Coagulation assay device and method

Abstract

To provide a method and device for determining the activity of coagulation factors in diluted capillary whole blood, citrated whole blood, and citrated plasma.SOLUTION: Latex microparticles that are not reacted, exposed, or coupled with proteins are used. These unreacted particles retain the bioactive substance adsorption properties when they are either dried in a carbohydrate matrix or used in a liquid reagent system. When in use, the particles can adsorb biomolecules. This action enhances and improves the reactivity of the particle surface to anticoagulants contained in whole blood and plasma, particularly when used in diluted samples / diluent environments. The process involves presenting a mixture of reactive proteins and particles for various activators used in coagulation assays. The latex microparticles are utilized in a turbidity measurement type bioassay of the present invention in which the typical optical properties of the sample solution remain clear without turbidity.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] 1. Field of the Invention The present invention relates generally to methods and devices for determining the activity of clotting factors in diluted capillary whole blood, citrated whole blood, and citrated plasma. [Background technology]

[0002] 2. Description of the Prior Art Methods and devices for determining clotting factor activity in diluted capillary whole blood, citrated whole blood, and citrated plasma frequently involve physicians measuring a patient's international normalized ratio (INR) level during a prothrombin time (PT) test. This type of bioanalysis is designed to measure how long it takes a patient's blood to clot. This test ensures that the patient is receiving the dosage and type of medication that will prevent blood clots from forming and causing deep vein thrombosis (DVT), pulmonary embolism (PE), stroke, heart attack, and more. Such medications work by blocking the formation of vitamin K-dependent clotting factors, substances in the blood that cause clotting. If the INR score is too low, the patient may be at risk for blood clots. However, if the INR is too high, the patient may experience bleeding. A typical INR score ranges between 2 and 3. The "ideal" INR score may vary between patients.

[0003] How frequently a patient should be tested can vary depending on how stable their INR is over time. According to the American Heart Association (AHA), patients should be tested at least monthly, and in some cases as often as twice weekly. This testing involves frequently drawing blood and sending it in for analysis by an in vitro diagnostic analyzer.

[0004] In vitro diagnostic analyzers have been available for several decades. The market for these types of analyzers has typically been for use in central laboratories. Central laboratories have typically been able to test for a wide variety of biomedical species in a patient's blood and / or blood plasma. Recently, there appears to be an ongoing shift for such testing from central laboratory testing to point-of-care sites within hospitals. This shift allows for more rapid test data results, which can be important in the diagnosis and treatment of certain conditions.

[0005] Point-of-care testing plays a vital 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 staff. To meet this demand, manufacturers have had to miniaturize analyzers and simplify testing procedures so that only minimal training is required to perform the procedure.

[0006] One key feature common to all point-of-care analyzers is that they must be either handheld and / or portable. Examples of such point-of-care analyzers include, but are not limited to, the OptiCCA and Omni9 critical care analyzers from Roche Diagnostics, a division of Hoffmann-La Roche, the Stat Profile Ultra-C from Nova Biomedical Corporation, the CRT from Nova Biomedical Corporation, and the Dimension RxL from Dade Behring, a division of Siemens Healthcare Diagnostics.

[0007] More recently, there has been a further shift toward testing in the doctor's office or in a laboratory located at the doctor's office. As testing moves away from central laboratories, new single-use medical devices are being developed to meet this need.

[0008] In a clinic environment, there are numerous devices that utilize capillaries to collect fingerstick samples for analysis. The capillaries can be either glass or plastic. Typical analyses are for species such as HbA1c, lipids, etc. Once the sample is collected, these capillary-based collection devices are loaded into an analytical cartridge, which is then loaded into an instrument for analysis. At this point, two known bioassays used for diagnostic purposes will be discussed with reference to their original patent documents.

[0009] First, Patent Document 1 discloses stabilized beads comprising latex particles having carboxylic acid groups and a stabilizer functionally coupled to the latex particles, which can completely or substantially prevent degradation or inactivation of a diagnostic agent in close proximity to the stabilizer. The stabilized beads may further comprise at least one of human serum albumin (HSA), bovine serum albumin (BSA), or a linker group coupled to the latex particles.

[0010] Second, Patent Document 2 discloses a method and test kit for performing a simple detection assay for the fibrin breakdown product D-dimer. This utilizes purified fragment E of human fibrinogen attached to a solid phase for direct chemical binding of D-dimer from biological samples. Fragment E can be conjugated to latex carrier particles, and an agglutination assay can be performed. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] US Patent Application Publication No. 2011 / 0196085 [Patent Document 2] European Patent No. 0655627 [Patent Document 3] U.S. Patent No. 10,117,615 Summary of the Invention [Problem to be solved by the invention]

[0012] Advantages and Differences of the Present Invention over Known Prior Art The above-mentioned portions of the prior art have not proven fully satisfactory for meeting all of the industry's requirements. In dehydrated cells, inaccuracies have been found to be associated with automated measurements of mean cellular hemoglobin concentration. In clotting, additional mixing and hydration time, etc., can induce uncontrolled procoagulant factor activation. Utilizing a dry, liquid, or dry and liquid reagent approach in accordance with the present invention provides greatly increased capacity for particle and reagent assays compared to prior art methods. [Means for solving the problem]

[0013] To enhance the capabilities of immunodiagnostics, which have been achieved with varying degrees of success prior to this invention, the present invention utilizes a carbohydrate matrix to preserve the functionality of certain microparticles. Specifically, the present invention utilizes latex microparticles that have not been reacted, exposed, or coupled to proteins. These unreacted particles retain their bioactive substance adsorption properties when dried in the carbohydrate matrix or used in a liquid reagent system. During use, the particles can adsorb biomolecules. This action favors and enhances the reactivity of the particle surface with procoagulant factors contained in whole blood and plasma, especially when used in a dilute sample / diluent environment. This process involves the presentation of protein and particle mixtures reactive to various activating factors used in clotting assays. The latex microparticles are used in the turbidimetric bioassays of the present invention, in which the typical optical characteristics of the sample solution are clear rather than turbid.

[0014] The present invention addresses the failures of the prior art by providing a novel application of modified methodology to diluted lysed whole blood samples or plasma matrices, where latex particles provide a method for clot detection. Such a method allows binding and adsorption applications to be applied in clinical testing settings, reducing manufacturing time, cost, and, at the end-user stage, incubation reaction time and device footprint or size. Both intact latex microparticles and particles with surface groups such as sulfate or amidine are effective for the present invention. Appropriate buffer and temperature conditions are also required for each microparticle type to work in a diluted blood assay scheme.

[0015] Together, the biomolecules, microparticles, specialized buffers, and assay temperatures are suitable for rapid protein absorption for use in rapid clinical assays. Retaining this property is paramount for functionality. In addition, particle dispersion also plays a major role. Uniform dispersion in solution allows for rapid reaction and consistent analytical quantification of agglutination. Thus, retaining both of these properties is essential for the development of any reliable test component.

[0016] The present invention provides a process methodology for drying uncoated, unconjugated, protein-free latex microparticles of polystyrene, either plain or bearing surface functional groups. The dispersed particles allow for the adsorption of quantifiable analytical protein biomarkers, which are then available for ligand attachment. In particular, the carbohydrate matrix supports the adsorption of biomarker proteins while simultaneously allowing rapid dissolution and uniform dispersion of the particles.

[0017] It is an object of the present invention to provide a liquid microparticle reagent for ease of use in certain bioassays. It is another object of the present invention to provide a methodology for the use of dry or liquid latex microparticles in coagulation diagnostic products. It is a further object of the present invention to provide a methodology for simultaneous hemoglobin detection and quantitative correction of clotting time values.

[0018] It is yet another aim of the present invention to provide a new method for evaluation of the extrinsic coagulation pathway and monitoring oral anticoagulant therapy (OAT).

[0019] The methods of the present invention provide assays for whole blood or plasma clotting time, along with hemoglobin measurement and correction for whole blood samples. Various embodiments use adjustments to dilution levels, temperature, particle type, and buffer components to vary the overall bioassay time. These embodiments use flexible dry, liquid, or dry and liquid matrices in conjunction with specific microparticles. However, it should be understood that all assay components can be adjusted to provide the most representative timing scheme for coagulation bioassays according to the methods of the present invention. Overall, the final whole blood or plasma sample dilution ratio should be in the range of 1 part to 50 to 75 for coagulation assays performed between 33°C and 38°C.

[0020] The present invention achieves these and other goals by providing a disposable bioassay diagnostic cartridge for monitoring anticoagulant activity. The disposable cartridge can have a first well containing a quantity of matrix (either dry or liquid matrix) and a second well containing microparticles. The microparticles can be of at least one surface type: unreacted plain (textile), uncoated latex with sulfate, carboxylate, and amidine groups that retain activity. The cartridge can further have a third well containing a quantity of an activator, which can be thromboplastin, 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, or celite.

[0021] The matrix may be a liquid carbohydrate matrix and / or a dry matrix having at least one of NaCl, PEG, TWEEN®, and CaCl2. The disposable cartridge may have an integrated cuvette capable of supporting dual optical detection readings. The integrated cuvette may have a first wall capable of supporting a first optical detection reading with a first LED at 530 nm; and a second wall capable of supporting a second optical detection reading with a second LED at 660 nm.

[0022] The present invention achieves another goal by providing an all-inclusive coagulation bioassay diagnostic kit with all required components, with the exception of the analyzer itself. Such a kit may include a fingerstick, a pipette, bioassay components, and an optical cuvette. To ensure hygienic delivery to the user, the fingerstick, pipette, bioassay components, and optical cuvette may be contained in a sanitary and sealed container bearing an identifier, such as a barcode, that can be scanned by the analyzer. The bioassay components may have a matrix and microparticles, where the microparticles may be uncoated latex having at least one surface type selected from the group consisting of unreacted plain, sulfate, carboxylate, and amidine chemical structures that retain activity.

[0023] Further coagulation bioassays according to the present invention can include a carbohydrate matrix and microparticles in the carbohydrate matrix. The bioassay can also include 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 phospholipids, calcium ions, tissue factor, silica, kaolin, and celite. The matrix of the bioassay can be either a dry matrix or a liquid matrix, and the carbohydrate matrix can include Maltrin 250, sucrose, or isomalt. The microparticles of this type of bioassay can have a diameter of approximately 10 nm to 150 nm and can be in a 1%, 2%, 4%, 8%, or 10% weight by volume (%W / V) solution.

[0024] Using the above bioassay, the present invention provides a method for obtaining clotting time measurements for any of the following blood sample types: diluted lysed whole blood, whole blood (straight from a fingerstick), plasma, citrated blood, and / or mixed blood and plasma. Such a method would then include the steps of selecting a microparticle matrix 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 carbohydrate matrix when dry or liquid, preferably selected from the group consisting of unreacted plain, sulfate, carboxylate, and amidine chemical structures that retain activity. This microparticle matrix can then be applied to a blood sample as a reagent; a clotting time measurement of diluted lysed whole blood or plasma can then be obtained by optically detecting INR. Alternatively, a separate reagent can also be added to the reaction mixture to activate natural clotting substrates in the blood sample. The separate reagent will preferably be 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 phospholipids, calcium ions, tissue factor, silica, Kaolin®, or Celite.

[0025] Then, by simultaneously obtaining optical density readings at two different wavelengths, the clotting time measurement can be corrected for the hemoglobin concentration of the sample. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is an illustration of the method of the present invention. [Figure 1a] FIG. 1a is an illustration of a user selecting a bioassay cartridge kit of the present invention for use with a scanner as shown. [Figure 2]FIG. 2 is an illustration of a user scanning an identifier on a cartridge of the present invention. [Figure 3] FIG. 3 is an illustration of a user using a fingerstick to obtain a blood sample according to the method of the present invention. [Figure 4] FIG. 4 is an illustration of a user removing a sampler from a cartridge of the present invention. [Figure 5] FIG. 5 is an illustration of a user loading a blood sample into a sampler from a cartridge of the present invention. [Figure 6] FIG. 6 is an illustration of a user returning a loaded sampler with a blood sample to a cartridge of the present invention. [Figure 7] FIG. 7 is an illustration of a user loading a cartridge of the present invention with a loaded sampler into an analytical device. [Figure 8] FIG. 8 is a cross-sectional image of a cartridge according to one embodiment of the present invention. [Figure 9] FIG. 9 is a cross-sectional image of a cartridge according to a further embodiment of the present invention. [Figure 10] FIG. 10 is a diagram of the automated steps according to the method of one embodiment of the present invention. [Figure 11] FIG. 11 is a schematic diagram of a further discussion of one step of the method shown in FIG. [Figure 12] FIG. 12 is a diagram of a further discussion of the steps of the method shown in FIG. [Figure 13] FIG. 13 is a schematic diagram of a further discussion of another step of the method shown in FIG. [Figure 14] FIG. 14 is a graph illustrating the effect of hemoglobin on prothrombin time versus INR values, with and without correction according to the present method. [Figure 15] FIG. 15 is a graph illustrating the effect of hemoglobin on delta prothrombin time versus INR values, with and without correction according to the present method. [Figure 16]FIG. 16 is a graph illustrating a bioassay using microparticles with and without bovine serum albumin (BSA) surface groups against a standard sample. [Figure 17] FIG. 17 is a graph illustrating a bioassay using microparticles with and without bovine serum albumin (BSA) surface groups on abnormal samples. [Figure 18] FIG. 18 is a graph illustrating the results of a prothrombin time assay of a first embodiment using a first amidine particle dilution and a first matrix by measuring optical density at 660 nm versus time. [Figure 19] FIG. 19 is a graph illustrating the results of a prothrombin time assay of the second embodiment using a first sulfate particle dilution and a first matrix by measuring optical density at 660 nm versus time. [Figure 20] FIG. 20 is a graph illustrating the results of a prothrombin time assay of the second embodiment using a first surface-free microparticle dilution and a first matrix by measuring optical density at 660 nm versus time. [Figure 21] FIG. 21 is a graph illustrating the results of a prothrombin time assay of the fourth embodiment using a second amidine particle dilution and a second matrix by measuring optical density at 660 nm versus time. [Figure 22] FIG. 22 is a graph illustrating the results of a prothrombin time assay of the fifth embodiment using a second sulfate particle dilution and a second matrix by measuring optical density at 660 nm versus time. [Figure 23] FIG. 23 is a graph illustrating the results of a prothrombin time assay of the sixth embodiment using a second surface-free microparticle dilution and a second matrix by measuring optical density at 660 nm versus time. [Figure 24]FIG. 24 is a graph illustrating the results of a bioassay using sulfate microparticle dilution according to the present invention on standard whole blood by measuring optical density at 660 nm versus time. [Figure 25] FIG. 25 is a graph illustrating the results of a bioassay using sulfate microparticle dilution according to the present invention on abnormal whole blood by measuring optical density at 660 nm versus time. [Figure 26] FIG. 26 is a graph illustrating the determination of the level of hemoglobin present in the bioassay of FIG. 25 in accordance with the present invention by measuring the optical density at 520 nm. [Figure 27] FIG. 27 is a graph illustrating the correction of the raw prothrombin time calculated in FIG. 25 as a result of adjustment by the determined hemoglobin value from FIG. [Figure 28] FIG. 28 is a graph illustrating the determination of the bioassay INR values ​​from FIG. 25 based on the adjusted prothrombin times provided in FIG. [Figure 29] FIG. 29 is a graph illustrating the results of a bioassay using carboxyl microparticle dilutions according to the present invention on standard whole blood by measuring optical density at 660 nm versus time. [Figure 30] FIG. 30 is a graph illustrating the results of a bioassay using carboxyl microparticle dilutions according to the present invention on abnormal whole blood by measuring optical density at 660 nm versus time. [Figure 31] FIG. 31 is a graph illustrating the results of a prothrombin time assay of two embodiments of the present invention using different temperatures by measuring optical density at 660 nm versus time. [Figure 32] FIG. 32 is a graph illustrating the results of a prothrombin time assay of two embodiments of the present invention using different temperatures by measuring optical density at 660 nm versus time. [Figure 33]FIG. 33 is a graph illustrating and comparing the results of a standard blood bioassay to a Coumadin present blood bioassay by measuring optical density at 660 nm versus time. [Figure 34] FIG. 34 is a graph illustrating and comparing the results of a thrombin time bioassay with standard citrated blood versus standard citrated plasma by measuring optical density at 660 nm versus time. [Figure 35] FIG. 35 is a graph illustrating the results of a standard control of an activated partial thromboplastin time bioassay. [Figure 36] FIG. 36 is a graph illustrating the results of an activated partial thromboplastin time bioassay with abnormal controls for comparison. [Figure 37] FIG. 37 is a graph illustrating the results of a bioassay for activated partial thromboplastin time by a standard plasma-based one-stage factor VIII assay by measuring optical density at 660 nm versus time. [Figure 38] Figure 38 is a graph illustrating and comparing the results of an activated partial thromboplastin time bioassay with a one-stage factor assay for Factor VIII by measuring optical density at 660 nm versus time. Abnormal plasma APTT and one-stage mixing of APTT are compared. DETAILED DESCRIPTION OF THE INVENTION

[0027] Preferred embodiments of the present invention are discussed with reference to Figures 1 through 38. As discussed above, the present invention provides processes, methodologies, systems, and devices relating to methods and devices for determining the activity of clotting factors in diluted capillary whole blood, citrated whole blood, and citrated plasma.

[0028] Overview At this point, an overview of the overall method 100, system cartridge 2, and manual and automated portions of the bioassay will be discussed from various perspectives with reference to the indicated Figures 1-12.

[0029] Overview of the method from the user's perspective At this point, an overview of the user's perspective will be discussed with reference to Figures 1-7. As shown therein, initially, a bioassay cartridge or cartridge kit is selected by the user 102. After manually selecting a bioassay cartridge 102, the cartridge may be scanned by the analyzer to identify the selected bioassay 103. Obtaining a blood sample 104 may then involve a simple fingerstick 105 to provide the required blood sample size. The ease with which a blood sample can be obtained at this step demonstrates one of the benefits of the system of the present invention over other prior art systems and methods that require venipuncture or other collection of large blood sample sizes.

[0030] If the bioassay is initially identified, the capillary sampler can be removed from the cartridge 106, filled with a blood sample 107, and returned to the designated cartridge 108, all within five seconds of performing the fingerstick 105. After returning the filled sampler to the cartridge 108, the identifier is scanned by the analyzer 103, and the cartridge can be inserted into the analyzer 109. After loading the cartridge into the analyzer, the automated process begins 110. Then, depending on the selected bioassay, the automated process will involve the automated steps of adding 112, mixing 114, measuring 116, incubating 117, correcting 118, and reporting the results to the user or other designated person 119, according to a predetermined sequence of bioassay components.

[0031] Depending on the system involved and the purpose of the bioassay, the automated process 110 may include a further step 120 of automatically adjusting the amount of drug prescribed and / or the amount of drug provided by a built-in drug delivery system (not shown).

[0032] FIG. 1A shows an illustration of a cartridge kit after user selection 102. The cartridge kit includes a pre-packaged bioassay cartridge 2 and fingerstick 4 for single use in an analyzer system 1. The cartridge kit also preferably includes a sterile pipette 5, a sampler 8, and a cuvette 7, which are discussed in more detail below with respect to FIGS. 8-9. An exemplary analyzer system 1 capable of performing the bioassays described herein is the ALLEGRO® analyzer by Nova Biomedical Corporation. An exemplary sampler and cartridge base that can be used with the cartridge 2 described herein are described in more detail in U.S. Patent No. 6,249,397 to Nova Biomedical Corporation.

[0033] Turning now to Figures 2-7, which further illustrate the use of certain components of the cartridge kit from a user's perspective. Specifically, Figure 2 illustrates a user having an analyzer 1 identify a bioassay method by scanning a selected cartridge 2 of the present invention. Then, Figure 3 illustrates a user gaining access to a blood sample 60 by using a fingerstick 4 provided with the cartridge kit. Then, Figure 4 shows a user removing a sampler 8 from the cartridge 2. Next, Figure 5 shows a user loading the sampler 8 with the blood sample 60 accessed by the fingerstick 4. Thereafter, Figure 6 illustrates a user returning the loaded sampler 8, along with the blood sample 60 obtained from the fingerstick 4, to the cartridge 2 of the present invention. Finally, Figure 7 shows a user loading the cartridge 2 with the loaded sampler 8 into the analyzer 1, thereby initializing the selected and identified bioassay. By providing these components as a combined kit in a single package 9, the present invention both reduces overall procedure time and reduces user error.

[0034] Bioassay Cartridge Overview An exemplary cartridge kit that can be used in the systems and methodologies of the present invention can include a self-contained, single-use, disposable, integrated bioassay cartridge 2 as now described with further reference to FIGS.

[0035] The first cartridge 2 embodiment illustrated in Figure 8 is prepared with bioassay components for only a single type of bioassay according to one embodiment of the present invention. Alternatively, the second cartridge embodiment illustrated in Figure 9 is a multi-purpose cartridge 2' pre-loaded with multiple components for at least two types of bioassays according to a further embodiment of the present invention.

[0036] Both Figures 8 and 9 illustrate a cartridge 2 having an identifier 3, such as a barcode, that can identify a particular type of cartridge (and therefore bioassay) to an analytical device 1. This identifier 3 may be visible on a visible exterior surface of the cartridge 2 or on the exterior surface of a main package 9. This package 9 may contain the cartridge 2, a fingerstick 4, a sterile pipette 5, a sampler 8, and a cuvette 7. Both Figures 8 and 9 illustrate a cartridge 2 in which the capillary sampler 8 is a removable component of the cartridge 2 itself. Also, both Figures 8 and 9 illustrate a cartridge 2 with an integral cuvette 7 having sidewalls that can facilitate optical measurements. However, it is also contemplated that the package 9 may contain cartridges that require a separate, different cuvette 7 to be provided in addition to the cartridge.

[0037] 8 and 9 also both illustrate a cartridge 2 having a series of wells 6 that are preloaded with components of an assay according to the methods of the present invention. In both types of cartridges, a selection stage 102 (discussed above with reference to FIGS. 1-7) involves selecting the desired bioassay cartridge. After selecting the desired cartridge / assay 102, the cartridge is scanned 103, filled 107, and then inserted 109 into the system.

[0038] From the user's perspective, the only difference between using the first and second types of cartridges begins at this point. With the first cartridge, the user can then manually select an automated process option 110 by selecting one of several bioassay options via manual user input from the analyzer's access control panel. Meanwhile, with the second cartridge type, cartridge insertion 109 alone is sufficient to trigger the automated process 110. With this second cartridge type, from the user's perspective, the bioassay cartridge alone is "selected." However, before the user can select a bioassay option, the bioassay cartridge must first be prepared, and prior to preparation, the bioassay components themselves must first be selected. With further reference to Figures 10-13 and initially to Figure 1, several bioassay cartridge component options are further discussed below.

[0039] Consider now more specifically elements of the cartridge 2 that are not necessarily observed by the user once the sampler 8 has been filled and placed back into the cartridge 2 107. As can be seen in Figures 8 and 9 (although not numbered), the sampler 8 has a capillary element that is inserted through a corresponding capillary receiving hole in the upper surface of the stepped protrusion of the cover protrusion of the disposable test cartridge 2 and then secured to the stepped protrusion.

[0040] During the insertion and set process, the capillary tube of the sampler 8 is inserted through a hole in the lower section located at the top end of the capillary wiper. Because the cross-sectional area of ​​the hole in the lower section is smaller than the cross-sectional area of ​​the capillary tube, the hole in the lower section acts like a squeegee on the outer surface of the capillary tube, preventing any sample inadvertently placed on the outer surface of the capillary tube from entering and depositing in the chamber 6 of the cartridge 2.

[0041] Also, to aid in accuracy, the capillary wipers of these cartridges 2 remove any sample 60 from the outer surface of the capillary tubes, thereby preventing erroneous results from "overfilling" the appropriate wells 6' of the test cartridge 2 with sample 60. Similarly, because the capillary tubes are not wiped by the user, there is little or no likelihood of inadvertently removing any sample 60 within the capillary tubes, which could lead to erroneous results from "underfilling" the wells 6' of the test 2 cartridge with sample 60.

[0042] Automated Process Overview The cartridge 2 is then inserted into the point-of-care analyzer 1 for the automated testing portion 110 of the blood sample 60. At this point, this will be discussed further with reference to Figure 10. Once inside the analyzer 1, the automated arm of the analyzer uncovers the cartridge 2 by unlocking the releasable tabs, with the cover and sample 60 still seated within with the capillary sampler 8.

[0043] The analyzer then uses a small tip on the cartridge cover to pierce the seal of each of wells 6, 6', 6'', etc. The seals on this cartridge 2 can be foil seals or other covers, as long as they are capable of preventing cross-contamination of the contents of the wells during transport. These seals should also be capable of reducing uncontrolled water vapor dilution and evaporation.

[0044] The automated arm then removes the pipette tip 5 from the first well 6. The pipette tip 5 is then used by the analytical device 1 to transfer the sample and other bioassay components to the appropriate wells for mixing according to the bioassay methods described herein.

[0045] If a dry microparticle matrix 11 is used, the pipette 5 draws additional diluent 90 from the fourth well 6''' and adds it to the microparticles 11 in the third well 6'' according to the protocol, where mixing and agitation occurs to form diluted particles 11. Then, regardless of what type of matrix is ​​used, the pipette tip 5 draws a selected amount of the (now diluted) microparticles 11 from the third well 6'' into the second well 6'.

[0046] The cover of the cartridge 2 is then placed back onto the cartridge and the pipettor engages the capillary sampler 8, which adds 112 the sample 60 to the second well 6' where the diluted selected microparticles 11 await. The arm of the analyzer 1 then removes the cover of the cartridge 2 again, and the sample 60 and diluted selected particles 11 are then mixed 114 by pipetting up and down for a first predetermined time, forming a mixture 121 of microparticles 11 and sample 60.

[0047] The pipette tip 5 then draws up a specified amount of the selected formed matrix 40 from the fifth well 6'''' and then, in a second addition step 112, adds the matrix 40 to the second well 6', which already contains a mixture 121 of sample 60 and diluted microparticles 11. The matrix 40 and mixture 121 are then mixed 114, for example by pipetting up and down, in the second well 6' for a second predetermined time period to form a solution 122 (having matrix 40, sample 60, and microparticles 11).

[0048] The pipette 5 then draws 112 a predetermined amount of solution 122 (containing matrix 40, sample 60, and microparticles 11) from the second well 6' and adds this solution 122 to the seventh well 7 of the cartridge 2 containing the selected reagent 80. The solution 122 (microparticles, matrix, sample) and reagent 80 are then mixed for a third predetermined time to form a co-mixture 123 (now containing matrix 40, sample 60, microparticles 11, and reagent 80).

[0049] 8 and 9, this seventh well 7 is an integral optical detection cuvette 7 having walls of sufficient transparency, viscosity, and thickness to ensure that optical detection can occur at this point without further movement of the co-mixture 123. The optical density of the co-mixture 123 will then be measured 112, 112' at least twice, as discussed in more detail further below.

[0050] To begin the first measurement 112, a first light emitting device (LED) of the analyzer 1 is turned on and light from the first LED at a wavelength of 660 nm is transmitted through a first wall of the integrated cuvette 7. The light is then transmitted through the co-mixture 123 and a second wall of the integrated cuvette 7. The light is then detected by a photodetector of the analyzer. Successive readings are collected over a predetermined amount of time to provide first coagulation data 124.

[0051] In the second measurement 112', a second light-emitting device (LED) of the analyzer 1 is turned on, and light from the second LED at a second wavelength of 530 nm is transmitted through a first wall of the integrated cuvette 7. The light is then transmitted through the co-mixture 123 and again through a second wall of the integrated cuvette 7. The light is then collected by a photodetector of the analyzer. Only a single reading is required to provide hemoglobin level data 125. Although these measurements are referred to as first and second, it should be recognized that the order can be reversed. Alternatively, instead of providing the measurements sequentially, they can be provided contemporaneously or even partially simultaneously.

[0052] As will be discussed further below, the results from the optical detection 116, 116' are then used 118 to correct the clotting time results 126. It is these corrected results 126 that are then reported to the user.

[0053] Overview of Bioassay Components The present invention provides a bioassay cartridge 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 cartridge formation, while in other embodiments, the microparticle matrix 10 is formed after addition 112 of the blood sample 60 during the bioassay process 110.

[0054] Microparticle matrix 10 contains microparticles 11, which can be plain (textile) or uncoated, unconjugated, protein-free latex on polystyrene microparticles with surface functionalized groups. Matrix 40 is generally composed of carbohydrate 46, which aids in the adsorption of biomarker proteins while simultaneously allowing for rapid dissolution and uniform dispersion of microparticles 11. The dispersed nature of microparticles 11 in matrix 40 allows for the adsorption of quantifiable analytical protein biomarkers, which are then available for ligand attachment.

[0055] Microparticles 11 used in the present invention include polystyrene microparticles 18 having surface active groups such as amidine 22 and sulfate 24. Microparticles 11 of the present invention have diameter sizes 26 ranging from 20 nm to 800 nm or more. A preferred range of particle diameters 26 is 40 nm to 150 nm, with the most preferred diameters 26 ranging from 75 to 125 nm.

[0056] Amidine Microparticles One embodiment of the present invention uses amidine particles 22 having a diameter 26 of 95 nm at a dilution 11 of 0.080 weight to volume % (% W / V) 28. The optical density 31 of dilution 11 was measured at 660 nm to have an optical density value of 0.19.

[0057] The amidine microparticles 22 of the present invention have diameter sizes 26 ranging from 20 nm to 800 nm or more. A preferred range of particle diameters 26 is 40 nm to 150 nm, with more preferred diameters 26 ranging from 75 to 125 nm; most preferred diameters are in the range of 90 nm to 98 nm. The amidine microparticles 22 have dilutions 11 ranging from 0.006% weight by volume (%W / V) to 8% weight by volume (%W / V), more preferably from 0.010% weight by volume (%W / V) to 0.20% weight by volume (%W / V), and most preferably 0.080% weight by volume (%W / V).

[0058] The present bioassay method using amidine latex particles 22 requires a dilution 11 having a total dilution ratio 30 of microparticles 22 to dihydrogen monoxide ranging from 1:10 to 1:400; more preferably ranging from 1:20 to 1:150.

[0059] Amidine latex particles 22 that can fulfill the goals of the bioassay method of the present invention when prepared according to the methods of the present invention discussed herein can include high activity latex beads provided by Invitrogen®.

[0060] Sulfate Microparticles Another embodiment of the present invention uses sulfuric acid microparticles 24 having a diameter of 110 nm at a 0.044 weight to volume % (% W / V) dilution. The optical density of dilution 11 was measured at 660 nm to be 0.21.

[0061] Other sulfate microparticles 24 of the present invention have diameter sizes 26 ranging from 20 nm to 800 nm or more. A preferred range of sulfate particle 24 diameters 26 is 40 nm to 150 nm, with a more preferred diameter 26 range of 75 to 125 nm; and a most preferred diameter 26 range of 90 nm to 110 nm.

[0062] According to some embodiments, the sulfate microparticles 24 have a dilution 11 ranging from 0.001% weight by volume (%W / V) to 12% weight by volume (%W / V), more preferably from 0.01% weight by volume (%W / V) to 8% weight by volume (%W / V), and most preferably 0.016% weight by volume (%W / V).

[0063] The present bioassay method using sulfate microparticles 24 requires a dilution ratio 30 of microparticle 24 to dihydrogen monoxide ranging from 1:50 to 1:2000; more preferably, having a ratio ranging from 1:100 to 1:1000; most preferably, a ratio of 1:500.

[0064] Sulfate latex particles 24 that can achieve the goals of the present invention when prepared according to the methods of the present invention discussed herein can include high activity latex beads available from Invitrogen®.

[0065] Surface-free microparticles Another embodiment of the present invention uses surface-free microparticles 20 having a diameter 26 of 96 nm in a dilution 11 having a weight to volume % (% W / V) 28 of 0.067. This dilution has a dilution ratio 30 of 1 to 150. The optical density 31 of dilution 11 was measured at 660 nm to be a value of 0.21.

[0066] Other surface-free microparticles 20 of the present invention have diameter sizes 26 ranging from 20 nm to 800 nm or more. The surface-free microparticles 20 have a preferred range of diameters 26 of 40 nm to 150 nm, with more preferred diameters 26 ranging from 75 to 125 nm; and most preferred diameters 26 ranging from 90 nm to 110 nm. According to some embodiments, the surface-free microparticles 20 have a dilution 11 ranging from 0.001% weight by volume (%W / V) to 2% weight by volume (%W / V), more preferably from 0.01% weight by volume (%W / V) to 0.2% weight by volume (%W / V), and most preferably 0.016% weight by volume (%W / V).

[0067] 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; more preferably in the range of 1:100 to 1:1000.

[0068] Surface-free particles that may meet the requirements of the microparticles of the present invention when prepared according to the methods of the present invention discussed herein may include plain microparticles provided by Varian Labs®.

[0069] Carboxyl Microparticles Another embodiment of the present invention uses carboxyl latex microparticles having a diameter of 103 nm in a 0.016% weight to volume (% W / V) solution. The optical density of dilution 11 was measured at 660 nm to be 0.08.

[0070] Other carboxyl microparticles 25 of the present invention have diameter sizes 26 ranging from 20 nm to 800 nm or more. A preferred range of carboxyl particle 25 diameters 26 is 40 nm to 150 nm, with more preferred diameters 26 ranging from 75 nm to 125 nm; most preferred diameters are in the range of 90 nm to 110 nm.

[0071] According to some embodiments, the carboxyl microparticles 24 are in a solution 28 having a concentration ranging from 0.001% weight by volume (%W / V) to 2% weight by volume (%W / V), more preferably from 0.005% weight by volume (%W / V) to 0.1% weight by volume (%W / V), and most preferably 0.016% weight by volume (%W / V).

[0072] The present bioassay method using carboxyl microparticles 25 requires a dilution 11 having a dilution ratio 30 of microparticles 25 to diluent 33 in the range of 1:50 to 1:2000; more preferably in the range of 1:100 to 1:1000.

[0073] Carboxyl latex particles 24 that can achieve the goals of the present invention when prepared according to the methods of the present invention discussed herein can include high activity latex beads available from Invitrogen®.

[0074] Dry Matrix Generally, the bioassay methods of the present invention can utilize most latex particle suspensions with plain or functionalized surfaces. Some embodiments use a matrix with a reagent attenuator, such as a surfactant. Two such reagent attenuators that have been tested include polysorbate-type nonionic surfactants and octylphenol ethoxylate surfactants. Polysorbate-type nonionic and octylphenol ethoxylate surfactants that can meet the goals of the present invention when prepared by the methods of the present invention discussed herein can include surfactants provided by the Tween® and Triton® families.

[0075] Some dry matrices according to the present invention include carbohydrates, carbohydrate derivatives, and mixtures that create an environment that protects the particles from harmful temperatures and allows for rapid rehydration and uniform dispersal upon addition of fluids such as buffers, diluted samples, or other fluid reagents.

[0076] Carbohydrates and their derivatives are preferred compounds used to dry particles and provide stabilization during the drying process for the bioassay methods discussed herein. These reagents are prepared with water. However, low molarity buffers are also used in other embodiments, examples of which include glycine and bicine. The percentage concentration of the stabilizer ranges from 2 to 25%, with a preferred range of 5 to 10%.

[0077] Compounds used in dry matrices include: glycine, bicine, sodium chloride, n-octenylsuccinic anhydride, polyvinyl alcohol-polyethylene glycol graft copolymer, maltodextrin, α-(1,6)-linked maltotriose, α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside, water-soluble glucose polymers obtained from acid and / or enzymatic hydrolysis of starch in the presence of water, polysaccharide polymers, polyethylene glycol, polyethylene glycol hydroxystearate (15), povidone, sucrose, sorbitol, polyoxyethylene ester of 12-hydroxystearic acid, and 1-O-alpha-D-glucopyranosyl-D-mannitol.

[0078] Publicly available versions of these compounds that can meet the goals of the present invention include HiCap100®, Kollicoat®, Maltrin 250®, pullulan, trehalose, Solutol® Plus, and Solutol®. Preferred embodiment solutions are Lab9101®, Maltrin 250®, Trehalose®, and sucrose, with the most preferred matrices being Maltrin®, sucrose, and isomalt. While in embodiments using dried sulfate microparticles, the preferred carbohydrates are sucrose or isomalt.

[0079] Liquid Matrix The same particle suspensions described above for each of the dry matrices can also be made in more dilute forms using so-called "liquid" matrices for ease of dispensing and blotting. For example, if a dry matrix formulation consists of 25 uL of 1:18 sulfate latex with 10% sucrose, a similar liquid matrix would have reagents in the range of 50 uL of 1:9 sulfate latex with 5% sucrose. In other embodiments, the liquid matrix is ​​formed from a lyse diluent.

[0080] Compounds used in the liquid matrix of this embodiment include: glycine, bicine, sodium chloride, n-octenylsuccinic anhydride, polyvinyl alcohol-polyethylene glycol graft copolymer, maltodextrin, α-(1,6)-linked maltotriose, α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside, water-soluble glucose polymers obtained from acid and / or enzymatic hydrolysis of starch in the presence of water, polysaccharide polymers, polyethylene glycol, polyethylene glycol (15) hydroxystearate, povidone, sucrose, sorbitol, polyoxyethylene esters 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 simethicone diluted with injection-grade purified water to a concentration supporting a defoaming performance of less than 15 seconds in a USP assay.

[0081] A select few publicly available versions of these compound products that may meet the goals of the present invention may include HiCap100®, KollicoatIR®, Lab9101®, Maltrin 250®, Pullulan®, Trehalose®, Solutol Plus®, Solutol®, FoamAWAY®, and Sorbital®.

[0082] reagent The bioassay methods of the present invention use a variety of reagents 80 or activators. Compounds used as activators in this embodiment include: thrombokinase, thromboplastin, tissue thromboplastin factor III, platelet tissue factor, thrombokinase, thrombozyme, tissue factor, enzyme-generating substances, ellagic acid, activated partial thromboplastin, thrombin, factor II, factor VII, factor I, factor X, factor XII, activated protein C, negatively charged phospholipids, calcium ions, alumina silicate clay, silicon oxide, silica, celite, and povidone.

[0083] Selected commercially available versions of these compounds and products that, when properly prepared, may meet the goals of the present invention include Kaolin®, Innovin® Thromboplastin 82, APTT-XL 84, and Siemens® Thrombin 86.

[0084] These reagents or activators may be dried to ensure additional stability while stored for transport prior to the actual bioassay. Then, during the assay, these reagents 80 may be diluted with a diluent 90, such as DH2O, in various volumes 94, ranging from 20 uL to 400 uL, more preferably from 50 uL to 300 uL, and even more preferably from 50 uL to 100 uL, to various concentrations 96. Still other embodiments use CaCl2 or other diluents, as discussed in specific embodiments below.

[0085] Optical density & correction 10, the principles of coagulometric (turbidimetric) clot detection are used in the present invention and system to measure 116, 116' and record the amount of time required for a plasma or whole blood sample 60 to clot. This technique assesses clot initiation 132 and clotting endpoint 134 by measuring the change 135 in optical density 130 detected over time.

[0086] In a medium where fibrinogen is converted to fibrin, clot formation 124 is inferred and "detected" based on the principle that any light passing through this medium will be absorbed by the fibrin strands. Therefore, as fibrin clot formation progresses over time, light absorption increases, resulting in a change 135 in optical density 130.

[0087] Then, in each of the bioassays discussed herein, after collecting and mixing the sample as discussed above, light is transmitted from the source through the co-mixture 123. The transmitted light is then directed to a photodetector, which is positioned at 180° incidence relative to the source. A first corresponding electrical signal output from the photodetector in first measurement 116' determines the level of hemoglobin 125 using optical detection.

[0088] The light transmittance of the co-mixture 123 is again measured 116 by the photodetector over a predetermined time period, which generates a second corresponding electrical signal output, and both the first and second corresponding electrical signal outputs from the photodetector vary according to the detected light.

[0089] The signal output is processed by the software through a series of algorithms to determine the associated freezing point, clot initiation 132, and formation 134. Briefly, changes 135 in the optical density 130 of the latex-microparticle co-mixture 123 signal are used to indicate clot initiation 132 and formation 134.

[0090] Reduced procedure times The present invention seeks to address several sources of laboratory error in clot detection procedures. Because clot formation is time dependent, one of the largest sources of laboratory error in clot detection procedures is due to the time lapse between sampling 104 and measuring 116.

[0091] One of the principles of the present invention is to address the failure of the prior art to address the increased number of errors that can occur due to the passage of time between sample acquisition 104 and detection (measurement) 116. By packaging the assay components as an all-inclusive cartridge 2, providing pre-selected components for the assay methodology, the present invention expedites the pre-detection stage.

[0092] Another way the present invention achieves this goal is by providing a bioassay that can achieve accurate results by using a whole blood sample without the need to separate red blood cells from the plasma prior to testing. Currently available point-of-care analyzers require the use of blood plasma as the sample. This requires separation of red blood cells from the plasma of the blood sample before obtaining test results, further extending the period between sample acquisition and testing.

[0093] It has been discovered that one of the reasons current analyzers require blood plasma is due to the inability of prior art automated systems and assays to provide self-correcting analysis. The level of hemoglobin in a blood sample will have an impact on the rate of clot initiation. The inventors have discovered that failure to adjust the optical density signal output to account for variations in individual hemoglobin content can result in errors in correctly identifying clot initiation and formation.

[0094] To address this potential source of error in the prior art, the present invention provides a bioassay method for automatic correction of hemoglobin. In the described clotting assay embodiment using whole blood, the optical density of the co-mixture is measured at a visible wavelength. Hemoglobin levels are determined by sequentially measuring the sample optical density at another visible wavelength. The hemoglobin measurement is then used to correct clotting time for the sample's true adjusted plasma value. To illustrate the benefits of this automatic correction, the effect of hemoglobin on prothrombin time INR values ​​with and without correction is illustrated in Figure 14.

[0095] Specifically, the graph in Figure 14 depicts the effect of various levels of hemoglobin (hematocrit effect) on the INR of a standard sample. As shown, hemoglobin ranged from 0 to 23 gm / dl. A standard INR ranges from 0.8 to 1.3 INR units. Moderate oral anticoagulation therapy results in an INR of 1.8 to 2.8 INR units. As hemoglobin increases, the INR increases due to the reduced plasma fraction in the whole blood sample. With an additional LED in the visible range, the INR can be corrected for the detected hemoglobin. The figure also shows the INR corrected for hemoglobin for comparison.

[0096] The present invention helps expand the capabilities of the instrument to include multi-signal measurement devices, such as, but not limited to, multi-wavelength light-emitting diodes (LEDs). Measuring the optical density of the co-mixture to read the assay is preferably performed at visible wavelengths, in the range of 620 nm to 700 nm, more preferably between 650 and 680 nm, even more preferably between 658 nm and 668 nm, and most preferably at 660 nm. Measuring the optical density of the sample to read the hemoglobin level is preferably performed at visible wavelengths, in the range of 500 nm to 550 nm, more preferably between 510 nm and 545 nm, even more preferably between 520 nm and 540 nm, and most preferably at 530 nm.

[0097] Table 1 shows the data illustrated in Figure 14 and identifies sample IDs, hemoglobin levels, and initial uncorrected INRs: TIFF0007732719000001.tif58161

[0098] Table 2 shows data identifying sample ID, hemoglobin levels, and corrected initial INR, which are also illustrated in Figure 14: TIFF0007732719000002.tif58161

[0099] Next, the difference between plasma INR value(s) obtained by a pre-approved PT / INR analyzer is illustrated in Figure 15, with and without hemoglobin correction according to the present method. Again, hemoglobin values ​​ranged from 0 to 23 g / dl.

[0100] Table 3 shows the data illustrated in Figure 15 and identifies the sample ID, plasma INR, and the difference from the plasma INR values ​​before and after correction for hemoglobin (delta INR). TIFF0007732719000003.tif43161

[0101] Standard prothrombin time / INR It should be understood that a standardized INR is used in various embodiments of the bioassay of the present invention. To account for differences in the thromboplastin used, which can result in variability in output, a standardized prothrombin time INR is used. This INR-corrected measurement (or standardization) is developed from the prothrombin time, the sensitivity index of the thromboplastin, and the mean prothrombin time.

[0102] Specifically, in mathematical formulation, this normalization can be written as the following calculation: INR=(PT / MT) ISI where in the above formula, INR means normalized value; PT means prothrombin test time; ISI means thromboplastin sensitivity index; MT means mean prothrombin time derived from 20 standard samples.

[0103] Specific Bioassays of the Invention At this point, we turn to Figures 16-24, which illustrate the results of particular bioassays conducted in accordance with embodiments of the present invention. Unless specifically stated otherwise, these bioassay results illustrate optical density readings at 660 nm using kinetics mode over time (in seconds).

[0104] Bioassay using tissue thromboplastin The results of different bioassays B1-B20 according to the methods of the present invention using various matrices for various microparticle dilutions are illustrated in Figures 16-33, with a discussion of each provided below. Each of these bioassays B1-B20 used a tissue thromboplastin reagent. Specifically, the reagents selected for these bioassays were tissue thromboplastin and CaCl2 in the amounts listed in each of the protocols below.

[0105] B1-B4 bioassays using BSA-coated latex and plain microparticles At this point, we turn to Figures 16 and 17 which illustrate the results of four bioassays B1 to B4 according to the present invention.

[0106] The first bioassay, B1, used microparticle dilution 11, which had microparticles 27 with bovine serum albumin (BSA) surface groups. The microparticles were diluted 1:500 with 0.02% sodium azide in water to a total microparticle concentration of 0.016%. The optical density of dilution 11 was measured at 660 nm after dilution and had an optical density value of 0.08. This bioassay, B1, further used matrix 40, which had 0.17 M glycine, 1.0 M NaCl, and 1% simethicone at pH 10.0 diluted with water. This bioassay, B1, was performed with standard citrated whole blood samples, B1.

[0107] In comparison, a second bioassay, B2, had the same protocol as bioassay B20 above, only B2 was performed with microparticle dilution 11 having microparticles 20 (without BSA surface groups). The selected reagents 80 for this bioassay were tissue thromboplastin and CaCl2.

[0108] As shown in Figure 16, the B1 assay, an attempt using BSA microparticles known to work according to the prior art discussed above, did not detect a consistent change in optical density over time, and therefore no change in clotting time, clotting end time, or clotting was detected in the B1 assay.

[0109] 16, in a B2 assay using the inventive concepts according to the novel methods discussed herein, clotting can be seen to begin 132 at about 4 seconds with an OD value of 0.1342, and clotting can be seen to be complete 134 at about 10 seconds with an OD value of 0.1907. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.0565.

[0110] To confirm this difference, these bioassays B1 and B2 were repeated again, this time with abnormal blood samples, and for clarity are designated as third and fourth bioassays B3 and B4, respectively.

[0111] As shown in Figure 17, the B3 assay, which again attempts to use BSA microparticles known to work according to the prior art discussed above, did not detect a consistent change in optical density over time, and therefore no change in clotting initiation time, clotting end time, or clotting was detected in the B3 assay.

[0112] Also, as shown in Figure 17, in the B4 assay using the method of the present invention, clotting can be seen to begin 132 at about 31 seconds, with an OD value of 0.1164, and clotting can be seen to be complete 134 at about 59 seconds, with an OD value of 0.2180. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.1016.

[0113] Protocols for bioassays B1-B4 are provided in Table 16 below: TIFF0007732719000004.tif34161

[0114] Table 5 below shows optical density result data using kinetics mode at 660 nm over time for the above described bioassays B1 to B4 according to the present invention: TIFF0007732719000005.tif126161TIFF0007732719000006.tif219161TIFF0007732719000007.tif58161

[0115] B5-B10 bioassays with polyethylene glycol matrices. At this point, we turn to Figures 18-23, which illustrate the results of six bioassays B5-B10 according to various embodiments of the present invention.

[0116] Specifically, Figure 18 illustrates the results of a first bioassay B5 according to the present invention, which had microparticles 22 with amidine surface groups having a diameter 26 of 95 nm and used a microparticle dilution 11 having a 0.080 weight to volume % (% W / V) 28. This bioassay B5 further used a matrix 40 having 0.17 M glycine, 1.29 M NaCl, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0117] The optical density of the B5 dilution was measured at 660 nm and had an optical density value 31 of 0.19. As shown in Figure 18, in the B5 assay, clotting can be seen to begin 132 at about 5 seconds with an OD value of 1.153 and clotting can be seen to be complete 134 at about 50 seconds with an OD value of 1.164. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.011.

[0118] 19 illustrates the results of a second bioassay embodiment B6, which used microparticles having sulfate surface groups 24 with a diameter 26 of 110 nm and a microparticle dilution 11 having a 0.044 weight-to-volume 28. This embodiment B6 further used a matrix 40 having 0.17 M glycine, 1.29 M NaCl, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0119] The optical density of the dilution was measured at 660 nm and had an optical density value of 0.21. As shown in Figure 19, in the B6 assay, clotting can be seen to begin 132 at about 5 seconds with an OD value of 0.564 and clotting can be seen to be complete 134 at about 70 seconds with an OD value of 1.000. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.436.

[0120] Turning now to Figure 20 for a graphical illustration of the results of a third bioassay embodiment B7, which used a microparticle dilution 11 having 0.067% weight to volume of surface group-free 20 microparticles having a diameter 26 of 96 nm. This dilution had a microparticle-to-diluent dilution ratio of 1 to 150. This embodiment B7 further used a matrix having 0.17 M glycine, 1.29 M NaCl, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0121] The optical density of the diluted solution was measured at 660 nm after dilution and had an optical density value of 0.21. As shown in Figure 20, in the B7 assay, clotting can be seen to begin 132 at about 5 seconds, with an OD value of 0.514, and clotting can be seen to be complete 134 at about 85 seconds, with an OD value of 1.215. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.701.

[0122] 21 then provides an illustration of the results of a fourth bioassay embodiment B8, which used a microparticle dilution 11 having microparticles with amidine surface groups 22 having a diameter 26 of 95 nm. The microparticle dilution had a 0.080 weight to volume % 28. The optical density 31 of dilution 11 was measured to have an optical density value of 0.19 at 660 nm.

[0123] This embodiment B8 used a matrix 40 having 0.17 M glycine 47 at pH 7.0 with 1.29 M NaCl. The matrix 40 further includes carbohydrate derivatives in the form of 1% Tween® 20 and 10% polyethylene glycol (PEG) 20K.

[0124] As shown in Figure 21, in the B8 assay, clotting can be seen to begin 132 at about 15 seconds with an OD value of 0.633 and clotting can be seen to be complete 134 at about 65 seconds with an OD value of 1.081. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.448.

[0125] Next, Figure 22 provides an illustration of the results of a fifth bioassay embodiment B9 using microparticle dilution 11. This dilution 11 has microparticles with sulfate surface groups 24 and a diameter 26 of 110 nm. The microparticles 24 in dilution 11 were present at 0.044% weight to volume of the dilution. Specifically, this dilution 11 was diluted with water at a particle-to-water ratio of 1 to 180. The optical density of the diluted solution was measured at 660 nm after dilution and had an optical density value of 0.21. This embodiment B5 further used a matrix 40 having 0.17 M glycine, 1.29 M NaCl, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0126] As shown in Figure 22, in the B9 assay, clotting can only be detected when the data is analyzed and enhanced as shown (all data is provided in Table 7 below). However, when enhanced, clotting can be seen to begin 132 at approximately 25 seconds, with an OD value of 0.3780, and clotting can be seen to be complete 134 at approximately 85 seconds, with an OD value of 0.3830. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.005.

[0127] 23 then provides an illustration of the results of a sixth bioassay embodiment B10, which used microparticle dilution 11 with 0.067% weight to volume of surface group-free 20 microparticles with a diameter of 96 nm. The final dilution had a microparticle-to-water dilution ratio of 1:150.

[0128] The optical density 31 of dilution 11 was measured at 660 nm after dilution to have an optical density value 31 of 0.21. This embodiment further used a matrix 40 having 0.17 M glycine, 1.29 M NaCl, and 10% polyethylene glycol (PEG) 20K at pH 7.0.

[0129] As shown in Figure 23, in the B10 assay, clotting can only be detected when the data is analyzed and enhanced as shown (all data is provided in Table 7 below). However, when enhanced, clotting can be seen to begin immediately 132, with an OD value of 0.3780, and clotting can be seen to be complete in about 85 seconds 134, with an OD value of 0.3860. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.008.

[0130] The protocols for all six embodiments B5 to B10 listed above are shown in Table 4 below: TIFF0007732719000008.tif34161

[0131] The general methodology for each of these bioassays B1-B6 was identical and were generally prepared as discussed above with reference to FIG.

[0132] Table 5 shows corrected optical density result data using kinetics mode at 660 nm over one and a half minutes for each of the above six described bioassay embodiments according to the present invention: TIFF0007732719000009.tif116161

[0133] B11-B12 bioassay with simethicone matrix At this point, we turn to Figure 24, which illustrates the results of a first bioassay B11 in accordance with the present invention using a microparticle dilution 11 having microparticles 24 with sulfate surface groups having a diameter of 100 nm. The microparticles were diluted 1:500 with 0.02% sodium azide in water to a total microparticle concentration of 0.016%.

[0134] The optical density of Dilution 11 was measured at 660 nm after dilution and had an optical density value of 0.08. This Bioassay B11 further used a matrix 40 having 0.17 M glycine, 1.29 M NaCl, and 1% simethicone diluted with water of injection grade purified water at a concentration that supports an antifoaming performance of less than 15 seconds in the USP assay by its commercial identity of FoamAWAY®. This Bioassay B11 was performed with a standard sample 60.

[0135] As shown in Figure 24, in the B11 assay, clotting can be seen to begin 132 at approximately 3 seconds with an OD value of 0.1516, and clotting can be seen to be complete 134 at approximately 65 seconds with an OD value of 0.2356. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.084.

[0136] In comparison, Figure 25 illustrates the results of bioassay B12, which had the same protocol as bioassay B11 above, but was performed with only abnormal samples at this point. As shown in Figure 25, in the B12 assay, clotting can be seen to begin 132 at about 30 seconds at this point, with an OD value of 0.1528, and clotting can be seen to be complete 134 at about 65 seconds, with an OD value of 0.3215. The change in absorbance 135 over time is the change in optical density value, with a difference in OD value of 0.1687.

[0137] Protocols for Bioassays B11 and B12 are provided in Table 16 below: TIFF0007732719000010.tif34161

[0138] Table 9 below shows optical density result data using kinetics mode at 660 nm for bioassays B11-B12 described above in accordance with the present invention over 1 minute for bioassay B12 and over 20 seconds for B11: TIFF0007732719000011.tif122161TIFF0007732719000012.tif219161TIFF0007732719000013.tif33161

[0139] Although these optical density values ​​are shown here, it should be understood that these values ​​are not normally presented to the user. Instead, according to the method discussed above, the clotting time is corrected using the hemoglobin measurement for the true plasma value after sample adjustment. This corrected clotting time is then used to report the normalized INR, as discussed above.

[0140] For example, in the above abnormal blood bioassay B12, a first optical density measurement 116' was taken to determine the hemoglobin content. The optical density detected at 530 nm was 1.5135, and this optical density value 140 was compared to a predetermined relationship 141 of OD values ​​to known hemoglobin values ​​to determine the specific hemoglobin level 142 present in the sample being analyzed. Again, this level is not normally reported to the client or user, but this analysis is illustrated in FIG. 26. This specific HGB level 142 for bioassay B11 was then stored in system memory until after the second set of optical density measurements 116 was completed.

[0141] As discussed above with reference to Figure 25, for the B12 assay, at this hemoglobin level, clotting began at a first time (30 seconds) with an OD value of 132, 0.1528, and clotting ended at a second time (65 seconds) with an OD value of 134, 0.3215. The change in absorbance 135 due to delta time (35 seconds) was a difference in the first OD value of 0.1687. For various bioassays of the present method, the change in absorbance 135 due to a change in time resulting in a difference in OD value ranges between 0.005 and 1.0. More preferably, the difference in OD value is at least 0.2, and when necessary, the difference in OD value is at least 0.08. This change in absorbance 135 can be used to calculate the PT value 136 (47 seconds, which was accompanied by an absorbance OD value of 0.23715).

[0142] The system then uses this original PT value 136 to derive a particular HGB level 142, as determined by reference to Figure 24. A predetermined relationship 137 is then used to determine an adjusted or corrected PT value 138. Specifically, Figure 27 illustrates the correction of the uncorrected PT value 136 with the predetermined relationship 137 corresponding to the hemoglobin level 142 (12.3) of the sample 60. This relationship 137 is then used to provide the corrected PT value 138. Here, for the hemoglobin level 142, the relationship 137 is mathematically: CPT=PT*SQRT(C / HGB) 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 a particular sample 60.

[0143] Figure 28 then illustrates the calculation of INR from the corrected PT138. Specifically, this calculation is: INR=(PT / MT) ISI where in the above formula applied to data obtained with B12, PT means the prothrombin-corrected test time of 138 (50.1 seconds); ISI means the sensitivity index of the thromboplastin used (0.98, no associated unit); MT means the mean prothrombin time from 20 standard samples (10.2 seconds); and INR means the normalized value of 144 (4.8, no associated unit). In common practice, only the corrected PT value (50.1 seconds) and INR value (4.8, no associated unit) will be reported to the user.

[0144] B13-B14 bioassay using carboxyl microparticles At this point, we turn to Figure 29, which illustrates the results of bioassay B13 in accordance with the present invention, which used a microparticle dilution 11 having microparticles 25 with carboxyl surface groups having a diameter of 103 nm at a 0.016% weight to volume dilution. This bioassay method using carboxyl microparticles 25 requires a dilution 11 having a microparticle 25 to dihydrogen monoxide 33 dilution ratio 30 ranging from 1:50 to 1:2000; more preferably ranging from 1:100 to 1:1000.

[0145] The optical density of the diluted solution was measured at 660 nm after dilution and had an optical density value of 0.08. This bioassay B13 further used a matrix having 0.17 M glycine at pH 10.0, 1 M NaCl, and 1% simethicone diluted with water of injection-grade purified water at a concentration that supports an antifoaming performance of less than 15 seconds in the USP assay by its commercial identity of FoamAWAY®. This bioassay B13 was performed with standard sample 60.

[0146] As shown in Figure 29, in the B13 assay, clotting can be seen to begin 132 at about 5 seconds with an OD value of 0.1304, and clotting can be seen to be complete 134 at about 20 seconds with an OD value of 0.1826. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.0522.

[0147] In comparison, Figure 30 illustrates the results of bioassay B14, which had the same protocol as bioassay B13 above, but this time with only abnormal samples. As shown in Figure 30, in the B14 assay, clotting can be seen to begin 132 at approximately 40 seconds, with an OD value of 0.1218, and clotting can be seen to be complete 134 at approximately 68 seconds, with an OD value of 0.2027. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.0811.

[0148] Protocols for bioassays B13 and B14 are provided in Table 10 below. TIFF0007732719000014.tif34161

[0149] Table 11 below shows optical density result data using kinetics mode at 660 nm for bioassays B13-B14 described above in accordance with the present invention over 1 minute for bioassay B13 and over 20 seconds for B14: TIFF0007732719000015.tif78161TIFF0007732719000016.tif220161TIFF0007732719000017.tif95161

[0150] B15-B18 bioassays with different operating temperatures Typically, many bioassays according to the present invention are performed at physiological temperature, but this is not always the case. It should be understood that, in general, most temperatures are applicable to the methods of the present invention. However, adjusting this variable in the bioassays discussed above introduces potential variability, which would generally alter the bioassay results. Therefore, temperature variability should not be introduced without addressing these variability, especially in coagulation assays where precise timing can impact clinical outcomes.

[0151] Figures 31 and 32 illustrate the results of four bioassay embodiments B15-B18 of the present invention using two different microparticle matrices at two different temperatures. Protocols for all four bioassay embodiments are provided in Table 12 below: TIFF0007732719000018.tif34161

[0152] Specifically, Bioassay B15 used microparticle dilution 11, which had microparticles with amidine surface groups 22 having a diameter of 95 nm. At 0.080% weight to volume, Dilution 11 had microparticles 22 diluted with water at a 1:50 ratio. The optical density of the diluted solution was measured at 660 nm after dilution and had an optical density value of 0.19. Bioassay B15 used matrix 40, which had 0.17 M glycine, 0.29 M NaCl, and 10% polyethylene glycol (PEG) 20K at pH 7.0. Bioassay B15 was performed at a temperature of 22°C.

[0153] As shown in Figure 31, in the B15 assay, clotting can be seen to begin almost immediately at about 4 seconds 132 with an OD value of 1.079, and clotting can be seen to be complete at about 100 seconds 134 with an OD value of 1.748. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.669.

[0154] Also shown in Figure 31 is a graphical representation of the results of a similar bioassay B16. This bioassay B16 also used a microparticle diluent 11 having microparticles 22 with amidine surface groups having a diameter of 95 nm. The microparticles 22, present at 0.080% weight to volume, were diluted with water at a dilution ratio 30 of 1:50. The optical density 31 of the dilution was measured at 660 nm after dilution and had an optical density value 31 of 0.19. This bioassay B16 also used a matrix 40 having 0.17 M glycine, 0.29 M NaCl, and 10% polyethylene glycol (PEG) 20K at pH 7.0. However, unlike bioassay B15, this bioassay B16 was performed at an operating temperature of 37°C.

[0155] Also, as shown in Figure 31, in the B16 assay, clotting values ​​are more difficult to discern, but clotting can again be seen to begin almost immediately, at about 4 seconds 132', with an OD value of 1.023. Unlike the B15 assay, the B16 assay appears to complete clotting quickly, as clotting appears to be complete in about 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 value difference of only 0.087.

[0156] 32, there is shown a graphical representation of the results of another pair of bioassays B17 and B18 in accordance with the present invention. In the first bioassay, B17, microparticle dilution 11 utilized microparticles 22 having amidine surface groups with a diameter 26 of 0.95 nm. Microparticles 22, diluted 1:50 with water, were present in dilution 11 at 0.080% weight to volume for this bioassay, B17.

[0157] The optical density 31 of dilution 11 was measured at 660 nm and had an optical density value of 0.19. This bioassay B17 used a matrix 40 with 0.17 M glycine, 0.29 M NaCl, 10% polyethylene glycol (PEG) 20K, and 1% Tween® 20 at pH 7.0. Bioassay B17 was performed at an operating temperature of 22°C.

[0158] As shown in Figure 32, in the B17 assay, clotting can be seen to begin 132 at approximately 12 seconds with an OD value of 0.997, and clotting can be seen to be complete 134 at approximately 58 seconds with an OD value of 1.772. The change in absorbance 135 over time is the change in optical density value with a difference in OD value of 0.775.

[0159] A second bioassay B18 according to the present invention employed a microparticle dilution using microparticles 22 with 0.080% weight to volume amidine surface groups and a diameter of 0.95 nm. Dilution 11 had a dilution ratio of 1:50. The optical density of the diluted solution was measured at 660 nm after dilution and had an optical density value of 0.19. Bioassay B18 also employed a matrix having 0.17 M glycine, 0.29 M NaCl, 10% polyethylene glycol (PEG) 20K, and 1% Tween® 20 at pH 7.0. However, unlike bioassay B17, bioassay B18 was performed at an operating temperature of 37°C.

[0160] Also shown in Figure 32, for the B18 assay, the clotting values ​​are more difficult to discern, but clotting can be seen to begin again at about 20 seconds 132', with an OD value of 0.653. The B18 assay appears to complete clotting 134' at about 60 seconds, with an OD value of 1.054. The change in absorbance 135' over time is a change in optical density value with only an OD value difference of 0.401.

[0161] The general methodology for each of these bioassays B15-B18 was identical and were generally prepared as discussed above with reference to FIG.

[0162] Table 13 below shows corrected optical density result data using kinetics mode at 660 nm over several minutes for each of the above described bioassays B15-B18 in accordance with the present invention: TIFF0007732719000019.tif18161TIFF0007732719000020.tif176161

[0163] B19-B20 bioassay for monitoring anticoagulant use At this point, we turn to Figure 33, which illustrates the results of prothrombin time bioassays B19 and B20, which measure the extrinsic pathway of coagulation and provide multistep monitoring of oral anticoagulant use. These bioassays B11 and B12 are prothrombin time assays illustrating the results of optical density measurements versus time in seconds for the first assay B19 of the method of the present invention on a standard whole blood sample versus the second assay B20 of the method of the present invention on a sample from a patient taking the oral anticoagulant Coumadin®.

[0164] Coumadin® (also known as warfarin) inhibits vitamin K synthesis and therefore the half-life of Factor VII. Factor VII levels were assayed and corrected in this graph using the prothrombin time test and dual wavelength correction as described herein.

[0165] As illustrated in Figure 33, standard clotting times 132 are typically seen to begin after approximately 15 to 20 seconds, where clot formation is initiated by the extrinsic clotting pathway via Factor VII activation, as discussed above. Specifically, as illustrated in Figure 33, assay B19 of the standard blood sample begins clotting 132 at approximately 15 seconds, while assay B20 of the Coumadin sample begins clotting 132' at approximately 100 seconds.

[0166] In both of these assays B19 and B20, microparticle dilutions 11 used microparticles 22 with amidine surface groups at 0.080% weight to volume and 95 nm diameter 26. These microparticle dilutions 11 were diluted with water at a ratio of 1 to 50. The optical density of the dilutions was measured at 660 nm after dilution and had an optical density value of 0.19.

[0167] As shown in Figure 33, in the B19 assay, clotting can be seen to begin 132 at about 15 seconds with an OD value of 0.989, and clotting can be seen to be complete 134 at about 90 seconds with an OD value of 1.700. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.711.

[0168] Also, in the B20 assay shown in Figure 33, clotting can be seen to begin 132' at about 50 seconds with an OD value of 0.919, and clotting can be seen to be complete 134' at about 180 seconds with an OD value of 1.6290. The change in absorbance 135' over time is a change in optical density value with a difference in OD value of 0.7100.

[0169] Protocols for Bioassays B19 and B20 are provided in Table 14 below: TIFF0007732719000021.tif34161

[0170] The general method for each of these bioassays B19-B20 was identical and were generally prepared as discussed above with reference to Figure 10. Table 15 shows the optical density data using kinetics mode at 660 nm over 2 minutes for the first bioassay B19 with standard citrated blood and the second bioassay B20 with Coumadin citrated blood: TIFF0007732719000022.tif47161TIFF0007732719000023.tif190161

[0171] Bioassays using thrombin The results of different bioassays B21-B22 according to the methods of the present invention using various matrices for various microparticle dilutions are illustrated in Figure 34, and a discussion of each is provided below. Each of these bioassays B21-B22 used a thrombin reagent. Specifically, the reagents selected for these bioassays were tissue thromboplastin and CaCl2 in the amounts listed in each of the protocols below.

[0172] B21-B22 bioassay for measuring fibrinogen levels At this point, we turn to Figure 34, which illustrates the results of thrombin time (TT) assays B21 and B22, which directly measure fibrinogen levels and function and will also determine whether thrombin inhibitors are present in the sample. Figure 34 illustrates the results of measuring optical density versus time in seconds for a first TT assay B21 of the method of the invention with 30 uL thrombin reagent in citrated plasma versus a second TT assay B22 with 20 uL thrombin reagent and citrated blood according to the method of the invention.

[0173] Protocols for Bioassays B15 and B16 are provided in Table 16 below: TIFF0007732719000024.tif34161

[0174] Specifically, these bioassays B21 and B22 used a microparticle diluent 11 having microparticles 24 with sulfate surface groups at 0.2% weight to volume and a diameter 26 of 110 nm. Both bioassays B21 and B22 used a matrix 40 having 0.17 M glycine, 1.0 M NaCl at pH 10.0. The first bioassay B21 used 30 uL of Siemens® Thrombin Time (TT) Reagent, and the second bioassay B22 used 20 uL of Siemens® Thrombin Time (TT) Reagent.

[0175] Some thrombin inhibitors that can be used in the methods of the present invention are unfractionated heparin, low molecular weight heparin, and direct antithrombin oral anticoagulants, including, but not limited to, hirudin, rivaroxaban, apixaban, dabigatran, and argatroban. The general method for each of these bioassays B21-B22 is identical and was generally prepared as discussed above with reference to Figure 10.

[0176] As shown in Figure 34, in the B21 assay, clotting can be seen to begin 132 at about 5 seconds with an OD value of 0.1829, and clotting can be seen to be complete 134 at about 100 seconds with an OD value of 0.3481. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.1652.

[0177] Also, in the B22 assay shown in Figure 34, clotting can be seen to begin 132' at about 5 seconds with an OD value of 0.1357, and clotting can be seen to be complete 134' at about 80 seconds with an OD value of 0.2491. The change in absorbance 135' over time is the change in optical density value with a difference in OD value of 0.1134.

[0178] Table 17 shows optical density data using kinetics mode at 660 nm over 3 minutes for the first bioassay B21 with 30 uL thrombin reagent and the second bioassay B22 with 20 uL thrombin reagent. TIFF0007732719000025.tif225161TIFF0007732719000026.tif74161

[0179] Bioassay using activated partial thromboplastin The results of different bioassays B23-B27 according to the methods of the present invention using various matrices for various microparticle dilutions are illustrated in Figures 35-38, and a discussion of each is provided below. Each of these bioassays B23-B27 used an activated partial thromboplastin reagent. Specifically, the selected reagents 80 for these bioassays were activated partial thromboplastin and CaCl2 in the amounts listed in each of the protocols below.

[0180] B23-B24 bioassay for the intrinsic pathway At this point, we turn to Figures 35 and 36, which illustrate the results of activated partial thromboplastin time (APPT) bioassays B23 and B24, which measure the intrinsic pathway of coagulation in a single step. Specifically, Figure 35 illustrates the results of measuring optical density versus time in seconds for the first APTT bioassay B23 of the method of the present invention for a normal control. Figure 36 then illustrates the results of measuring optical density versus time in seconds for the second APTT bioassay B24 of an abnormal control according to the method of the present invention.

[0181] Bioassays B23 and B24 both used microparticle dilution 11 with 0.392% weight by volume microparticles 24 with sulfate surface groups having a diameter of 110 nm. The dry matrix discussed above with 20 μL of 0.005 M CaCl2 was used. In a separate well, 100 μL of APTT-XL reagent was diluted with 150 μL of distilled water. The general method for each of these bioassays B23-B24 was identical and generally prepared as discussed above with reference to FIG. 10.

[0182] As shown in Figure 35, in the B23 assay, clotting can be seen to begin 132 at approximately 13 seconds with an OD value of 0.5524, and clotting can be seen to be complete 134 at approximately 150 seconds with an OD value of 0.9315. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.3791.

[0183] As shown in Figure 36, in the B24 assay, clotting can be seen to begin 132 at approximately 65 seconds with an OD value of 0.5111 and clotting can be seen to be complete 134 at approximately 240 seconds with an OD value of 0.9097. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.3986.

[0184] Protocols for Bioassays B23 and B24 are provided in Table 19 below: TIFF0007732719000027.tif34161

[0185] Table 19 shows the optical density data using kinetics mode at 660 nm over 3 minutes for the first bioassay B23 with normal controls and the second bioassay B24 with abnormal controls: TIFF0007732719000028.tif152161TIFF0007732719000029.tif189161

[0186] One-stage APTT-based bioassay of factors B25-B27 Specifically, Figures 37 and 38 illustrate the results of three bioassays, B25, B26, and B27, for factor-based one-stage APTT assays for measuring intrinsic pathway activity and activity levels of blood coagulation. The first, bioassay B25, uses an APTT on normal plasma, the second, bioassay B26, uses an APTT on abnormal 99.9% Factor VIII-deficient plasma, and the third, bioassay B27, is an APTT one-stage factor assay with a normal / abnormal mix of plasma.

[0187] Specifically, Figures 37 and 38 illustrate the results of bioassays B25, B26, and B27, which used microparticle dilution 11, which had microparticles 24 at a 0.044% particle concentration and sulfate surface groups with a diameter of 110 nm. This dilution 11 had microparticles diluted with water at a ratio of 1 to 180. The optical density of dilution 11 was measured at 660 nm and had an optical density value of 0.21. Each of these bioassays further used matrix 40, which had 0.17 M glycine and 1.0 M NaCl at pH 10.0.

[0188] Factors XII, XI, IX, and VIII are measured directly by these assays. In addition, factors X, V, and II are measured because they participate in a common cascade of pathways leading to final coagulation. Partial thromboplastin is formed from the sample by adding common surface activators for factor XII, along with calcium and phospholipids. Activators include, but are not limited to, kaolin, celite, and ellagic acid. The single-stage factor assays according to the methods of the present invention titrate the individual factor levels of a sample by comparing them to standard curves derived from dilutions of samples depleted of known factors and standard samples.

[0189] The general method for bioassays B25 and B26 was prepared generally as discussed above with reference to Figure 10. Single-factor bioassay B27 uses the same general method discussed in Figure 10, but additionally has a known amount of dried plasma depleted of a single known factor in an additional well 6''' to attenuate the response of sample 60. According to the methods disclosed herein, the actual response level of sample 60 is measured 116, corrected 118, and reported 119 using a standard curve provided by the software.

[0190] As shown in Figure 37, in the B25 assay, clotting can be seen to begin 132 at about 30 seconds, with an OD value of 0.0340, and clotting can be seen to be complete 134 at about 100 seconds, with an OD value of 0.3530. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.3190.

[0191] As shown in Figure 38, in the B26 assay, clotting can be seen to begin 132 at about 100 seconds with an OD value of 0.3140 and clotting can be seen to be complete 134 at about 200 seconds with an OD value of 0.8440. The change in absorbance 135 over time is a change in optical density value with a difference in OD value of 0.3986.

[0192] Also, in the B27 assay shown in Figure 38, clotting can be seen to begin 132' at about 50 seconds, with an OD value of 0.1820, and clotting can be seen to be complete 134' at about 110 seconds, with an OD value of 0.5480. The change in absorbance 135' over time is a change in optical density value with a difference in OD value of 0.3660.

[0193] Protocols for all three bioassays B25-B27 are provided in Table 20 below: TIFF0007732719000030.tif39161

[0194] Table 21 below shows corrected optical density result data using kinetics mode at 660 nm over 5 minutes for each of the above described bioassays B25-B27 in accordance with the present invention: TIFF0007732719000031.tif177161

[0195] List of elements with reference numbers The following reference numbers will be adhered to herein to refer to numbered elements in the drawings of the present application: [Explanation of symbols]

[0196] 1 Analyzer 2 cartridges 3. Barcode 4 finger sticks 5 Sterile pipettes 6 wells 7 cuvettes 8. Sampler 9 Main Package 10 Microparticle Matrix 11 Microparticle Dilution 18 Functional Group Types 20 Plain (fabric) 22 Amidine 24 Sulfate group Size 26 28 % weight to volume (%W / V) 30 dilution ratio 31 Optical Density Values 32 quantity 33 Diluents 40 Matrix 42 Wet Matrix 44 Dry Matrix 46 carbohydrates 47 Glycine 48 quantity 50 acidity 52 NaCl 54 PEG 56 TWEEN® 60 blood samples 61 Citrated Blood 62 Whole blood 63 Plasma 64 mixed 65 quantity 68 Microparticle Mixture 70 temperature 80 Reagents / Activators 82 Thromboplastin 84 APTT 86 Thrombin 88 Ellagic acid 90 Diluents 92 CaCl2 100 ways 102 Cartridges / Bioassays Selected 103 Scan the cartridge with the analyzer 104 Obtain a blood sample 105 Do Fingersticks 106 Remove the cartridge and the provided capillary sampler 107 Touch the sampler to the blood and fill the sampler 108 Return the sampler to the cartridge 109 Insert the cartridge into the analyzer 110 Automated Bioassay Process Steps Add 112 components 114 Mixing / Stirring Components 115 Incubation 116 measurements 118 Correction 119 Report Results 120 adjustment 121 Mixture 122 Solution 123 Comixture 124 Optically Detected Clot Formation 125 Optically Detected Hemoglobin Levels 126 Corrected Clotting Time 130 optical density 132 Coagulation begins 134 Clotting ends 135 Change in absorption = change in optical density over time 136 Uncorrected PT 137 HGB Relationship 138 Corrected PT 140 OD value detected at 530nm 141 Predetermined relationship of OD values ​​to HGB 142 Specific HGB Levels

[0197] conclusion While preferred embodiments of the present invention have been described herein, the above description is merely illustrative. Further modifications of the invention disclosed herein will occur to those skilled in the art. All such modifications are deemed to be within the scope of the present invention as defined by the appended claims.

Claims

1. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; The matrix comprises at least one of glycine, sodium chloride, and 1% simethicone. The disposable bioassay diagnostic cartridge.

2. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the cuvette is formed integrally with the body of the disposable bioassay diagnostic cartridge such that the cuvette cannot be separated from the body of the disposable bioassay diagnostic cartridge without damaging the disposable bioassay diagnostic cartridge; The disposable bioassay diagnostic cartridge.

3. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the cuvette is formed separate from the body of the disposable bioassay diagnostic cartridge so that the cuvette can be separated from the body of the disposable bioassay diagnostic cartridge without damaging the disposable bioassay diagnostic cartridge; The disposable bioassay diagnostic cartridge.

4. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the first optical detection reading uses a first light having a wavelength in the range between 500 nm and 550 nm, and the second optical detection reading uses a second light having a wavelength in the range between 620 nm and 700 nm; The disposable bioassay diagnostic cartridge.

5. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the first optical detection reading uses a first light having a wavelength in the range between 510 nm and 545 nm, and the second optical detection reading uses a second light having a wavelength in the range between 650 nm and 680 nm; The disposable bioassay diagnostic cartridge.

6. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the first optical detection reading uses a first light having a wavelength in the range between 520 nm and 540 nm, and the second optical detection reading uses a second light having a wavelength in the range between 658 nm and 668 nm; The disposable bioassay diagnostic cartridge.

7. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the first optical detection reading uses a first light having a wavelength of 530 nm, and the second optical detection reading uses a second light having a wavelength of 660 nm; The disposable bioassay diagnostic cartridge.

8. The disposable bioassay diagnostic cartridge of claim 7, wherein at least one of the first light and the second light comprises a light-emitting diode (LED) light.

9. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; The blood sample is selected from the group consisting of diluted lysed whole blood, diluted capillary whole blood, whole blood, plasma, citrated blood, citrated whole blood, citrated plasma, and mixed blood and plasma; The disposable bioassay diagnostic cartridge.

10. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, said disposable bioassay diagnostic cartridge comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the disposable bioassay diagnostic cartridge is configured to perform only a single type of bioassay; The disposable bioassay diagnostic cartridge.

11. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, said disposable bioassay diagnostic cartridge comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; The disposable bioassay diagnostic cartridge is configured to perform at least two different types of bioassays. The disposable bioassay diagnostic cartridge.

12. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, said disposable bioassay diagnostic cartridge comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; a fourth well holding a quantity of diluent; The disposable bioassay diagnostic cartridge.

13. A disposable bioassay diagnostic cartridge as described in claim 12, wherein the disposable bioassay diagnostic cartridge is configured such that when the disposable bioassay diagnostic cartridge is operated to monitor anticoagulant activity, at least some of the matrix and at least some of the diluent are mixed together in a third well of the disposable bioassay diagnostic cartridge.

14. A disposable bioassay diagnostic cartridge for monitoring anticoagulant activity, said disposable bioassay diagnostic cartridge comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; The matrix includes a carbohydrate or carbohydrate derivative that aids in the adsorption of the biomarker protein. The disposable bioassay diagnostic cartridge.

15. The disposable bioassay diagnostic cartridge of claim 14, wherein the carbohydrate or carbohydrate derivative further allows for dissolution and uniform dispersion of the plurality of microparticles.

16. A disposable bioassay diagnostic cartridge for monitoring anticoagulant drug activity, said disposable bioassay diagnostic cartridge comprising: a first well holding a quantity of matrix, the matrix being either a dry matrix or a liquid matrix; a second well holding a plurality of microparticles, the microparticles being unreacted, uncoated latex having at least one surface type selected from the group consisting of sulfate groups, carboxylate groups, and amidine groups; a third well; and a cuvette containing a quantity of activator; Including, the activator is selected from the group consisting of thromboplastin, 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 Celite; wherein the cuvette is a first wall configured to facilitate a first optical detection readout therethrough for monitoring anticoagulant activity; and a second wall configured to facilitate a second optical detection readout therethrough for monitoring anticoagulant activity; and and the disposable bioassay diagnostic cartridge is configured such that, when operating the disposable bioassay diagnostic cartridge to monitor anticoagulant activity, at least some of the matrix, at least some of the plurality of microparticles, and the blood sample are first mixed together in a third well of the disposable bioassay diagnostic cartridge, and subsequently, at least some of the resulting mixture and at least some of the activator are then mixed together in a cuvette of the disposable bioassay diagnostic cartridge; the plurality of microparticles comprising polystyrene; The disposable bioassay diagnostic cartridge.

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