Compositions for detecting hyperfibrinolysis and arrest of fibrinolysis and uses thereof

Abstract

The present disclosure provides compositions for hemostasis blood testing, as well as related methods, systems, kits, and sample holders. The compositions include any one or more of (a) a plurality of phospholipids, (b) a plurality of activators, (c) a blood coagulation initiator, and (d) plasmin or a precursor thereof. In some embodiments, the composition further includes one or more of (e) a buffer, (f) a sugar, (g) a protein concentration reference standard, and (h) CaCl2.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of the earlier filing date of U.S. Provisional Patent Application Ser. No. 63 / 651,747, entitled "Compositions for Detection of Hyperfibrinolysis and Fibrinolytic Shutdown and Uses Thereof," filed on May 24, 2024, which is incorporated herein by reference in its entirety. Background Technology

[0003] Fibrinolytic activation is almost universal after severe trauma. Systemic hyperfibrinolysis is a key component of acute traumatic coagulopathy (ATC) and is associated with poor clinical outcomes, although there is controversy regarding optimal treatment strategies. Furthermore, treatment triggers are complicated by the lack of sensitive and rapid diagnostic tools, and there are discrepancies between diagnoses of hyperfibrinolysis based on viscoelasticity assays and those based on biomarkers of fibrinolysis. Current diagnostic methods appear capable of detecting the most severe forms of hyperfibrinolysis but are relatively insensitive to moderate but clinically significant fibrinolytic activation. Rapid diagnosis of hyperfibrinolysis would facilitate timely intervention, guide the administration of antifibrinolytic drugs in treating bleeding in trauma patients, and reduce mortality. Therefore, there is an urgent need for technologies for the rapid and accurate diagnosis of hyperfibrinolysis and / or fibrinolysis. Summary of the Invention

[0004] This disclosure provides compositions, methods, and systems for the rapid detection of hyperfibrinolysis and cessation of fibrinolysis in blood samples (e.g., whole blood or plasma).

[0005] This document provides compositions for hemostasis blood tests comprising one or more, for example, two, three, or all four of the following: (a) a phospholipid component; (b) a surfactant; (c) a blood coagulation initiator, such as tissue factor; and / or (d) plasmin or a precursor thereof. In some embodiments, the compositions for hemostasis blood tests comprise one or more of the following: (a) a phospholipid component; (b) a surfactant; (c) a blood coagulation initiator, such as tissue factor; and (d) plasmin or a precursor thereof. In some embodiments, the compositions may further comprise one or more of the following: (e) a buffer; (f) a stabilizer; (g) a protein concentration reference standard; and (h) a salt, such as a calcium salt, for example, CaCl2.

[0006] In some embodiments, the phospholipid component is present at a concentration of about 0.001 mg / mL to about 1.0 mg / mL. In some embodiments, the phospholipid component comprises multiple phospholipids. In some embodiments, the surfactant is present at a concentration of about 0.01% to about 1.0%. In some embodiments, the blood coagulation initiator comprises tissue factor, and the tissue factor is present at a concentration of about 0.000001 mg / mL to about 0.001 mg / mL. In some embodiments, the plasmin or its precursor is present at a concentration of about 2.0 μg / mL to about 24 μg / mL. In some embodiments, the surfactant comprises silica particles.

[0007] This document also provides a method for detecting hemostasis disorder in a subject, the method comprising (a) obtaining a blood sample from the subject; (b) contacting at least a portion of the blood sample with a assay reagent, wherein the assay reagent comprises any of the compositions described herein, thereby producing a assay sample; (c) performing a blood coagulation assay on the assay sample and measuring a time parameter of the blood coagulation assay; and (d) comparing the time parameter with a reference coagulation time, wherein a higher time parameter compared to the reference coagulation time indicates that the subject has a hemostasis disorder.

[0008] In some embodiments, the higher time parameter is at least one second or more higher than the reference coagulation time. In some embodiments, a lower time parameter compared to the reference coagulation time indicates that the subject does not have hyperfibrinolysis or cessation of fibrinolysis.

[0009] In some embodiments, the assay reagent is placed in a sample holder. In some embodiments, the assay reagent is a dried reagent. In some embodiments, the blood coagulation assay includes a microfluidic device-based assay.

[0010] In some embodiments, the method further includes treating the subject based on the subject being determined to have a hemostatic disorder, such as hyperfibrinolysis. For example, the subject may be treated by administering empirical tranexamic acid, ε-aminocaproic acid, or other lysine analogs, or a blood transfusion may be provided.

[0011] This document also provides a sample holder for blood coagulation assays, the sample holder comprising an assay reagent comprising any of the compositions described herein.

[0012] In some embodiments, the sample holder includes a cartridge or cuvette. In some embodiments, the assay reagent is provided in the sample holder in a dry form. In some embodiments, the blood coagulation assay detects hemostatic defects in the blood sample. In some embodiments, the blood sample is a whole blood sample.

[0013] In some embodiments, the sample holder includes a first reagent comprising (a) a phospholipid component; (b) a surfactant; (c) a blood coagulation initiator, such as tissue factor; and (d) plasmin or a precursor thereof; and a second reagent (e.g., a control) comprising (a) a phospholipid component; (b) a surfactant; and (c) a blood coagulation initiator, such as tissue factor, without any plasmin or plasmin precursor.

[0014] This document also provides systems or kits for detecting hemostasis disorders in blood samples, including any of the sample holders described herein.

[0015] This document also provides a kit comprising a first reagent comprising (a) a phospholipid component; (b) a surfactant; (c) a blood coagulation initiator, such as tissue factor; and (d) plasmin or a precursor thereof; and a second reagent (e.g., a control) comprising (a) a phospholipid component; (b) a surfactant; and (c) a blood coagulation initiator, such as tissue factor, without plasmin. This document also provides a kit comprising a first reagent comprising (a) a phospholipid component; (b) a surfactant; (c) a blood coagulation initiator, such as tissue factor; and (d) plasmin or a precursor thereof; and a second reagent (e.g., a control) comprising (a) a phospholipid component; (b) a surfactant; and (c) a blood coagulation initiator, such as tissue factor, without plasmin.

[0016] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. It will be further understood that the terms “comprising” and / or “including” or “containing” and / or “comprise”, when used in this specification, specify the presence of the stated feature, integer, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated enumerated items.

[0017] As used herein, the term "approximately," when used to refer to values, means a value that is similar to the reference value in the context. Generally, those skilled in the art will understand that, in the appropriate context, the degree of similarity is ±10%. Unless otherwise explicitly stated from the context, all numerical values ​​provided herein are modified by the term "approximately."

[0018] As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject suffers from a relevant disease, condition, or symptom. In some embodiments, the subject is susceptible to a disease, condition, or symptom. In some embodiments, the subject displays one or more signs, symptoms, or characteristics of a disease, condition, or symptom. In some embodiments, the subject does not display any symptoms or characteristics of a disease, condition, or symptom. In some embodiments, the subject is a person with one or more characteristics of susceptibility or risk to a disease, disorder, or symptom. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has received and / or has received a diagnostic and / or treatment. In some embodiments, the subject is a human.

[0019] 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 to which this invention pertains. While similar or equivalent methods and materials may be used to carry out the invention as described herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including its definitions, shall prevail. Furthermore, the materials, methods, and embodiments described are illustrative only and not restrictive.

[0020] Details of one or more embodiments of the invention are set forth in the following drawings and description. Other features, objects, and advantages of the invention will be apparent from the description, drawings, and claims. Attached Figure Description

[0021] These and other features and advantages of the invention will be more fully understood by referring to the following detailed description and the accompanying drawings.

[0022] Figure 1 This is a graph showing the results from the first assay, in which the assay reagents included phospholipids, silica, and tissue factor. (GEM) 100 Whole Blood Hemostasis System (by Instrumentation Laboratory Company (d / b / a) The determination was performed at Bedford, Mass. (“Werfen”).

[0023] Figure 2 This is a graph showing the results from the second assay, in which the assay reagents included silica and tissue factor. (GEM) The measurements were performed on the instrument. Detailed Implementation

[0024] While various embodiments of the invention have been shown and described herein, those skilled in the art will understand that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the inventable embodiments described herein may be employed.

[0025] Acute traumatic coagulopathy (ATC) is a response to trauma or severe trauma in which normal blood clotting is disrupted. ATC is characterized by impaired thrombin production, dysfunctional platelets, increased anticoagulant activity, and fibrinolytic dysregulation. Fibrinolytic dysregulation, i.e., deviation from physiological fibrinolysis, can take the form of hyperfibrinolysis or fibrinolysis cessation. Hyperfibrinolysis, due to overactivation of the fibrinolytic system (including overactivation of plasmin), leads to excessive breakdown of fibrin clots and is a key component of ATC. Hyperfibrinolysis can cause bleeding and uncontrolled bleeding. Conversely, in fibrinolysis cessation, fibrinolysis is severely inhibited or absent, resulting in persistent clots or uncontrolled growth. Fibrinolysis cessation (or hypofibrinolysis) can lead to organ ischemia, multiple organ failure, and death. In addition to occurring after trauma, hyperfibrinolysis and / or fibrinolysis cessation can be observed during or after obstetric hemorrhage, liver transplantation, or administration of exogenous tissue plasminogen activator (tPA).

[0026] Hyperfibrinolysis and cessation of fibrinolysis are each associated with adverse clinical outcomes. Uncertainty exists regarding the optimal treatment strategy. Additionally, decisions about when to initiate patient care and what care to initiate, such as determining if a therapeutic trigger has been met, can be complicated by the lack of sensitive and rapid diagnostic tools. For example, there can be discrepancies between a diagnosis of hyperfibrinolysis based on viscoelasticity measurements and a diagnosis based on biomarkers of fibrinolysis. Current diagnostic methods appear capable of detecting the most severe forms of hyperfibrinolysis but are relatively insensitive to moderate but clinically significant fibrinolytic activation. Rapid diagnosis of hyperfibrinolysis would facilitate timely intervention and guide the administration of antifibrinolytic drugs in treating bleeding in trauma patients, reducing mortality. Therefore, there is an urgent need for improved diagnostic methods for hyperfibrinolysis and / or fibrinolysis, including faster and more accurate diagnoses.

[0027] Uncontrolled massive bleeding and subsequent coagulation system disturbances are major challenges in the management of surgical and severely injured patients. Under physiological conditions, coagulation activators and inhibitors regulate a sensitive balance between clot formation and fibrinolysis. In some cases, excessive and diffuse bleeding is caused by systemic activation of fibrinolysis, i.e., hyperfibrinolysis (HF). Uncontrolled HF is associated with high mortality. Conversely, cessation of fibrinolysis has also been observed as a pathological phenomenon that can lead to organ failure and death in many injured and / or critically ill patients. There is an increasing need for timely and reliable hemostatic blood tests (e.g., routine coagulation tests (CCT) or viscoelasticity tests (VET)) that can examine the processes of clot formation and fibrinolysis in whole blood and plasma products.

[0028] However, the main limitation of current techniques is that they typically require 60-90 minutes to obtain results on fibrinolytic activity, and using these current assays, the magnitude of the differences between patients with normal fibrinolysis, hyperfibrinolysis, and cessation of fibrinolysis is usually very small, but clinically significant. Therefore, improved compositions and methods are needed.

[0029] This document provides compositions for hemostasis blood tests comprising (a) optionally, a phospholipid component (e.g., one or more synthetic phospholipids), (b) optionally, a surfactant (e.g., silica particles), (c) a blood coagulation initiator (e.g., tissue factor, such as recombinant human tissue factor (RTF)); and (d) plasmin or a precursor thereof (e.g., plasminogen). In some embodiments, the compositions may further comprise one or more of the following: (e) a buffer (e.g., HEPES buffer), (f) a stabilizer, such as one or more different sugars (e.g., trehalose), (g) a protein concentration reference standard (e.g., bovine serum albumin (BSA)), and (h) CaCl2.

[0030] This document also provides methods, systems, and kits for detecting hyperfibrinolysis or cessation of fibrinolysis in subjects, using any of the compositions described herein.

[0031] This document describes various non-limiting aspects of such compositions, and they can be used in any combination without limitation. Additional aspects of the methods for preparing and using said compositions, including various components, are known in the field.

[0032] Composition for detecting hemostatic disorders

[0033] Hemostasis is the process that stops bleeding from blood vessels. As a balanced and strictly regulated process that depends on the complex balance between coagulation and fibrinolysis, hemostasis is essential to human health. Fibrinolysis is a physiological process that maintains the patency of microvessels by breaking down excess fibrin clots.

[0034] Hyperfibrinolysis is a coagulation disorder characterized by excessive clot degradation, which is associated with a doubling of mortality in injured patients. Hyperfibrinolysis is a state of overactivation of the fibrinolytic pathway, where excessive activation or impaired clearance or inactivation of plasminogen can lead to excessive proteolysis of both fibrin and fibrinogen. In some embodiments, hyperfibrinolysis has been observed in trauma, obstetric hemorrhage, during liver transplantation, and after administration of exogenous tissue plasminogen activator (tPA).

[0035] Conversely, cessation of fibrinolysis is an acute injury resulting from fibrinolysis. It is common in the later stages following trauma and is also associated with increased mortality. In some embodiments, cessation of fibrinolysis can lead to multi-system organ failure associated with hypercoagulability and / or microvascular occlusion.

[0036] In some embodiments, hemostasis blood tests can be used to evaluate whole blood hemostasis by assessing specific components (e.g., plasma components) and / or portions (e.g., coagulation) of the hemostasis process. In some embodiments, hemostasis blood tests may include routine coagulation tests (CCT), including... The test measures the time required for blood clotting. In some embodiments, conventional coagulation assays may include prothrombin time (PT) / international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, and platelets (PLTs). In some embodiments, hemostasis blood tests may include viscoelasticity testing (VET), including rotational thrombus elastography (ROTEM), thromboelastography (TEG), and acoustic estimation of elasticity via resonance sonorheometry.

[0037] However, routine hemostasis testing can typically take approximately 60–90 minutes before results indicating fibrinolytic activity are obtained. In some cases, tranexamic acid is an antifibrinolytic agent, which is administered to control bleeding and slow the breakdown of the blood clot. However, in trauma patients, tranexamic acid can be administered blindly because it needs to be administered within 3 hours of the injury to be effective.

[0038] This disclosure describes compositions, methods, systems, and kits that can reduce the turnaround time of conventional hemostasis assays.

[0039] This document provides compositions for hemostasis blood tests comprising (a) one or more different phospholipids (e.g., synthetic phospholipids), (b) one or more different surfactants (e.g., silica particles), (c) a blood coagulation initiator (e.g., tissue factor, such as recombinant tissue factor); and (d) plasmin or a precursor thereof (e.g., plasminogen). In some embodiments, the compositions may further comprise one or more of (e) a buffer (e.g., HEPES buffer), (f) a stabilizer (e.g., trehalose), (g) a protein concentration reference standard (e.g., bovine serum albumin (BSA)), and (h) CaCl2.

[0040] Phospholipids

[0041] The compositions described herein may include phospholipid components, which may include one or more different types of phospholipids. In some embodiments, one of the multiple phospholipids is derived from a natural source. In some embodiments, one of the multiple phospholipids includes a synthetic phospholipid. For example, the phospholipid may be selected from one or more of glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin), acetal phospholipids (e.g., plasmenylcholine and plasmenylethanolamine), lysophosphatidylcholine, lysophosphatidic acid, and sphingomyelin.

[0042] In some embodiments, the composition comprises one of the plurality of phospholipids at a concentration of about 0.001 mg / mL to about 1.0 mg / mL (e.g., about 0.005 mg / mL to about 1.0 mg / mL, about 0.01 mg / mL to about 1.0 mg / mL, about 0.05 mg / mL to about 1.0 mg / mL, about 0.1 mg / mL to about 1.0 mg / mL, about 0.25 mg / mL to about 1.0 mg / mL, about 0.5 mg / mL to about 1.0 mg / mL, about 0.75 mg / mL to about 1.0 mg / mL, about 0. 0.001 mg / mL to about 0.75 mg / mL, about 0.005 mg / mL to about 0.75 mg / mL, about 0.01 mg / mL to about 0.75 mg / mL, about 0.05 mg / mL to about 0.75 mg / mL, about 0.1 mg / mL to about 0.75 mg / mL, about 0.25 mg / mL to about 0.75 mg / mL, about 0.5 mg / mL to about 0.75 mg / mL, about 0.001 mg / mL to about 0.5 mg / mL, about 0.005 mg / mL to about 0.5 mg / mL, about 0.01 mg / mL To about 0.5 mg / mL, about 0.05 mg / mL to about 0.5 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, about 0.25 mg / mL to about 0.5 mg / mL, about 0.001 mg / mL to about 0.25 mg / mL, about 0.005 mg / mL to about 0.25 mg / mL, about 0.01 mg / mL to about 0.25 mg / mL, about 0.05 mg / mL to about 0.25 mg / mL, about 0.1 mg / mL to about 0.25 mg / mL, about 0.001 mg / mL to about 0.1 mg / mL Approximately 0.005 mg / mL to approximately 0.1 mg / mL, approximately 0.01 mg / mL to approximately 0.1 mg / mL, approximately 0.05 mg / mL to approximately 0.1 mg / mL, approximately 0.001 mg / mL to approximately 0.05 mg / mL, approximately 0.005 mg / mL to approximately 0.05 mg / mL, approximately 0.01 mg / mL to approximately 0.05 mg / mL, approximately 0.001 mg / mL to approximately 0.01 mg / mL, approximately 0.005 mg / mL to approximately 0.01 mg / mL, or approximately 0.001 mg / mL to approximately 0.0005 mg / mL).

[0043] surface activator

[0044] The compositions described herein may also include one or more different types of surfactants. Surfactants comprise particles or molecules that promote blood clotting. For example, surfactants can promote blood clotting by providing a platform for promoting the assembly and activation of clotting factors, particularly within the intrinsic pathway. For instance, blood in contact with a surfactant can promote the activation of factor XII (Hageman factor) into factor XIIa (part of the cascade of activating factors XI and IX), ultimately leading to thrombin production and fibrin formation.

[0045] Examples of surfactants include, but are not limited to, glass, silica, kaolin, bentonite, diatomaceous earth, thrombin, snake venom, and ellagic acid. In some embodiments, various surfactants include silica particles. In some embodiments, the surfactant includes a silica dispersion. In examples, the silica may be a fumed silica formulation, for example, available from Cabot Corporation (Boston, Mass.) under the trade name... The fumed silica formulation obtained from the aqueous dispersion at 1030K has approximately 30% solids.

[0046] In some embodiments, the composition comprises a surfactant, wherein the surfactant comprises silica particles at concentrations of: about 0.01% (w / w) to about 1.0% (w / w) (e.g., about 0.01% (w / w) to about 0.75% (w / w), about 0.01% (w / w) to about 0.5% (w / w), about 0.01% (w / w) to about 0.25% (w / w), about 0.01% (w / w) to about 0.1% (w / w), about 0.01% (w / w) to about 0.05% (w / w), about 0.05% (w / w) to about 1.0% (w / w), about 0.05% (w / w) to about 0.75% (w / w), about 0.05% (w / w) to about 0.5% (w / w), about 0.05% (w / w) to about 1.0% (w / w), about 0.05% (w / w) to about 0.75% (w / w), about 0.05% (w / w) to about 0.5% (w / w), about 0.05% (w / w) to about 0.5% (w / w). (w) to about 0.25% (w / w), about 0.05% (w / w) to about 0.1% (w / w), about 0.1% (w / w) to about 1.0% (w / w), about 0.1% (w / w) to about 0.75% (w / w), about 0.1% (w / w) to about 0.5% (w / w), about 0.1% (w / w) to about 0.25% (w / w), about 0.25% (w / w) to about 1.0% (w / w), about 0.25% (w / w) to about 0.75% (w / w), about 0.25% (w / w) to about 0.5% (w / w), about 0.5% (w / w) to about 1.0% (w / w), about 0.5% (w / w) to about 0.75% (w / w), or about 0.75% (w / w) to about 1.0% (w / w).

[0047] Blood coagulation initiator

[0048] The compositions described herein may include a blood coagulation initiator or a variety of different types of blood coagulation initiators. Examples of blood coagulation initiators include tissue factor (TF) and thrombin. More specifically, the blood coagulation initiator may be tissue factor. The blood coagulation initiator may be recombinant human tissue factor (RTF), such as that provided by Werfen. The reagent series is named after the product name. The provided RTF.

[0049] Tissue factor (TF), also known as factor III or CF142, is a major cellular initiator of blood clotting. Under normal physiological conditions, tissue factor is expressed by cells within subendothelial tissues (such as smooth muscle and fibroblasts) and is not exposed to circulating blood. When vascular injury occurs, TF becomes exposed to the blood flow and binds, for example, to circulating factor VII, forming the TF-factor VIIa complex. This complex activates factors IX and X, triggering a cascade that leads to thrombin production and ultimately the formation of a stable fibrin clot.

[0050] In some embodiments, the blood coagulation initiator includes a blood coagulation initiator from a natural source. In some embodiments, the blood coagulation initiator includes a blood coagulation initiator from a mammal (e.g., a rabbit). In some embodiments, the blood coagulation initiator includes a synthetic blood coagulation initiator. In some embodiments, the blood coagulation initiator includes a recombinant blood coagulation initiator.

[0051] In some embodiments, the composition comprises a blood coagulation initiator at concentrations of about 0.000001 mg / mL to about 0.001 mg / mL (e.g., about 0.000005 mg / mL to about 0.001 mg / mL, about 0.00001 mg / mL to about 0.001 mg / mL, about 0.00005 mg / mL to about 0.001 mg / mL, about 0.0001 mg / mL to about 0.001 mg / mL, about 0.0005 mg / mL to about 0.001 mg / mL, about 0.000001 mg / mL to about 0.0005 mg / mL, about 0.000005 mg / mL to about 0.0005 mg / mL, about 0.00001 mg / mL to about 0.0005 mg / mL, about 0. ... mg / mL to about 0.0005 mg / mL, about 0.000001 mg / mL to about 0.0001 mg / mL, about 0.000005 mg / mL to about 0.0001 mg / mL, about 0.00001 mg / mL to about 0.0001 mg / mL, about 0.00005 mg / mL to about 0.0001 mg / mL, about 0.000001 mg / mL to about 0.00005 mg / mL mg / mL, about 0.000005 mg / mL to about 0.00005 mg / mL, about 0.00001 mg / mL to about 0.00005 mg / mL, about 0.000001 mg / mL to about 0.00001 mg / mL, about 0.000005 mg / mL to about 0.00001 mg / mL, or about 0.000001 mg / mL to about 0.000005 mg / mL).

[0052] fibrinolytic enzyme

[0053] The compositions described herein may include plasmin or a precursor thereof. In some embodiments, the compositions include plasmin or plasminogen activator. Plasmin is an enzyme that degrades fibrin into fibrin degradation products during fibrinolysis, and plasminogen activator is an inactive precursor of plasmin that is enzymatically converted into plasmin by plasminogen activator (e.g., tissue plasminogen activator (tPA) and urokinase). In some embodiments, the compositions include plasminogen activator and plasminogen activator. In some embodiments, the composition comprises a plasminogen activator at a concentration of about 2.0 μg / mL to about 24 μg / mL (e.g., about 2.0 μg / mL to about 20 μg / mL, about 2.0 μg / mL to about 15 μg / mL, about 2.0 μg / mL to about 10 μg / mL, about 2.0 μg / mL to about 8.0 μg / mL, about 2.0 μg / mL to about 6.0 μg / mL, about 2.0 μg / mL to about 4.0 μg / mL, about 4.0 μg / mL to about 24 μg / mL, about 4.0 μg / mL to about 20 μg / mL, about 4.0 μg / mL to about 15 μg / mL, about 4.0 μg / mL to about 10 μg / mL, about 4.0 μg / mL to about 8.0 μg / mL, about 4.0 μg / mL to about 6.0 μg / mL, about 6...). 0.0 μg / mL to about 24 μg / mL, about 6.0 μg / mL to about 20 μg / mL, about 6.0 μg / mL to about 15 μg / mL, about 6.0 μg / mL to about 10 μg / mL, about 6.0 μg / mL to about 8.0 μg / mL, about 8.0 μg / mL to about 24 μg / mL, about 8.0 μg / mL to about 20 μg / mL, about 8.0 μg / mL to about 15 μg / mL, about 8.0 μg / mL to about 10 μg / mL, about 10 μg / mL to about 24 μg / mL, about 10 μg / mL to about 20 μg / mL, about 10 μg / mL to about 15 μg / mL, about 15 μg / mL to about 24 μg / mL, or about 20 μg / mL to about 24 μg / mL).

[0054] buffer

[0055] In some embodiments, the compositions described herein may further include a buffer. In some embodiments, the buffer includes Good's buffers, wherein the Good's buffer may include MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, citric acid, phosphoric acid, acetic acid, imidazole, barbiturates, GTA, or any combination thereof. In some embodiments, the buffer includes HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer.

[0056] In some embodiments, the composition comprises a buffer at a concentration of about 1 mM to about 500 mM (e.g., about 5 mM to about 500 mM, about 10 mM to about 500 mM, about 20 mM to about 500 mM, about 40 mM to about 500 mM, about 60 mM to about 500 mM, about 80 mM to about 500 mM, about 100 mM to about 500 mM, about 120 mM to about 500 mM, about 140 mM to about 500 mM, about 160 mM to about 500 mM, about 180 mM to about 500 mM, about 200 mM to about 500 mM, about 250 mM to about 500 mM, about 300 mM to about 500 mM, about 350 mM to about 500 mM, about 400 mM). M to approximately 500mM, approximately 450mM to approximately 500mM, approximately 1mM to approximately 450mM, approximately 5mM to approximately 450mM, approximately 10mM to approximately 450mM, approximately 20mM to approximately 450mM, approximately 40mM to approximately 450mM, approximately 60mM to approximately 450mM, approximately 80mM to approximately 450mM, approximately 100mM to approximately 450mM, approximately 120mM to approximately 450mM, approximately 140mM to approximately 450mM, approximately 160mM to approximately 450mM, approximately 180mM to approximately 450mM, approximately 200mM to approximately 450mM, approximately 250mM to approximately 450mM, approximately 300mM to approximately 450mM, approximately 350mM to approximately 450mM, approximately 400mM to approximately 450mM 0mM, approximately 1mM to approximately 400mM, approximately 5mM to approximately 400mM, approximately 10mM to approximately 400mM, approximately 20mM to approximately 400mM, approximately 40mM to approximately 400mM, approximately 60mM to approximately 400mM, approximately 80mM to approximately 400mM, approximately 100mM to approximately 400mM, approximately 120mM to approximately 400mM, approximately 140mM to approximately 400mM, approximately 160mM to approximately 400mM, approximately 180mM to approximately 400mM, approximately 200mM to approximately 400mM, approximately 250mM to approximately 400mM, approximately 300mM to approximately 400mM, approximately 350mM to approximately 400mM, approximately 1mM to approximately 350mM, approximately 5mM to approximately 350mM, approximately 10mM Approximately 350 mm, approximately 20 mm to approximately 350 mm, approximately 40 mm to approximately 350 mm, approximately 60 mm to approximately 350 mm, approximately 80 mm to approximately 350 mm, approximately 100 mm to approximately 350 mm, approximately 120 mm to approximately 350 mm, approximately 140 mm to approximately 350 mm, approximately 160 mm to approximately 350 mm, approximately 180 mm to approximately 350 mm, approximately 200 mm to approximately 350 mm, approximately 250 mm to approximately 350 mm, approximately 300 mm to approximately 350 mm, approximately 1 mm to approximately 300 mm, approximately 5 mm to approximately 300 mm, approximately 10 mm to approximately 300 mm, approximately 20 mm to approximately 300 mm, approximately 40 mm to approximately 300 mm, approximately 60 mm to approximately 300 mm.Approximately 80mm to approximately 300mm, approximately 100mm to approximately 300mm, approximately 120mm to approximately 300mm, approximately 140mm to approximately 300mm, approximately 160mm to approximately 300mm, approximately 180mm to approximately 300mm, approximately 200mm to approximately 300mm, approximately 250mm to approximately 300mm, approximately 1mm to approximately 250mm, approximately 5mm to approximately 250mm, approximately 10mm to approximately 250mm, approximately 20mm to approximately 250mm, approximately 40mm to approximately 250mm, approximately 60mm to approximately 250mm, approximately 80mm to approximately 250mm, approximately 100mm to approximately 250mm, approximately 120mm to approximately 250mm, approximately 140mm to approximately 250mm, approximately 160mm M to approximately 250mM, approximately 180mM to approximately 250mM, approximately 200mM to approximately 250mM, approximately 1mM to approximately 200mM, approximately 5mM to approximately 200mM, approximately 10mM to approximately 200mM, approximately 20mM to approximately 200mM, approximately 40mM to approximately 200mM, approximately 60mM to approximately 200mM, approximately 80mM to approximately 200mM, approximately 100mM to approximately 200mM, approximately 120mM to approximately 200mM, approximately 140mM to approximately 200mM, approximately 160mM to approximately 200mM, approximately 180mM to approximately 200mM, approximately 1mM to approximately 180mM, approximately 5mM to approximately 180mM, approximately 10mM to approximately 180mM, approximately 20mM to approximately 180mM, approximately 40mM to approximately 180mM, approximately 60mM to approximately 180mM, approximately 80mM to approximately 180mM, approximately 100mM to approximately 180mM, approximately 120mM to approximately 180mM, approximately 140mM to approximately 180mM, approximately 160mM to approximately 180mM, approximately 1mM to approximately 160mM, approximately 5mM to approximately 160mM, approximately 10mM to approximately 160mM, approximately 20mM to approximately 160mM, approximately 40mM to approximately 160mM, approximately 60mM to approximately 160mM, approximately 80mM to approximately 160mM, approximately 100mM to approximately 160mM, approximately 120mM to approximately 160mM, approximately 140mM to approximately 160mM, approximately 1mM to approximately 140mM, approximately 5mM to approximately 140mM M, approximately 10mM to approximately 140mM, approximately 20mM to approximately 140mM, approximately 40mM to approximately 140mM, approximately 60mM to approximately 140mM, approximately 80mM to approximately 140mM, approximately 100mM to approximately 140mM, approximately 120mM to approximately 140mM, approximately 1mM to approximately 120mM, approximately 5mM to approximately 120mM, approximately 10mM to approximately 120mM, approximately 20mM to approximately 120mM, approximately 40mM to approximately 120mM, approximately 60mM to approximately 120mM, approximately 80mM to approximately 120mM, approximately 100mM to approximately 120mM, approximately 1mM to approximately 100mM, approximately 5mM to approximately 100mM, approximately 10mM to approximately 100mM, approximately 20mM to approximately 100mMApproximately 40 mm to approximately 100 mm, approximately 60 mm to approximately 100 mm, approximately 80 mm to approximately 100 mm, approximately 1 mm to approximately 80 mm, approximately 5 mm to approximately 80 mm, approximately 10 mm to approximately 80 mm, approximately 20 mm to approximately 80 mm, approximately 40 mm to approximately 80 mm, approximately 60 mm to approximately 80 mm, approximately 1 mm to approximately 60 mm, approximately 5 mm to approximately 60 mm, approximately 10 mm to approximately 60 mm mM, approximately 20mM to approximately 60mM, approximately 40mM to approximately 60mM, approximately 1mM to approximately 40mM, approximately 5mM to approximately 40mM, approximately 10mM to approximately 40mM, approximately 20mM to approximately 40mM, approximately 1mM to approximately 20mM, approximately 5mM to approximately 20mM, approximately 10mM to approximately 20mM, approximately 1mM to approximately 10mM, approximately 5mM to approximately 10mM, or approximately 1mM to approximately 5mM).

[0057] In some embodiments, the buffer may have a pH of 4.0 to 9.0 (e.g., 4.5 to 9.0, 5.0 to 9.0, 5.5 to 9.0, 6.0 to 9.0, 6.5 to 9.5, 7.0 to 9.0, 7.5 to 9.0, 8.0 to 9.0, 8.5 to 9.0, 4.0 to 8.5, 4.5 to 8.5, 5.0 to 8.5, 5.5 to 8.5, 6.0 to 8.5, 6.5 to 8.5, 7.0 to 8.5, 7.5 to 8.5, 8.0 to 8.5, 4.0 to 8.0, 4.5 to 8.0, 5.0 to 8.0, 5.5 to 8.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 ... 0.5 to 8.0, 4.0 to 7.5, 4.5 to 7.5, 5.0 to 7.5, 5.5 to 7.5, 6.0 to 7.5, 6.5 to 7.5, 7.0 to 7.5, 4.0 to 7.0, 4.5 to 7.0, 5.0 to 7.0, 5.5 to 7.0, 6.0 to 7.0, 6.5 to 7.0, 4.0 to 6.5, 4.5 to 6.5, 5.0 to 6.5, 5.5 to 6.5, 6.0 to 6.5, 4.0 to 6.0, 4.5 to 6.0, 5.0 to 6.0, 5.5 to 6.0, 4.0 to 5.5, 4.5 to 5.5, 5.0 to 5.5, 4.0 to 5.0, 4.5 to 5.0, or 4.0 to 4.5).

[0058] stabilizer

[0059] In some embodiments, the composition may further comprise one or more stabilizers. In some embodiments, the composition may further comprise one or more stabilizers, wherein the stabilizer may comprise a disaccharide. In some embodiments, the composition may further comprise one or more stabilizers, wherein the stabilizer may comprise a sugar. In some embodiments, the stabilizer may comprise glucose, α-D-mannopyranoside, lactose, cellobiose, mannose, maltose, inositol, sucrose, inulin, fructose, or dextran. In some embodiments, the stabilizer acts as a preservative and protects components such as proteins during lyophilization or drying. In some embodiments, the stabilizer comprises trehalose. In some embodiments, the composition comprises a concentration of about 0.005 g / mL to about 1.0 g / mL (e.g., about 0.01 g / mL to about 1.0 g / mL, about 0.05 g / mL to about 1.0 g / mL, about 0.1 g / mL to about 1.0 g / mL, about 0.5 g / mL to about 1.0 g / mL, about 0.005 g / mL to about 0.5 g / mL, about 0.01 g / mL to about 0.5 g / mL, about 0.05 g / mL). Stabilizers of about 0.5 g / mL, about 0.1 g / mL to about 0.5 g / mL, about 0.005 g / mL to about 0.1 g / mL, about 0.01 g / mL to about 0.1 g / mL, about 0.05 g / mL to about 0.1 g / mL, about 0.005 g / mL to about 0.05 g / mL, about 0.01 g / mL to about 0.05 g / mL, or about 0.0005 g / mL to about 0.01 g / mL.

[0060] Protein Concentration Reference Standard

[0061] In some embodiments, the composition may further comprise a protein concentration reference standard. In some embodiments, the protein concentration reference standard comprises albumin, such as bovine serum albumin (BSA). In some embodiments, the protein concentration reference standard may comprise synthetic polymers, recombinant human albumin, and other non-animal-derived proteins or synthetic substitutes. In some embodiments, the protein concentration reference standard may comprise sericin or casein.

[0062] In some embodiments, the composition comprises a protein concentration reference standard (e.g., BSA) at a concentration of about 0.0001 mg / mL to about 5.0 mg / mL (e.g., about 0.0001 mg / mL to about 2.5 mg / mL, about 0.0001 mg / mL to about 1.0 mg / mL, about 0.0001 mg / mL to about 0.5 mg / mL, about 0.0001 mg / mL to about 0.1 mg / mL, about 0.0001 mg / mL to about 0.05 mg / mL, about 0.0001 mg / mL to about 0.01 mg / mL, about 0.0001 mg / mL to about 0.005 mg / mL, about 0.0001 mg / mL to about 0.001 ... 1 mg / mL to about 0.0005 mg / mL, about 0.0005 mg / mL to about 5.0 mg / mL, about 0.0005 mg / mL to about 2.5 mg / mL, about 0.0005 mg / mL to about 1.0 mg / mL, about 0.0005 mg / mL to about 0.5 mg / mL, about 0.0005 to about 0.1 mg / mL, about 0.0005 to about 0.05 mg / mL, about 0.0005 mg / mL to about 0.01 mg / mL, about 0.0005 mg / mL to about 0.005 mg / mL, about 0.0005 mg / mL to about 0.001 mg / mL, about 0.001 mg / mL to about 5.0 mg / mL, about 0.00 1 mg / mL to about 2.5 mg / mL, about 0.001 mg / mL to about 1.0 mg / mL, about 0.001 mg / mL to about 0.5 mg / mL, about 0.001 to about 0.1 mg / mL, about 0.001 to about 0.05 mg / mL, about 0.001 mg / mL to about 0.01 mg / mL, about 0.001 mg / mL to about 0.005 mg / mL, about 0.005 mg / mL to about 5.0 mg / mL, about 0.005 mg / mL to about 2.5 mg / mL, about 0.005 mg / mL to about 1.0 mg / mL, about 0.005 mg / mL to about 0.5 mg / mL, about 0.005 to about 0.1 mg / mL, about 0.005 to about 0.05 mg / mL, about 0.005 mg / mL to about 0.01 mg / mL, about 0.01 mg / mL to about 5.0 mg / mL, about 0.01 mg / mL to about 2.5 mg / mL, about 0.01 mg / mL to about 1.0 mg / mL, about 0.01 mg / mL to about 0.5 mg / mL, about 0.01 to about 0.1 mg / mL, about 0.01 to about 0.05 mg / mL, about 0.05 mg / mL to about 5.0 mg / mL, about 0.05 mg / mL to about 2.5 mg / mL, about 0.05 mg / mL to about 1.0 mg / mL, about 0.05 mg / mL to about 0.5 mg / mL, about 0.05 to about 0.1 mg / mL, about 0.1 mg / mL to about 5.0 mg / mL, about 0.1 mg / mL to about 2.5 mg / mL, about 0.1 mg / mL to about 1.0 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, about 0.5 mg / mL to about 5.0 mg / mL, about 0.5 mg / mL to about 2.5 mg / mL, about 0.5 mg / mL to about 1.0 mg / mL, about 1.0 mg / mL to about 5.0 mg / mL, about 1.0 mg / mL to about 2.5 mg / mL, or about 2.5 mg / mL to about 5.0 mg / mL.

[0063] Salt

[0064] In some embodiments, the composition may further comprise salts, such as calcium salts, such as calcium chloride (CaCl2), calcium gluconate, calcium citrate, calcium lactate, calcium acetate, calcium gluconate, calcium aspartate; magnesium salts, such as magnesium sulfate, magnesium chloride, magnesium carbonate, magnesium acetate, or magnesium orotate; or zinc salts, such as zinc chloride, zinc sulfate, zinc gluconate, zinc acetate, or zinc stearate; or iron salts, such as ferric chloride, ferric sulfate, ferric gluconate, ferric fumarate, or ferric citrate. In some embodiments, the composition comprises a calcium salt at a concentration of about 0.0005 g / mL to about 0.05 g / mL (e.g., about 0.001 g / mL to about 0.05 g / mL, about 0.005 g / mL to about 0.05 g / mL, about 0.01 g / mL to about 0.05 g / mL, about 0.0005 g / mL to about 0.01 g / mL, about 0.001 g / mL to about 0.01 g / mL, about 0.005 g / mL to about 0.01 g / mL, about 0.0005 g / mL to about 0.005 g / mL, about 0.001 g / mL to about 0.005 g / mL, or about 0.0005 g / mL to about 0.001 g / mL).

[0065] Methods for detecting hemostasis disorders

[0066] This article provides a method for detecting hemostasis disorders (e.g., hyperfibrinolysis or cessation of fibrinolysis) in a subject, comprising (a) obtaining a blood sample (e.g., whole blood or plasma) from the subject; (b) contacting at least a portion of the blood sample with an assay reagent, wherein the assay reagent comprises any of the compositions described herein, thereby producing an assay sample; (c) measuring a time parameter in the assay sample by performing a blood coagulation assay on the assay sample; and (d) comparing the time parameter with a reference coagulation time, wherein a higher time parameter compared to the reference coagulation time indicates that the subject has hyperfibrinolysis or cessation of fibrinolysis.

[0067] In some embodiments, alternative methods for detecting hemostasis disorders in a subject may include (a) obtaining a blood sample (e.g., whole blood or plasma) from the subject; (b) contacting a first portion of the blood sample (e.g., whole blood or plasma) with a first assay reagent, wherein the first assay reagent comprises any of the compositions described herein but does not contain plasmin or plasminogen activator, thereby generating a first assay sample; (c) contacting a second portion of the blood sample (e.g., whole blood or plasma) with a second assay reagent, wherein the second assay reagent comprises (i) the composition from step b and (ii) plasmin or plasminogen activator, thereby generating a second assay sample; (d) measuring a first time parameter in the first assay sample and a second time parameter in the second assay sample by performing a coagulation assay on the first and second assay samples; and (e) analyzing the first and second time parameters, wherein a second time parameter that is higher or equal to the first blood time parameter indicates that the subject has a hemostasis disorder. In some embodiments, the method may be implemented using two devices: a first device for contacting a first portion of the blood sample and a second device for testing a second portion of the blood sample. In some other embodiments, the method can be implemented on a single device having two or more channels, with a first channel for contacting the first portion of the blood sample and a second channel for testing the second portion of the blood sample. The blood sample can be any blood sample, such as whole blood or plasma.

[0068] As used herein, the term "clotting time" or "blood clotting time" refers to the time until evidence of clot formation is present in a blood sample (e.g., whole blood or plasma). The term "time parameter" as used herein refers to the clotting time or maximum time (e.g., time out of range) used to determine hemostatic impairment. The term "reference clotting time" as used herein refers to the clotting time of normal blood containing plasmin or plasminogen activator. In some embodiments, the reference clotting time may be the average of one or more clotting times determined from one or more different normal blood samples.

[0069] In some embodiments, if no coagulation time is detected during the test, the maximum time is used as the reference coagulation time. In some embodiments, the method includes measuring a time parameter in the test sample and comparing the time parameter to a reference coagulation time to determine whether the subject has a hemostatic disorder (e.g., hyperfibrinolysis or cessation of fibrinolysis). In some embodiments, a higher time parameter compared to a reference coagulation time indicates that the subject has hyperfibrinolysis or cessation of fibrinolysis, wherein the higher time parameter is a time parameter that is higher when compared to the reference coagulation time. In some embodiments, the higher time parameter is about 60 seconds or longer. In some embodiments, the higher time parameter is about 80 seconds or longer (e.g., about 100 seconds, about 120 seconds, about 3 minutes, about 5 minutes, about 10 minutes, or about 30 minutes or longer). In some implementations, the higher time parameter is at least one second or more higher than the reference solidification time (e.g., at least 5 seconds or more, at least 10 seconds or more, at least 20 seconds or more, at least 30 seconds or more, at least 60 seconds or more, at least 120 seconds or more, at least 180 seconds or more, at least 240 seconds or more, or at least 300 seconds or more).

[0070] In some embodiments, a lower blood clotting time compared to a reference clotting time indicates that the subject does not have hyperfibrinolysis or cessation of fibrinolysis, wherein the lower time parameter is a time parameter lower than the reference clotting time. In some embodiments, the lower blood clotting time is about 50 seconds or less. In some embodiments, the lower blood clotting time is about 40 seconds or less (e.g., about 30 seconds, about 20 seconds, about 15 seconds, about 10 seconds, or about 5 seconds).

[0071] In some embodiments, the assay reagent used in any of the methods described herein may be disposed in a sample holder (i.e., placed therein). The sample holder includes a support or sample handling device configured to receive any sample or reagent required to perform the assay. In some embodiments, the sample holder may include a box or cup (e.g., a consumable cup). In some embodiments, the assay reagent disposed in the sample holder is a dried reagent, wherein the assay reagent is dispensed and dried onto the sample holder.

[0072] In some embodiments, the methods described herein include performing a blood coagulation assay on a test sample, wherein the blood coagulation assay includes a microfluidic device-based assay. In some embodiments, the blood coagulation assay can be performed on a microfluidic device-based assay, wherein the microfluidic device is a handheld device, such as a GEM provided by Werfen. 100 and Signature Elite instruments.

[0073] In some embodiments, the method for detecting hyperfibrinolysis or cessation of fibrinolysis in a subject can be performed over a duration of less than 5 minutes. In some embodiments, the duration is less than 4 minutes. In some embodiments, the duration is less than 3 minutes. In some embodiments, the duration is less than 2 minutes. In some embodiments, the duration is less than 1 minute.

[0074] In some embodiments, the method described herein may further include treating the subject based on the subject being determined to have hyperfibrinolysis or cessation of fibrinolysis. In some embodiments, the subject is identified as having hyperfibrinolysis. In some embodiments, treating a subject already identified as having hyperfibrinolysis may include administering an antifibrinolytic agent. In some embodiments, the treatment of the subject already identified as having hyperfibrinolysis may include administering... (tranexamic acid)(TXA) or (Aminocaproic acid). In some embodiments, the subject is identified as having fibrinolysis cessation. In some embodiments, the treatment of the subject who has been identified as having fibrinolysis cessation may include the application of a sequential compression device (e.g., applied to the subject's arm or leg), heparin, or... (Synthetic testosterone) IM. In some embodiments, ε-aminocaproic acid or other lysine analogues may be administered, or blood transfusions may be provided to treat these hemostatic disorders.

[0075] Systems / Reagents for Detecting Hemostasis Disorders

[0076] This document provides a system or kit for detecting hemostatic disorders (e.g., hyperfibrinolysis or cessation of fibrinolysis) in blood samples (e.g., whole blood or plasma), said system or kit comprising a sample holder containing an assay reagent comprising any of the compositions described herein.

[0077] This document also provides a sample holder for blood coagulation assays, the sample holder comprising an assay reagent comprising any of the compositions described herein. In some embodiments, the sample holder comprises a box or a small cup (e.g., a consumable cup).

[0078] In some embodiments, the kit may include two or more sample holders. A first sample holder may include a first assay reagent, wherein the assay reagent comprises any of the compositions described herein but does not contain plasmin or plasminogen activator. A second sample holder may include a second assay reagent, wherein the assay reagent comprises any of the compositions described herein and contains plasmin or plasminogen activator.

[0079] In some embodiments, the sample holder is configured to include an assay reagent, wherein the assay reagent is a drying reagent in the sample holder. In some embodiments, the assay reagent may be lyophilized. In some embodiments, the assay reagent may be dried (e.g., by airflow under mild heating (e.g., below 70°C), or in a desiccator at approximately room temperature or under mild heating). In some embodiments, the assay reagent may comprise a dried powder, a film, or dried beads.

[0080] In some embodiments, the sample holder includes a channel containing an assay reagent, wherein the assay reagent includes any of the compositions described herein. In some other embodiments, the sample holder includes a first channel and a second channel. The first channel includes a first assay reagent, wherein the first assay reagent includes any of the compositions described herein, but does not contain plasmin or plasminogen activator. The second channel includes a second assay reagent, which includes the same assay reagent as in the first channel, but further includes plasmin or plasminogen activator.

[0081] In some embodiments, the method can be implemented with two devices: a first device for contacting a first portion of a blood sample (e.g., whole blood or plasma), and a second device for testing a second portion of the blood sample. In some other embodiments, the method can be implemented on a single device having two or more channels, with a first channel for contacting the first portion of the blood sample and a second channel for testing the second portion of the blood sample.

[0082] In some embodiments, the sample holder is configured for a blood coagulation assay, wherein the blood coagulation assay detects hemostatic impairment in the blood sample (e.g., hyperfibrinolysis or cessation of fibrinolysis). In some embodiments, the blood sample is a whole blood sample. In some embodiments, the blood sample is a plasma sample. In some embodiments, the blood sample is derived from a human subject.

[0083] Coagulation testing system

[0084] In a non-limiting example, the system described in U.S. Patent No. 10,175,225 (“Blood Testing System and Method”), issued January 8, 2019, can be used to perform the coagulation assay, the contents of which are incorporated herein by reference. U.S. Patent No. 10,175,225 describes a blood testing system comprising an analyzer console and one or more cartridges for performing tests, including the use of liquid thrombin reagents (such as those described herein) to determine the amount of fibrinogen in a test sample.

[0085] Examples of systems that can provide coagulation tests using a cup and needle configuration include those provided by Werfen. sigma and Delta system. The coagulation characteristics can be used by the associated analyzer console to perform the process described herein for determining the amount of fibrinogen in the mixture and / or determining the amount of thrombin inhibitor in the mixture.

[0086] In another non-limiting example, a mechanical clumping detection system (e.g., the one provided by Werfen) can be used. A whole blood hemostasis system (100) is described, and a mechanical clot detection method is used to perform the coagulation assay, wherein a whole blood sample is introduced into a test chamber, and the system monitors the formation of fibrin clots by detecting changes in the movement of a steel ball within the chamber to determine the coagulation time. The operation of the mechanical clot detection system is described in U.S. Patent No. 11,242,848, issued February 8, 2022 (“Methods of Operating a Pump to Reduce or Eliminate Pump Backlash Errors”), the contents of which are incorporated herein by reference.

[0087] In another non-limiting example, the coagulation assay may be performed using the system described in U.S. Patent No. 11,366,093, issued June 21, 2022 (“Disposable System for Analysis of Hemostatic Function”), the contents of which are incorporated herein by reference. That patent describes a system provided by Hemosonics, LLC (Durham, NC). Hemostasis analyzer.

[0088] In another non-limiting instance, the aforementioned provided by Haemonetics Corporation (Boston, Mass.) may be used. 6s box or the aforementioned The coagulation assay is performed using a system based on an active tip box. This system includes a box comprising one or more chambers that can hold a mixture of a test sample, such as blood, and the liquid thrombin reagent described herein. Testing in the 6s system utilizes optical analysis combined with resonant frequency technology to determine coagulation characteristics based on mixtures such as TTC. More specifically, in using the aforementioned... In the example of the 6s box system, such as As stated in the USFDA 501K summary, "the The 6S technology is based on a disposable cartridge containing up to four independent measurement cells. Each cell consists of a short, vertically oriented injection molded tube (ring). In the 6-second hemostasis system, coagulation detection is performed optically. A piezoelectric actuator vibrates the measuring cell via a motion trajectory formed by the sum of sinusoidal curves of different frequencies. This movement of the measuring cell causes motion in the meniscus of the sample, which is detected by a photodiode. The resulting motion of the meniscus is optically detected and analyzed by the instrument to calculate the sample's resonant frequency and elastic modulus (stiffness). The resonant frequency is determined by performing a Fast Fourier Transform (FFT) on the meniscus motion data. The analyzer detects the harmonic motion of the suspended blood droplet in response to external vibrations. As the sample transitions from a liquid to a gel state during coagulation, the elastic modulus (stiffness) increases, and the resonant frequency also increases. The 6s hemostasis analyzer measures these changes in resonant frequency during coagulation and dissolution. The coagulation characteristics determined by the 6s technique can be used by the analyzer to perform the methods described herein to determine the amount of fibrinogen in the mixture and / or the amount of thrombin inhibitor in the mixture.

[0089] Examples of parameters that can generate test result values ​​by these exemplary viscoelasticity testing instruments include, but are not limited to, the following clot initiation parameters, clot kinetic parameters, clot firmness parameters, and clot dissolution parameters.

[0090]

[0091]

[0092] for and CT refers to clotting time; CFT refers to clot formation time; α angle refers to the angle tangent to the coagulation curve at a point with a range of two millimeters (mm); A5 refers to the clot hardness range five minutes after CT; A10 refers to the clot hardness range ten minutes after CT; A20 refers to the clot hardness 20 minutes after CT; MCF refers to maximum clot hardness; ML refers to maximum dissolution; LI30 refers to the dissolution index 30 minutes after CT; LI45 refers to the dissolution index 45 minutes after CT; LI60 refers to the dissolution index 60 minutes after CT; LOT refers to dissolution onset time; LT refers to dissolution time.

[0093] for CT refers to clotting time; R-time refers to reaction time; K-time refers to kinetic time; α angle refers to the rate of fibrin accumulation and aggregation, and is closely related to K-time; A5 refers to the clot hardness range five minutes after CT; A10 refers to the clot hardness range ten minutes after CT; MA refers to maximum range; ML refers to maximum dissolution; LY30 refers to dissolution 30 minutes after MA; LY60 refers to dissolution 60 minutes after MA.

[0094] for CT refers to clotting time without heparinase; CTH refers to clotting time with heparinase; CTR refers to the clotting time ratio; CS refers to clot stiffness; PCS refers to the contribution of platelets to clot stiffness; FCS refers to the contribution of fibrinogen to clot stiffness; CSL refers to the stability of clot dissolution.

[0095] It can be used in GEM These parameters are obtained by performing an activated clotting time (ACT) test on the system.

[0096] This disclosure can be implemented using various hemostasis assays. Those skilled in the art will understand that various hemostasis assays exist.

[0097] Standard coagulation tests include the D-dimer test, prothrombin time (PT) test, international normalized ratio (INR) blood test, activated partial thromboplastin clotting time (aPTT) test, thrombin time (TT) test, dilute thrombin time (dTT) test, calibrated direct oral anticoagulant (DOAC) test, platelet function test, and platelet count test. The parameters can be obtained from one or more of these tests.

[0098] Platelet function testing is included in VerifyNow. TM The system includes aspirin and P2Y12 response unit (PRU) tests. Platelet function tests may include... Platelet test, multi-platelet test Platelet mapping test and platelet function analyzer (PFA) 100 / 200 test. The parameters can be obtained from one or more of these tests.

[0099] Two measures that can be used to understand the effectiveness of the compositions and methods described herein are the percentage of clot readings and the difference in readings.

[0100] The percentage of clot readings (clot reading %) reflects the ratio of test readings (T) with plasmin to control readings (C) without plasmin, and can be calculated using Equation 1:

[0101]

[0102] The difference in readings reflects the absolute difference between the test reading (T) with plasmin and the control reading (C) without plasmin, and can be calculated using Equation 2:

[0103] Reading difference = | Test result reading with plasmin -

[0104] Test results without plasmin | (Equation 2)

[0105] Compared to a clot-free cup (control), normal healthy blood samples are expected to produce lower clot readings on a clot-containing cup (test). Blood samples from patients at risk of hyperfibrinolysis or cessation of fibrinolysis will produce equal or longer clot readings on a clot-containing cup (test) compared to a clot-free cup (control). Therefore, it is preferable to maximize the difference between the T / C (i.e., percentage of clot readings) of healthy individuals and the T / C of patients with hyperfibrinolysis. The larger this difference, the more likely the T / C of healthy individuals and the T / C of patients with hyperfibrinolysis will be statistically clearly distinguishable in clinical trials, with fewer false positive or false negative results. TC (i.e., reading difference) is a substitute representation of this data, representing an absolute difference, while T / C represents a relative difference. Again, it is preferable to maximize the absolute difference to achieve improved test performance.

[0106] Example

[0107] The present disclosure is further described in the following embodiments, which do not limit the scope of the present disclosure as described in the claims.

[0108] Example 1 - Reagents including phospholipids / silica / tissue factor

[0109] Blood samples from healthy patients were used for screening tests. Each blood sample was tested in parallel using a cup without plasmin and a cup containing three levels of plasmin.

[0110] In GEM On the instrument, citric acidified blood samples were tested separately using kits containing zero plasmin and specific levels of plasmin to obtain a pair of test results. The results were generated using the ratio of the test results with plasmin to the test results without plasmin. The composition of the reagents used in the assay is described in Table 1.

[0111] Table 1. Composition of the reagents in the solution

[0112]

[0113] Using the GEM The instrument determines the test results for each of the measurements described in Table 1. The test results are reported in Table 2.

[0114] Table 2. Average test results based on two replicates

[0115]

[0116]

[0117] **Indicates an out-of-range high reading.

[0118] The percentage of clot readings and the reading difference were calculated as described above. The results for the percentage of clot readings are shown in Table 3 and... Figure 1 The reading differences are recorded in Table 4.

[0119] Table 3. Clot readings (%) relative to the control without plasmin.

[0120]

[0121] The data of 17.0 ug / mL uses 200% of the nominal data to indicate a high reading. Actual test data showing a reading that is high outside the range means the data is outside the measurement range.

[0122] Table 4. Differences in plasmin levels between those with and without plasmin (absolute numbers)

[0123]

[0124] The average percentage of clot readings for the three concentration levels of plasmin, with the quantitative results shown in Table 3 (9.7, 11.4, and 13.6 μg / mL), was 47%. The absolute difference in the average readings was approximately 42 seconds. The figures (…) Figure 1 The figure shows the percentage of average clot readings of the test reagent relative to the negative control reagent at a range of plasmin concentration levels for blood samples from five different healthy donors.

[0125] Example 2 - Reagents containing tissue factor

[0126] Blood samples from healthy patients were used for screening tests. Each blood sample was tested in parallel using a cup without plasmin and a cup containing three levels of plasmin.

[0127] The determination was performed according to the method described herein, wherein in GEM Blood samples from three donors were tested on the instrument to obtain test results. The composition of the reagents used in the determination is described in Table 5.

[0128] Table 5. Composition of the reagents in the solution

[0129]

[0130] Determine the test results for each of the determinations shown in Table 6. Calculate the % clot reading results and show them in Table 7 and... Figure 2 The results of the reading differences are calculated and shown in Table 8.

[0131] Table 6. Average test results based on three repetitions

[0132]

[0133] **Indicates out of range - high reading

[0134] Table 7. Clot Readings (%)

[0135]

[0136] The data of 17.0 ug / mL uses 200% of the nominal data to indicate a high reading. Actual test data showing a reading that is high outside the range means the data is outside the measurement range.

[0137] Table 8. Differences in plasmin levels between those with and without plasmin (absolute numbers)

[0138]

[0139]

[0140] For Example 2, the overall average percentage of clot readings was 74%. The overall average reading difference was approximately 13 seconds.

[0141] Example 3 - Reagents including phospholipids / tissue factors

[0142] Blood samples from healthy patients were used for screening tests. Each blood sample was tested in parallel using a cup without plasmin and a cup containing three levels of plasmin.

[0143] The determination was performed according to the method described herein, wherein in GEM Blood samples from five donors were tested on instrument 100 to obtain test results. The composition of the reagents used in the assay is described in Table 9.

[0144] Table 9. Composition of the reagents in the solution

[0145]

[0146] The test results for each of the measurements are determined as shown in Table 10. The percentage of clot readings is determined as shown in Table 11. The reading differences are shown in Table 12. The average percentage of clot readings is 95%. The absolute value of the reading differences is approximately 2 seconds.

[0147] Table 10. Average test results based on three repetitions

[0148]

[0149] Table 11. Clot Readings (%)

[0150]

[0151] Table 12. Differences in plasmin levels between those with and without plasmin (absolute numbers)

[0152]

[0153]

[0154] Example 4 - Reagents including silica / tissue factor

[0155] Blood samples from healthy patients were used for screening tests. Each blood sample was tested in parallel using a cup without plasmin and a cup containing five levels of plasmin.

[0156] The determination was performed according to the method described herein, wherein in GEM Blood samples from five donors were tested on instrument 100 to obtain test results. The composition of the reagents used in the assay is described in Table 13. The test results are shown in Table 14.

[0157] Table 13. Composition of the reagents in the solution

[0158]

[0159] The test results are shown in Table 14. The results show poor accuracy, with an average coefficient of variation (CV%) greater than 20%. Due to the extensive data, the average percentage of clot readings was not calculated. It is not desirable to be bound by theory; it is believed that the selection of other components in the formulation influences the silica gel and causes aggregation.

[0160] Table 14. Average test results based on three repetitions

[0161]

[0162] **Indicates out of range - high reading

[0163] Results analysis of Examples 1-4

[0164] The tests described in Examples 1-4 demonstrate that the compositions and methods described herein can be used to test for hemostatic disorders, such as hyperfibrinolysis and cessation of fibrinolysis. The results of the mean percentage of clot readings and the absolute difference in mean readings measured in each of Examples 1-4 (summarized in Table 14) show that the reagents used in Example 1 (including phospholipids, silica, and tissue factor) provided the lowest mean percentage of clot readings (47%) and the largest difference in mean readings (42 seconds). These results indicate that the reagents used in Example 1 are superior to those in Examples 2-4. Specifically, a lower mean percentage of clot readings and a higher difference in mean readings are preferred because they indicate greater sensitivity and specificity for the assay in diagnosing hyperfibrinolysis or cessation of fibrinolysis.

[0165] As discussed above, the greater the gap between healthy individuals and patients with hyperfibrinolysis, the more likely the T / C ratios of healthy individuals and patients with hyperfibrinolysis will be statistically clearly distinguishable in clinical trials, with fewer false positive or false negative results. The calculation of TC is an alternative representation of this data, representing the absolute difference, while T / C represents the relative difference. Again, maximizing the absolute difference is preferable for improved test performance.

[0166] Therefore, the results demonstrate that reagents containing phospholipids, silica, and tissue factor are superior to tissue factor (without phospholipids or silica), phospholipids and tissue factor (without silica), or silica and tissue factor (without phospholipids).

[0167] Table 14

[0168]

[0169] **Useful results cannot be obtained due to poor accuracy.**

[0170] Other implementation plans

[0171] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Variations, alterations, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A composition for a hemostatic blood test, the composition comprising: (a) Phospholipid components; (b) Surface activator; (c) Blood coagulation initiators; and (d) Plasmin or its precursor.

2. The composition according to claim 1, further comprising one or more of the following: (e) Buffer; (f) Stabilizers; (g) Protein concentration reference standard; and (h)CaCl2.

3. The composition according to claim 1 or 2, wherein the phospholipid component comprises a variety of phospholipids present at a concentration of about 0.001 mg / mL to about 1.0 mg / mL.

4. The composition according to any one of claims 1-3, wherein the surfactant is present at a concentration of about 0.01% to about 1.0%.

5. The composition according to any one of claims 1-4, wherein the blood coagulation initiator is a tissue factor, and the tissue factor is present at a concentration of about 0.000001 mg / mL to about 0.001 mg / mL.

6. The composition according to any one of claims 1-5, wherein the plasmin or its precursor is present at a concentration of about 2.0 μg / mL to about 24 μg / mL.

7. The composition according to any one of claims 1-6, wherein the surfactant comprises silica particles.

8. A method for detecting hemostasis disorder in a subject, the method comprising: (a) Obtaining a blood sample from the subject; (b) Contacting at least a portion of the blood sample with a assay reagent, wherein the assay reagent comprises the composition of any one of claims 1-7, thereby producing an assay sample; (c) Perform a blood coagulation assay on the test sample and measure the time parameters of the blood coagulation assay; and (d) The time parameter is compared with a reference coagulation time, wherein a time parameter that is higher than the reference coagulation time indicates that the subject has a hemostatic disorder.

9. The method of claim 8, wherein the higher time parameter is at least one second or more than the reference solidification time.

10. The method of claim 8 or 9, wherein a time parameter lower than the reference coagulation time indicates that the subject does not have hyperfibrinolysis or cessation of fibrinolysis.

11. The method according to any one of claims 8-10, wherein the assay reagent is placed in a sample holder.

12. The method according to any one of claims 8-11, wherein the assay reagent is a dried reagent.

13. The method according to any one of claims 8-12, wherein the blood coagulation assay comprises a microfluidic device-based assay.

14. The method according to any one of claims 8-13, the method further comprising treating the subject based on the subject being determined to have a hemostatic disorder.

15. A sample holder for blood coagulation assay, wherein the sample holder comprises an assay reagent comprising a composition according to any one of claims 1-7.

16. The sample holder of claim 15, wherein the sample holder comprises a box or a small cup.

17. The sample holder according to claim 15 or 16, wherein the assay reagent is provided in the sample holder in a dry form.

18. The sample holder according to any one of claims 15-17, further comprising a control reagent, said control reagent comprising: a. Phospholipid components; b. Surface activators; and c. Blood clotting initiator.

19. The sample holder according to any one of claims 15-18, wherein the blood coagulation assay detects hemostatic impairment in the blood sample.

20. The sample holder of claim 19, wherein the blood sample is a whole blood sample.

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