Coagulation assay device and method thereof

Abstract

The present invention relates to a method and apparatus for determining the activity of clotting factors in diluted capillary whole blood, citrated whole blood and citrated plasma. The invention also includes the detection of hemoglobin content in the whole blood sample to correct for clotting time so that the clotting time values are not affected by hemoglobin and hematocrit.

Description

BACKGROUND 1. TECHNICAL FIELD

[0002] The present invention relates generally to a method and apparatus for determining coagulation factor activity in diluted capillary whole blood, citrated whole blood, and citrated plasma.

[0003] 2. Prior art description

[0004] Methods and apparatus for determining coagulation factor activity in diluted capillary whole blood, citrated whole blood, and citrated plasma generally involve a physician measuring a patient's International Normalized Ratio (INR) level in a Prothrombin Time-International Normalized Ratio (INR) test. This type of biological analysis is designed to measure the time it takes for a patient's blood to clot. The test ensures that the dose and type of medication a patient is receiving prevents the formation of blood clots and prevents conditions such as deep vein thrombosis (DVT), pulmonary embolism (PE), stroke, heart attack, etc. This class of medication works by blocking the formation of vitamin K-dependent coagulation factors, which are substances in the blood that cause clotting. If the INR fraction is too low, the patient can be at risk for a blood clot. However, if the INR is too high, the patient can also bleed. A typical INR fraction is between 2 and 3. The "ideal" INR fraction can vary from patient to patient.

[0005] How often a patient should be tested depends on how stable their INR is over a period of time. According to the American Heart Association (AHA), a patient should be tested at least once a month, and in some cases, as often as twice a week. This testing typically involves drawing blood and analyzing it with an in vitro diagnostic analyzer.

[0006] In vitro diagnostic analyzers have been around for decades. These types of analyzers are typically used in central laboratories. Central laboratories are able to perform tests on a variety of biomedical substances in a patient's blood and / or plasma. Recently, this testing seems to be shifting from central laboratory testing to point-of-care testing within a hospital. This shift provides faster test data results, which is important for the diagnosis and treatment of certain diseases.

[0007] Point-of-care testing plays an important role in the management of critically ill patients and is widely used in operating rooms, emergency rooms, and intensive care units. These tests are no longer performed exclusively by skilled medical technicians, but also by multi-skilled personnel including nurses, respiratory therapists, paramedics, physicians, and other medical personnel. To meet this demand, manufacturers have had to downsize the analyzers and simplify the testing procedures so that only minimal training is required to perform the testing procedures.

[0008] A common key feature of all point-of-care analyzers is that they must be portable and / or transportable. Examples of such point-of-care analyzers include, but are not limited to, the Opti CCA and Omni 9 Critical Care Analyzers by Roche Diagnostics (a division of Hoffmann-La Roche, Inc.), the Stat Profile Ultra C by Nova Biomedical, the CRT by Nova Biomedical, and the Dimension RxL by Dade Behring Diagnostics (a division of Siemens Medical Diagnostics).

[0009] More recently, a further shift is occurring, where testing is performed in the physician's office or in a laboratory located within the physician's office. As testing moves away from the central laboratory, new single-use medical devices have been developed to meet this need.

[0010] In the physician's office environment, there are a number of devices that utilize capillaries to collect a finger stick sample for analysis. The capillaries can be glass or plastic. Typical analyses are for substances such as HbAlc, lipids, etc. After sample collection, these capillary-based collection devices are loaded into an analysis cartridge, which is then loaded into an instrument for analysis. Two known bioassays for diagnostic purposes will now be discussed with reference to their original patent documents.

[0011] First, U.S. Patent Document US 2011 / 0196085 Al discloses a stabilized bead comprising a latex particle having a carboxyl group, and a stabilizing agent functionally coupled to the latex particle, wherein the stabilizing agent is capable of completely or substantially preventing degradation or inactivation of a diagnostic agent in proximity to the stabilizing agent. The stabilized bead can further comprise at least one of human serum albumin (HSA), bovine serum albumin (BSA), or a linker coupled to the latex particle.

[0012] Second, European Patent Document EP 0655627 discloses a method and test kit for simple detection of the fibrinolysis product D-dimer, which method utilizes a purified human fibrinogen fragment E attached to a solid phase for direct chemical binding of D-dimer in a biological sample. The fragment E can be conjugated to latex carrier particles and an agglutination assay is performed. SUMMARY

[0013] Advantages and differences of the invention relative to known prior art

[0014] It has been found that the above-mentioned parts of the prior art do not fully satisfy all the requirements of the industry. Inaccuracy has been found to be associated with the automated measurement of the average cell hemoglobin concentration in dehydrated cells. Uncontrolled activation of procoagulants is induced by additional mixing and hydration times during the coagulation process. In comparison with the methods of the prior art, the method according to the present application provides great capacity for particle and reagent detection using dry, liquid or dry and liquid reagents.

[0015] The present application employs a carbohydrate matrix to preserve the functionality of specific microparticles to improve the capacity of immunodiagnosis, which has been successful to varying degrees prior to the present application. Specifically, the present application employs latex microparticles that have not been reacted, contacted or coupled with proteins. These unreacted particles preserve their bioactive adsorption properties when dried in a carbohydrate matrix or used in a liquid reagent system. When used, these particles are capable of adsorbing biomolecules. This action promotes and enhances the reactivity of the particle surface to procoagulants contained in whole blood and plasma, particularly when used in a diluted sample / diluent environment. This process includes the presentation of a mixture of reactive proteins and particles, as well as the presentation of various activators used in coagulation assays. Latex microparticles are used in the turbidimetric bioassay of the present application, in which the typical optical property of the sample solution is clear and not turbid.

[0016] The present application addresses the failures of the prior art by applying a new improved methodology to a diluted, lysed whole blood sample or plasma matrix, in which latex particles provide a method of coagulation detection. The method allows for the combination of adsorption to be applied to the point of clinical testing, reducing production time, cost, incubation reaction time at the end user stage, and the footprint or size of the instrument. Both untreated latex microparticles and particles with surface groups such as sulfate or amidine are effective for the present application. Each microparticle type also requires appropriate buffers and thermal conditions to work in a diluted blood assay protocol.

[0017] The biomolecules, microparticles, specialized buffers and detection temperatures work together to allow rapid protein adsorption for rapid clinical testing. In addition, the dispersibility of the particles also plays an important role. Uniform dispersibility in solution allows for rapid reaction and consistent clot analysis quantification. Therefore, it is mandatory to preserve both of these characteristics for the development of any reliable detection assembly.

[0018] The present application provides a process for dry polystyrene uncoated, unbound, unproteinized latex microparticles with common substances or surface functionalized groups. The dispersed particles allow the adsorption of quantifiable analytical protein biomarkers, which can then be used for ligand attachment. In particular, a matrix composed of carbohydrates favors the adsorption of biomarker proteins, while allowing rapid dissolution and uniform dispersion of the particles.

[0019] It is an object of the present invention to provide a liquid microparticle reagent for use in certain biological assays. It is another object of the present invention to provide a method for using dried or liquid latex microparticles in diagnostic coagulation products. It is another object of the present invention to provide a method for simultaneously performing a hemoglobin measurement and a quantitative correction of coagulation time values.

[0020] It is a further object of the present invention to provide a novel method for assessing the extrinsic coagulation pathway and monitoring oral anticoagulant therapy (OAT).

[0021] The method of the present invention provides a method for detecting coagulation time of whole blood or plasma, performing a hemoglobin measurement and correction on a whole blood sample. In various embodiments, the overall biological assay time is varied by adjusting dilution levels, temperature, particle type, and buffer composition. These embodiments employ flexible dried, liquid, or dried and liquid matrices and specific microparticles. However, it should be understood that all detection components can be adjusted to provide the most representative time profile for a coagulation biological assay according to the method of the present invention. Generally, for coagulation assays performed between 33°C and 38°C, the final whole blood or plasma sample dilution ratio should be in the range of 50 to 75 parts.

[0022] The present invention achieves these and other objects by providing a disposable biological assay diagnostic kit for monitoring anticoagulant activity. The disposable kit can be provided with a first well containing an amount of a matrix, which can be a dried matrix or a liquid matrix; a second well containing microparticles, which can be uncoated latex microparticles having at least one surface type: unreacted normal, sulfate, carboxylate, and amidine chemistries with preserved activity. The kit can also be provided with a third well having an amount of an activator, which can be thromboplastin, thrombin, ellagic acid, activated partial thromboplastin time, factor II, factor VII, factor I, factor X, factor XII, activated protein C, snake venom, negatively charged phospholipid, calcium ion, tissue factor, silica, kaolin, celite.

[0023] The matrix can be a liquid carbohydrate matrix, or a dried matrix containing at least one of sodium chloride, polyethylene glycol, Tween, and calcium chloride. The disposable kit can be provided with an integrated cuvette capable of facilitating dual optical detection readings. The integrated cuvette can have a first wall capable of facilitating a first optical detection reading by a first 530 nm LED; and a second wall capable of facilitating a second optical detection reading by a second 660 nm LED.

[0024] The present invention achieves other objects by providing a full package coagulation bioassay diagnostic kit that has all the necessary components in addition to the analyzer itself. The kit can include a swab, pipette, bioassay components, and optical cuvette. To ensure hygienic delivery to the user, the swab, pipette, bioassay components, and optical cuvette can be housed in a hygienic and sealed container that is provided with an identifier, such as a bar code that can be scanned by the analyzer. The bioassay components can have a substrate and microparticles, where the microparticles can be uncoated latex with at least one surface type selected from the group consisting of unreacted normal, sulfate, carboxylate, and amidine chemistries.

[0025] Another coagulation bioassay according to the present invention can have a carbohydrate substrate and microparticles in the carbohydrate substrate. This bioassay can also have an amount of an activator, such as thromboplastin, thrombin, ellagic acid, activated partial thromboplastin, Factor II, Factor VII, Factor I, Factor X, Factor XII, activated protein C, snake venom, negatively charged phospholipid, calcium ion, tissue factor, silica, kaolin, and celite. The substrate of the bioassay can be a dry substrate or a liquid substrate, and the carbohydrate substrate can contain maltose 250, sucrose, or isomaltose. The microparticles of this type of bioassay can have a diameter from 10 nm to 150 nm, and the microparticles can be in a 1% by volume solution, a 2% by volume solution, a 4% by volume solution, an 8% by volume solution, or a 10% by volume solution.

[0026] By employing the above biological assay, the present application seeks to provide a method to obtain a coagulation time measurement of a blood sample type of diluted, lysed whole blood, whole blood (directly from a finger stick), plasma, citrated blood and / or any one of a mixture of blood and plasma. The method comprises the steps of selecting a microparticle substrate having a carbohydrate matrix and a plurality of microparticles in the carbohydrate matrix. The microparticles are preferably uncoated latex having at least one surface type in the dry or liquid carbohydrate matrix, the at least one surface type preferably selected from the group consisting of unreacted normal, sulfate, carboxylate and a chemically active amidine structure. This microparticle substrate can then be used as a reagent with the blood sample; the coagulation time measurement of one of diluted, lysed whole blood or plasma is then obtained by optical detection of INR. Alternatively, a separate reagent can also be added to the reaction mixture to activate the natural coagulation substrates in the blood sample. The separate reagent is preferably an activator such as thromboplastin, thrombin, ellagic acid, activated partial thromboplastin time, factor II, factor VII, factor I, factor X, factor XII, activated protein C, snake venom, negatively charged phospholipid, calcium ion, tissue factor, silica, kaolin, celite.

[0027] The coagulation time measurement of the sample can then be corrected for the hemoglobin concentration of the sample by simultaneously obtaining optical density readings at two different wavelengths. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1a A method of the present application is shown;

[0029] Figure 1b A user has selected the biological assay kit set of the present application for use with the scanner shown;

[0030] Figure 2 A user scans the identifier of the kit of the present application;

[0031] Figure 3 A user uses a finger stick to obtain a blood sample according to the method of the present application;

[0032] Figure 4 A user retrieves a lancing device from the kit of the present application;

[0033] Figure 5 A user fills the lancing device with a blood sample into the kit of the present application;

[0034] Figure 6 A user replaces the filled lancing device with a blood sample back into the kit of the present application;

[0035] Figure 7is a schematic of a user placing the inventive cartridge with the sampler into the analyzer;

[0036] Figure 8 is a cross-sectional view of a cartridge according to one embodiment of the present invention;

[0037] Figure 9 is a cross-sectional view of a cartridge according to another embodiment of the present invention;

[0038] Figure 10 is a schematic of the automated steps of the method according to one embodiment of the present invention;

[0039] Figure 11 is a schematic of one step of the method shown in Figure 1a further observation;

[0040] Figure 12 is a schematic of a step of the method shown in Figure 11 further observation;

[0041] Figure 13 is a schematic of another step of the method shown in Figure 1a further observation;

[0042] Figure 14 shows the effect of hemoglobin on the ratio of prothrombin time to INR with and without correction according to the present method;

[0043] Figure 15 shows the effect of hemoglobin on the ratio of delta prothrombin time to INR with and without correction according to the present method.

[0044] Thromboplastin with BSA vs. normal

[0045] Figure 16 is a schematic of a bioassay of a normal sample using microparticles with and without a bovine serum albumin (BSA) surface group;

[0046] Figure 17 is a schematic of a bioassay of an abnormal sample using microparticles with and without a bovine serum albumin (BSA) surface group.

[0047] Thromboplastin with amidines vs. sulfates and normal

[0048] Figure 18 is a plot of optical density versus time at 660 nm showing the results of a prothrombin time assay of a first embodiment using a first amidine-based diluent and a first substrate;

[0049] Figure 19is a graph showing the results of a prothrombin time assay using the second embodiment of the first sulfate particle diluent and the first substrate by measuring the optical density at 660 nm versus time;

[0050] Figure 20 is a graph showing the results of a prothrombin time assay using the second embodiment of the first surface free micro particle diluent and the first substrate by measuring the optical density at 660 nm versus time;

[0051] Figure 21 is a graph showing the results of a prothrombin time assay using the fourth embodiment of the second amidine particle diluent and the second substrate by measuring the optical density at 660 nm versus time;

[0052] Figure 22 is a graph showing the results of a prothrombin time assay using the fifth embodiment of the second sulfate particle diluent and the second substrate by measuring the optical density at 660 nm versus time;

[0053] Figure 23 is a graph showing the results of a prothrombin time assay using the sixth embodiment of the second surface free micro particle diluent and the second substrate by measuring the optical density at 660 nm versus time.

[0054] Thromboplastin with simethicone

[0055] Figure 24 is a graph showing the results of a biological assay of normal whole blood using the sulfate micro particle diluent according to the present application by measuring the optical density at 660 nm versus time;

[0056] Figure 25 is a graph showing the results of a biological assay of abnormal whole blood using the sulfate micro particle diluent according to the present application by measuring the optical density at 660 nm versus time;

[0057] Figure 26 shows the hemoglobin value present in a biological assay according to the present application by measuring the optical density at 520 nm; Figure 25

[0058] Figure 27 is a graph showing the results of a biological assay according to the present application by measuring the optical density at 520 nm; Figure 25 Figure 26

[0059] Figure 28 shows the results of a biological assay according to the present application by measuring the optical density at 520 nm; Figure 27 ​​​Adjusted prothrombin time determination Figure 25 INR value in biological assay.

[0060] Thromboplastin with carboxyl

[0061] Figure 29 Results of biological assay of normal whole blood using carboxymethylcellulose diluent according to the present application are shown by measuring the optical density at 660 nm versus time;

[0062] Figure 30 Results of biological assay of abnormal whole blood using carboxymethylcellulose diluent according to the present application are shown by measuring the optical density at 660 nm versus time.

[0063] Thromboplastin at different temperatures

[0064] Figure 31 Results of prothrombin time determination using two embodiments of the present application at different temperatures are shown by measuring the optical density at 660 nm versus time;

[0065] Figure 32 Results of prothrombin time determination using two embodiments of the present application at different temperatures are shown by measuring the optical density at 660 nm versus time.

[0066] Thromboplastin with coumarin

[0067] Figure 33 Results of biological assay of normal blood and biological assay of blood with coumarin are shown and compared by measuring the optical density at 660 nm versus time.

[0068] Thrombin

[0069] Figure 34 Results of thrombin time biological assay of normal citrated plasma and thrombin time biological assay of normal citrated blood are shown and compared by measuring the optical density at 660 nm versus time.

[0070] Activated partial thromboplastin

[0071] Figure 35 Graphical representation of activated partial thromboplastin time biological assay of normal control results;

[0072] Figure 36 Results of activated partial thromboplastin time biological assay and abnormal control are shown for comparison;

[0073] Figure 37A biological assay of activated partial thromboplastin time is shown, where a one-stage factor assay for Factor VIII is performed with normal plasma by measuring optical density at 660 nm versus time;

[0074] Figure 38 A biological assay of activated partial thromboplastin time is shown and compared to a one-stage factor assay for Factor VIII, where APTT abnormal plasma and APTT one-stage mix are compared by measuring optical density at 660 nm versus time. DETAILED DESCRIPTION

[0075] The preferred embodiments of the present application are discussed with reference to FIGS. 1-38. As discussed above, the present application provides a process, method, system and apparatus relating to a method and apparatus for determining the activity of coagulation factors in diluted capillary whole blood, citrated whole blood and citrated plasma.

[0076] General overview

[0077] The general overview of the entire method 100, system kit 2 and manual and automated portions of the biological assay will now be discussed from different perspectives with reference to FIGS. 1-12.

[0078] General overview of the method from the user's perspective

[0079] The general overview from the user's perspective will now be discussed with reference to FIGS. 1-7. As shown, initially, the user 102 selects a biological assay kit or kit package. After manually selecting a biological assay kit 102, the kit can be scanned by the analyzer to identify the selected biological assay 103. Then, obtaining a blood sample 104 can involve a simple finger stick 105 to provide the required blood sample size. The ease of obtaining a blood sample at this step shows one of the advantages of the present system over prior art systems and methods that require venipuncture or other large size blood sample collection.

[0080] If the biological assay is initially identified, the capillary sampler can be removed from the kit 106, filled with the blood sample 107 and replaced into the designated kit 108, all within five seconds of operating the finger stick 105. After replacing the filled sampler 108 into the kit, the identifier can be scanned 103 by the analyzer and the kit inserted into the analyzer 109. After the kit is loaded into the analyzer, the automated process 110 begins. Depending on the selected biological assay, the automated process will involve automated steps of adding 112, mixing 114, measuring 116, incubating 117, correcting 118 and reporting 119 the results to the user or other designated person according to the designated order of the biological assay components.

[0081] Depending on the system involved and the purpose of the bioassay, the automated process 110 can also include a step of automatically adjusting 120 the prescribed amount of drug and / or the amount of drug provided by a built-in drug delivery system (not shown).

[0082] Figure 1b is an illustration of the kit set after user selection 102. The kit set includes a bioassay kit 2 and a pre-packaged swab 4, which can be used separately with the analyzer system 1. The kit set preferably also includes a sterilized pipette 5, a sampler 8, and cuvettes 7, which will be discussed in more detail below. Figures 8-9 The exemplary analyzer system 1 capable of performing the bioassays described herein is the ALLEGRO® Analyzer by Nova Biomedical. Nova Biomedical’s U.S. Patent 10,117,615 provides a more comprehensive description of an exemplary sampler and kit base that can be used with the kit 2 described herein. TM The exemplary analyzer system 1 capable of performing the bioassays described herein is the ALLEGRO® Analyzer by Nova Biomedical. Nova Biomedical’s U.S. Patent 10,117,615 provides a more comprehensive description of an exemplary sampler and kit base that can be used with the kit 2 described herein.

[0083] Next turning to Figures 2-7 which further illustrates the use of the specific components of the kit set from the user’s perspective. Specifically, Figure 2 shows the user having the analyzer 1 identify the bioassay method by scanning the kit 2 of the present invention. Then, Figure 3 shows the user obtaining a blood sample 60 by using the swab 4 provided with the kit set. Then, Figure 4 shows the user removing the sampler 8 from the kit 2. Next, Figure 5 shows the user filling the sampler 8 with the blood sample 60 taken from the swab 4. After that, Figure 6 shows the user replacing the filled sampler 8 with the blood sample 60 taken from the swab 4 back into the kit 2 of the present invention. Finally, Figure 7 shows the user placing the kit 2 with the filled sampler 8 into the analyzer 1, thereby initializing the selected and identified bioassay. By providing these components as a unified set in a single package 9, the present invention both reduces the overall procedure time and reduces user error.

[0084] General overview of the bioassay kit

[0085] An exemplary kit set that can be used with the system and method of the present invention can include a stand-alone, disposable, integrated bioassay kit 2, as will now be further described with reference to Figure 8 , 9 .

[0086] According to one embodiment of the present invention, Figure 8The first kit 2 embodiment shown in the middle is prepared with bioassay components for only a single type of bioassay. In addition, Figure 9 The second kit embodiment shown is a multi-purpose kit 2' pre-loaded with components for at least two types of bioassays, according to a further embodiment of the application.

[0087] Figure 8 and Figure 9 Both show a kit 2 with an identifier 3, such as a bar code, which enables the analyzer 1 to identify the specific type of kit (and thus also the bioassay). The identifier 3 can be visible along the visible outer surface of the kit 2, or along the outer surface of the main package 9. The package 9 can contain the kit 2, a swab 4, a sterile pipette 5, a sampler 8 and cuvettes 7. Figure 8 and Figure 9 Both show a kit 2 in which the capillary sampler 8 is a detachable component of the kit 2 itself. Figure 8 and Figure 9 Also shown is a kit 2 with integrated cuvettes 7, the side walls of which facilitate optical measurements. However, it is also envisaged that the package 9 can contain a kit for which a separate different cuvette 7 needs to be provided outside the kit.

[0088] Figure 8 , 9 Also shown is a kit 2 with a series of wells 6 pre-loaded with detection components according to the method of the application. For both types of kits, the selection stage 102 (discussed above with reference to Figures 1-7) involves selecting the desired bioassay kit. After the desired kit / detection method has been selected 102, the kit is scanned 103, filled 107 and then inserted into the system 109.

[0089] The only difference in using the first and second kits from the user's perspective is from now on. With the first kit, the user can select one of several bioassay options by manual user input through an access control panel on the analyzer, thereby manually selecting the automatic process option 110. For the second kit type, merely inserting the kit 109 is sufficient to trigger the automatic process 110. For this second kit type, from the user's perspective, only the "selection" of the bioassay kit is required. However, before the user selects the bioassay option, the bioassay kit must be prepared, and before the preparation, the bioassay components themselves must be selected. The several bioassay kit component options are further discussed below with reference to the initial Figure 1a and further with reference to Figures 10-13

[0090] ​Now let's look more specifically at the components of sampler 2, which the user may not necessarily be able to observe once sampler 8 is filled with 107 and returned to kit 2. From Figure 8 , Figure 9 As can be seen (although not numbered), the sampler 8 has a capillary element that is inserted through a corresponding capillary receiving hole on the top surface of the stepped extension of the cap extension of the disposable test kit 2, and then placed in the stepped extension.

[0091] During insertion and setup, the capillary of sampler 8 is inserted through the lower orifice located at the top of the capillary wiper. Because the cross-sectional area of ​​the lower orifice is smaller than that of the capillary, the lower orifice acts like a scraper against the outer surface of the capillary, preventing any sample accidentally placed on the outer surface of the capillary from entering and depositing into the cavity 6 of kit 2.

[0092] Furthermore, the capillary wiper of these kits 2 can remove any sample 60 from the outer surface of the capillary, thereby improving accuracy. Therefore, erroneous results can be avoided by "overfilling" sample 60 in the appropriate wells 6' of kit 2. Similarly, since the user does not wipe the capillary, the possibility of any sample 60 being inadvertently removed from the capillary is nonexistent or extremely rare, which could result in sample 60 "underfilling" the wells 6' in the detection kit 2 and leading to erroneous results.

[0093] General overview of the automated process

[0094] Then, reagent kit 2 is inserted into the nursing point analyzer 1 to automatically test blood sample 60 110. Further reference will now be made. Figure 10 Discussion is underway. Once inside analyzer 1, the analyzer's automated arm disconnects the lid of kit 2 by unlocking the releasable tag, while the capillary sampler 8 and sample 60 remain enclosed within the lid.

[0095] The analyzer then uses a small tip on the reagent kit lid to pierce the seals at 6", 6", 6", etc., of each well. The seals for this reagent kit 2 can be aluminum foil seals or other types of seals, as long as they prevent cross-contamination of the contents of the wells during transport. These seals should also reduce uncontrolled water vapor dilution and evaporation.

[0096] The automated arm then retrieves the pipette tip 5 from the first well 6. The analyzer 1 then uses the pipette tip 5 to transfer the sample and other bioassay components into the appropriate wells for mixing according to the bioassay method described herein.

[0097] If a dry microparticle matrix 11 is used, the pipette tip 5 aspirates additional diluent 90 from the fourth well 6"' and adds it to the microparticles 11 in the third well 6" according to protocol, where it is mixed and stirred to form diluted particles 11. Then, regardless of the type of matrix used, the pipette tip 5 aspirates a selected number of (now diluted) microparticles 11 from the third well 6" into the second well 6'.

[0098] The cap of kit 2 is then reattached to the kit, and the pipette is connected to the capillary sampler 8, thereby adding sample 60 112 into the second well 6', where the diluted selected microparticles 11 await. Then, the robotic arm of analyzer 1 removes the cap of kit 2 again, and then mixes sample 60 and diluted selected particles 11 114 by up-and-down pipetting, forming a mixture 121 of microparticles 11 and sample 60 within a first predetermined time.

[0099] Then, pipette tip 5 aspirates a specified amount of the selected molding matrix 40 from the fifth well 6"", and in the second addition step 112, the matrix 40 is added to the second well 6', which already contains a mixture 121 of sample 60 and diluted microparticles 11. Then, the matrix 40 and mixture 121 are mixed 114, for example, by up-and-down aspiration within the second well 6' for a second predetermined time, to form a solution 122 (containing matrix 40, sample 60, and microparticles 11).

[0100] Next, pipette 5 aspirates a predetermined amount of solution 122 (comprising matrix 40, sample 60, and microparticles 11) from well 6' and adds solution 122 to well 7 of kit 2, which contains selected reagent 80. Solution 122 (microparticles, matrix, sample) and reagent 80 are then mixed for a third predetermined time to form mixture 123 (now containing matrix 40, sample 60, microparticles 11, and reagent 80).

[0101] If using Figure 8 and Figure 9 The preferred kit 2 shown includes a seventh well 7 that is also an integrated optical detection cuvette 7 with walls of sufficient transparency, viscosity, and thickness to ensure that optical detection can now be performed without further displacement of the mixture 123. As will be discussed in further detail below, the optical density of the mixture 123 will be measured at least twice, 112 and 112'.

[0102] Upon initiation of the first measurement 112, a first light emitting device (LED) of the analyzer 1 is turned on, and light from the first LED is transmitted through a first wall of the integrated cuvette 7 at a wavelength of 660 nm. The light then passes through the mixture 123 and through a second wall of the integrated cuvette 7 until the light is detected by a light detector of the analyzer. Successive readings are collected over a predetermined time to provide first coagulation data 124.

[0103] For the second measurement 112', a second light emitting device (LED) of the analyzer 1 is turned on, and light from the second LED is transmitted through the first wall of the integrated cuvette 7 at a second wavelength of 530 nm. The light then passes through the mixture 123 and through the second wall of the integrated cuvette 7 back until the light is collected by the light detector of the analyzer. Only one reading is needed to provide hemoglobin level data 125. It should be appreciated that while these measurements are referred to as first and second measurements, the order can be reversed. Alternatively, the measurements can be provided out of sequence, or even simultaneously, or partially simultaneously.

[0104] As will be discussed further below, the results from the optical detection 116, 116' are then used to correct 118 the coagulation time results 126. These corrected results 126 are then reported to the user.

[0105] General overview of the bioassay assembly

[0106] The present invention provides a bioassay kit 2 having at least one of several microparticle matrices 10, which will now be discussed. In some embodiments, the microparticle matrix 10 is formed prior to formation of the kit, and in other embodiments, the microparticle matrix 10 is formed after the blood sample 60 is added 112 in the bioassay process 110.

[0107] The microparticle matrix 10 has microparticles 11, which can be uncoated, unbound, unproteinated latex or common material, or have surface functionalized groups on polystyrene microparticles. The matrix 40 is typically comprised of a carbohydrate 46 that facilitates adsorption of biomarker proteins while allowing rapid dissolution and uniform dispersion of the microparticles 11. The dispersion of the microparticles 11 in the matrix 40 allows for adsorption of quantifiable analytical protein biomarkers that can then be used for ligand attachment.

[0108] The microparticles 11 used in the present invention include polystyrene microparticles 18 with surface active groups such as amidine 22 and sulfate 24. The microparticles 11 of the present invention have a diameter 26 in the range of from 20 nm to 800 nm or more. The preferred range of particle diameter 26 is from 40 nm to 150 nm, with the most preferred range of diameter 26 being from 75 to 125 nm.

[0109] Amidine microparticles

[0110] One embodiment of the present application employs amidine microparticles 22 having a diameter 26 of 95 nm in a dilution 11 having a container weight 28 of 0.080%. The optical density 31 of the dilution 11 is measured at 660 nm and has an optical density value of 0.19.

[0111] The diameters 26 of the amidine microparticles 22 of the present application range from 20 nm to 800 nm or more. The diameters 26 of the particles preferably range from 40 nm to 150 nm, more preferably the diameters 26 range from 75 to 125 nm; and most preferably the diameters 26 range from 90 nm to 98 nm. The amidine microparticles 22 are employed in a dilution 11 having a container weight 28 ranging from 0.006 to 8%, more preferably a container weight 28 ranging from 0.010 to 0.20%, and most preferably a container weight 28 of 0.080%.

[0112] The present bioassay method employing amidine latex particles 22 requires a dilution 11 wherein the total dilution ratio 30 of microparticles 22 to dihydrogen monoxide ranges from 1 : 10 to 1 :400; and more preferably from 1 :20 to 1 : 150.

[0113] Amidine latex particles 22 that can satisfy the objectives of the present bioassay method can include high activity latex beads provided by Invitrogen Corporation when prepared according to the methods of the present application as discussed herein. TM

[0114] Sulfate microparticles

[0115] Another embodiment of the present application employs sulfate microparticles 24 having a diameter of 110 nm in a dilution having a container weight of 0.044%. The optical density of the dilution 11 is measured at 660 nm and has an optical density value of 0.21.

[0116] Other sulfate microparticles 24 of the present application have diameters 26 ranging from 20 nm to 800 nm or more. The diameters 26 of the sulfate particles 24 preferably range from 40 nm to 150 nm, more preferably the diameters 26 range from 75 to 125 nm; and most preferably the diameters 26 range from 90 nm to 110 nm.

[0117] According to some embodiments, the sulfate microparticles 24 are employed in a dilution 11 having a container weight ranging from 0.001% to 12%, more preferably a container weight ranging from 0.01% to 8%, and most preferably a container weight of 0.016%.

[0118] ​The present bioassay method using sulfate microparticles 24 requires a dilution ratio 30 having a ratio of microparticles 24 to dihydrogen monoxide in the range of 1 :50 to 1 :2000; more preferably in the range of 1 :100 to 1 :1000; and most preferably at a ratio of 1 :500.

[0119] When prepared according to the methods of the present invention as discussed herein, the sulfate latex microparticles 24 that can satisfy the objectives of the present invention can include high activity latex beads supplied by Invitrogen TM Corporation.

[0120] Surface free microparticles

[0121] Another embodiment of the present invention is the use of surface free microparticles 20 having a diameter 26 of 96 nm in a dilution 11 having a container weight 28 of 0.067%. The dilution ratio 30 of this dilution is 1 to 150. The optical density 31 of the dilution 11 is measured at 660 nm and has a value of 0.21.

[0122] Other surface free microparticles 20 of the present invention have diameters 26 in the range of from 20 nm to 800 nm or more. Preferred diameters 26 of the surface free microparticles 20 are in the range of 40 nm to 150 nm, more preferably diameters 26 are between 75 and 125 nm; and most preferably diameters 26 are between 90 nm and 110 nm. According to some embodiments, the surface free microparticles 20 are in a dilution 11 having a container weight in the range of 0.001% to 2%, more preferably in the range of 0.01% to 0.2%, and most preferably in a dilution 11 having a container weight of 0.016%.

[0123] The present bioassay method using surface free microparticles 20 requires a dilution 11 having a dilution ratio 30 of microparticles 20 to dihydrogen monoxide 33 in the range of 1 :50 to 1 :2000; and more preferably in the range of 1 :100 to 1 :1000.

[0124] When prepared according to the methods of the present invention as discussed herein, the surface free microparticles that can satisfy the objectives of the present invention can include ordinary microparticles supplied by Varian Laboratories TM Corporation.

[0125] Carboxyl microparticles

[0126] Another embodiment of the present invention is the use of carboxyl latex microparticles having a diameter of 103 nm in a solution having a container weight of 0.016%. The optical density of the dilution 11 is measured at 660 nm and has a value of 0.08.

[0127] The diameter 26 of the other carboxyl microparticles 25 of the present invention range from 20 nm to 800 nm or greater. The preferred range of the diameter 26 of the carboxyl microparticles 25 is 40 nm to 150 nm, with a more preferred diameter 26 between 75 nm and 125 nm; and a most preferred diameter between 90 nm and 110 nm.

[0128] According to some embodiments, the carboxyl microparticles 24 are in a solution 28 having a volume concentration ranging from 0.001% to 2%, more preferably in a solution 28 having a volume concentration ranging from 0.005% to 1%, and most preferably in a dilution 11 having a volume concentration of 0.016%.

[0129] The present bioassay method using the carboxyl microparticles 25 requires a dilution 11 wherein the dilution ratio 30 of the microparticles 25 to the dilution 33 is in the range of 1 :50 to 1 :2000; and more preferably in the range of 1 : 100 to 1 : 1000.

[0130] When prepared according to the methods of the present invention as discussed herein, the carboxyl latex particles 24 that satisfy the present invention can include high activity latex beads provided by Invitrogen TM Corporation.

[0131] Dry matrix

[0132] In general, the bioassay method of the present invention can utilize most common or functionalized surface latex particle suspensions. Some embodiments employ a matrix with reagent attenuating agents, such as surfactants. Two reagent attenuating agents that are detected include polysorbate non-ionic surfactants and octylphenol ethoxylate surfactants. When prepared according to the methods of the present invention as discussed herein, the polysorbate non-ionic surfactants and octylphenol ethoxylate surfactants that satisfy the objectives of the present invention can include surfactants provided by Tween TM and Triton TM X-100 families.

[0133] Some dry matrices according to the present invention include carbohydrates, carbohydrate derivatives, and mixtures that create an environment that protects the particles from adverse temperatures and allows for rapid rehydration and even dispersion upon the addition of fluids such as buffers, dilution samples, or other liquid reagents.

[0134] Carbohydrates and their derivatives are the preferred compounds for drying the particles and providing stability to the bioassay method as discussed herein during the drying process. These reagents are prepared in water. However, low molar buffers are also used in other embodiments, examples of which include glycine and diglycine. The percentage concentration of the stabilizer ranges from 2-25%, with a preferred range of 5-10%.

[0135] Compounds used for the dry base include: glycine, diglycine, sodium chloride, octenyl succinic anhydride, polyvinyl alcohol-polyethylene glycol graft copolymer, maltodextrin, alpha-(1,6)-linked maltotriose, alpha-D-glucopyranosyl-(1→1)-alpha-D-glucopyranoside, water-soluble glucose polymers obtained by hydrolysis of starch with acid and / or enzymes in the presence of water, polysaccharide polymers, polyethylene glycol, polyethylene glycol (15)-hydroxystearate, povidone, sucrose, sorbitol, polyoxyethylene esters of 12-hydroxystearic acid, and 1-O-alpha-D-glucopyranosyl-D-mannitol.

[0136] Disclosed versions of these compound products that can meet the objectives of the present application can include HiCap 100 TM , Kollicoat IR TM , Maltrin 250 TM , Pullulan, Trehalose, Solutol TM Plus, and Solutol TM . A preferred embodiment is Lab 9101 TM , Maltrin 250, Trehalose TM , and sucrose. The most preferred base is Maltrin TM , sucrose, and isomalt. And for embodiments using dry sulfate microparticles, the preferred carbohydrate is sucrose or isomalt.

[0137] Liquid matrix

[0138] The same particle suspensions of each of the dry bases described above can also be made in a more dilute form with so-called "liquid" bases to facilitate dosing and pipetting. For example, when the dry base formulation consists of 25 uL of 1:18 sulfate latex with 10% sucrose, a similar liquid base formulation would range from 50 uL of 1:9 sulfate latex with 5% sucrose. In other embodiments, the liquid base is formed from a lysed diluent.

[0139] Compounds used in the liquid base of the present embodiments include: glycine, diglycine, sodium chloride, octenyl succinic anhydride, polyvinyl alcohol-polyethylene glycol graft copolymer, maltodextrin, alpha-(1,6)-linked maltotriose, alpha-D-glucopyranosyl-(1→1)-alpha-D-glucopyranoside, water soluble glucose polymers obtained by hydrolysis of starch with acid and / or enzymes in the presence of water, polysaccharide polymers, polyethylene glycol, polyethylene glycol (15)-hydroxystearate, povidone, sucrose, sorbitol, polyoxyethylene ester of 12-hydroxystearic acid, 1-O-alpha-D-glucopyranosyl-D-mannitol, polyethylene glycol (PEG), PEG 6K, PEG 12K and PEG 20K, polysorbate-type nonionic surfactants, octylphenol ethoxylate surfactants and / or dimethicone, diluted with purified water for injection to a concentration that supports a defoaming performance of less than 15 seconds in the USP test.

[0140] Disclosed versions of these compound products that can meet the objectives of the present invention include HiCap 100 TM , Kollicoat IR TM , Lab 9101 TM , Maltrin 250 TM , Pullulan TM , Trehalose TM , Solutol Plus TM , Solutol TM , Foam AWAY TM , and Sorbital TM .

[0141] Reagents

[0142] The biological assay method of the present invention employs various reagents 80 or activators. Compounds used as activators in embodiments of the present invention include: thromboplastin, thromboplastinogen, tissue thromboplastin factor III, platelet tissue factor, thromboplastin, thrombokinase, tissue factor, thromboplastin, ellagic acid, activated partial thromboplastin time, thrombin, factor II, factor VII, factor I, factor X, factor XII, activated protein C, negatively charged phospholipid, calcium ion, aluminum silicate clay, silicon oxide, silicon dioxide, diatomaceous earth, and povidone.

[0143] When properly prepared, selected few commercially available forms of these compounds and products that can meet the objectives of the present invention can include kaolin TM , Innovin TM thromboplastin 82, APTT-XL 84, and Siemens TM , thrombin 86.

[0144] Prior to actual biological assays, these reagents or activators can be in a dry state at the time of storage shipment to ensure additional stability. These reagents 80 can then be diluted during the assay process with diluent 90, such as DH20 and the like, in the range of 20 uL to 400 uL, more preferably in the range of 50 uL to 300 uL, and in the range of 50 uL to 100 uL, at various concentrations 96 and various amounts 94. Some embodiments employ calcium chloride or other diluents, which will be discussed in the specific embodiments below.

[0145] Optical density and calibration

[0146] As initially discussed, the present invention and system uses the principle of coagulation (turbidity) detection to measure 116, 116' and record the time required for the coagulation of a plasma or whole blood sample 60. This technique assesses the onset of coagulation 132 and the endpoint of coagulation 134 by measuring the change 135 in optical density 130 over time of detection. Figure 10 The formation 124 of coagulation is inferred and detected according to the principle that in a medium where fibrinogen is converted to fibrin, any light passing through the medium is absorbed by the fibrin strands. Thus, over time, as the fibrin clot forms, the absorption of light increases, resulting in a change 135 in optical density 130.

[0147] For each of the biological assays discussed herein, as described above, after the sample is collected and mixed, light is transmitted from the source through the mixture 123. The transmitted light is then directed onto a light detector, which is at a 180° angle of incidence from the light source. The first corresponding electrical signal output of the photodetector output is measured 116' using optical detection to determine the level of hemoglobin 125.

[0148] The transmittance of light through the mixture 123 over a predetermined time is again measured 116 by the photodetector, thereby producing a second corresponding electrical signal output. Both the first and second corresponding electrical signal outputs of the photodetector vary according to the detected light.

[0149] The signal outputs are processed by software and through a series of algorithms to determine the relevant coagulation points, onset 132 and formation 134 of coagulation. In short, the change 135 in optical density 130 of the latex microparticle mixture 123 signal is used to indicate the onset 132 and formation 134 of coagulation.

[0150]

[0151] Reduced operating procedure run time

[0152] ​The present invention addresses several laboratory error sources in coagulation testing procedures. Since clot formation is dependent on the passage of time, one of the largest laboratory error sources in coagulation testing procedures is the passage of time between sampling 104 and measurement 116.

[0153] One of the principles of the present invention is to address the problem of increased error due to the passage of time between sampling 104 and testing (measurement) 116 that the prior art has failed to address. By pre-packaging the assay components in one all-inclusive kit 2, and providing pre-selected components for the assay method, the present invention accelerates the pre-testing phase.

[0154] Another way in which the present invention achieves this goal is by providing an assay method that is able to obtain accurate results using a whole blood sample without the need to separate the red blood cells from the plasma prior to testing. Currently available point-of-care analyzers require the use of plasma as the sample. This requires the separation of the red blood cells from the plasma in the blood sample prior to obtaining the test result, and further extends the time between sampling and testing.

[0155] One of the reasons that current analyzers require plasma is due to the fact that prior art automated systems and testing methods are not able to provide self-correcting analysis. The level of hemoglobin in the blood sample will have an effect on the rate of initiation of clotting. The present inventors have discovered that if the optical density signal output is not adjusted to account for variations in the amount of hemoglobin in the individual, errors will result in the correct identification of the initiation and formation of the clot.

[0156] To address the errors of the prior art, the present invention provides an automated correction for hemoglobin in the assay method with respect to this potential source of error. For the described embodiments of the coagulation assay using whole blood, the optical density of the mixture is measured at a visible wavelength. Subsequently, the hemoglobin level is determined by measuring the optical density of the sample at another visible wavelength. The clotting time is then corrected for the hemoglobin measurement to obtain an adjusted true plasma value for the sample. To illustrate the benefits provided by this automated correction, the effect of hemoglobin on the prothrombin time INR value (with and without correction) is illustrated in Figure 14

[0157] In particular, Figure 14 ​The effect of different levels of hemoglobin (hematocrit effect) on INR of normal samples is shown in the middle. As shown, the range of hemoglobin is from 0 to 23 gm / dl. The range of normal INR is 0.8 to 1.3 INR units. Moderate oral anticoagulation therapy results in INR from 1.8 to 2.8 INR units. As hemoglobin increases, INR also increases due to the decrease in plasma fraction in the whole blood sample. With additional LEDs in the visible range, INR can be corrected based on the detected hemoglobin. The graph also shows the corrected INR for hemoglobin for comparison.

[0158] The present application advantageously extends the functionality of the instrument to include multiple signal measurement devices, such as but not limited to multiple wavelengths of light emitting diodes (LEDs). The measurement of optical density of the mixture to read the test result is preferably performed at a visible wavelength in the range of 620 nm to 700 nm, more preferably in the range of 650 to 680 nm, even more preferably in the range of 658 nm to 668 nm, and most preferably at 660 nm. The measurement of optical density of the sample to read the hemoglobin level is preferably performed at a visible wavelength in the range of 500 nm and 550 nm, more preferably in the range of 510 nm and 545 nm, even more preferably in the range of 520 nm and 540 nm, and most preferably at 530 nm.

[0159] Table 1 shows data in Figure 14 determining sample ID, hemoglobin level, and initial INR before correction.

[0160]

[0161] Table 2 shows data shown in Figure 14 determining sample ID, hemoglobin level, and initial INR after correction.

[0162]

[0163] Next, as shown in Figure 15 , the difference between the plasma INR values obtained on a pre-approved PT / INR analyzer with and without correction for hemoglobin according to the present method is illustrated graphically. Again, the range of hemoglobin values is 0 to 23 g / dl.

[0164] Table 3 shows data in Figure 15 determining sample ID, plasma INR, difference in plasma INR values (DELTA INR) before and after correction for hemoglobin.

[0165]

[0166] Standardized thromboplastin time / INR

[0167] It should be understood that the standardized INR is employed in each of the various embodiments of the biological assays of the present application. The standardized prothrombin time INR is used to account for differences in thromboplastin used, which cause variations in the output. This INR correction measurement (or standardization) is made from the prothrombin time, the sensitivity index of the thromboplastin, and the mean prothrombin time.

[0168] Specifically, in mathematical terms, this standardization can be written as follows:

[0169] INR = (PT / MT) ISI

[0170] where, in the above expression, INR represents the standardized value; PT represents the prothrombin test time; ISI represents the sensitivity index of the thromboplastin; and MT represents the mean prothrombin time derived from 20 normal samples.

[0171] Particular bioassays of the invention

[0172] Now turning to Figures 16-24 , the results of specific biological assays performed in accordance with embodiments of the present application are shown. Unless otherwise specified, these biological assay results illustrate the optical density readings (in seconds) at 660 nm using the kinetic mode as a function of time.

[0173] Bioassay method using tissue thromboplastin

[0174] The results of different biological assays B1-B20 using different substrates for various microparticle dilutions in accordance with the methods of the present application are shown in Figures 16-33 and discussed below. Each of these biological assays B1-B20 employed a tissue thromboplastin reagent. Specifically, the reagents 80 selected for these biological assays were tissue thromboplastin and calcium chloride, in the amounts listed in each of the protocols below.

[0175] B1-B4 bioassay with BSA-coated latex and normal microparticles

[0176] Now turning to Figure 16 and Figure 17 , the results of four biological assays B1-B4 in accordance with the present application are shown in graphical form.

[0177] The first bioassay, B1, used microparticle diluent 11, in which microparticles 27 had bovine serum albumin (BSA) surface groups. The microparticles were diluted in water with 0.02% sodium azide at a ratio of 1:500, resulting in a total microparticle concentration of 0.016%. The optical density of diluent 11 was measured at 660 nm after dilution, and the value was 0.08. Bioassay B1 further used matrix 40, which contained 0.17M glycine at pH 10.0, 1.0M sodium chloride, and 1% dimethyl silicone oil diluted with water. Bioassay B1 was performed using a standard citric acid-treated whole blood sample 60.

[0178] In contrast, the protocol for the second bioassay B2 is the same as that for bioassay B20, except that B2 is performed using a microparticle diluent 11 containing microparticles 20 (without BSA surface groups). The reagent 80 selected for the bioassay is tissue coagulation activating enzyme and calcium chloride.

[0179] like Figure 16 As shown, for the B1 assay, which attempts to use BSA microparticles known according to the prior art discussed above, no consistent change in optical density over time was detected. Therefore, the B1 assay did not detect changes in coagulation initiation time, coagulation end time, or coagulation.

[0180] Similarly, Figure 16 As shown, for the B2 determination using the concept of the present invention according to the new method discussed herein, it can be seen that coagulation begins at approximately 4 seconds 132 with an OD value of 0.1342, and coagulation ends at approximately 10 seconds 134 with an OD value of 0.1907. The change in absorption 135 over time is the change in optical density value, with an OD value difference of 0.0565.

[0181] To confirm this difference, these bioassays B1 and B2 were repeated, this time using abnormal blood samples, and for clarity, they were named the third and fourth bioassays, B3 and B4, respectively.

[0182] like Figure 17 As shown, for the B3 assay, it was again attempted to use BSA microparticles known from the prior art as discussed above, and no consistent change in optical density over time was detected. Therefore, the B3 assay did not detect changes in coagulation initiation time, coagulation end time, or coagulation.

[0183] Similarly, Figure 17 As shown, for the B4 determination using the method of the present invention, coagulation can be seen to begin at approximately 31 seconds (132), with an OD value of 0.1164, and coagulation can also be seen to end at approximately 59 seconds (134), with an OD value of 0.2180. The change in absorption (135) over time is the change in optical density value, with an OD value difference of 0.1016.

[0184] The B1-B4 bioassay protocol is provided in Table 4 below:

[0185]

[0186] Table 5 below shows the optical density results of the above-described bioassays B1-B4 using kinetic mode at 660 nm over a period of time according to the present invention:

[0187]

[0188]

[0189]

[0190] B5-B10 bioassay using polyethylene glycol matrix

[0191] Looking at it now Figures 18-23 They illustrate, in a graphical manner, the results of six bioassays B5-B10 according to various embodiments of the present invention.

[0192] Specifically Figure 18 The results of a first bioassay B5 according to the present invention are illustrated in the figure, which uses a microparticle diluent 11 containing microparticles 22 having amidine surface groups with a diameter of 95 nm and a bulk density of 0.080%. The bioassay B5 further employs a matrix 40 having 0.17 M glycine, 1.29 M sodium chloride, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0193] The optical density of the diluted solution of B5 was measured at 660 nm, and its optical density value was 0.19. For example... Figure 18 As shown, for the B5 assay, coagulation begins at approximately 5 seconds (132) with an OD value of 1.153, and ends at approximately 50 seconds (134) with an OD value of 1.164. The change in absorbance (135) over time represents the change in optical density, with an OD value difference of 0.011.

[0194] Figure 19 The results of the second bioassay embodiment B6 are illustrated in the figure. This embodiment uses a microparticle diluent 11 containing microparticles with sulfate surface groups 24 having a diameter of 26 of 110 nm and a bulk density of 28 of 0.044. Embodiment B6 further uses a matrix 40 containing 0.17 M glycine, 1.29 M sodium chloride, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0195] The optical density of the diluent was measured at 660 nm, and its value was 0.21. For example... Figure 19 As shown, for the B6 assay, coagulation begins at approximately 5 seconds (132), with an OD value of 0.564, and ends at approximately 70 seconds (134), with an OD value of 1.000. The change in absorbance (135) over time represents the change in optical density, with an OD value difference of 0.436.

[0196] Next, let's look at... Figure 20 The results of the third bioassay embodiment B7 are illustrated graphically. This embodiment uses a microparticle diluent 11 containing microparticles with a diameter of 96 nm and a bulk density of 0.067%, lacking surface groups 20. The dilution ratio of the microparticles to the diluent is 1:150. Embodiment B7 further uses a matrix comprising 0.17 M glycine, 1.29 M sodium chloride, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0197] The optical density of the diluent was measured at 660 nm, and its value was 0.21. For example... Figure 20 As shown, for the B7 measurement, coagulation begins at approximately 5 seconds (132) with an OD value of 0.514, and ends at approximately 85 seconds (134) with an OD value of 1.215. The change in absorbance (135) over time corresponds to the change in optical density, with an OD value difference of 0.701.

[0198] Figure 21 The results of the fourth bioassay implementation scheme B8 are then provided, which employs a microparticle diluent 11 having microparticles containing amidine surface groups 22 and having a diameter 26 of 95 nm. The bulk density 28 of the microparticle diluent is 0.080%. The optical density 31 of the diluent 11 was measured at 660 nm, and its optical density value was 0.19.

[0199] This embodiment B8 employs a matrix 40 having 0.17M glycine 47 and 1.29M sodium chloride at pH 7.0. The matrix 40 further comprises 10% polyethylene glycol (PEG) 20K and 1% a carbohydrate derivative in the form of Tween 20.

[0200] like Figure 21 As shown, for the B8 measurement, coagulation begins at approximately 15 seconds (132) with an OD value of 0.633, and ends at approximately 65 seconds (134) with an OD value of 1.081. The change in absorbance (135) over time represents the change in optical density, with an OD value difference of 0.448.

[0201] Next, Figure 22 Results for a fifth bioassay embodiment B9 are shown, which employed a microparticle diluent 11. The diluent 11 had microparticles 24 with sulfate surface groups 20 and a diameter 26 of 110 nm. The microparticles 24 within the diluent 11 were present in the diluent at a concentration of 0.044%. Specifically, the diluent 11 was diluted with water at a ratio of 1 part particles to 180 parts water. The optical density of the diluent 11 was measured after dilution at 660 nm, and the optical density value was 0.21. The embodiment B5 further employed a substrate 40 having 0.17 M glycine, 1.29 M sodium chloride, and 10% polyethylene glycol (PEG) 20K at a pH of 7.0.

[0202] As Figure 22 shown, for the B9 assay, clotting was only detected when the data was analyzed and enhanced (the full data is provided in Table 7 below). However, when enhanced, clotting was seen to begin 132 at approximately 25 seconds with an OD value of 0.3780, and clotting was seen to end 134 at approximately 85 seconds with an OD value of 0.3830, and the change in absorbance 135 over time was a change in optical density value, with an OD value difference of 0.005.

[0203] Then, Figure 23 A graph of the results for a sixth bioassay embodiment B10 is provided, which employed a microparticle diluent 11 having microparticles 24 without surface groups 20 and a diameter of 96 nm at a concentration of 0.067%. In the final diluent, the dilution ratio of microparticles to water was 1 to 150.

[0204] The optical density 31 of the diluent 11 was measured after dilution at 660 nm, and the optical density value 31 was 0.21. The embodiment further employed a substrate 40 having 0.17 M glycine, 1.29 M sodium chloride, and 10% polyethylene glycol (PEG) 20K at a pH of 7.0.

[0205] As Figure 23 shown, for the B10 assay, clotting was only detected when the data was analyzed and enhanced (the full data is provided in Table 7 below). However, when enhanced, clotting was seen to begin 132 immediately with an OD value of 0.3780, and clotting was seen to end 134 at approximately 85 seconds with an OD value of 0.3860, and the change in absorbance 135 over time was a change in optical density value, with an OD value difference of 0.008.

[0206] The protocols for all six embodiments B5-B10 listed above are shown in Table 6 below:

[0207]

[0208] The general procedure for these biological assays B1-B6 was the same and was prepared as described above Figure 10

[0209] Table 7 shows the data for the corrected optical density results at 660 nm using the kinetic mode over one minute and a half for each of the six described biological assay embodiments according to the present application:

[0210]

[0211]

[0212] B11-B12 bioassay using simethicone

[0213] Now looking at Figure 24 which shows in graphical fashion the results of a first biological assay B11 according to the present application, which employed a microparticle diluent 11 having microparticles 24 with sulfate surface groups and a diameter of 100 nm. The microparticles were diluted in water at a ratio of 1 to 500 with 0.02% sodium azide, for a total concentration of microparticles of 0.016%.

[0214] The optical density of the diluent 11 was measured after dilution at 660 nm and was found to be 0.08. The biological assay B11 further employed a matrix 40 having 0.17 M glycine, 1.29 M sodium chloride and 1% dimethyl silicone oil at a pH of 10.0, diluted with USP grade purified water to a concentration that supports a defoaming performance of less than 15 seconds in the USP test, which is commercially identified as Foam AWAY® TM The biological assay B11 was performed with a normal sample 60.

[0215] As Figure 24 can be seen for the B11 assay, clotting was seen to begin at about 3 seconds 132 with an OD value of 0.1516 and clotting was seen to end at about 65 seconds 134 with an OD value of 0.2356, the change in absorbance 135 over time is the change in optical density value, the difference in OD value is 0.084.

[0216] In contrast, Figure 25 which shows in graphical fashion the results of a biological assay B12, which was identical in protocol to the above described biological assay B11, except that now an abnormal sample was employed for the assay. As Figure 25 ​As shown, for the B12 assay, clotting can now be seen to have begun at about 30 seconds 132 with an OD value of 0.1528, and clotting can also be seen to have ended at about 65 seconds 134 with an OD value of 0.3215, the change in absorbance 135 over time is the change in optical density, the difference in OD values is 0.1687.

[0217] The protocol for biological assays B11, B12 is provided below in Table 8:

[0218]

[0219] The following Table 9 shows the data for the optical density results at 660 nm using the kinetic mode for the above biological assays B11-B12 according to the present application, for more than one minute for biological assay B12, and for more than 20 seconds for B11:

[0220]

[0221]

[0222]

[0223] It should be understood that while these optical density values are shown here, these values are not typically presented to the user. Rather, the clotting time is corrected for the adjusted true plasma value of the sample using the measurement of hemoglobin according to the methods discussed above. This corrected clotting time is then used to report the standardized INR discussed above.

[0224] For example, for the abnormal blood biological assay B12 discussed above, in order to determine the hemoglobin content, a first optical density measurement 116' is taken. The optical density detected at 530 nm is 1.5135, this optical density value 140 is compared to a predetermined relationship 141 of OD values and known hemoglobin values to determine the specific hemoglobin level 142 present in the sample being analyzed. Again, while this level is not typically reported to the customer or user, this analysis is described in detail in Figure 26 The specific hemoglobin level 142 for biological assay B11 is then stored in the system memory until the second set of optical density measurements 116 are completed.

[0225] As discussed above with reference to Figure 25For the B12 assay in question, the clot began at the first time (30 seconds) 132 with an OD value of 0.1528 and ended at the second time (65 seconds) 134 with an OD value of 0.3215. The change in absorbance 135 over the incremental time (35 seconds) was the difference in the first OD value, which was 0.1687. In various biological assays of the method of the present application, the difference in the OD value resulting from the change in absorbance 135 over time is in the range of 0.005 to 1.0. More preferably, the difference in the OD value is at least 0.2, and, if necessary, at least 0.08. This change in absorbance 135 is used to calculate the PT value 136 (47 seconds, which is associated with an absorbance OD value of 0.23715).

[0226] The system then uses this initial PT value 136 and retrieves the reference Figure 24 corresponding to the specific HGB level 142 determined. A predetermined relationship 137 is then used to determine an adjusted or corrected PT value 138. Specifically, Figure 27 shows the uncorrected PT value 136 being corrected by the predetermined relationship 137 corresponding to the hemoglobin level 142 (12.3) of the sample 60. This relationship 137 is then used to provide a corrected PT value 138. Where the relationship 137 is mathematically expressed as:

[0227] CPT = PT * SQRT(C / HGB)

[0228] where CPT is the corrected prothrombin time 138; PT is the uncorrected prothrombin time 136; C is the hemoglobin constant; and HGB is the specific hemoglobin level 142 associated with the particular sample 60.

[0229] Then, Figure 28 shows the calculation of the INR from the corrected PT 138. Specifically, the calculation is:

[0230] INR = (PT / MT) ISI

[0231] Applying the above formula to the data obtained for B12, PT represents the corrected prothrombin test time 138 (50.1 seconds); ISI represents the sensitivity index of the thromboplastin used (0.98 arbitrary units); MT represents the mean prothrombin time (10.2 seconds) derived from 20 normal samples; and INR represents the normalized value 144 (4.8 arbitrary units). In general, only the corrected PT value (50.1 seconds) and the INR value (4.8 arbitrary units) are reported to the user.

[0232] B13-B14 bioassay using carboxyl microparticles

[0233] Now consider Figure 29 which graphically illustrates the results of the biological assay B13 according to the present application, which employed a microparticle diluent 11 containing microparticles 25 having a diameter of 103 nm and carboxyl surface groups in a diluent having a concentration of 0.016%. The present biological assay B13 employing carboxyl microparticles 25 required that the diluent 11 have a dilution ratio 30 of microparticles 25 to dihydrogen monoxide 33 in the range of 1 :50 to 1 :2000; more preferably in the range of 1 :100 to 1 :1000.

[0234] The diluted solution was measured for optical density at 660 nm and had an optical density value of 0.08. The biological assay B13 also employed a substrate having 0.17 M glycine, 1 M sodium chloride and 1% dimethyl silicone oil at a pH of 10.0, diluted with USP grade purified water to a concentration that provided a defoaming performance of less than 15 seconds in the support USP assay, which is commercially identified as Foam AWAY® TM The biological assay B13 was performed with a normal sample 60.

[0235] As Figure 29 can be seen for the B13 assay, clotting was seen to begin 132 at about 5 seconds with an OD value of 0.1304, and clotting was seen to end 134 at about 20 seconds with an OD value of 0.1826, the change in absorbance 135 over time being the change in optical density value, the difference in OD value being 0.0522.

[0236] In contrast, Figure 30 which graphically illustrates the results of the biological assay B14, which had the same protocol as the biological assay B13 described above, except that it was now performed with an abnormal sample. As Figure 30 can be seen for the B14 assay, clotting was seen to begin 132 at about 40 seconds with an OD value of 0.1218, and clotting was seen to end 134 at about 68 seconds with an OD value of 0.2027, the change in absorbance 135 over time being the change in optical density value, the difference in OD value being 0.0811.

[0237] Table 10 provides the B13, B14 biological assay protocols:

[0238]

[0239]

[0240] The following Table 11 shows the data for the optical density results at 660 nm using the kinetic mode for the biological assays B13-B14 described above according to the present application, for more than one minute for the biological assay B13 and for more than 20 seconds for B14:

[0241]

[0242]

[0243]

[0244] B15-B18 bioassay at different operating temperatures

[0245] Typically, many bioassays according to the invention are performed at physiological temperatures, but this is not always the case. It should be understood that, in general, most temperatures are suitable for the methods of the invention. However, adjusting this variable using the bioassays discussed above will introduce potential variations that will generally alter the results of the bioassays. Therefore, temperature variations should not be introduced without considering these variations, especially in coagulation assays where precise timing can affect clinical outcomes.

[0246] Figure 31 and 32 The results of four bioassay embodiments B15-B18 of the present invention are illustrated in the figures, using two different microparticle matrices at two different temperatures. Protocols for all four bioassay embodiments are provided in Table 12 below:

[0247]

[0248]

[0249] Specifically, bioassay B15 uses a microparticle diluent 11 containing microparticles with amidine surface groups 22 having a diameter of 95 nm. At a bulk density of 0.080%, the diluent 11 contains microparticles 22 diluted with water at a ratio of 1:50. The optical density of the diluted solution was measured at 660 nm, and the optical density value was 0.19. Bioassay B15 uses a matrix 40 containing 0.17 M glycine, 0.29 M sodium chloride, and 10% polyethylene glycol (PEG) 20K at a pH of 7.0. Bioassay B15 is performed at a temperature of 22°C.

[0250] like Figure 31 As shown, for the B15 assay, clotting can be seen to begin almost immediately 132, with an OD value of 1.079 at approximately 4 seconds, and clotting can also be seen to end at approximately 100 seconds 134, with an OD value of 1.748. The change in absorbance 135 over time is the change in optical density value, with an OD value difference of 0.669.

[0251] exist Figure 31A similar illustration of the results for bioassay B16 can also be seen. Bioassay B16 also uses microparticle diluent 11, which contains microparticles with amidine surface groups 22 having a diameter of 95 nm. The microparticles 22 are diluted with water at a dilution ratio of 1:50 30 when the bulk density is 0.080%. The optical density 31 of the diluted sample is measured at 660 nm, and the optical density value 31 is 0.19. Bioassay B16 also uses matrix 40, which contains 0.17 M glycine, 0.29 M sodium chloride, and 10% polyethylene glycol (PEG) 20K at a pH of 7.0. However, unlike bioassay B15, this bioassay B16 is performed at an operating temperature of 37°C.

[0252] like Figure 31 As shown, for the B16 assay, the coagulation value is more difficult to distinguish, although coagulation can be seen to begin almost immediately at approximately 4 seconds (132'), with an OD value of 1.023. Unlike the B15 assay, the B16 assay appears to complete coagulation quickly, as coagulation seems to end at approximately 40 seconds (134'), with an OD value of 1.110. The change in absorbance over time (135') is a change in optical density value, with an OD difference of only 0.087.

[0253] Next turn Figure 32 The results of another pair of bioassays B17 and B18 according to the invention are illustrated in the figure. In the first bioassay B17, the microparticle diluent 11 uses microparticles 22 having amidine-like surface groups and a diameter 26 of 0.95 nm. Diluted with water at a ratio of 1:50, the microparticles 22 in bioassay B17 are present in the diluent 11 at a bulk density of 0.080%.

[0254] The optical density 31 of dilution 11 was measured at 660 nm, and its value was 0.19. This bioassay B17 used matrix 40, which contained 0.17 M glycine, 0.29 M sodium chloride, 10% polyethylene glycol (PEG) 20K, and 1% Tween 20 at pH 7.0. Bioassay B17 was performed at an operating temperature of 22 °C.

[0255] like Figure 32 As shown, for the B17 assay, coagulation begins at approximately 12 seconds (132), with an OD value of 0.997, and ends at approximately 58 seconds (134), with an OD value of 1.772. The change in absorbance (135) over time represents the change in optical density, with an OD value difference of 0.775.

[0256] The second bioassay B18 according to the present invention uses a microparticle diluent employing microparticles 22 having amidine surface groups with a diameter of 0.95 nm and a bulk density of 0.080%. The dilution ratio of diluent 11 is 1:50. The optical density of the diluted solution was measured at 660 nm, and the optical density value was 0.19. This bioassay B18 also uses a matrix having a pH of 7.0, consisting of 0.17 M glycine, 0.29 M sodium chloride, 10% polyethylene glycol (PEG) 20K, and 1% Tween 20. However, unlike bioassay B17, bioassay B18 is performed at an operating temperature of 37°C.

[0257] like Figure 32 As shown, for the B18 assay, the coagulation value is more difficult to distinguish, although coagulation can be seen to restart at approximately 20 seconds (132'), with an OD value of 0.653. The B18 assay appears to end coagulation at approximately 60 seconds (134'), with an OD value of 1.054. The change in absorbance over time (135') is a change in optical density value, with an OD value difference of only 0.401.

[0258] The general methods for these bioassays of B15-B18 are the same, and are generally followed as mentioned above. Figure 10 Let's get ready.

[0259] Table 13 below shows data on the corrected optical density results at 660 nm using kinetic mode over several minutes for each of the above-described bioassays B15-B18 according to the present invention:

[0260]

[0261]

[0262] B19-B20 bioassay for monitoring the use of anticoagulants

[0263] Looking at it now Figure 33 The results of prothrombin time bioassays B19 and B20 are illustrated graphically and used to determine the extrinsic pathway of coagulation in a multi-step process and to monitor the use of oral anticoagulants. These bioassays B11 and B12 are prothrombin time assays, illustrating the first assay B19 of the present invention in normal whole blood samples with the use of the oral anticoagulant Coumadin. TM The second method of the present invention for measuring the optical density of B20 in patient samples versus time (in seconds).

[0264] Coumadin TM(Also known as warfarin) inhibits the synthesis of vitamin K, thus inhibiting the half-life of factor VII. As described in this article, the level of factor VII was determined and corrected using a prothrombin time assay and dual-wavelength correction in this figure.

[0265] like Figure 33 As shown, normal clotting time 132 typically begins after approximately 15 to 20 seconds. As mentioned above, clotting formation is initiated via the extrinsic clotting pathway activated by factor VII. Specifically, as... Figure 33 As shown, the normal blood sample test method B19 begins to clot after approximately 15 seconds 132, and the Coumadin sample test method B20 begins to clot after approximately 100 seconds 132'.

[0266] For both B19 and B20 assays, microparticle diluent 11 used microparticles 22, which had amidine-based surface groups with a diameter of 26 nm (95 nm) and a bulk density of 0.080%. Microparticle diluent 11 was diluted with water at a ratio of 1:50. The optical density of the diluent was measured at 660 nm, and the value was 0.19.

[0267] like Figure 33 As shown, for the B19 assay, coagulation begins at approximately 15 seconds (132) with an OD value of 0.989, and ends at approximately 90 seconds (134) with an OD value of 1.700. The change in absorbance (135) over time represents the change in optical density, with an OD value difference of 0.711.

[0268] Similarly, Figure 33 As shown, for the B20 measurement, coagulation begins at approximately 50 seconds (132') with an OD value of 0.919, and ends at approximately 180 seconds (134') with an OD value of 1.6290. The change in absorbance over time (135') represents the change in optical density, with an OD value difference of 0.7100.

[0269] The bioassay protocols for B19 and B20 are shown in Table 14 below:

[0270]

[0271] The general methods for these bioassays of B19-B20 are the same, and are generally followed as mentioned above. Figure 10 Prepare accordingly. Table 15 shows the optical density data of the first bioassay B19 with normal citric acid-treated blood and the second bioassay B20 with coumarin citric acid-treated blood, measured in kinetic mode within two minutes at 660 nm:

[0272]

[0273]

[0274] Bioassay using thrombin

[0275] The results of the different bioassays B21-B22 using different dilutions of different microparticles of different matrices according to the method of the present application are illustrated in Figure 34 and discussed below for each bioassay. Each of these bioassays B21-B22 used a thrombin reagent. Specifically, the reagent 80 chosen for these bioassays was Tissue Thromboplastin and calcium chloride, in the amounts listed in each of the protocols below.

[0276] B21-B22 bioassay for measuring fibrinogen levels

[0277] Now turning to Figure 34 which illustrates the results of the thrombin time (TT) assays B21, B22, which directly measure the level and function of fibrinogen and will also determine the presence of a thrombin inhibitor in the sample. Figure 34 illustrates the optical density versus time (in seconds) measurements for the first TT assay B21 using 30 uL of the thrombin reagent on citrated plasma and the second TT assay B22 using 20 uL of the thrombin reagent and citrated blood according to the method of the present application.

[0278] The protocols for the bioassays B15, B16 are provided below in Table 16:

[0279]

[0280] Specifically, these bioassays B21, B22 used a microparticle diluent 11 having microparticles 24 having a sulfate surface group with a diameter 26 of 110 nm and a volume fraction of 0.2%. Both bioassays B21, B22 used a matrix 40 having 0.17 M glycine and 1.0 M sodium chloride with a pH of 10.0. The first bioassay B21 used 30 uL of Siemens Tm Thrombin Time (TT) reagent and the second bioassay B22 used 20 uL of Siemens Tm Thrombin Time (TT) reagent.

[0281] Some thrombin inhibitors that can be used with the method of the present application are unfractionated heparin, low molecular weight heparin, and oral anticoagulants of direct anti- thrombin agents, including but not limited to hirudin, rivaroxaban, apixaban, dabigatran, and argatroban. The general methods for these bioassays B21-B22 are the same and are generally prepared as discussed above Figure 10 .

[0282] AsFigure 34 For the B21 assay, clotting can be seen to begin at about 5 seconds 132 with an OD value of 0.1829, and clotting can be seen to end at about 100 seconds 134 with an OD value of 0.3481, the change in absorbance 135 over time is the change in optical density, the difference in OD values is 0.1652.

[0283] Also as shown in Figure 34 For the B22 assay, clotting can be seen to begin at about 5 seconds 132' with an OD value of 0.1357, and clotting can be seen to end at about 80 seconds 134' with an OD value of 0.2491, the change in absorbance 135' over time is the change in optical density, the difference in OD values is 0.1134.

[0284] Table 17 shows the optical density data measured at 660 nm using the kinetic mode for the first biological assay B21 using 30 uL of thrombin reagent and the second biological assay B22 using 20 uL of thrombin reagent over three minutes:

[0285]

[0286]

[0287] Bioassay using activated partial thromboplastin

[0288] The results of different biological assays B23-B27 using different dilutions of different microparticles of different substrates according to the method of the present application are illustrated in Figures 35-38 and are discussed below for each biological assay. Each of these biological assays B23-B27 used an activated partial thromboplastin reagent. Specifically, the reagent 80 selected for these biological assays was activated partial thromboplastin and calcium chloride, the amounts of which are listed in each of the protocols below.

[0289] B23-B24 bioassay for the intrinsic pathway

[0290] Now turning to Figure 35 and 36 which illustrate the results of activated partial thromboplastin time (APTT) biological assays B23, B24, which are used to measure the single step execution of the intrinsic pathway of coagulation. Specifically, Figure 35 shows the optical density versus time (in seconds) measurements for a first APTT biological assay B23 for a normal control in the method of the present application. Then, Figure 36 shows the optical density versus time (in seconds) measurements for a second APTT biological assay B24 for an abnormal control according to the method of the present application.

[0291] Both bioassays B23, B24 used microparticle diluent 11 having a microparticle 24 with a surface group having a diameter of 110 nm at a concentration of 0.392%. A dry matrix was used as described above with 20 uL of 0.005 M calcium chloride. In another well, 100 uL of APTT-XL reagent was diluted with 150 uL of distilled water. The general method for these bioassays B23-B24 was the same and was prepared as described above in Figure 10 .

[0292] As shown in Figure 35 , for the B23 assay, clotting was seen to begin at about 13 seconds 132 with an OD value of 0.5524 and to end at about 150 seconds 134 with an OD value of 0.9315. The change in absorbance 135 over time is the change in optical density values and the difference in OD values is 0.3791.

[0293] As shown in Figure 36 , for the B24 assay, clotting was seen to begin at about 65 seconds 132 with an OD value of 0.5111 and to end at about 240 seconds 134 with an OD value of 0.9097. The change in absorbance 135 over time is the change in optical density values and the difference in OD values is 0.3986.

[0294] Table 18 below provides the protocol for bioassays B23, B24:

[0295]

[0296] Table 19 shows the optical density data measured at 660 nm using kinetic mode for three minutes for the first bioassay B23 using normal controls and the second bioassay B24 using abnormal controls:

[0297]

[0298]

[0299] B25-B27 bioassay based on a one-stage APTT factor

[0300] In particular, Figure 37 and 38 the results of three bioassays B25, B26 and B27 based on one stage APTT factors are shown in graphical form to measure the activity and level of activity of the intrinsic pathway of coagulation. The first bioassay B25 uses APTT of normal plasma, the second bioassay B26 uses APTT of abnormal plasma with 99.9% factor VIII deficiency and the third bioassay B27 is a one stage factor assay using APTT of normal / abnormal mixed plasma.

[0301] In particular, Figure 37 and 38 The results of biological assays B25, B26, B27 are shown by way of illustration, which employed microparticle diluent 11 having a microparticle 24 concentration of 0.044% microparticles 24 having surface groups with a diameter of 110 nm. The microparticles of the diluent 11 were diluted with water at a ratio of 1 to 180. The optical density of the diluent 11 was measured at 660 nm, which had an optical density value of 0.21. These biological assays further employed a substrate 40 having 0.17 M glycine and 1.0 M sodium chloride at a pH of 10.0.

[0302] Factor XII, XI, IX and VIII can be directly measured using these assay methods. In addition, factor X, V, II are also measured as they participate in the common pathway cascade leading to final clotting. Partial thromboplastin is formed from the sample by addition of calcium and phospholipid, among other common surface activators of factor XII. The single stage factor assays performed according to the method of the present application titrate the individual factor levels of the sample by comparing this level to a standard curve obtained from dilutions of samples known to lack the factor and normal samples.

[0303] The general method of biological assays B25, B26 was prepared generally as discussed above with reference to Figure 10 The one stage factor biological assay B27 used the same general method as discussed in Figure 10 but additionally dried a known amount of single known factor deficient plasma in an extra well 6'" to attenuate the reaction of the sample 60. Using the standard curve provided in the software, the actual reaction level in the sample 60 was measured 116, corrected 118 and reported 119 according to the methods disclosed herein.

[0304] As Figure 37 shown for the B25 assay, clotting can be seen to begin 132 at approximately 30 seconds with an OD value of 0.0340 and to end 134 at approximately 100 seconds with an OD value of 0.3530, the change in absorbance 135 over time is the change in optical density value, the difference in OD value is 0.3190.

[0305] As Figure 38 shown for the B26 assay, clotting can be seen to begin 132 at approximately 100 seconds with an OD value of 0.3140 and to end 134 at approximately 200 seconds with an OD value of 0.8440, the change in absorbance 135 over time is the change in optical density value, the difference in OD value is 0.3986.

[0306] Also asFigure 38 For the B27 assay, coagulation can be seen to start at about 50 seconds 132' with an OD value of 0.1820, and coagulation can be seen to end at about 110 seconds 134' with an OD value of 0.5480, the change in absorbance 135' over time is the change in optical density value, the OD value difference is 0.3660.

[0307] Table 20 below provides the protocol for all three biological assays B25-B27:

[0308]

[0309] Table 21 below shows the corrected optical density results for each of the above biological assays B25-B27 using the kinetic mode at 660 nm over 5 minutes according to the present application:

[0310]

[0311]

[0312] List of figures

[0313] The following reference numbers are used in the description to refer to those reference elements in the attached drawings of the present application.

[0314] 1 Analyzer 9 Main package

[0315] 2 Kit 10 Particulate matrix

[0316] 3 Barcode 11 Particulate diluent

[0317] 4 Sticks 18 Functional group type

[0318] 5 Sterilized pipette 20 Common substance

[0319] 6 Wells 22 Amidine

[0320] 7 Cuvette 24 Sulfate

[0321] 8 Sampler 26 Size

[0322] 28 Bulk density 102 Select kit / biological assay

[0323] 30 Dilution ratio 103 Scan kit with analyzer

[0324] 31 Optical density value 104 Take blood sample

[0325] 32 Quantity 105 Operate stick

[0326] 33 Diluent 106 Take capillary sampler with kit

[0327] 40 matrix 107 contact the sampler with the blood, inject the sampler with the blood

[0328] 42 wet matrix 108 replace the sampler of the cartridge

[0329] 44 dry matrix 109 insert the cartridge into the analyzer

[0330] 46 carbohydrate 110 automated bioassay process steps

[0331] 47 glycine 111 add components

[0332] 48 quantity 112 mix / agitate components

[0333] 50 acidity 113 measure

[0334] 52 sodium chloride 114 incubate

[0335] 54 polyethylene glycol 115 measure

[0336] 56 tween 116 correct

[0337] 60 blood sample 117 report results

[0338] 61 citrated blood 118 adjust

[0339] 62 whole blood 119 mixture

[0340] 63 plasma 120 solution

[0341] 64 mix 121 mixture

[0342] 65 quantity 122 optically detect thrombus formation

[0343] 68 microparticle mixture 123 optically detect hemoglobin levels

[0344] 70 temperature 124 correct clotting time

[0345] 80 reagent / activator 125 start of clotting

[0346] 82 thromboplastin 126 end of clotting

[0347] 84 activated partial thromboplastin time 127 change in absorbance = optical density over time

[0348] 86 thrombin 128 uncorrected prothrombin time

[0349] 88 ellagic acid 129 HGB relationship

[0350] 90 diluent 130 optical density

[0351] 92 calcium chloride 138 corrected PT

[0352] 100 method 140 OD value measured at 530 nm

[0353] 141 predetermined relationship of OD value to hemoglobin 142 specific hemoglobin level

[0354] Conclusion

[0355] While preferred embodiments of the application have been described herein, the above description is illustrative only. Further modifications of the application disclosed herein will occur to those skilled in the art. All modifications are believed to fall within the scope of the application as defined by the following claims.

Claims

1. A disposable bioassay diagnostic kit for monitoring the activity of an anticoagulant, said disposable bioassay diagnostic kit comprising: a first well containing an amount of a substrate, said substrate being a dry substrate or a liquid substrate; a second well containing a plurality of microparticles, wherein said plurality of microparticles is an uncoated latex having at least one surface type; a third well; and a cuvette containing an amount of an activator, said activator being selected from the group consisting of thromboplastin, thrombin, ellagic acid, activated partial thromboplastin time, factor II, factor VII, factor I, factor X, factor XII, activated protein C, snake venom, negatively charged phospholipid, calcium ion, tissue factor, silicon dioxide, kaolin, diatomaceous earth, wherein said cuvette has: - a first side wall configured to facilitate the performance of a first optical detection reading for monitoring the activity of an anticoagulant; and - a second side wall configured to facilitate the performance of a second optical detection reading for monitoring the activity of an anticoagulant; wherein said disposable bioassay diagnostic kit is configured such that during its operation for monitoring the activity of an anticoagulant: - at least part of said substrate, at least part of said plurality of microparticles and a blood sample are first mixed together within said third well; and - at least part of the resulting mixture and at least part of said activator are subsequently mixed together within said cuvette; wherein said first optical detection reading is performed by a first 500-550 nm light emitting diode; and said second optical detection reading is performed by a second 620-700 nm light emitting diode. Said substrate comprises at least one of sodium chloride, polyethylene glycol, a surfactant, a carbohydrate and calcium chloride.

2. The disposable bioassay diagnostic kit according to claim 1, wherein, 3. A bioassay diagnostic kit comprising: a swab, a pipette, a kit, a bioassay component and an optical cuvette, wherein the swab, the pipette, the bioassay component and the optical cuvette are contained and sealed within the kit, the outer surface of said kit further being provided with an identifier; wherein said bioassay component comprises: a substrate; a plurality of microparticles, wherein said plurality of microparticles is an uncoated latex having at least one surface type; and an activator, said activator being selected from the group consisting of thromboplastin, thrombin, ellagic acid, activated partial thromboplastin time, factor II, factor VII, factor I, factor X, factor XII, activated protein C, snake venom, negatively charged phospholipid, calcium ion, tissue factor, silicon dioxide, kaolin, diatomaceous earth; wherein during the operation of said kit of said bioassay diagnostic kit, at least part of said substrate, at least part of said plurality of microparticles and a blood sample are first mixed together within at least one well of said kit, at least part of the resulting mixture being placed within a cuvette of said kit, wherein said cuvette has: - a first side wall configured to facilitate the performance of a first optical detection reading for monitoring the activity of an anticoagulant; and - a second side wall configured to facilitate the performance of a second optical detection reading for monitoring the activity of an anticoagulant; ​ The first optical detection reading is performed using a first 500-550 nm light-emitting diode; and the second optical detection reading is performed using a second 620-700 nm light-emitting diode.

4. A coagulation bioassay device, comprising: A matrix comprising at least one of glycine, sodium chloride, and 1% dimethyl silicone oil; Multiple microparticles suspended in a microparticle matrix, wherein the multiple microparticles are uncoated latex having at least one surface type; as well as A certain amount of activator, wherein the activator is selected from the group consisting of thrombin, thrombin, ellagic acid, activated partial thromboplastin, factor II, factor VII, factor I, factor X, factor XII, activated protein C, snake venom, negatively charged phospholipids, calcium ions, tissue factor, silica, kaolin, and diatomaceous earth. In the coagulation bioassay procedure, at least a portion of the matrix, at least a portion of the plurality of microparticles, and the blood sample are first mixed together in at least one well of a kit containing the matrix, the plurality of microparticles, and the activator. At least a portion of the resulting mixture and at least a portion of the activator are then mixed together in a cuvette of the kit, wherein the cuvette has: - A first sidewall configured to facilitate the execution of an initial optical detection reading for monitoring anticoagulant activity; and - A second sidewall configured to facilitate the execution of a second optical detection reading in order to monitor anticoagulant activity; The first optical detection reading is performed using a first 500-550 nm light-emitting diode; and the second optical detection reading is performed using a second 620-700 nm light-emitting diode.

5. The coagulation bioassay device of claim 4, wherein, The at least one surface type is selected from the group consisting of sulfates, amidines, and carboxylates.

6. The coagulation bioassay device of claim 4, wherein, The matrix is ​​either a dry matrix or a liquid matrix.

7. The coagulation bioassay device of claim 4, wherein, Each of the plurality of microparticles has a diameter of 10 nm to 150 nm.

8. The coagulation bioassay device according to claim 4, wherein each of the plurality of microparticles has a diameter of 90 nm to 110 nm.

9. The coagulation bioassay device of claim 4, wherein the plurality of microparticles are in a solution selected from the group consisting of: 0.006% per volume weight, 0.01% per volume weight, and 0.08% per volume weight.

10. A method for obtaining a coagulation time measurement using diluted, lysed whole blood or plasma, comprising the steps of: A matrix and a plurality of microparticles are mixed together in at least one well of a kit, the kit comprising the matrix and the plurality of microparticles, wherein the plurality of microparticles are uncoated latex having at least one surface type; The resulting mixture is used as a reagent in combination with one of the diluted, lysed whole blood or plasma. A separate activator is added to the mixture to activate a natural coagulation substrate in diluted, lysed whole blood or plasma; obtaining a clot time measurement of the diluted, lysed whole blood or one of plasma by taking a first optical detection reading through a first side wall of a cuvette of the kit; determining a hemoglobin level of the diluted, lysed whole blood or one of plasma by taking a second optical detection reading through a second side wall of the cuvette of the kit; and correcting the clot time measurement by adjusting the hemoglobin level of the diluted, lysed whole blood or one of plasma; wherein the activator is selected from the group consisting of thromboplastin, thrombin, ellagic acid, activated partial thromboplastin time, factor II, factor VII, factor I, factor X, factor XII, activated protein C, snake venom, negatively charged phospholipid, calcium ion, tissue factor, silica, kaolin, and celite; wherein the first optical detection reading is taken by a first 500-550 nm light emitting diode and the second optical detection reading is taken by a second 620-700 nm light emitting diode.

11. The method of claim 10, wherein, obtaining a clot time measurement of the diluted, lysed whole blood or one of plasma includes: repeatedly measuring the optical density of the diluted, lysed whole blood or one of plasma at a first wavelength over a period of time.

12. The method of claim 11, wherein, determining a hemoglobin level of the diluted, lysed whole blood or one of plasma includes: measuring the optical density of the diluted, lysed whole blood or one of plasma at a second wavelength.

13. The method of claim 12, wherein, the first wavelength ranges from 620 nm to 700 nm and the second wavelength ranges from 500 nm to 550 nm.

14. The method of claim 10, wherein, obtaining a clot time measurement of the diluted, lysed whole blood or one of plasma includes: obtaining a difference in optical density value of at least 0.

08.

15. The method of claim 10, wherein, the matrix is a liquid matrix and includes 0.17 M glycine, 1.29 M sodium chloride, and 1% dimethyl silicone oil diluted with injectable grade purified water at a pH of 10.0.

Images