Polyvinyl sulfonate-treated platelets for the detection of antiplatelet factor 4 antibodies

Abstract

Described herein are methods and kits for the detection of platelet-activating pathogenic antibodies in samples from patients suspected of having platelet-activating antibodies or non-HIT anti-PF4 antibody mediated syndromes using platelets and polyvinyl sulfonate or salts thereof. Upon platelet activation, thrombospondin-1 (TSP1), a platelet granule protein, is released and can be detected using a TSP1 antigen capture enzyme linked immunosorbent assay (ELISA) to accurately measure platelet activation.

Description

[0001] 211509-0006-W001

[0002] POLYVINYL SULFON ATE-TREATED PLATELETS FOR THE DETECTION OF ANTIPLATELET FACTOR 4 ANTIBODIES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 737,015, filed December 20, 2024, which is incorporated by reference herein in its entirety.

[0005] FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under grant number HL147734 awarded by the National Institutes of Health Small Business Innovation Research Program. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin, caused by antibodies to platelet factor 4 (PF4) in which affected subjects experience platelet activation and develop thrombocytopenia. A subset of these individuals experiences arterial and / or venous thrombosis, which in severe cases can be life-threatening. Experts believe that up to 20,000 people per year develop HIT in the U.S., leading to more than 5 deaths every day. Because early diagnosis and treatment can reduce morbidity, it is important that a timely and accurate diagnosis of HIT be made. An accurate diagnosis of HIT requires attention to both clinical findings and laboratory test results.

[0009] Conventional treatment for patients suspected of having HIT includes the immediate cessation of heparin followed by prompt administration of a non-heparin, alternative anticoagulant such as a direct thrombin inhibitor. Such treatments involve additional hospitalization, a considerable expense, and a risk of severe bleeding of 1% per treatment day. Thus, an accurate diagnosis of HIT and characterization of thrombosis risk is critical for effective patient management.

[0010] Among available tests for HIT antibody detection, the serotonin release assay (SRA) is considered by many to correlate best with a clinical picture typical of HIT and is often used as the “gold standard” test for HIT diagnosis. The SRA is performed by loading platelets with serotonin, treating the platelets with low or high concentrations of heparin with a HIT-suspected patient sample and quantifying the amount of serotonin released into the supernatants of the mixture. High amounts of serotonin released in the low heparin concentration condition and low amounts of serotonin in the high heparin condition is indicative of HIT. The SRA is performed routinely only 211509-0006-W001 in a few specialized laboratories because of the use of radioactivity, labor intensiveness, reliance on fresh platelets, and technical demands of the assay.

[0011] In addition to HIT, several other non-heparin-induced thrombotic thrombocytopenias mediated by anti-PF4 antibodies have been recently identified, including but not limited to spontaneous HIT, vaccine-induced immune thrombotic thrombocytopenia (VITT), spontaneous VITT, adenovirus-associated thrombotic thrombocytopenia, and monoclonal gammopathy of thrombotic significance (MGTS). Detection of these disorders can be challenging due to limited access to platelet activation- based functional diagnostic assays. Herein, these disorders are described as non-HIT anti-PF4 antibody mediated syndromes.

[0012] It has been previously shown that fresh and cryopreserved platelets are capable of being activated by HIT antibodies in the presence of PF4 or unfractionated heparin. See e.g., Sheridan et al., Blood 67(1): 27-30 (1986); Samuelson-Bannow et al., Blood 137(8): 1082-1089 (2021); Kanack et al., Blood 140(25): 2722-2729 (2022). However, PF4 and fresh platelets have key limitations: PF4 is expensive; requires recombinant protein production or extensive purification from human platelets; and needs to self-associate in the right format (as a tetramer) for the HIT antibody detection assay to work. In addition, the need to draw fresh platelets is a challenge, particularly for smaller laboratories. There are challenges with the use of heparin as well: unfractionated heparin (and low molecular weight heparin) is a heterogeneous animal (typically porcine)-derived glycosaminoglycan, and often suffers from significant lot-to-lot variability in molecular size, sulfation, and carbohydrate composition.

[0013] What is critically needed are methods and kits for the accurate, yet technically simple detection of platelet-activating pathogenic antibodies in samples from subjects suspected of having HIT or non-HIT anti-PF4 antibody mediated syndromes.

[0014] SUMMARY

[0015] One embodiment described herein is a method for identifying platelet-activating antibodies in a sample from a subject suspected of having platelet-activating antibodies, the method comprising: obtaining a sample comprising serum, plasma, or fractionated blood from a subject suspected of having platelet-activating antibodies; obtaining platelets from one or more normal subjects; combining the platelets from one or more normal subjects with the sample from the subject suspected of having platelet-activating antibodies and polyvinyl sulfonate (PVS) to form a combined sample, and incubating the combined sample for a period of time; measuring platelet activation in the combined sample; wherein platelet activation in the combined sample is indicative of the presence of platelet-activating antibodies in the sample from the subject suspected of 211509-0006-W001 having platelet-activating antibodies. In one aspect, the platelet-activating antibodies comprise platelet-activating heparin-induced thrombocytopenia (HIT) antibodies. In another aspect, the platelet-activating antibodies comprise platelet-activating non-HIT anti-platelet factor 4 (PF4) antibodies. In another aspect, the PVS is polyvinyl sulfonic acid sodium salt. In another aspect, the platelets from one or more normal subjects are treated with the PVS prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are combined with the sample from the subject suspected of having platelet-activating antibodies prior to combining with the PVS. In another aspect, a concentration of the PVS in the combined sample is about 0.01 g / mL to about 200 pg / mL. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 3 hr. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 5.0 x 106platelets / pL. In another aspect, measuring platelet activation in the combined sample comprises one or more of: analyzing the release of platelet granule components; detecting the expression of cell surface markers that are expressed upon platelet activation; detecting increase in expression of p-selectin on the platelet; detecting an increase in binding of annexin V to the platelet, detecting an increased proteolytic cleavage of the platelet membrane protein Fc gamma RII (CD32); detecting a change in platelet shape; detecting an increased level of ionized calcium in the platelet cytoplasm; detecting changes in expression levels of one or more CD markers found on activated platelets; detecting a change in platelet integrin conformation; detecting a change in platelet membrane potential; detecting a change in platelet impedance; detecting platelet agglutination; detecting platelet aggregation; detecting activated-platelet metabolic state; or detecting activated-platelet metabolites or byproducts. In another aspect, measuring platelet activation in the combined sample comprises analyzing the release of platelet granule components comprising thrombospondin-1 (TSP1). In another aspect, measuring platelet activation in the combined sample comprises using flow cytometry, radioimmunoassays (RIA), or enzyme-linked immunosorbent assays (ELISAs). In another aspect, the cell surface markers comprise one or more of selectins, integrins, or immunoglobulins. In another aspect, the cell surface markers comprise p-selectin. In another aspect, the method further comprises combining heparin or a heparin-like compound with the combined sample. In another aspect, the heparin or heparin-like compound has a final concentration of at least about 0.05 U / mL. In another aspect, the platelets from one or more normal subjects comprise one or more of isolated platelets, 211509-0006-W001 platelet-rich plasma, or washed platelets. In another aspect, the platelets from one or more normal subjects have been stabilized for storage by one or more of cooling, freezing, chemical storage, or lyophilization. In another aspect, the platelets from one or more normal subjects have been cryopreserved. In another aspect, the platelet activation is compared to one or more controls comprising blood from a normal subject, historical data, blood from a subject diagnosed as having platelet-activating antibodies, or a recombinant platelet-activating antibody. In another aspect, the subject is determined as having HIT or a non-HIT anti-PF4 antibody mediated syndrome. In another aspect, the non-HIT anti-PF4 antibody mediated syndrome comprises one or more of vaccine-induced immune thrombotic thrombocytopenia (VITT); spontaneous VITT; monoclonal gammopathy of thrombotic / thrombocytopenic significance (MGTS); Adenovirus associated thrombocytopenia; anti-PF4 immunothrombosis without proximate heparin, adenovirus vector vaccine exposure; or antisense oligonucleotide (ASO)-induced thrombocytopenia, or anti-PF4 effects of monoclonal antibody drugs / drug candidates.

[0016] Another embodiment described herein is a kit comprising: cryopreserved platelets; polyvinyl sulfonate or a salt thereof; buffers and reagents; heparin; positive and negative control samples; and optionally, packaging, labels, or instructions for use.

[0017] DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows cryopreserved platelets were recovered by washing and treated with PVS for 20 minutes. Following the 20-minute PVS incubation, platelets were treated with eight different HIT serum samples or six different normal serum samples for 30 minutes. The supernatant was collected by centrifugation and assayed for thrombospondin-1 (TSP1) in an antigen capture ELISA. The x-axis depicts the HIT (closed circles) or Normal (open squares) sample groups, the y-axis depicts the optical density of the TSP1 ELISA, and the horizontal lines depict the mean of the sample groups.

[0019] FIG. 2 shows cryopreserved platelets were recovered by washing and pre-treated with PVS (left) or PVS and high concentrations (100 U / mL) unfractionated heparin (right) for 20 minutes. Following this incubation, platelets were treated with a recombinant positive control HIT antibody (closed bars) or normal serum (open bars) for 30 minutes. The supernatant was collected by centrifugation and assayed for TSPI in an antigen capture ELISA. Results shown are mean of duplicate determinations + 1 standard deviation. The y-axis depicts the optical density of the TSP1 ELISA. The high-heparin concentration condition was used to confirm specificity of platelet activation (i.e., that platelet activation was not induced by non-anti-PF4 antibodies such as anti-HLA antibodies). 211509-0006-W001

[0020] FIG. 3 shows cryopreserved platelets were recovered by washing and co-incubated with PVS and HIT (closed bars) or normal (open bars) serum for between 40-80 minutes (left data set) or first pre-incubated with PVS for 20 minutes followed by incubation (right data set) with HIT (closed bars) or normal (open bars) samples for 30 minutes. The supernatant was collected by centrifugation and assayed for TSP1 in an antigen capture ELISA. Results shown are mean of duplicate determinations + 1 standard deviation. The y-axis depicts the optical density of the TSP1 ELISA.

[0021] FIG. 4 shows three cryopreserved platelet samples were pooled, recovered by washing and co-incubated with PVS, and seven HIT or seven normal serum for 70 minutes. The supernatant was collected by centrifugation and assayed for TSPI in an antigen capture ELISA. The x-axis depicts the HIT (closed circles) or Normal (open squares) sample groups, the y-axis depicts the optical density of the TSP1 ELISA, and the horizontal lines depict the mean of the sample groups.

[0022] FIG. 5 shows three cryopreserved platelet samples, recovered by washing and co- incubated with PVS, and blood samples from two patients with non-HIT anti-PF4 antibody mediated syndromes (closed bars) or one normal control serum (open bar) for 70 minutes. The supernatant was collected by centrifugation and assayed for TSPI in an antigen capture ELISA. Results shown are mean of duplicate determinations + 1 standard deviation. The y-axis depicts the optical density of the TSP1 ELISA. The horizontal dotted line separates non-HIT anti-PF4 antibody syndrome samples from normal control serum.

[0023] FIG. 6A-B show flow chart diagrams of exemplary methods for identifying plateletactivating antibodies in a subject as described herein. FIG. 6A shows that platelets from a normal subject are first treated with PVS, and this PVS-treated platelet sample is then combined and incubated with a blood sample from a subject suspected of having platelet-activating antibodies before measuring platelet activation. FIG. 6B shows that platelets from a normal subject are first combined with a blood sample from a subject suspected of having platelet-activating antibodies, and this combined sample is then treated with PVS and incubated before measuring platelet activation.

[0024] DETAILED DESCRIPTION

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid 211509-0006-W001 chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0026] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0027] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0028] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0029] As used herein, the term “or” can be conjunctive or disjunctive.

[0030] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0031] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0032] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean 211509-0006-W001 within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0033] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0034] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15- 30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0035] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0036] As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0037] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. In one aspect, the subject is suspected of having pathogenic antibodies or platelet-activating antibodies. A “subject suspected of having platelet-activating antibodies” or “test subject” as used herein refers to a subject exhibiting clinical findings indicative of pathogenic antibodies, including, for example, a below-normal platelet count, a decrease in platelet count, enlargement or extension of a previously diagnosed blood clot, or the development of a new blood clot elsewhere in the body. Additional symptoms indicative of pathogenic antibodies include fever, rash, chills, 211509-0006-W001 high blood pressure, shortness of breath and chest pain. In one embodiment, the plateletactivating antibody is an antibody capable of causing HIT or non-HIT anti-PF4 antibody mediated syndromes. In another embodiment, the platelet-activating antibodies comprise plateletactivating heparin-induced thrombocytopenia HIT antibodies. A “normal subject” refers to a subject not suffering from any aliment or not suspected of having platelet-activating antibodies.

[0038] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0039] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0040] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of’ or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0041] “Platelets,” also known as “thrombocytes,” as used herein refers to the anucleate fragments of megakaryocytes involved in blood coagulation, hemostasis, and blood thrombus formation. Human platelets are routinely isolated through a variety of methods including platelet apheresis, plateletpheresis, gel filtration, or differential centrifugation. Isolated platelets would be suitable, however, platelets from other sources, including washed platelets, unwashed platelets, platelet rich plasma or purified platelets, could also be used. In one embodiment, the platelets have been stabilized for storage by one or more of cooling, freezing, chemical storage, or lyophilization.

[0042] “Platelet activation” as used herein refers to the response of platelets when platelets encounter a “platelet activator” molecule that triggers activation, such as platelet activating 211509-0006-W001 antibodies, Thrombin Receptor Activating Peptide (TRAP), adenosine diphosphate (ADP), arachidonic acid, epinephrine, collagen, thrombin, thromboxane A2 (TxA2), thromboxane A2 (TXA2) mimetic U46619, calcium ionophore A23187, ristocetin, rhodocytin, among others. Platelet activation results in various changes to the platelets, including, for example, changes in markers associated with platelet activation, exocytosis of the dense granules and alpha granules, activation of the membrane enzyme phospholipase A2, changes in shape, aggregation, agglutination, changes in membrane potential, changes in integrin conformation, inter alia. Upon activation, platelets release granule contents including adenosine triphosphate (ATP), adenosine diphosphate (ADP), 5-hydroxytryptamine (serotonin), thrombospondin (e.g., thrombospondin-1 (TSP1)), fibrinogen, CXCL12, thromboxanes, among other metabolites and proteins, which may be assessed by using different methodologies such as immunological assay, high-performance liquid chromatography (HPLC), fluorescence microscopy, or flow cytometry. Upon activation, platelets have a change in surface expression various markers including of P-Selectins, CD34, CD41, CD61 , phosphatidyl serine, among others which may be assessed by using different methodologies such as immunological assay, high-performance liquid chromatography (HPLC), fluorescence microscopy, or flow cytometry. An “effective amount” as used herein refers to an amount of a compound that is sufficient to affect the desired outcome. In one embodiment, platelets are activated by administering an effective amount of one or more platelet activators as described herein.

[0043] Platelet activation levels may be measured using any method known in the art, such as, for instance, measuring levels of a marker found on or released from activated platelets in a test subject’s blood sample as compared to levels of activated platelets in a normal subject’s sample (e.g., a negative control sample) or a subject know to have pl ate let- activating antibodies or HIT (e.g., a positive control sample). Any marker known to be found on or released from activated platelets may be used to measure platelet activation, including, for example, one or more CD markers found on activated platelets (e.g., CD9, CD31 , CD36, CD41 , CD42, CD49b, CD61 , CD62P, CD63, CD107, their isoforms, or any marker present on the outside of platelets), including the marker CD62P, also known as p-selectin. In addition, platelet activation can be measured by measuring any increased binding of immunoglobulin, phosphatidyl serine expression, platelet aggregation, intracellular levels of ionized calcium, changes in integrin conformation, release of platelet granule contents, changes in platelet membrane potential or platelet impedance, levels of Fc gamma receptor 2 cleavage fragments, or shape change of platelets.

[0044] “Increased platelet activation” as used herein refers to an increase in platelet activation that is significantly different than that of the baseline platelet activation in a normal subject’s blood 211509-0006-W001 sample or in comparision to known values or historical data obtained using the assays described herein. An increase in platelet activation is indicative of the patient having platelet-activating antibodies, such as antibodies that cause HIT or non-HIT anti-PF4 antibody mediated syndromes. In one embodiment, the increase in platelet activation is at least two or three times the amount of baseline platelet activation in normal subject blood samples or historical data obtained for normal subjects using the methods described herein.

[0045] A “sample” as used herein refers to a specimen or culture obtained from a subject. Biological samples can be obtained from subjects and encompass fluids, solids, tissues, and gases. In one embodiment, the subject’s sample is a blood sample. Blood samples include whole blood, plasma, serum, blood products such as platelet-rich plasma, or fractionated blood components, such as one of the Cohn fractions I— IV, or an antibody fraction. Typically, a sample of about 0.2 mL is needed for the method described herein.

[0046] “Instructions for use” as used herein refers to a publication, diagram, or any other medium of expression which is used to provide instructions or steps for performing the methods described herein. The instructions for use can be provided in printed form, affixed to a container which contains the kit materials, shipped together with the kit, or provided at an internet site.

[0047] “Identifying,” “detecting,” or “diagnosing” as used herein refers to classifying a subject as having a pathology or a symptom, determining a severity of the pathology (grade or stage), monitoring pathology progression, or forecasting an outcome of a pathology or prospects of recovery. In one embodiment, a subject is identified or diagnosed as having platelet-activating antibodies in its blood. In one aspect, the platelet-activating antibodies comprise plateletactivating heparin-induced thrombocytopenia antibodies or non-HIT anti-PF4 antibody mediated syndromes. The identification or detection of platelet-activating heparin-induced thrombocytopenia antibodies is indicative or diagnostic of the subject having heparin-induced thrombocytopenia (HIT) or non-HIT anti-PF4 antibody mediated syndromes.

[0048] “Heparin-induced thrombocytopenia” or “HIT” as used herein refers to an adverse reaction to heparin, in which affected subjects produce platelet-activating antibodies that bind complexes of heparin / platelet membrane components and other molecules such as PF4, IL-8 and NAP-2, resulting in a prothrombotic and thrombocytopenic condition that can be life-threatening.

[0049] As used herein, “non-HIT anti-PF4 antibody mediated syndromes” refers to disorders where subjects develop HIT-like symptoms such as thrombocytopenia occurring in the absence of heparin exposure. These syndromes may include spontaneous HIT, vaccine-induced immune thrombotic thrombocytopenia (VITT); spontaneous VITT; monoclonal gammopathy of thrombotic / thrombocytopenic significance (MGTS); Adenovirus associated thrombocytopenia; 211509-0006-W001 anti-PF4 immunothrombosis without proximate heparin, adenovirus vector vaccine exposure; or antisense oligonucleotide (ASO)-induced thrombocytopenia, or anti-PF4 effects of monoclonal antibody drugs / drug candidates. See Kanack et al., Am. J. Hematol. 97(5): 519-526 (2022); Kanack et al., Blood 141 (14): 1772-1776 (2023); Warkentin et al., N. Engl. J. Med. 10;389(6): 574-577 (2023); Schdnborn et al., Blood 142(26): 2305-2314 (2023); Benson et al., N. Engl. J. Med. 379(1): 22-31 (2018); and Loberg et al., MAbs 13(1): 1887628 (2021), each of which is incorporated by reference for such teachings.

[0050] “Heparin-induced” as used herein refers to antibodies that result from exposure to heparin or those that recognize blood factors complexed with heparin as in spontaneous HIT, as indicative of HIT.

[0051] As used herein “heparin-like compound” refers to a compound with a high negative charge such as a polyanion, a heparin derivative, a chemically modified heparin, a heparin-like glycosaminoglycan molecule, a proteoglycan containing multiple heparin or heparin-like glycosaminoglycans, lower-molecular-weight heparin, a synthetic glycosaminoglycan comprising at least 15 sugar units, or a synthetic heparin-like glycosaminoglycan any of which could be connected directly or through a spacer / linker molecule to a core molecule.

[0052] As used herein “polyvinylsulfonic acid,” “polyvinyl sulfonate,” or “PVS” refers to a o=s=o

[0053] I > hydrophilic polymer having the linear formula (C2H3O3S)n and structure O , with typical molecular weights of 130 to 5000 g / mol. PVS is typically supplied as an alkaline salt, such as sodium or potassium, as a 30-40% by mass solution in water with a molecular weight of 2000- 5000 g / mol.

[0054] Described herein are methods and kits for the detection of platelet-activating pathogenic HIT or non-HIT anti-PF4 antibodies in samples from patients suspected of HIT or non-HIT anti- PF4 antibody mediated syndromes using platelets and polyvinyl sulfonate or salts thereof. Upon platelet activation, thrombospondin-1 (TSP1), a platelet granule protein, is released and can be detected using an easy-to-use TSP1 antigen capture enzyme linked immunosorbent assay (ELISA).

[0055] Methods for Identifying Platelet-Activating Antibodies

[0056] One embodiment described herein is a method for identifying platelet-activating antibodies in a sample from a subject suspected of having platelet-activating antibodies, the method comprising: obtaining a sample comprising serum, plasma, or fractionated blood from a subject 211509-0006-W001 suspected of having platelet-activating antibodies; obtaining platelets from one or more normal subjects; combining the platelets from one or more normal subjects with the sample from the subject suspected of having platelet-activating antibodies and polyvinyl sulfonate (PVS) to form a combined sample, and incubating the combined sample for a period of time; measuring platelet activation in the combined sample; wherein platelet activation in the combined sample is indicative of the presence of platelet-activating antibodies in the sample from the subject suspected of having platelet-activating antibodies. In one aspect, the platelet-activating antibodies comprise platelet-activating heparin-induced thrombocytopenia (HIT) antibodies. In another aspect, the platelet-activating antibodies comprise platelet-activating non-HIT anti-platelet factor 4 (PF4) antibodies. In another aspect, the PVS is polyvinyl sulfonic acid sodium salt. In another aspect, the PVS is polyvinyl sulfonic acid potassium salt. In another aspect, the platelets from one or more normal subjects are treated with the PVS prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are combined with the sample from the subject suspected of having platelet-activating antibodies prior to combining with the PVS. In another aspect, a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 200 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 170 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 140 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 110 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 80 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 50 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 20 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 30 pg / mL to about 200 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 60 pg / mL to about 200 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 90 pg / mL to about 200 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 120 pg / mL to about 200 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 150 pg / mL to about 200 pg / mL. In another aspect, a concentration of the PVS in the combined sample is about 180 pg / mL to about 200 pg / mL. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 15 min to about 2 hr prior to combining with the sample from the subject 211509-0006-W001 suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 30 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 45 min to about 2 hr prior to combining with the sample from the subject suspected of having plateletactivating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 60 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 75 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 90 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 105 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 105 min prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 90 min prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 75 min prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 60 min prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 45 min prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 30 min prior to combining with the sample from the subject suspected of having platelet-activating antibodies. In another aspect, the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 15 min prior to combining with the sample from the subject suspected of having plateletactivating antibodies. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 3 hr. In another aspect, the combined sample is incubated for a 211509-0006-W001 period of time of about 35 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 55 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 75 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 95 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 115 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 135 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 155 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 175 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 3 hr. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 160 min. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 140 min. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 120 min. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 100 min. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 80 min. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 60 min. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 40 min. In another aspect, the combined sample is incubated for a period of time of about 15 min to about 20 min. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pLto about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.5 x 106platelets / pL to about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 1.0 x 106platelets / pL to about 5.0 x io6platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 1.5 x 106platelets / pLto about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 2.0 x 106platelets / pL to about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 2.5 x 106platelets / pL to about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 3 x 106platelets / pL to about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 3.5 x 106platelets / pLto about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is 211509-0006-W001 about 4 x 106 piatelets / pLto about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 4.5 x 106platelets / pLto about 5.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 4.5 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 4.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 3.5 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pLto about 3.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 06platelets / pL to about 2.5 x io6platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 2.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pLto about 1.5 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 1.0 x 106platelets / pL. In another aspect, a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 0.5 x 106platelets / pL. In another aspect, measuring platelet activation in the combined sample comprises one or more of: analyzing the release of platelet granule components; detecting the expression of cell surface markers that are expressed upon platelet activation; detecting increase in expression of p-selectin on the platelet; detecting an increase in binding of annexin V to the platelet, detecting an increased proteolytic cleavage of the platelet membrane protein Fc gamma RII (CD32); detecting a change in platelet shape; detecting an increased level of ionized calcium in the platelet cytoplasm; detecting changes in expression levels of one or more CD markers found on activated platelets; detecting a change in platelet integrin conformation; detecting a change in platelet membrane potential; detecting a change in platelet impedance; detecting platelet agglutination; detecting platelet aggregation; detecting activated-platelet metabolic state; or detecting activated-platelet metabolites or byproducts. In another aspect, measuring platelet activation in the combined sample comprises analyzing the release of platelet granule components comprising thrombospondin-1 (TSP1). In another aspect, measuring platelet activation in the combined sample comprises using flow cytometry, radioimmunoassays (RIA), or enzyme-linked immunosorbent assays (ELISAs). In another aspect, the cell surface markers 211509-0006-W001 comprise one or more of selectins, integrins, or immunoglobulins. In another aspect, the cell surface markers comprise p-selectin. In another aspect, the method further comprises combining heparin or a heparin-like compound with the combined sample. In another aspect, the heparin or heparin-like compound has a final concentration of at least about 0.05 U / mL. In another aspect, the platelets from one or more normal subjects comprise one or more of isolated platelets, plateletrich plasma, or washed platelets. In another aspect, the platelets from one or more normal subjects have been stabilized for storage by one or more of cooling, freezing, chemical storage, or lyophilization. In another aspect, the platelets from one or more normal subjects have been cryopreserved. In another aspect, the platelet activation is compared to one or more controls comprising blood from a normal subject, historical data, blood from a subject diagnosed as having platelet-activating antibodies, or a recombinant platelet-activating antibody. In another aspect, the subject is determined as having HIT or a non-HIT anti-PF4 antibody mediated syndrome. In another aspect, the non-HIT anti-PF4 antibody mediated syndrome comprises one or more of vaccine-induced immune thrombotic thrombocytopenia (VITT); spontaneous VITT; monoclonal gammopathy of thrombotic / thrombocytopenic significance (MGTS); Adenovirus associated thrombocytopenia; anti-PF4 immunothrombosis without proximate heparin, adenovirus vector vaccine exposure; or antisense oligonucleotide (ASO)-induced thrombocytopenia, or anti-PF4 effects of monoclonal antibody drugs / drug candidates.

[0057] Kits

[0058] Another embodiment described herein is a kit comprising: cryopreserved platelets; polyvinyl sulfonate or a salt thereof; buffers and reagents; heparin; positive and negative control samples; and optionally, packaging, labels, or instructions for use.

[0059] In various aspects, the cryopreserved platelets in the kit are from one or more normal subjects and have been stabilized for storage by cooling, freezing, chemical storage, or lyophilization. In another aspect, the cryopreserved platelets are provided at a concentration of about 0.1 x 106platelets / pL to about 5.0 x 106platelets / pL. In another aspect, the polyvinyl sulfonate is polyvinyl sulfonic acid sodium salt or polyvinyl sulfonic acid potassium salt. In another aspect, the polyvinyl sulfonate is provided at a concentration suitable to achieve a final concentration of about 0.01 pg / mL to about 200 pg / mL in the combined sample. In another aspect, the buffers and reagents comprise one or more of platelet wash buffer, platelet activation buffer, ELISA wash buffer, or detection reagents. In another aspect, the buffers and reagents comprise a platelet wash buffer containing NaCI, KCI, NaHCCh, sodium citrate, glucose, and trehalose. In another aspect, the buffers and reagents comprise a platelet activation buffer 211509-0006-W001 containing NaCI, Na2HPC>4, KH2PO4, trehalose, and bovine serum albumin. In another aspect, the heparin is unfractionated heparin provided at a concentration suitable to achieve a final concentration of at least about 0.05 U / mL in the combined sample. In another aspect, the positive control samples comprise serum, plasma, or fractionated blood from subjects diagnosed as having platelet-activating antibodies, or recombinant platelet-activating antibodies. In another aspect, the negative control samples comprise serum, plasma, or fractionated blood from normal subjects. In another aspect, the kit may further comprise detection reagents for measuring platelet activation, including antibodies for detecting thrombospondin-1 (TSP1), p-selectin, or other platelet activation markers. In another aspect, the kit may further comprise reagents for enzyme-linked immunosorbent assays (ELISAs), flow cytometry, or radioimmunoassays (RIA). In another aspect, the kit components may be provided in separate containers suitable for storage and use in clinical or research laboratory settings.

[0060] In various aspects, the disclosed kits may comprise packaging, labels, or instructions for use. The instructions for use may provide step-by-step protocols for performing the methods described herein for identifying platelet-activating antibodies. The information and instructions may be in the form of words, pictures, or both, and the like. Instructions included in kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written on printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. As used herein, the term “instructions” may also include the address of an internet site that provides the instructions.

[0061] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the 211509-0006-W001 other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0062] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0063] Clause 1. A method for identifying platelet-activating antibodies in a sample from a subject suspected of having platelet-activating antibodies, the method comprising: obtaining a sample comprising serum, plasma, or fractionated blood from a subject suspected of having platelet-activating antibodies; obtaining platelets from one or more normal subjects; combining the platelets from one or more normal subjects with the sample from the subject suspected of having platelet-activating antibodies and polyvinyl sulfonate (PVS) to form a combined sample, and incubating the combined sample for a period of time; measuring platelet activation in the combined sample; wherein platelet activation in the combined sample is indicative of the presence of plateletactivating antibodies in the sample from the subject suspected of having plateletactivating antibodies.

[0064] Clause 2. The method of clause 1 , wherein the platelet-activating antibodies comprise platelet-activating heparin-induced thrombocytopenia (HIT) antibodies.

[0065] Clause 3. The method of clause 1 or 2, wherein the platelet-activating antibodies comprise platelet-activating non-HIT anti-platelet factor 4 (PF4) antibodies.

[0066] Clause 4. The method of any one of clauses 1-3, wherein the PVS is polyvinyl sulfonic acid sodium salt.

[0067] Clause 5. The method of any one of clauses 1-4, wherein the platelets from one or more normal subjects are treated with the PVS prior to combining with the sample from the subject suspected of having platelet-activating antibodies.

[0068] Clause s. The method of any one of clauses 1-5, wherein the platelets from one or more normal subjects are combined with the sample from the subject suspected of having platelet-activating antibodies prior to combining with the PVS.

[0069] Clause 7. The method of any one of clauses 1-6, wherein a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 200 pg / mL. 211509-0006-W001

[0070] Clause 8. The method of clause 5, wherein the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies.

[0071] Clause 9. The method of any one of clauses 1-8, wherein the combined sample is incubated for a period of time of about 15 min to about 3 hr.

[0072] Clause 10. The method of any one of clauses 1-9, wherein a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 5.0 x 106platelets / pL.

[0073] Clause 11. The method of any one of clauses 1-10, wherein measuring platelet activation in the combined sample comprises one or more of: analyzing the release of platelet granule components; detecting the expression of cell surface markers that are expressed upon platelet activation; detecting increase in expression of p-selectin on the platelet; detecting an increase in binding of annexin V to the platelet, detecting an increased proteolytic cleavage of the platelet membrane protein Fc gamma RII (CD32); detecting a change in platelet shape; detecting an increased level of ionized calcium in the platelet cytoplasm; detecting changes in expression levels of one or more CD markers found on activated platelets; detecting a change in platelet integrin conformation; detecting a change in platelet membrane potential; detecting a change in platelet impedance; detecting platelet agglutination; detecting platelet aggregation; detecting activated-platelet metabolic state; or detecting activated-platelet metabolites or byproducts.

[0074] Clause 12. The method of clause 11 , wherein measuring platelet activation in the combined sample comprises analyzing the release of platelet granule components comprising thrombospondin-1 (TSP1).

[0075] Clause 13. The method of clause 11 , wherein measuring platelet activation in the combined sample comprises using flow cytometry, radioimmunoassays (RIA), or enzyme-linked immunosorbent assays (ELISAs).

[0076] Clause 14. The method of clause 11 , wherein the cell surface markers comprise one or more of selectins, integrins, or immunoglobulins.

[0077] Clause 15. The method of clause 11 , wherein the cell surface markers comprise p-selectin.

[0078] Clause 16. The method of any one of clauses 11-16, further comprising combining heparin or a heparin-like compound with the combined sample.

[0079] Clause 17. The method of clause 16, wherein the heparin or heparin-like compound has a final concentration of at least about 0.05 U / mL. 211509-0006-W001

[0080] Clause 18. The method of any one of clauses 1-17, wherein the platelets from one or more normal subjects comprise one or more of isolated platelets, platelet-rich plasma, or washed platelets.

[0081] Clause 19. The method of any one of clauses 1-18, wherein the platelets from one or more normal subjects have been stabilized for storage by one or more of cooling, freezing, chemical storage, or lyophilization.

[0082] Clause 20. The method of any one of clauses 1-19, wherein the platelets from one or more normal subjects have been cryopreserved.

[0083] Clause 21. The method of any one of clauses 1-20, wherein the platelet activation is compared to one or more controls comprising blood from a normal subject, historical data, blood from a subject diagnosed as having platelet-activating antibodies, or a recombinant platelet-activating antibody.

[0084] Clause 22. The method of any one of clauses 1-21 , wherein the subject is determined as having HIT or a non-HIT anti-PF4 antibody mediated syndrome.

[0085] Clause 23. The method of clause 22, wherein the non-HIT anti-PF4 antibody mediated syndrome comprises one or more of vaccine-induced immune thrombotic thrombocytopenia (VITT); spontaneous VITT; monoclonal gammopathy of thrombotic / thrombocytopenic significance (MGTS); Adenovirus associated thrombocytopenia; anti-PF4 immunothrombosis without proximate heparin, adenovirus vector vaccine exposure; or antisense oligonucleotide (ASO)-induced thrombocytopenia, or anti-PF4 effects of monoclonal antibody drugs / drug candidates.

[0086] Clause 24. A kit comprising: cryopreserved platelets; polyvinyl sulfonate or a salt thereof; buffers and reagents; heparin; positive and negative control samples; and optionally, packaging, labels, or instructions for use. 211509-0006-W001

[0087] EXAMPLES

[0088] Example 1

[0089] Pre-treatment of Cryopreserved Platelets with PVS Followed by HIT or Normal Serum

[0090] Platelets were cryopreserved as previously described at a concentration of 1.5 x 106platelets / pL. See e.g., Int. Pat. App. Pub. No. WO 2022 / 225624 A 1 and Kanack et al., Blood 140(25): 2722-2729 (2022), each of which are incorporated by reference herein for such teachings. 500 pL aliquots of cryopreserved platelets were thawed at 37 °C for 3 minutes. Platelets were isolated by centrifugation (1000 x g, 5 minutes) and resuspended in 1 mL of Platelet Wash Buffer (108 mM NaCI, 3.8 mM KCI, 1.7 mM NaHCOs, 22.9 mM sodium citrate, 27.8 mM glucose, 50 mM trehalose, pH 6.5). 166.6 pL aliquots of platelets in wash buffer were isolated by centrifugation (1000 x g, 5 minutes) and resuspended in 92.5 pL of a PVS Platelet Activation Buffer containing 155 mM NaCI, 2.97 mM Na2HPC>4, 1.06 mM KH2PO4, 50 mM trehalose, 0.15 mM bovine serum albumin, and 2.16 pg / mL PVS, and incubated for 20 minutes. Following the 20-minute incubation, 7.5 pL of HIT or normal serum was added to the PVS treated platelets and incubated for 30 minutes. The supernatant of each reaction was collected by centrifugation (1000 x g, 5 minutes). The supernatants were diluted 1 :625 into 1 x ELISA Wash Buffer (ImmunoChemistry Technologies; Product No. 652) containing 1 pg / mL of a horseradish peroxidase (HRP) labelled anti-TSP1 monoclonal antibody. 100 pL of the diluted supernatant was added in duplicate to an ELISA plate coated with 1 pg / mL of an anti-TSP1 monoclonal antibody (developed by Retham Technologies Inc.) and incubated for 90 minutes. Following 90- minute incubation, the ELISA plate was washed 4 times with 300 pL of ELISA wash buffer. 100 pL of TMB substrate was added and incubated for 20 minutes. Finally, 50 pL of 1 M H2SO4 was added to stop the color development and absorbance was measured at 450 nm with a subtraction at 650 nm.

[0091] Example 2 Pre-treatment of Cryopreserved Platelets with PVS, or PVS with high concentration Heparin Followed by Recombinant HIT-Like Positive Control Antibody or Normal Serum

[0092] Platelets were cryopreserved as described in Example 1. 500 pL aliquots of cryopreserved platelets were thawed at 37 °C for 3 minutes. Platelets were isolated by centrifugation and resuspended in 1 mL of Platelet Wash Buffer. 166.6 pL aliquots of platelets in wash buffer were isolated by centrifugation and resuspended in 92.5 pL of a PVS Platelet Activation Buffer containing 2.16 pg / mL PVS or 2.16 pg / mL PVS and 100 U / mL unfractionated heparin and incubated for 20 minutes. Following the 20-minute incubation, 7.5 pL of Recombinant 211509-0006-W001

[0093] H IT-Like Control Antibody (50 pg / mL, final) or a normal serum was added to the PVS or PVS and heparin treated platelets and incubated for 30 minutes. The supernatant of each reaction was collected by centrifugation. The supernatants were diluted 1 :625 into ELISA Wash Buffer containing 1 pg / mL of an HRP labelled anti-TSP1 monoclonal antibody. 100 pL diluted supernatant was added in duplicate to an ELISA plate coated with 1 pg / mL of an anti-TSP1 monoclonal antibody and incubated for 90 minutes. Following 90-minute incubation, the ELISA plate was washed 4 times with 300 pL of ELISA Wash Buffer. 100 pL of TMB substrate was added and incubated for 20 minutes. Finally, 50 pL of 1 M H2SO4was added to stop the color development and absorbance was measured at 450 nm with a subtraction at 650 nm.

[0094] Example 3

[0095] Co-incubation of Cryopreserved Platelets with PVS, and HIT or Normal Serum

[0096] Platelets were cryopreserved as described above in Example 1. 500 pL aliquots of cryopreserved platelets were thawed at 37 °C for 3 minutes. Platelets were isolated by centrifugation and resuspended in 1 mL of Platelet Wash Buffer. 166.6 pL aliquots of platelets in wash buffer were isolated by centrifugation and resuspended in 92.5 pL of a PVS Platelet Activation Buffer containing 2.16 pg / mL PVS. 7.5 pL of a HIT or normal serum was immediately added and the mixture was incubated for 40, 50, 60, 70, or 80 minutes. The supernatant of each reaction was collected by centrifugation. The supernatants were diluted 1:625 into ELISA Wash Buffer containing 1 pg / mL of an HRP labelled anti-TSP1 monoclonal antibody. 100 pL of diluted supernatant was added in duplicate to an ELISA plate coated with 1 pg / mL of an anti-TSP1 monoclonal antibody and incubated for 90 minutes. Following 90-minute incubation, the ELISA plate was washed 4 times with 300 pL of an ELISA Wash Buffer. 100 pL of TMB substrate was added and incubated for 20 minutes. Finally, 50 pL of 1 M H2SO4 was added to stop the color development and absorbance was measured at 450 nm with a subtraction at 650 nm.

[0097] Example 4

[0098] Co-incubation of Pooled Cryopreserved Platelets with PVS, and HIT or Normal Serum

[0099] Platelets were cryopreserved as described above in Example 1. 500 pL aliquots of cryopreserved platelets from three donors were thawed at 37 °C for 3 minutes. Platelets were isolated by centrifugation and resuspended in 1 mL of Platelet Wash Buffer and mixed to create a pool. 166.6 pL aliquots of pooled platelets in Platelet Wash Buffer were isolated by centrifugation and resuspended in 92.5 pL of a PVS Platelet Activation Buffer containing 2.16 pg / mL PVS. 7.5 pL of a HIT serum, normal serum, or serum from patients with non-HIT anti-PF4 211509-0006-W001 antibody mediated syndromes were immediately added, and the mixture was incubated for 40, 50, 60, 70, or 80 minutes. The supernatant of each reaction was collected by centrifugation. The supernatants were diluted 1:625 into an ELISA Wash Buffer containing 1 pg / mL of an HRP labelled anti-TSP1 monoclonal antibody. 100 pL diluted supernatant was added in duplicate to an ELISA plate coated with 1 pg / mL of an anti-TSP1 monoclonal antibody and incubated for 90 minutes. Following 90-minute incubation, the ELISA plate was washed 4 times with 300 pL of ELISA Wash Buffer. 100 pL of TMB substrate was added and incubated for 20 minutes. Finally, 50 pL of 1 M H2SO4was added to stop the color development and absorbance was measured at 450 nm with a subtraction at 650 nm.

[0100] Example 5

[0101] As shown in FIG. 1 , PVS pre-treated platelets are strongly activated by each of the eight HIT serum samples, but minimal / background platelet activation is observed in the presence of any of the six normal serum samples. This demonstrates that PVS-treated platelets can act as a target for HIT antibodies present in blood samples from patients with HIT.

[0102] As shown in FIG. 2, a recombinant HIT-Like positive control antibody was used to replicate these findings and to demonstrate that activation of PVS-treated platelets can be inhibited by high concentrations of unfractionated heparin. This serves as a confirmation step to exclude other possibilities of platelet activation such as anti-HLA antibodies.

[0103] In an effort to optimize this method / technology for detection of HIT antibodies, coincubation of platelets, PVS, and patient blood sample was compared to pre-incubation of platelets and PF4 followed by addition of patient blood sample. As shown in FIG. 3, co-incubation of platelets, PVS, and serum is superior to segmented incubation from a signal-to-noise standpoint, and the 70-minute co-incubation time was optimal compared to the other tested timepoints.

[0104] These optimized conditions (70-minute platelet- PVS-patient blood sample co-incubation) were tested using a pool of three cryopreserved platelets and a panel of seven HIT and seven normal serum samples. All seven HIT normal samples activated platelets strongly, while minimal / background activation was observed with the normal samples resulting in clear differentiation of the two sample groups, as shown in FIG. 4.

[0105] As shown in FIG. 5, two samples from patients with non-HIT anti-PF4 antibody mediated syndromes activate PVS treated platelets.

Claims

211509-0006-W001CLAIMSWhat is claimed:

1. A method for identifying platelet-activating antibodies in a sample from a subject suspected of having platelet-activating antibodies, the method comprising: obtaining a sample comprising serum, plasma, or fractionated blood from a subject suspected of having platelet-activating antibodies; obtaining platelets from one or more normal subjects; combining the platelets from one or more normal subjects with the sample from the subject suspected of having platelet-activating antibodies and polyvinyl sulfonate (PVS) to form a combined sample, and incubating the combined sample for a period of time; measuring platelet activation in the combined sample; wherein platelet activation in the combined sample is indicative of the presence of plateletactivating antibodies in the sample from the subject suspected of having plateletactivating antibodies.

2. The method of claim 1 , wherein the platelet-activating antibodies comprise plateletactivating heparin-induced thrombocytopenia (HIT) antibodies.

3. The method of claim 1 , wherein the platelet-activating antibodies comprise plateletactivating non-HIT anti-platelet factor 4 (PF4) antibodies.

4. The method of claim 1 , wherein the PVS is polyvinyl sulfonic acid sodium salt.

5. The method of claim 1 , wherein the platelets from one or more normal subjects are treated with the PVS prior to combining with the sample from the subject suspected of having platelet-activating antibodies.

6. The method of claim 1 , wherein the platelets from one or more normal subjects are combined with the sample from the subject suspected of having platelet-activating antibodies prior to combining with the PVS.

7. The method of claim 1 , wherein a concentration of the PVS in the combined sample is about 0.01 pg / mL to about 200 pg / mL.211509-0006-W0018. The method of claim 5, wherein the platelets from one or more normal subjects are treated with the PVS for about 1 min to about 2 hr prior to combining with the sample from the subject suspected of having platelet-activating antibodies.

9. The method of claim 1 , wherein the combined sample is incubated for a period of time of about 15 min to about 3 hr.

10. The method of claim 1 , wherein a final concentration of the platelets from one or more normal subjects in the combined sample is about 0.1 x 106platelets / pL to about 5.0 x 106platelets / pL.

11. The method of claim 1 , wherein measuring platelet activation in the combined sample comprises one or more of: analyzing the release of platelet granule components; detecting the expression of cell surface markers that are expressed upon platelet activation; detecting increase in expression of p-selectin on the platelet; detecting an increase in binding of annexin V to the platelet, detecting an increased proteolytic cleavage of the platelet membrane protein Fc gamma RII (CD32); detecting a change in platelet shape; detecting an increased level of ionized calcium in the platelet cytoplasm; detecting changes in expression levels of one or more CD markers found on activated platelets; detecting a change in platelet integrin conformation; detecting a change in platelet membrane potential; detecting a change in platelet impedance; detecting platelet agglutination; detecting platelet aggregation; detecting activated-platelet metabolic state; or detecting activated-platelet metabolites or byproducts.

12. The method of claim 11 , wherein measuring platelet activation in the combined sample comprises analyzing the release of platelet granule components comprising thrombospondin-1 (TSP1).

13. The method of claim 11 , wherein measuring platelet activation in the combined sample comprises using flow cytometry, radioimmunoassays (RIA), or enzyme-linked immunosorbent assays (ELISAs).211509-0006-W00114. The method of claim 11, wherein the cell surface markers comprise one or more of selectins, integrins, or immunoglobulins.

15. The method of claim 11, wherein the cell surface markers comprise p-selectin.

16. The method of claim 1 , further comprising combining heparin or a heparin-like compound with the combined sample.

17. The method of claim 16, wherein the heparin or heparin-like compound has a final concentration of at least about 0.05 U / mL.

18. The method of claim 1 , wherein the platelets from one or more normal subjects comprise one or more of isolated platelets, platelet-rich plasma, or washed platelets.

19. The method of claim 1, wherein the platelets from one or more normal subjects have been stabilized for storage by one or more of cooling, freezing, chemical storage, or lyophilization.

20. The method of claim 1 , wherein the platelets from one or more normal subjects have been cryopreserved.

21. The method of claim 1 , wherein the platelet activation is compared to one or more controls comprising blood from a normal subject, historical data, blood from a subject diagnosed as having platelet-activating antibodies, or a recombinant platelet-activating antibody.

22. The method of claim 1 , wherein the subject is determined as having HIT or a non-HIT anti- PF4 antibody mediated syndrome.

23. The method of claim 22, wherein the non-HIT anti-PF4 antibody mediated syndrome comprises one or more of vaccine-induced immune thrombotic thrombocytopenia (VITT); spontaneous VITT; monoclonal gammopathy of thrombotic / thrombocytopenic significance (MGTS); Adenovirus associated thrombocytopenia; anti-PF4 immunothrombosis without proximate heparin, adenovirus vector vaccine exposure; or antisense oligonucleotide211509-0006-W001(ASO)-induced thrombocytopenia, or anti-PF4 effects of monoclonal antibody drugs / drug candidates.

24. A kit comprising: cryopreserved platelets; polyvinyl sulfonate or a salt thereof; buffers and reagents; heparin; positive and negative control samples; and optionally, packaging, labels, or instructions for use.

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