area of ​​the solid support to prevent nonspecific binding with the reagent. Non-limiting blocking agents are albumin, polyethylene glycol, and casein. In a specific embodiment, the blocking agent is albumin.

[0068] The method may further comprise another step before (i), which comprises activating the solid support. Activation may be performed by washing with a weakly acidic buffer (eg, pH range 5 to 6).

[0069] As described above, in most embodiments, the solid support contains functional groups. Supports containing active functional groups are commercially available. For example, a wide variety of functionalized particles of various sizes and forms are readily available on the market. If the solid support does not contain functional groups, the first step of the disclosed method may include functionalizing the solid support. Technicians can perform this step according to standard procedures, depending on the desired functional groups.

[0070] The third aspect of the present disclosure relates to a reagent obtained by the above method, wherein the reagent can bind to an anti-PF4 antibody.

[0071] The present disclosure also provides compositions comprising the disclosed agents. In some embodiments, the compositions further comprise other components, which may be selected from buffers, stabilizing compounds, preservatives (eg, antimicrobial agents), and combinations thereof.

[0072] A further aspect of the present disclosure relates to the use of the reagents of the first or third aspects of the present disclosure for detecting anti-PF4 antibodies or for diagnosing thrombotic events that are not induced by heparin, such as vaccine-induced thrombotic thrombocytopenia (VITT) and other thrombotic events that are not induced by heparin or vaccination. "Thrombotic events that are not induced by heparin" should be understood as thrombotic events caused by the activation of platelets and other blood cells or endothelial cells by anti-PF4 antibodies, which are produced in response to unknown triggers other than PF4 alone or heparin. Non-limiting examples of thrombotic events that are not induced by heparin are VITT, spontaneous HIT-like syndrome and other unidentified thrombosis. The term "spontaneous HIT-like" has been used to refer to conditions in which patients develop thrombocytopenia and thrombosis associated with platelet-activating anti-PF4 antibodies even in the absence of close heparin exposure. Obviously, the serum of these patients is able to activate platelets in the absence of heparin, that is, the serum contains anti-PF4 heparin-independent antibodies. The present disclosure shows that some patients with spontaneous HIT-like syndrome have both anti-PF4 heparin-dependent (anti-PF4 / H) and anti-PF4 heparin-independent (anti-PF4) antibodies, although some patients only exhibit anti-PF4 heparin-independent (anti-PF4) antibodies alone (see Figure 7). In a specific embodiment, the reagent of the first or third aspect of the present disclosure is used to diagnose VITT. In a specific embodiment, the reagent of the first or third aspect of the present disclosure is used to diagnose VITT caused by COVID-19 vaccination. In another embodiment, the reagent of the first or third aspect of the present disclosure is used to diagnose thrombotic events not caused by heparin or vaccination, such as, for example, spontaneous HIT-like syndrome. In a specific embodiment, the reagent of the first or third aspect of the present disclosure is used to diagnose unidentified thrombosis. All embodiments described above for the reagents of the first and third aspects of the present disclosure also apply to their uses.

[0073] In patients with thrombocytopenia and catastrophic venous and arterial thrombosis, the presence of anti-PF4 heparin-dependent and heparin-independent antibodies should be considered as a potential cause, even in the absence of proximal heparin exposure. This is highly relevant because these patients require adjunctive therapy, for example, with IVIG. This is now possible for the first time with this approach.

[0074] It is further pointed out that if Figure 8 As shown, severe, atypical manifestations of HIT ("autoimmune HIT") are associated with both HIT-like (heparin-dependent) and VITT-like (heparin-independent) antibodies. Again, the key concept herein is that the presence of anti-PF4 heparin-independent antibodies requires anticoagulation plus adjunctive therapy, i.e., high-dose IVIG, to degrade the severe anti-PF4 IgG-mediated hypercoagulable state. In one embodiment, the reagents of the first or third aspects of the present disclosure can be used to diagnose atypical HIT.

[0075] The seventh and eighth aspects of the present disclosure relate to in vitro methods for detecting anti-PF4 antibodies and for diagnosing thrombotic events not induced by heparin, respectively. The method comprises (i) contacting a reagent as defined in the first and third aspects or a composition comprising the reagent with a biological sample obtained from a subject, and (ii) analyzing the sample to determine whether a complex has formed between the reagent and the anti-PF4 antibody, wherein the formation of the complex indicates that the sample contains anti-PF4 antibodies not induced by heparin, which in turn indicates a thrombotic event not induced by heparin. Non-limiting examples of thrombotic events not triggered by heparin are VITT, spontaneous HIT-like syndrome, and other unidentified thrombosis. In a specific embodiment, the method of the present disclosure is used to diagnose VITT. In a specific embodiment, the method of the present disclosure is used to diagnose VITT caused by COVID-19 vaccination. In a specific embodiment, the method of the present disclosure is used to diagnose thrombotic events not caused by heparin or vaccination, such as, for example, spontaneous HIT-like syndrome. In a specific embodiment, the method of the present disclosure is used to diagnose thrombotic complications of unknown origin. In a specific embodiment, the method of the present disclosure is used to diagnose atypical HIT. All of these conditions can be diagnosed by the present method because they present with anti-PF4 antibodies.All embodiments described above for the reagents of the first and third aspects of the disclosure also apply to these methods.

[0076] The biological sample is preferably whole blood or a portion of whole blood, such as plasma or serum. In certain embodiments, the biological sample is citrated plasma or serum. In other embodiments, the biological sample is plasma anticoagulated with a calcium chelator (e.g., EDTA) or an antithrombin agent (e.g., hirudin or PPACK) or an anticoagulant aptamer.

[0077] The diagnostic method of the present disclosure is based on the reagent of the present disclosure, which specifically binds to anti-PF4 antibodies when anti-PF4 antibodies are present in a sample. The reagent comprises a binding molecule (a) that can recognize and capture anti-PF4 antibodies. As shown in the following examples, the reagent of the present invention does not detect anti-PF4 / H (anti-PF4 antibodies induced by heparin) ( Figure 2 This allows for the differential diagnosis of HIT and non-heparin-induced thrombotic events.

[0078] In one embodiment, the method is used to differentially diagnose thrombotic events not induced by heparin. For example, the method is used to differentiate between HIT and thrombotic events not induced by heparin. In a specific embodiment, the method is used to differentiate between HIT and VITT, particularly HIT and VITT caused by COVID-19 vaccination.

[0079] The ninth aspect relates to a method for recommending or initiating a medical regimen for a subject suspected of having a thrombotic event, the method comprising detecting human anti-PF4 antibodies in a biological sample obtained from the subject by the method defined above, and recommending or initiating an appropriate medical regimen if human anti-PF4 antibodies are detected. As described above, the possibility of detecting the presence of anti-PF4 antibodies (heparin-independent) is of great significance to the clinician, who can choose the most appropriate treatment regimen accordingly. When a patient's sample tests positive using the methods disclosed herein (indicating the presence of anti-PF4 antibodies), this indicates a VITT or other thrombotic event not induced by heparin, which can be very serious. This information is valuable to the clinician, who can recommend auxiliary measures to downgrade FcγRIIa-dependent cellular activation that triggers a large hypercoagulable state. Typically, this will be achieved by administering intravenous immunoglobulin (IVIG). In addition, when a patient's sample tests positive using the methods disclosed herein, but a very specific method for detecting anti-PF4 / heparin antibodies (e.g. Acustar HIT-IgG (PF4-H) Determination and HIT-Ab (PF4-H) ) test negative, the thrombotic event is most likely not caused by heparin, and therefore, the clinician does not need to interrupt heparin therapy. In contrast, when a patient sample tests negative using the methods disclosed herein, but is positive using a very specific method for detecting anti-PF4 / heparin antibodies (e.g., Acustar HIT-IgG (PF4-H) Determination and HIT-Ab (PF4-H) ) tests positive, the thrombotic event is likely caused by heparin, and therefore, the clinician should clearly consider interrupting heparin treatment (possibly replacing it with another anticoagulant), and IVIG administration (or equivalent treatment) may be recommended. In addition, when a patient sample tests positive using the methods disclosed herein and anti-PF4 / heparin antibodies are also positive, the clinician should consider interrupting heparin treatment, possibly replacing it with another anticoagulant, and taking auxiliary measures to reduce FcγRIIa-dependent cellular activation that triggers a large hypercoagulable state (e.g., IVIG administration). The present disclosure also provides a method for treating a thrombotic event in a subject in need thereof, the method comprising performing the method of the eighth aspect to determine whether the patient has a thrombotic event that is not induced by heparin, and if so, administering an appropriate medical regimen, wherein the appropriate medical regimen may include heparin.

[0080] Also provided is an in vitro method for the differential diagnosis of HIT and non-heparin-induced thrombotic events, comprising (1) performing the in vitro method of the seventh aspect of the present disclosure and (2) performing an in vitro diagnosis of HIT, wherein when (1) is positive and (2) is negative, this indicates that the thrombotic event was not triggered by heparin and HIT can be ignored. If HIT is ignored, the medical regimen to be administered to the patient may include heparin. When (1) is negative and (2) is positive, this indicates HIT and the administration of heparin to the patient should be discontinued. The in vitro diagnosis of HIT can be performed, for example, by using, for example Acustar HIT-IgG (PF4-H) or HIT-Ab (PF4-H) Additionally or alternatively, in vitro diagnosis of HIT can be performed by functional assays.

[0081] As summarized above, the methods of the present disclosure are based on the binding of a reagent of the present disclosure to an anti-PF4 antibody, forming a detectable complex when the antibody is present in a sample. In certain embodiments, the in vitro methods disclosed above comprise: (i) contacting a reagent as defined in the first or third aspect, or a composition as defined in the fourth aspect, with a biological sample obtained from a subject to form an immune complex between the reagent and the anti-PF4 antibody (if present), and (ii) analyzing the sample to determine whether an immune complex has formed, wherein the formation of a complex indicates that the sample contains the anti-PF4 antibody.

[0082] The immune complex formed by the reagent and the anti-PF4 antibody can be directly detectable, for example, when the reagent emits a detectable signal that changes when it forms an immune complex with the anti-PF4 antibody. This can be achieved by selecting certain solid supports. Solid supports that emit detectable signals are known to those of skill in the art and can be used to prepare the reagents of the present disclosure. Non-limiting examples of such solid supports are gold nanoparticles and quantum dots. In another example, the immune complex formed by the reagent and the anti-PF4 antibody can be directly detectable in a turbidimetric assay. This can be achieved by using latex particles as the solid support. It can also be achieved by using polystyrene particles as the solid support.

[0083] In another embodiment, the in vitro method disclosed above further comprises contacting the mixture obtained from (i) with a tracer capable of binding to the captured anti-PF4 antibody. In a specific embodiment, the label is a detectable molecule selected from the group consisting of a chemiluminescent molecule, a chromogenic molecule, a fluorescent molecule, a radioactive molecule, a quantum dot, a colloidal gold, and a gold nanoparticle. In some embodiments, the label is a molecule that becomes detectable after contact with a trigger. In another embodiment, the tracer comprises an enzyme that can trigger a detectable change in a substrate present in the sample. In another embodiment, the tracer comprises a substrate that can undergo a detectable change, optionally in the presence of an appropriate trigger. In a specific embodiment, the tracer is a labeled anti-human IgG antibody. In a very specific embodiment, the tracer is an anti-human IgG antibody labeled with a chemiluminescent molecule. Chemiluminescent molecules are well known in the art and can include luminol, isoluminol, N-(4-aminobutyl)-N-ethylisoluminol (ABEI), acridine, etc. In certain embodiments, the in vitro method for detecting anti-PF4 antibodies or thrombotic events not induced by heparin further comprises contacting the mixture obtained from (i) with a tracer capable of binding to the captured anti-PF4 antibodies and a trigger that induces the tracer to undergo a detectable change.

[0084] The tenth aspect of the present disclosure provides a kit for performing any of the in vitro methods disclosed above (e.g., detecting anti-PF4 antibodies, or diagnosing thrombotic events not induced by heparin, or recommending a suitable medical regimen for a patient suffering from or suspected of having a thrombotic event). In a specific embodiment, the kit comprises in a separate container (i) a reagent as defined in the first or third aspect of the present disclosure, or a composition comprising a reagent as defined in the fourth aspect, and (ii) a tracer capable of binding to anti-PF4 antibodies. In another embodiment, the kit may further comprise a trigger solution for inducing a detectable change in the tracer. In a further embodiment, the kit may further comprise a buffer solution, a diluent, a stabilizer, a preservative and / or instructions for use for detecting anti-PF4 antibodies or diagnosing thrombotic events not induced by heparin.

[0085] The present disclosure also provides a kit for detecting anti-PF4 antibodies or diagnosing thrombotic events or recommending an appropriate medical regimen, all as disclosed above. In a specific embodiment, the kit comprises (i) a reagent as defined in the first or third aspect of the present disclosure, or a composition comprising a reagent as defined in the fourth aspect, and (ii) a tracer capable of binding to anti-PF4 antibodies in a separate container. In another embodiment, the kit may further comprise a trigger solution for inducing a detectable change in the tracer. In a further embodiment, the kit may additionally comprise a buffer solution, a diluent, a stabilizer, a preservative and / or instructions for use in any of the above methods.

[0086] In a specific embodiment, the reagents in the test kit or medicine box include particles as solid supports, such as magnetic particles. In another embodiment, the medicine box allows the sample to be subjected to automated testing to detect anti-PF4 antibodies or to diagnose thrombotic events as defined above, or to recommend appropriate treatment to the patient, as defined above. The medicine box can include all components required for the test, for example, the medicine box can include reagents in the form of a particle suspension, an assay buffer, a tracer, and a diluent. The reagents in the medicine box can be released from their containers and mixed with the sample, thereby achieving automated testing, which is a related advantage of the disclosed method. In a specific embodiment, the medicine box is used for automated testing using an automated instrument. The automated instrument is suitable for the medicine box and automatically detects the signal emitted by the tracer when anti-PF4 antibodies are present in the sample. In a specific embodiment, the automated instrument is an ACL AcuStar (Werfen).

[0087] For completeness, the present disclosure is further defined in the following numbered embodiments:

[0088] 1. A reagent comprising:

[0089] (a) a molecule that binds to an anti-PF4 antibody (binding molecule); and

[0090] (b) a solid support,

[0091] wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and wherein the binding molecule (a) does not comprise heparin or a heparin substitute.

[0092] 2. A reagent consisting essentially of:

[0093] (a) a molecule that binds to an anti-PF4 antibody (binding molecule); and

[0094] (b) a solid support,

[0095] wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and wherein the binding molecule (a) does not comprise heparin or a heparin substitute.

[0096] 3. The agent according to any one of the preceding embodiments, wherein the binding molecule is a PF4 protein.

[0097] 4. The agent according to the preceding embodiment, wherein the binding molecule is human PF4 protein.

[0098] 5. The agent according to any one of embodiments 1-2, wherein the binding molecule is a polypeptide that is at least 80%, at least 85% or at least 90% identical to human PF4 (SEQ ID NO: 1).

[0099] 6. The agent according to the preceding embodiment, wherein the binding molecule is a polypeptide having at least 95%, at least 97%, at least 98% or at least 99% identity to human PF4 (SEQ ID NO: 1).

[0100] 7. The agent according to any one of embodiments 1-2, wherein the binding molecule is an antibody binding fragment of the PF4 protein.

[0101] 8. The agent according to any one of embodiments 1-2, wherein the binding molecule is an anti-idiotypic antibody of an anti-PF4 antibody.

[0102] 9. An agent according to any one of embodiments 1-2, wherein the binding molecule is a polypeptide comprising a conformational epitope defined by residues R22, H23, E28, K46, N47, K50, K62 and K66 of SEQ ID NO: 1.

[0103] 10. The reagent according to any one of the preceding embodiments, wherein the solid support is a particle, in particular a microparticle or a nanoparticle.

[0104] 11. The reagent according to any one of the preceding embodiments, wherein the solid support comprises or consists essentially of a metal.

[0105] 12. The reagent according to the preceding embodiment, wherein the metal is a magnetic metal.

[0106] 13. The reagent according to the preceding embodiment, wherein the metal is selected from the group consisting of Fe2O3, Fe3O4 and FePt.

[0107] 14. The reagent according to any one of embodiments 1-10, wherein the solid support comprises or consists essentially of a polymer.

[0108] 15. The agent according to the preceding embodiment, wherein the polymer is selected from latex and polystyrene.

[0109] 16. The reagent according to any one of the preceding embodiments, wherein the PF4 is bound to the solid support via an amide bond, an amine bond or a thioether bond.

[0110] 17. The reagent according to the preceding embodiment, wherein the PF4 is bound to the solid support via an amide bond.

[0111] 18. The agent according to any one of the preceding embodiments, wherein the agent further comprises a spacer.

[0112] 19. The agent according to any one of the preceding embodiments, wherein the agent has the following structure:

[0113] SS-(A)-CO-NH-(A)-PF4, or

[0114] SS-(A)-S-(A)-PF4 SS-(A)-CO-S-(A)-PF4,

[0115] wherein SS is the solid support, A is a spacer that may or may not be present in the reagent, and PF4 is the binding molecule capable of binding to the anti-PF4 antibody.

[0116] 20. The reagent according to the preceding embodiment, wherein the reagent has the following structure: SS-CO-NR-PF4, wherein R is H or (C1-C4)alkyl.

[0117] 21. A method for preparing a reagent as defined in any one of the preceding claims, the method comprising:

[0118] (i) contacting the solid support with a solution comprising a in the presence of a cross-linking agent and optionally a catalyst, and

[0119] (ii) adding a molecule capable of binding to an anti-PF4 antibody (binding molecule),

[0120] Or, alternatively, the method comprises:

[0121] (i) contacting the solid support with a solution comprising molecules capable of binding to the anti-PF4 antibody (binding molecules) in the presence of a cross-linking agent and optionally a catalyst.

[0122] 22. The method according to the preceding embodiment, wherein the solid support contains functional groups selected from the group consisting of carboxyl, hydroxyl, sulfhydryl and amino functional groups.

[0123] 23. A method according to embodiment 21, wherein the solid support contains negatively charged functional groups.

[0124] 24. The method according to the preceding embodiment, wherein the solid support is carboxylated.

[0125] 25. The method according to any one of embodiments 21 to 24, wherein the cross-linking agent is a carbodiimide.

[0126] 26. The method according to any one of embodiments 21 to 24, wherein the cross-linking agent is an N-hydroxysuccinimide ester.

[0127] 27. The method according to any one of embodiments 21 to 24, wherein the cross-linking agent is maleimide.

[0128] 28. The method according to any one of embodiments 21-27, wherein the binding molecule is PF4 protein.

[0129] 29. The method according to the preceding embodiment, wherein the binding molecule is human PF4 protein.

[0130] 30. The method according to any one of embodiments 21-27, wherein the binding molecule is a polypeptide that is at least 80%, at least 85% or at least 90% identical to human PF4 (SEQ ID NO: 1).

[0131] 31. The method according to the preceding embodiment, wherein the binding molecule is a polypeptide that is at least 95%, at least 97%, at least 98% or at least 99% identical to human PF4 (SEQ ID NO: 1).

[0132] 32. The method according to any one of embodiments 21-27, wherein the binding molecule is an antibody binding fragment of the PF4 protein.

[0133] 33. The method according to any one of embodiments 21-27, wherein the binding molecule is an anti-idiotypic antibody to an anti-PF4 antibody.

[0134] 34. A method according to any one of embodiments 21-27, wherein the binding molecule is a polypeptide comprising a conformational epitope defined by residues R22, H23, E28, K46, N47, K50, K62 and K66 of SEQ ID NO: 1.

[0135] 35. The method according to any one of embodiments 21 to 34, wherein the solid support is a particle, in particular a microparticle or a nanoparticle.

[0136] 36. The method according to any one of embodiments 21-35, wherein the solid support comprises or consists essentially of a metal.

[0137] 37. The method according to the preceding embodiment, wherein the metal is a magnetic metal.

[0138] 38. The method according to the preceding embodiment, wherein the metal is a magnetic metal selected from the group consisting of Fe2O3, Fe3O4 and FePt.

[0139] 39. The method according to any one of embodiments 21-35, wherein the solid support comprises or consists essentially of a polymer.

[0140] 40. The method according to the preceding embodiment, wherein the polymer is selected from latex and polystyrene.

[0141] 41. The process according to any one of embodiments 21 to 40, wherein the process is carried out in the presence of a catalyst.

[0142] 42. The method according to the preceding embodiment, wherein the catalyst is N-hydroxysuccinimide.

[0143] 43. The method according to any one of embodiments 21-42, comprising the further step of adding a blocking agent.

[0144] 44. The method according to any one of embodiments 21 to 43, comprising an initial step of activating the solid support.

[0145] 45. A reagent obtainable by a method as defined in any one of embodiments 21-44.

[0146] 46. ​​A composition comprising an agent as defined in any one of embodiments 1-20 or 45.

[0147] 47. The composition according to the preceding embodiment, further comprising a component selected from the group consisting of a buffer, a stabilizing compound, a preservative (eg, an antimicrobial agent), and combinations thereof.

[0148] 48. Use of a reagent as defined in any one of embodiments 1-20 or 45 or a composition as defined in any one of embodiments 46-47 for detecting anti-PF4 antibodies.

[0149] 49. Use of an agent as defined in any one of embodiments 1-20 or 45 or a composition as defined in any one of embodiments 46-47 for diagnosing a thrombotic event not induced by heparin or for diagnosing atypical HIT.

[0150] 50. An in vitro method for detecting anti-PF4 antibodies, comprising:

[0151] (i) contacting an agent as defined in any one of embodiments 1-20 or 45 or a composition as defined in any one of embodiments 46-47 with a biological sample obtained from a subject, and

[0152] (ii) analyzing the sample to detect a complex formed by the reagent and the anti-PF4 antibody,

[0153] Wherein detection of the complex indicates that the sample contains anti-PF4 antibodies.

[0154] 51. An in vitro method according to the preceding claim, which does not detect anti-PF4 / H antibodies.

[0155] 52. In vitro methods for diagnosing thrombotic events not induced by heparin, comprising:

[0156] (i) contacting an agent as defined in any one of embodiments 1-20 or 45 or a composition as defined in any one of embodiments 46-47 with a biological sample obtained from a subject, and

[0157] (ii) analyzing the sample to detect a complex formed by the agent and the anti-PF4 antibody, wherein detection of the complex indicates a thrombotic event that is not induced by heparin.

[0158] 53. The method according to any one of embodiments 50-52, further comprising contacting the mixture obtained from (i) with a tracer, in particular a tracer capable of binding to said anti-PF4 antibody.

[0159] 54. The method according to the preceding embodiment, wherein the tracer comprises a detectable label.

[0160] 55. The method according to the preceding embodiment, wherein the detectable label is selected from the group consisting of a chemiluminescent molecule, a chromogenic molecule, a fluorescent molecule.

[0161] 56. The method according to the preceding embodiment, wherein the detectable label is a chemiluminescent molecule.

[0162] 57. The method according to any one of embodiments 53-56, wherein the tracer comprises a labeled anti-human IgG antibody.

[0163] 58. The method according to any one of embodiments 50-57, wherein the biological sample is selected from whole blood, plasma and serum.

[0164] 59. The method according to the preceding embodiment, wherein the biological sample is plasma or serum.

[0165] 60. The method according to any one of embodiments 52-59, which is used for differential diagnosis of thrombotic events not induced by heparin and thrombotic events induced by heparin.

[0166] 61. The method according to the preceding embodiment, wherein the heparin-induced thrombotic event is HIT.

[0167] 62. The method according to embodiment 60, wherein the thrombotic event not induced by heparin is selected from VITT and a thrombotic event not induced by vaccination.

[0168] 63. The method according to the preceding embodiment, wherein the thrombotic event not induced by heparin or vaccination is spontaneous HIT or unidentified thrombosis.

[0169] 64. The method according to any one of embodiments 60-63, further comprising performing an in vitro diagnosis of HIT.

[0170] 65. The method according to the preceding embodiment, wherein the Acustar HIT-IgG (PF4-H) Determination and HIT-Ab (PF4-H) The in vitro diagnosis of HIT is performed by means of a panel of immunoassays.

[0171] 66. The method of embodiment 64, wherein the in vitro diagnosis of HIT is performed by means of a functional assay.

[0172] 67. A kit comprising:

[0173] (i) an agent as defined in any one of embodiments 1-20 or 45 or a composition as defined in any one of embodiments 46-47, and

[0174] (ii) optionally, further components selected from the group consisting of a tracer capable of binding to an anti-PF4 antibody, an assay buffer, a diluent, and combinations thereof.

[0175] 68. A kit comprising:

[0176] (i) an agent as defined in any one of embodiments 1-20 or 45 or a composition as defined in any one of embodiments 46-47, and

[0177] (ii) optionally, further components selected from the group consisting of a tracer capable of binding to an anti-PF4 antibody, an assay buffer, a diluent, and combinations thereof.

[0178] 69. Use of a test kit as defined in embodiment 65 or a kit as defined in embodiment 66 for detecting anti-PF4 antibodies or for diagnosing thrombotic events not induced by heparin.

[0179] 70. A method for recommending or initiating a medical regimen for a subject suspected of having a thrombotic event, comprising:

[0180] (a) detecting human anti-PF4 antibodies in a biological sample obtained from a subject by the method defined in any one of embodiments 50-51, and

[0181] (b) If human anti-PF4 antibodies are detected, recommend or initiate appropriate medical treatment.

[0182] 71. The method according to any one of embodiments 70, wherein the medical regimen comprises measures to reduce FcγRIIa-dependent cellular activation that triggers a massive hypercoagulable state.

[0183] 72. The method according to the preceding embodiment, wherein said measures comprise administering intravenous immunoglobulin (IVIG).

[0184] 73. The method according to any one of embodiments 70-72, further comprising detecting anti-PF4 / H antibodies in the patient sample, wherein:

[0185] (a) When the sample is positive for anti-PF4 antibodies and negative for anti-PF4 / H antibodies, heparin may be administered, or

[0186] (b) When the sample is positive for anti-PF4 / H antibodies, heparin is discontinued.

[0187] Throughout the specification and claims, the word "comprising" and variations of the word are not intended to exclude other technical features, additives, components or steps. In addition, the word "comprising" includes the case of "consisting of..." Other objects, advantages and features of the present invention will be clear to those skilled in the art after reading the specification, or can be understood by practicing the present invention. The following examples and drawings are provided for illustration only, and they are not intended to limit the present invention. Reference symbols related to the drawings placed in brackets in the claims are only used to attempt to increase the intelligibility of the claims and should not be interpreted as limiting the scope of the claims. In addition, the present invention covers all possible combinations of the specific and preferred embodiments described herein. Example

[0188] 1. Magnetic Particle Preparation

[0189] 1.1. Carboxyl-modified magnetic particles (Dynabeads M-270 Carboxylic Acid) were washed with 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.8, for 10 minutes with constant mixing. After removing the supernatant using a magnet, the particle pellet was resuspended in EDAC working solution at room temperature (20-25°C) with constant mixing for 30 minutes. The activated particles were washed in coupling buffer (MES or PBS-buffered saline) and mixed with a defined volume of PF4 solution. The mixture was incubated at room temperature (20-25°C) with constant mixing for 2-3 hours. Immediately thereafter, the PF4-coupled particles were repositioned on a magnet to remove the supernatant and rinsed in phosphate-buffered saline containing BSA as a blocking molecule. After the final rinse cycle, the blocked particles were placed on a magnetic separator, the supernatant removed by magnetic separation, and the particles were resuspended in PBS-buffered saline containing BSA and stored at 2°C-8°C until use. These particles are referred to herein as VITT P3 or carboxy PF4 particles and contain PF4 covalently bound to magnetic particles.

[0190] 1.2 Preparation of PF4-adsorbed magnetic particles Protein adsorption was performed on magnetic particles at pH 7.5 (VITT P1) and pH 5.8 (VITT P2).

[0191] Carboxyl-modified magnetic particles (Dynabeads M-270 Carboxylic Acid) were washed with 50 mM phosphate buffer, pH 7.5 (VITTP1 particles) or 50 mM MES buffer, pH 5.8 (VITT P2) for 10 minutes with uniform mixing. After removing the supernatant using a magnet, the particle pellet was resuspended in PF4 solution (PF4 diluted in 50 mM phosphate buffer, pH 7.5 for VITT P1 or 50 mM MES, pH 5.8 for VITT P2). The mixture was incubated at room temperature (20-25°C) for 2-3 hours with constant mixing. Immediately thereafter, the PF4-coupled particles were repositioned on the magnet to remove the supernatant and rinsed in phosphate buffer containing BSA as a blocking molecule. After the final wash cycle, the blocked pellet was placed on a magnetic separator, the supernatant was removed by magnetic separation, and the pellet was resuspended in PBS-buffered saline containing BSA and stored at 2°C to 8°C until use.

[0192] Table 1. Main characteristics of magnetic particles used in the examples

[0193]

[0194] 2.VITT samples

[0195] Plasma samples from patients with suspected VITT were collected from the University of Greifswald ( Kindly provided by Dr. Andreas Greinacher, VITT and HIT Reference Laboratory, Greifswald. Cases have been identified as suspected VITT at the University Hospital Greifswald based on clinical presentation, radiographic evidence of thrombosis, platelet count results from local laboratories, coagulation parameters, and positive testing for PF4-dependent platelet-activating antibodies. Case definition was: manifestation between 5 and 28 days after the ChAdOx1 nCOV-19 vaccine; thrombosis and thrombocytopenia (platelets <150 x 109 / L), or isolated thrombocytopenia; and evidence of extreme activation of the coagulation system (D-dimer >4000 μg / L, or >2000 μg / L accompanied by a strong clinical index of suspicion). These cases were classified as unlikely, possible, or probable to have VITT. All samples analyzed in this study were collected before VITT treatment.

[0196] Plasma samples from patients diagnosed with HIT were kindly provided by Dr. Andreas Greinacher, University of Greifswald. The patients had been diagnosed with HIT by Dr. Andreas Greinacher, University of Greifswald, VITT and the Reference Laboratory for HIT.

[0197] Plasma samples from patients with thrombotic complications of unknown origin were kindly provided by Dr. Andreas Greinacher of the Reference Laboratories of the University of Greifswald, VITT, and HIT. The patients were studied by Dr. Andreas Greinacher of the Reference Laboratories of the University of Greifswald, VITT, and HIT, with HIT and VITT ignored after appropriate testing. Plasma from healthy donors was used as a control sample.

[0198] 3. VITT Determination

[0199] In ACL AcuStar TM Samples were analyzed on the system using the following assays: commercial anti-PF4 ELISA (LIFECODES PPF4 IgG assay), AcuStar TM HIT-IgG (PF4-H) (Werfen) and the particles obtained in part 1 (VITT assay). Use a commercial kit and follow the manufacturer's instructions. The following protocol was applied to the VITT assay:

[0200] 1. Pre-dilute 15 μl of the sample containing anti-PF4 antibody 10-fold with sample diluent (135 μl) and then mix with the assay reagent.

[0201] 2. Dispense 15 μl of pre-diluted sample into a dish and mix with 70 μl of assay buffer (AB) and 20 μl of magnetic particles (MP). Incubate the mixture for 8.5 minutes.

[0202] 3. Use a magnet to separate the formed complex (particles with anti-PF4 antibodies), aspirate the complex and wash with flushing solution. Repeat this washing step 3 times.

[0203] 4. Dispense 160 μl of tracer into the complex and incubate the mixture for 9.5 minutes. Separate the resulting complex (particles with antibody and tracer) using a magnet and wash four times with rinse solution.

[0204] 5. Dispense 190 μl of Trigger Solution A and 200 μl of Trigger Solution B to initiate the chemiluminescent reaction; and

[0205] 6. Report the results in relative light units (RLU).

[0206] The PF4 antibody concentration from ACL AcuStar was determined as a function of the concentration of anti-PF4 antibody (analyte) present in the sample. TM The RLU reading of the device. AcuStar HIT-IgG (PF4-H) , the signal (RLU) is proportional to the analyte concentration of the sample in the VITT assay.

[0207] 4. Results: The performance of the reagents disclosed herein for detecting VITT

[0208] like Figure 2 As shown in , the reagent of the present disclosure (VITT P3) (in which the PF4 protein alone is covalently bound to magnetic particles) accurately detected anti-PF4 antibodies in all VITT-positive plasma, but it detected almost no antibodies in samples from patients with HIT. Similarly, no reactivity was observed with plasma samples from healthy donors. A cutoff line (dashed line) can be established to determine when the VITT assay is positive only for VITT samples. These results indicate that the VITT assay based on the reagent of the present disclosure is highly specific for VITT detection and can distinguish between VITT and HIT. In addition, as expected, Acustar HIT-IgG (PF4 / H) The assay (which uses PF4 / PVS complexes on magnetic particles) was unable to detect vaccine-induced anti-PF4 antibodies in VITT samples. This was shown by Figure 3 , where it can also be seen that the plasma of all VITT patients showed a higher RLU response only when tested with VITT P3.

[0209] It was also found that covalent coupling of PF4 to the particles was essential for the detection of anti-PF4 antibodies in all VITT samples. Figure 4 This is demonstrated by the results shown in , where it can be observed that all VITT patient plasmas showed a high RLU response when tested with VITT P3 (PF4 protein covalently coupled to carboxylated-magnetic particles), while in contrast, VITTP1 and VITT P2 (PF4 protein adsorbed to carboxylated magnetic particles) recognized only one of the tested VITT patient samples. Again, none of the VITT plasmas were Acustar HIT-IgG (PF4 / H) These results were confirmed by comparing the results of the assay using VITT P3 particles and VITT P1 particles on plasma samples from a large number of patients suspected of VITT ( Figure 5 ).

[0210] Figure 6Two plasma samples from patients with thrombotic complications of unknown origin (HIT and VITT have been omitted) that have been Acustar HIT IgG(PF4 / H)( HIT) and VITTP3 assays / prototypes were analyzed. Figure 6 It can be seen that The Acustar HIT IgG assay did not detect anti-PF4 antibodies in these samples, whereas the VITT assay accurately detected anti-PF4 antibodies in two plasma samples.

[0211] 5. Performance of the reagent disclosed herein for detecting other thrombotic events not induced by heparin

[0212] Figure 7 Shown in AcuStar HIT-IgG (PF4-H) Results were presented for six patients with a VITT-like pattern (thrombocytopenia, thrombosis, strongly positive anti-PF4 / heparin IgG EIA and PIPA) using the VITT assay and the new VITT P3 assay / prototype, but without prior exposure to heparin or vaccination. Three patients had both anti-PF4 heparin-dependent and anti-PF4 heparin-independent antibodies. The other three of these patients were characterized by isolated anti-PF4 heparin-independent antibodies. The results are very relevant, as these patients may benefit from additional treatment with IVIG.

[0213] 6. Analysis of serum from patients with autoimmune HIT

[0214] The Acustar HIT-IgG (PF4 / H) and VITT P3 assays / prototypes were used to analyze the serum of patients clinically suspected of having aHIT. The results showed that 63% of aHIT patients had anti-PF4 antibodies ( Figure 8 ).

[0215] 7. Conclusion

[0216] VITT-like anti-PF4 antibodies are associated with severe immune thrombosis that is independent of heparin. Most of these patients have previously remained undiagnosed due to negative results of rapid HIT tests and heparin-dependent functional tests. The methods disclosed herein allow identification of these patients (also in prospective clinical studies), differentiation between HIT and VITT-like antibodies, and rapid clinical decision-making.

[0217] Citation List

[0218] Greinacher et al,"Thrombotic Thrombocytopenia after ChAdOx1 nCov-19Vaccination".DOI:10.1056 / NEJMoa2104840.

[0219] Platton et al,“Evaluation of laboratory assays for anti-plateletfactor 4antibodies after ChAdOx1 nCOV-19vaccination”.DOI:10.1111 / jth.15362.

[0220] Favaloro et al,2022“Antibodies against Platelet Factor 4and TheirAssociated Pathologies:From HIT / HITT to Spontaneous HIT-Like Syndrome,toCOVID-19,to VITT / TTS”DOI:10.3390 / antib1101000

[0221] Di Marco M,Shamsuddin S,Razak KA,et al.Overview of the main methodsused to combine proteins with nanosystems:absorption,bioconjugation,andencapsulation.Int J Nanomedicine.2010;5:37-49.

[0222] Biju V.Chem.Soc.Rev.,2014,43,744-764(DOI:10.1039 / C3CS60273G)

[0223] Huynh,A.et al.Nature volume 596,pages 565-569(2021)(DOI:10.1038 / s41586-021-03744-4)

Claims

1. An in vitro method for detecting anti-PF4 antibodies, the method comprising: (i) contacting a whole blood, plasma or serum sample obtained from a subject with a reagent comprising: (a) a binding molecule selected from the group consisting of a platelet factor 4 protein (PF4), a PF4 fragment capable of binding to an anti-PF4 antibody, and an anti-idiotypic antibody to an anti-platelet factor 4 antibody (anti-PF4 antibody); and (b) a solid support, wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and wherein the binding molecule (a) does not comprise heparin or a heparin substitute, and (ii) analyzing the sample to detect a complex formed by the reagent and the anti-PF4 antibody, wherein detection of the complex indicates that the sample contains the anti-PF4 antibody.

2. An in vitro method for diagnosing thrombotic events not induced by heparin, the method comprising: (i) contacting a whole blood, plasma or serum sample obtained from a subject with a reagent comprising (a) a binding molecule selected from the group consisting of a platelet factor 4 protein (PF4), a PF4 fragment capable of binding to an anti-PF4 antibody, and an anti-idiotypic antibody to an anti-platelet factor 4 antibody (anti-PF4 antibody); and (b) a solid support, wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and wherein the binding molecule (a) does not comprise heparin or a heparin substitute, and (ii) analyzing the sample to detect a complex formed by the agent and the anti-PF4 antibody, wherein detection of the complex indicates a thrombotic event that was not induced by heparin.

3. The in vitro method according to the preceding claim, wherein the thrombotic event not induced by heparin is selected from VITT and a thrombotic event not induced by vaccination.

4. In vitro method according to the preceding claim for the differential diagnosis of thrombotic events not induced by heparin and heparin-induced thrombotic thrombocytopenia (HIT).

5. The in vitro method according to the preceding claim, further comprising determining the absence of anti-PF4 heparin-dependent antibodies (anti-PF4 / H) in the sample.

6. The in vitro method according to any one of the preceding claims, further comprising contacting the mixture obtained from (i) with a tracer capable of binding to the anti-PF4 antibody.

7. An in vitro method according to the preceding claim, wherein the tracer comprises a labeled anti-human IgG antibody.

8. The in vitro method according to any one of the preceding claims, wherein the reagent consists essentially of: (a) a binding molecule selected from the group consisting of PF4, a PF4 fragment capable of binding to an anti-PF4 antibody, and an anti-idiotypic antibody of an anti-platelet factor 4 antibody (anti-PF4 antibody); and (b) a solid support, wherein the binding molecule (a) is covalently bound to the surface of the solid support (b), and wherein the binding molecule (a) does not include heparin or a heparin substitute.

9. The in vitro method according to any one of the preceding claims, wherein the binding molecule is PF4, in particular human PF4.

10. The in vitro method according to any one of the preceding claims, wherein the PF4 is bound to the solid support via an amide bond.

11. The in vitro method according to any one of the preceding claims, wherein the agent has the following structure: SS-(A)-CO-NH-(A)-PF4, or SS-(A)-S-(A)-PF4 SS-(A)-CO-S-(A)-PF4, Wherein SS is the solid support, A is a spacer which may or may not be present in the reagent, and PF4 is the binding molecule capable of binding to the anti-PF4 antibody.

12. The in vitro method according to any one of the preceding claims, wherein the solid support is a particle, in particular a microparticle or a nanoparticle.

13. The in vitro method according to any one of the preceding claims, wherein the solid support comprises or consists essentially of a magnetic metal.

14. A method for recommending or initiating a medical regimen for a subject suspected of having a thrombotic event, the method comprising (a) detecting human anti-PF4 antibodies in a biological sample obtained from the subject by the method defined in any one of claims 1, 6 to 13, and (b) if human anti-PF4 antibodies are detected, recommending or initiating an appropriate medical regimen.

15. The method according to claim 14, wherein the medical regimen comprises measures to downgrade FcγRIIa-dependent cellular activation triggering a massive hypercoagulable state, in particular, the measures comprise administration of intravenous immunoglobulin (IVIG).

16. The method according to any one of claims 14-15, further comprising detecting anti-PF4 / H antibodies in a sample from the patient, wherein: (a) when the sample is positive for anti-PF4 antibodies and negative for anti-PF4 / H antibodies, heparin may be administered, or (b) When the sample is positive for anti-PF4 / H antibodies, heparin is discontinued.

17. A test kit or a pharmaceutical kit comprising: (i) an agent as defined in any one of claims 1 to 6 to 13, (ii) a further component selected from the group consisting of a tracer capable of binding to the anti-PF4 antibody, an assay buffer, a diluent, and combinations thereof, and (iii) instructions for use in detecting anti-PF4 antibodies by a method as defined in any one of the preceding claims, or in diagnosing a thrombotic event not induced by heparin, or in recommending or initiating a medical regimen for a subject suspected of having had a thrombotic event.

18. A kit or reagent kit according to the preceding claim, wherein the solid support for the reagents is a particle.

19. Use of an agent as defined in any one of claims 1 to 6 to 13 or a kit or a pharmaceutical kit as defined in any one of claims 17 to 18 for detecting anti-PF4 antibodies, or for diagnosing a thrombotic event not induced by heparin, or for recommending or initiating a medical regimen for a subject suspected of having a thrombotic event.

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