Disposable system for analysis of hemostatic function

Abstract

A disposable system, in some embodiments, includes a multi-channel or multi-chamber test cartridge device configured to operate with a testing system for evaluation of hemostasis in a subject by in vitro evaluation of a test sample from the subject. The disposable system, in some embodiments, is configured to interrogate the test sample to evaluate clot stiffness, strength, or other mechanical properties of the test sample to assess the function of various physiological processes occur during coagulation and/or dissolution of the resulting clot.

Claims

1. A method comprising: receiving in a cartridge a sample from a sample holding tube; metering the sample in a plurality of metering chambers to form a plurality of metered samples; mixing each of the plurality of metered samples with a respective set of one or more reagents, or combinations of reagents, to form a respective mixed sample, wherein a first metered sample of the plurality of metered samples is mixed with a first set of one or more reagents or a combination of reagents to form a first mixed sample by repeatedly flowing, over a plurality of repeated cycles, the first metered sample (i) in a first direction along a mixing path defined (a) along a first metering chamber, (b) through a first set of one or more reagent pockets with the first set of one or more reagents or the combination of reagents located therein, and (c) along a first serpentine pathway in communication with the first metering chamber and the first set of one or more reagent pockets, until at least a portion of the first metered sample reaches a first detection zone located in, or after, the first serpentine pathway and (ii) in a second direction reversed to the first direction from the first detection zone through at least a portion of the first serpentine pathway toward the first metering chamber, wherein the first metering chamber is connected to a first testing chamber through the first serpentine pathway and the first set of one or more reagent pockets, and wherein the first metered sample is repeatedly flowing in the first and second direction over the mixing path from an application of a varying pressure from a measurement system at a first port in communication with the first serpentine pathway; driving the first mixed sample into the first testing chamber; and interrogating the first testing chamber by the measurement system to determine one or more viscoelastic properties of the first mixed sample, wherein the determined one or more viscoelastic properties of the first mixed sample is provided for an assessment of hemostasis of the sample.

2. The method of claim 1, further comprising: receiving the sample in a heating chamber configured to adjust a temperature of the sample from a first temperature to a second temperature, wherein the second temperature is different from the first temperature and associated with body temperature, and wherein the first metered sample is received from the heating chamber.

3. The method of claim 2, wherein the sample is moved into the heating chamber in response to a first applied positive or negative pressure that is applied by, or generated from, the measurement system.

4. The method of claim 2 further comprising: conditioning the sample in the heating chamber, wherein the first metered sample is mixed with the first set of one or more reagents following exit from the first metering chamber.

5. The method of claim 4, further comprising: isolating the first metered sample in the first metering chamber to prevent the first metered sample from contacting the first set of one or more reagents during the filling of the first metering chamber.

6. The method of claim 1, wherein the step of flowing the first metered sample comprises: applying a first positive or negative pressure from the measurement system at the first port in communication with the first serpentine pathway to move the first metered sample through the first serpentine pathway in the first direction; and applying a second positive or negative pressure that is opposite to the first positive or negative pressure from the measurement system to move the first metered sample through the first serpentine pathway in the second direction.

7. The method of claim 1, wherein the step of receiving the first mixed sample in the first testing chamber comprises: applying a negative or differential pressure via a second port, wherein the second port is in fluid communication with the first testing chamber.

8. The method of claim 7, wherein the first testing chamber is located downstream of the first serpentine pathway and the second port is located downstream of the first testing chamber.

9. The method of claim 1, wherein the first set of one or more reagent pockets includes the first set of one or more reagents, or the combination of reagents, wherein the first set of one or more reagents, or the combination of reagents, is in the form of one or more lyophilized beads.

10. The method of claim 9, wherein the first set of one or more reagent pockets associated with the first testing chamber comprises kaolin, calcium, buffers, and stabilizers, wherein the first testing chamber forms a part of a first test channel of the cartridge.

11. The method of claim 9, wherein a second set of one or more reagent pockets associated with a second test chamber comprises: a combination of kaolin, heparinase I, calcium, buffers and stabilizers; or a combination of thromboplastin, polybrene, calcium buffers, and stabilizers, wherein the second testing chamber forms a part of a second test channel of the cartridge.

12. The method of claim 9, wherein a third set of one or more reagent pockets associated with a third test chamber, comprises thromboplastin, polybrene, calcium, buffers, and stabilizers, wherein the third testing chamber forms a part of a third test channel of the cartridge.

13. The method of claim 9, wherein a third set of one or more reagent pockets associated with a third test chamber as a third test channel of the cartridge comprises thromboplastin, polybrene, at least one of abciximab and cytochalasin D, calcium, buffers, and stabilizers.

14. The method of claim 1, wherein the measurement system is configured to determine at least one of: a clot time-of the first mixed sample, a clot stiffness or a heparinase clot time of a second mixed sample, and a fibrinogen contribution of a third mixed sample.

15. The method of claim 1, wherein the measurement system is configured to determine: a clot stiffness, a clot stiffness change, and a clot reduction differential of the first mixed sample.

16. The method of claim 1, wherein the measurement system is configured to determine shear modulus of the first mixed sample as the determined one or more viscoelastic properties of the first mixed sample.

17. The method of claim 1, wherein the measurement system is configured to determine viscoelastic properties of a second mixed sample.

18. The method of claim 17, wherein the measurement system is configured to determine viscoelasticity properties of a third mixed sample.

19. The method of claim 1, wherein the first set of one or more reagent pockets associated with the first testing chamber comprises a coagulation activator selected from the group consisting of kaolin, Hageman factor, celite, glass, ellagic acid, micronized silica, tissue factor, recombinant tissue factor, thromboplastin, thrombin, factor Xa, reptilase, ecarin, and Russell's viper venom.

20. The method of claim 1, wherein a second set of one or more reagent pockets associated with a second test chamber comprises a platelet activator or inhibitor selected from the group consisting of abciximab, eptifibatide, tirofiban, roxifiban, orbofiban, cytochalasin D, blebbistatin, PAR1 inhibitors, PAR4 inhibitors, glycoprotein I.B. inhibitors, thrombin receptor activating peptide (TRAP), adenosine diphosphate (A.D.P.), and arachidonic acid.

21. The method of claim 1, wherein a third set of one or more reagent pockets associated with a third test chamber comprises a fibrinolytic function activator or inhibitor selected from the group consisting of tPA, uKA, streptokinase, TAFIa, plasmin/plasminogen, aprotinin, epsilon-aminocaproic acid, tranexamic acid, plasminogen activator inhibitor 1 (PAI1), α2-antiplasmin (α2-AP), plasmin-antiplasmin complexes, and carboxypeptidase inhibitor.

22. The method of claim 1, wherein a fourth set of one or more reagent pockets associated with a fourth test chamber comprises a reagent selected from the group consisting of FXIIIa inhibitor, Hexadimethrine bromide (polybrene), heparinase, ristocetin, heparin, low molecular weight heparin, corn trypsin inhibitor, adenosine, GPRP, calcium, fibronectin, collagen, epinephrine, immuno-detection reagent, direct thrombin inhibitor, factor Xa inhibitor, direct thrombin inhibitor, and thrombomodulin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments and together with the description, serve to explain the principles of the methods and systems:

(2) FIG. 1 shows a perspective view of an example biological sample input of a cartridge for use in a disposable system, in accordance with an illustrative embodiment.

(3) FIG. 2 shows a side cross-sectional view of the example biological sample input of FIG. 1 with a casing, in accordance with an illustrative embodiment.

(4) FIG. 3 shows a side cross-sectional view of the example biological sample input of FIG. 2 with a sample holding tube attached thereon, in accordance with an illustrative embodiment.

(5) FIG. 4 shows a detailed view of the example biological sample input of FIG. 3, in accordance with an illustrative embodiment.

(6) FIGS. 5A and 5B each shows the biological fluid pathways of four testing circuits (e.g., Hemostasis testing circuits) that are located on a sample preparation plane, in accordance with an illustrative embodiment. FIG. 5B further shows the cartridge body of FIG. 5A further coupled to a sample holding tube, in accordance with an illustrative embodiment.

(7) FIGS. 6A and 6B show a front perspective view and a back perspective view of FIGS. 5A, 5B, and 8 with labels corresponding to the heating chamber filling.

(8) FIGS. 6C and 6D show a front perspective view and a back perspective view of FIGS. 5A, 5B, and 8 with labels corresponding to the sample chamber filling.

(9) FIGS. 6E and 6F show a front perspective view and a back perspective view of FIGS. 5A, 5B, and 8 with labels corresponding to the sample mixing and test chamber filling.

(10) FIG. 7 shows the back-side of the cartridge of FIG. 5A and includes an interconnection plane that interfaces to the sample preparation plane, that collectively form the biological fluid pathways for the testing circuits, in accordance with an illustrative embodiment.

(11) FIG. 8 shows portions of the biological fluid pathways that are on the interconnection plane of FIG. 7, in accordance with an illustrative embodiment.

(12) FIG. 9 shows an example testing chamber section for use with the example cartridge, in accordance with an illustrative embodiment.

(13) FIG. 10 shows a cross-sectional view of an example testing chamber in the example testing chamber section, in accordance with an illustrative embodiment.

(14) FIG. 11 shows a detailed cross-sectional view of the example testing chamber of FIG. 10, in accordance with an illustrative embodiment.

(15) FIG. 12 shows a cross-sectional view of the disposable system operatively coupled to a measurement system, in accordance with an illustrative embodiment.

(16) FIG. 13 shows an example shear modulus versus time curve, in accordance with an illustrative embodiment.

(17) FIG. 14 shows an example of shear modulus curves obtained with an activator of coagulation and with and without a fibrinolysis inhibitor. A differential comparison of these curves can provide information about the fibrinolytic activity of the sample.

(18) FIG. 15 shows potential embodiments of differential metrics that can be measured from shear modulus curves obtained with an activator of coagulation and with and without a fibrinolysis inhibitor.

(19) FIG. 16 shows a photograph of an exemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9 for use in a disposable system, in accordance with an illustrative embodiment.

(20) FIG. 17 shows a front view of the exemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9 in accordance with an illustrative embodiment.

(21) FIG. 18 shows a front view of the exemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9, in accordance with an illustrative embodiments.

DETAILED DESCRIPTION

(22) The present invention now will be described more fully hereinafter with reference to specific embodiments of the invention. Indeed, the invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

(23) As used in the specification, and in the appended claims, the singular forms “a,” “an,” “the,” include plural referents unless the context clearly dictates otherwise.

(24) The term “comprising” and variations thereof as used herein are used synonymously with the term “including” and variations thereof and are open, non-limiting terms.

(25) As used throughout, by a “subject” is meant an individual. The subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The term does not denote a particular age or sex.

(26) The apparatus described here includes a single-use cartridge apparatus configured to facilitate in vitro assessment of one or more hemostatic functions. Hemostatic function refers to a functional role of various blood components such coagulation factors, fibrinogen, platelets, fibrinolytic factors, and components of the vasculature. The cartridge apparatus and associated measurement system, in some embodiments, are configured to assess hemostatic function by measuring changes in at least one mechanical property of the tested sample when such sample is exposed to one or more reagents. In some embodiments, the cartridge apparatus and its test chambers are configured to facilitate measurements of viscoelastic properties, e.g., based on interrogation using ultrasound pulses or ultrasonic energy. However, other interrogation systems may be used with a cartridge apparatus with the features described herein. Examples of other interrogation systems includes, for example, but not limited to, systems that employ cup/pin technologies (such as in the case of thromboelastography and thromboelastometry), oscillating piston to measure changes in mechanical impedance, optical sensing, fluorescence sensing, colorimetric sensing, aggregometry, resonance sensing, or electrical impedance sensing, among others.

(27) A broad array of reagents can be utilized in the cartridge apparatus, including intrinsic pathway activators (without limitations kaolin, Hageman factor, celite, glass, ellagic acid, micronized silica etc), extrinsic pathway activators (without limitations tissue factor, recombinant tissue factor, thromboplastin, etc), other coagulation activators (without limitations thrombin, factor Xa, reptilase, ecarin, Russell's viper venom or other snake venoms, etc), platelet activators or platelet inhibitors (without limitations GPIIb/IIIa inhibitors (such as abciximab, eptifibatide, tirofiban, roxifiban, orbofiban), cytochalasin D, blebbistatin, PAR1 inhibitors, PAR4 inhibitors, glycoprotein D3 inhibitors, TRAP, ADP, arachidonic acid, ADP inhibitors, non-steroidal anti-inflammatory drugs, etc.), fibrinolytic function activators or fibrinolytic function inhibitors (without limitations tPA, uKA, streptokinase, TAFIa, plasmin/plasminogen, aprotinin, epsilon-aminocaproic acid, tranexamic acid, plasminogen activator inhibitor 1 (PAI1), α2-antiplasmin (α2-AP), or plasmin-antiplasmin complexes, carboxypeptidase inhibitor, etc.), and others (FXIIIa inhibitors, Hexadimethrine bromide (polybrene), heparinase (e.g., heparinase I), ristocetin, heparin, low molecular weight heparin, corn trypsin inhibitor, adenosine, GPRP, calcium, fibronectin, collagen, epinephrine, immuno-detection reagents, direct thrombin inhibitors, factor Xa inhibitors, reagents aimed at reversing or eliminating the effects of the new oral anticoagulants (such as the direct thrombin inhibitors and the factor Xa inhibitors), thrombomodulin, etc.). Additional non-functional reagents could also be used to preserve the functionality of the other reagents (buffers and stabilizers for lyophilization or drying, dyes, etc.).

(28) Reagents, in some embodiments, are placed and stored in chambers (e.g., pockets located within a fluidic circuit) in the cartridge apparatus but in alternative embodiments reagents can be placed and stored in various chambers or fluidic channels in the fluidic circuit of the cartridge apparatus. A fluidic circuit generally refers to one or more fluidic pathways established between sample preparation and the one or more test chambers where samples are ultimately measured.

(29) In some embodiments, reagents are placed and stored in the cartridge apparatus in liquid forms or can be lyophilized in spheres (such as in the case of the Lyopheres™ produced by BioLyph LLC), lyophilized in films, lyophilized on the plastic surfaces, dried on the plastic surfaces, or spray coated, etc., in order to improve shelf-life stability. A person of ordinary skills in the art should recognize that these reagents are not fully inclusive and other reagents or reagent combinations that are inhibitors or activators of one or more hemostatic functions could be used in this cartridge.

(30) The cartridge apparatus disclosed here is a component of a measurement system (e.g., a hemostasis measurement system). The measurement system (also referred to as the instrument) includes at least an interface element which couples between the cartridge apparatus and a measuring element configured to measures viscoelastic properties or mechanical properties of a sample processed within the cartridge apparatus. The measured viscoelastic properties or mechanical properties are outputted as results to a user interface. An example user interface is described in commonly assigned U.S. Pub. No. 2011/0252352 to Viola et al., which is incorporated by reference herein in its entirety.

(31) In some embodiments, the interface element includes one or more heating and/or cooling elements.

(32) In some embodiments, the interface element includes a fluidic manifold that facilitate connection to one or more pump elements and one or more valves.

(33) In some embodiments, the interface element includes one or more sensors, e.g., configured to perform hemostasis measurements. The one or more sensors, in some embodiments, includes ultrasound sensors. In other embodiments, the one or more sensors includes other interrogative devices that is based on thromboelastography, thromboelastometry (e.g., a thromboelastography-based system or a thromboelastometry-based system), or that measures changes in mechanical impedance, changes in perturbation as observed via an optical-based system (e.g., having an optical sensor), fluorescence, colorimetric-based system, aggregometry-based system (e.g., having optical sensor, acoustic sensor, or electrodes that measure aggregation with the test sample), resonance-based system (e.g., having optical, acoustic, or mechanical position sensors that measures the sample when the sample is at, or near resonance), electrical impedance-based system (e.g., having electrodes configured to measure electrical impedance), or a combination thereof.

(34) In some embodiments, the interface element includes a mechanical clamp configured to position the cartridge apparatus in a desired orientation with respect to the components (the one or more sensors, the fluidic manifold, the heating and/or cooling elements, and etc.) of the measurement system. When the interface element is interfaced with the components of the measurement system, the cartridge apparatus, in some embodiments, is driven via a series of controlled actions orchestrated by the measurement system to prepare the test sample for measurement. In some embodiments, the preparation operations include sample aspiration of a sample from a sample container (also referred to as a sample holding tube), sample heating and/or cooling, sample metering, sample mixing with reagents, and sample measurement. Each step, with reference to various embodiments, is described below. After measurements are completed, the results are output in the instrument user interface.

(35) In some embodiments, the cartridge apparatus and its internal components are the only component that directly contact with a sample to be analyzed.

(36) In some embodiments, the cartridge includes computer readable information that can be optically or communicatively interrogated (e.g., RFID tags, computer readable medium such as flash ICs, QR codes, BAR codes, and etc.) and/or human readable information (e.g., labels).

(37) The various embodiments described below does not utilize any active valve element in the cartridge design, but instead relies on a fluidic manifold and one or more valves placed in the instrument. Fluid is moved through the various cartridge components via pressure differential and/or gravity and/or material properties (such as in the case of hydrophobicity or hydrophilicity) and/or capillary forces.

(38) In these embodiments, the cartridge is configured to couple with the instrument via one or more connection ports that are aligned via alignment slots. The connection ports include one or more pressure ports and one or more vent ports. However, in alternative embodiments, actuated valves (such as in the case of elastomeric valves) can be included in the cartridge design to control fluid flow. These valves are actuated, in some embodiments, by corresponding hardware and software components in the measurement system.

(39) The surface properties and texture of the cartridge surfaces in direct contact with the sample can be optimized to promote sample adhesion and/or sample flow. In some embodiments, the test chamber's interior surface and/or other interior surfaces of the fluidic circuit within the cartridge apparatus are plasma treated to optimize the surface energy and texture for adhesion of specific plasma proteins. In other embodiments, test chamber's interior surface and/or other interior surfaces of the fluidic circuit are treated with surface roughness texturing, material coating (such as in the case of gold plating), biological material coating (such as in the case of fibronectin or collagen coating, for example), raw material selection (e.g., use of specific plastic or other materials for the plate that does not require additional treatment), etc. Such treatments maybe performed independently, or in conjunction with, a plasma treatment. Similarly, the cartridge materials can be selected or manipulated to achieve the desired hydrophobicity or hydrophilicity. These properties can be changed by plasma treatment or by surface coatings.

(40) As described in more details below, the cartridge and the associated measurement system can utilize one or more sensors of one or more types (e.g., optics, pressure, ultrasound, etc.) as part of the automated operations of the cartridge. In addition, the outputs of such one or more sensor(s) can be further utilized to perform quality control checks. These checks may be performed before, during, or after cartridge testing to ensure function of one or more of the subsystems (for example, ultrasound or other interrogation system, fluidics, fluid level, clamping, cartridge positioning/orientation system, or temperature control), ensure the cartridge is functional, ensure correct sample preparation before measurements are performed or have been performed for the measurement, and may also be used to accept or reject a test result or even to abort testing before initiation of measurements.

(41) Note that in the discussion below a fluidic circuit includes a channel with fluidic component that connects one or more chambers together. Fluid circuit is also referred to as a testing channel in a multitude of channels that can be individually and controllably processed within a single cartridge apparatus.

(42) Cartridge Input Section

(43) FIGS. 1, 2, 3, and 4 are schematic illustrations of an example biological sample input section of a cartridge 100 for evaluating hemostasis. Specifically, FIG. 1 shows a perspective view of an example biological sample input of a cartridge for use in disposable system, in accordance with an illustrative embodiment. FIG. 2 shows a side view of the example biological sample input of FIG. 1 with a casing, in accordance with an illustrative embodiment. FIG. 3 shows a side cross-sectional view of the example biological sample input of FIG. 2 with a sample holding tube attached thereon, in accordance with an illustrative embodiment. FIG. 4 shows a detailed view of the example biological sample input of FIG. 3, in accordance with an illustrative embodiment. In alternative embodiments, the input section of the cartridge comprises a well to which fluid sample can be placed, for example, by way of a pipette or tube.

(44) In some embodiments, and as shown in FIG. 1, the cartridge 100 has a dual connection tab 28a, 28b for coupling the cartridge 100 to a sample container guide 1 (shown in FIG. 2). As shown in FIG. 2, the sample container guide 1, when mated with the cartridge 100, aligns a sample container 2 to a sample input port 3 of the cartridge 100. The cartridge 100 also includes an alignment tab 29 that is configured to slide into an alignment groove 30 of the sample container guide 1 to further stabilize the coupling of the sample container guide 1 to the cartridge 100. The sample container guide 1 can further provide a hard stop 5 (shown in FIG. 3) to hold the sample container 2 at the appropriate height to establish fluidic communication with the cartridge 100.

(45) In various embodiments, the sample container 2 is an evacuated tube (also referred to herein as the sample holding tube 2) such as a BD Vacutainer™ tube, and the sample input port 3 comprises one or more needles required for sample transferring 3a and venting 4 (see FIG. 1). Though shown as concentric in the figures, the needles can be configured to be concentric, side by side, or integrated. In some embodiments, and as shown in FIG. 1, the sample transferring needle 3a includes inlets (3b and 3d) and an outlet 3c that terminates in a sample inlet chamber 26 of the cartridge 100. The sample inlet chamber 26 is in fluid communication with an inlet pathway 8 that leads to a retention/heating chamber 6 (see FIG. 5A). In some embodiments, and as shown in FIG. 1, the venting needle 4 includes an outlet 4a that is configured to terminate within the sample container 2 when attached and is spaced apart from the inlet 3d so as to minimize bubbles being drawn into the inlets 3b and 3d. The venting needle 4 also has an inlet 4b that terminates in a venting inlet chamber 27 of the cartridge. The venting inlet chamber 27 is in fluid communication with a vent pathway 9 that, in some embodiments, terminates at a filter chamber 9a (shown in FIG. 5A) housing a filter. An alternative sample container 2 can be utilized, such as a syringe, which requires a luer lock connection on the cartridge 100. And, as noted above, in other embodiments, the input section of the cartridge comprises a well to which fluid sample can be placed, for example, by way of a pipette or tube.

(46) Vent Pathway

(47) FIGS. 5A, 5B, 7, and 8 are schematic illustrations of biological fluid pathways of the example cartridge 100 in accordance to an embodiment. Specifically, FIGS. 5A and 5B each shows the biological fluid pathways of four testing circuits (corresponding to test chambers 16a, 16b, 16c, and 16d, shown in FIG. 5A), also referred to herein as Hemostasis testing circuits, that are located on a sample preparation plane, in accordance with an illustrative embodiment. Though shown with four testing circuits, additional circuits or less may be included, including, e.g., two, three, five, six, seven, eight, and etc. FIG. 5B further shows of the cartridge body of FIG. 5A further coupled to a sample holding tube 2, in accordance with an illustrative embodiment. FIG. 7 shows the back-side of the cartridge of FIG. 5A and includes an interconnection plane that interfaces to the sample preparation plane, that collectively form the biological fluid pathways for the testing circuits, in accordance with an illustrative embodiment. FIG. 8 shows portions of the biological fluid pathways that are on the interconnection plane of FIG. 7, in accordance with an illustrative embodiment.

(48) As noted above, the biological fluid pathways are formed on, and across, multiple planes defined in the cartridge 100. A first plane of fluid pathways of the cartridge 100 is shown in FIGS. 5A and 5B. The first plane of fluid pathways of the cartridge 100 may alternatively be referred to as a front plane of the cartridge 100. FIGS. 7 and 8 each shows a second plane of fluid pathways of the cartridge 100 in which FIG. 8 shows the fluid pathways in the second plane isolated from the remainder of the structure of the cartridge 100 for ease of understanding. The second plane of fluid pathways of the cartridge 100 may alternatively be referred to as a back plane of the cartridge 100. The fluid pathways between the first and second planes are connected by fluidic vias that traverse across the various planes of the cartridge 100.

(49) As discussed above in relation to FIG. 1, in some embodiments, the venting inlet chamber 27 is in fluid communication with a vent pathway 9. The vent pathway 9 may terminate at a filter chamber 9a (shown in FIG. 5A), which may house a filter therein. The filter chamber 9a in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a vent port 22i (shown in FIG. 8) in the second plane of fluid pathways of the cartridge 100. As discussed in more detail below, cartridge 100 couples with the measurement system (also referred to herein as the instrument) via the vent port 22i to provide atmospheric pressure to vent pathway 9.

(50) Heating Chamber Pathway

(51) As discussed above in relation to FIG. 1, in some embodiments, the sample transferring needle outlet 3c terminates in a sample inlet chamber 26 of the cartridge 100. The sample inlet chamber 26 is in fluid communication with an inlet pathway 8. The sample inlet pathway 8 provides a fluid communication pathway between the sample inlet chamber 26 and a retaining/heating chamber 6 (also referred to herein as heating chamber 6 or as a “first chamber”) (shown in FIG. 5A). The label “first”, “second”, and “third” as used herein is provided merely as labels and do not intended to connote a sequence. The heating chamber 6 is configured to mate with a corresponding thermal regulating (e.g., heating/cooling) system in the measurement system to warm or cool the sample toward or to, or near, a pre-defined temperature.

(52) The heating chamber 6, as provided herein, facilitate uniform conditioning of the test fluid prior to the fluid be metered or aliquoted to their respective testing, thus reducing variability in the test sample that can affect subsequent measurements and analysis. The shape of the heating chamber 6 can be optimized for heating/cooling transfer, as in the case here in which a thin cross-section with thin walls is used. The materials of the cartridge 100 can also be optimized to facilitate heating/cooling. In some embodiments, the sample heating/cooling conditioning stage can also be implemented in one or more chamber/channels of the cartridge design and it is not limited to just occur within just the heating chamber 6. In some embodiments, a stirring, rotating, or oscillating element (not shown) can be placed in the heating chamber 6 that may be controlled by the measurement system to promote uniform temperature heating or cooling. In other embodiments, test fluid in the heating chamber 6 may be vibrated by the measurement system vibrating the cartridge 100 to promote uniform temperature conditioning of the test fluid.

(53) In some embodiments, temperature measurement is conducted of the test sample in the cartridge 100. To measure the temperature, a sensor can be incorporated in the measurement system or in the cartridge 100. In some embodiments, a thermistor or thermocouple can be placed in physical contact with the cartridge 100, or biological sample (such as blood). In other embodiments, an IR thermometer is pointed at the cartridge 100 or biological sample. In either case the cartridge 100 may incorporate a small well through which the incoming blood passes, rather than having direct contact with the blood. In some embodiments, the temperature of the test sample may be assessed at or near the heating chamber 6. In other embodiments, the temperature of the test sample may be assessed while the test sample is flowing through channels as it is directed toward the test chambers 16.

(54) Referring now to FIGS. 5A, 5B, and 8, the sample inlet pathway 8 terminates at a first corner 6a of the heating chamber 6, shown as the top left corner of heating chamber 6 in FIGS. 5A and 5B. In some embodiments, chambers along the fluid pathways are generally filled from the top as to prevent blood to backflow into the inlet. A fill outlet channel 10a extends from a second corner 6b of the heating chamber 6 opposite from the first corner 6a.

(55) The fill outlet channel 10a extends to a filter chamber 10 with a filter therein. The filter chamber 10 (e.g., as shown in FIGS. 5A and 5B) in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a heating-chamber fill channel 10b shown in the second plane of fluid pathways of the cartridge 100 (see FIG. 8). The fill conduit 10b is in fluid communication with a pressure port 22a (see also FIG. 8) that facilitate filling of the heating chamber 6. The conduit 10b is a part of a network of conduits used to consolidate pressure ports (e.g., 22a as discussed, as well as 22b-22i to be later discussed) of the cartridge 100 to one or more areas to which the measurement system can coupled with its pressure control interfaces. Such configuration reduces the complexity of the measurement system to control fluid movement within the cartridge 100. Indeed, the conduits that handles the control of movement of the fluid sample in the first plane of the cartridge 100 is primarily placed in the second plane of the cartridge 100. FIGS. 6A and 6B show a front perspective view and a back perspective view of FIGS. 5A, 5B, and 8 with additional labels corresponding to the description of this section.

(56) Heating Chamber Fill

(57) In operation, the instrument's fluid pump aspirates the sample through the input port 3 (see FIGS. 1-2) via the connection ports 22 (see FIG. 7) (also referred to herein as pressure ports) and into the heating chamber 6 (see FIG. 5A or 5B) of the cartridge 100. For example, the instrument's fluid pump may be in communication with and apply differential pressure (e.g., positive or negative) to a pressure port 22a (see FIG. 8). This in turn creates an applied pressure along the fill conduit 10b, within the heating chamber 6, and along the inlet pathway 8 to aspirate the sample into the heating chamber 6. At the same time, the inner vent needle 4 is linked to the isolated pathway 9 that receives atmospheric pressure from the instrument via the vent port 22i (see FIG. 8) to neutralize pressure in the sample container 2 as the sample is aspirated into the heating chamber 6 of the cartridge 100. During filling of the heating chamber 6, all other ports (e.g., 22b-221) are closed, e.g., by the measurement system.

(58) When the heating chamber 6 is filled, the filter within filter chamber 10 is clogged and creates a pressure spike that is detected by the instrument, causing the instrument to turn off the fluidic pump. The instrument may also close the vent port 22i or otherwise discontinue supplying atmospheric pressure via vent port 22i upon detecting the pressure spike. Alternative filling detection techniques could also be used, i.e., optical sensors placed at the desired fill level, volumetric control, fixed time of pressure alteration (negative and/or positive pressures), ultrasound detectors placed at the desired fill level, etc. The sample remains in the heating chamber until the desired temperature is reached, which can for example be at or near body temperature of a normal and typical subject (e.g., about 37° C. for a healthy person). In other instances, other desired temperatures may be warranted. The shape of the heating chamber 6 and the channels leading to the sample metering chambers 11 (described below) are configured so that bubbles that might be present in the fluid sample are trapped away from the rest of the fluidic circuit. The shape of the inlet pathway 8 includes an anti-siphon feature 8a (see FIGS. 5A and 5B) and is configured to reduce the occurrence of bubbles forming in the heating chamber 6 and prevent siphoning to and from the sample container 2. To this end, additional unprocessed test sample (e.g., un-warmed blood) is prevented from being siphoned into the heating chamber following the first drawn test sample being heated and/or cooled, e.g., when the processed test sample is pulled into the metering chamber 11 (also referred to herein as sample chamber 11 and “second” chamber). FIGS. 6A and 6B also show labels corresponding to the description of this section.

(59) Sample Aliquot (Metering) Chambers Pathway

(60) Referring to FIGS. 5A, 5B, and 8, a first side 6c of the heating chamber 6 (see FIG. 5A) extends between the first corner 6a and the second corner 6b.

(61) One or more of outlet ports 6e-6h (see FIG. 5A) are arranged along the length of the second side 6d of the heating chamber. Each of the one or more outlet ports 6e-6h may be arranged in a different one of the valleys along the second side 6d of the heating chamber in which the valleys direct the sample into conduits leading to each respective test channel. A test channel, in some embodiments, refers to the associated fluidic pathway structures and testing chamber, collectively, used to perform a measurement for a given aliquot sample. In some embodiments, and as shown in FIG. 5B, each of the one or more outlet ports 6e-6h in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a first end of a corresponding one or more channels 20a-20d (see FIG. 8) in the second plane of fluid pathways of the cartridge 100, collectively referred to as channels 20 (see FIG. 5B). A second end of each of the one or more channels 20a-20d (see FIG. 8) in the second plane of fluid pathways of the cartridge 100 is likewise in fluid communication with a first end of a corresponding one or more channels 11a-11d (see FIG. 5B) in the first plane of fluid pathways of the cartridge 100. A second end of each of the one or more channels 11a-11d terminates in a corresponding one or more sample chambers 11 (shown in duplicates (“×4”) in FIG. 5B with an “o” symbol therein). In the example shown in FIGS. 5A, 5B, 7, and 8, there are four sample chambers 11. More or fewer sample chambers 11 and corresponding fluid communication pathways with the heating chamber 6 may be present on the cartridge 100 in some configurations.

(62) The sample chambers 11 are fed by the one or more channels 20 originating from the bottom of the heating chamber 6. This geometric configuration avoids bubbles being drawn into the sample chambers 11 as the bubbles rise to the upper portion of the heating chamber 6.

(63) Each of the sample chambers 11 has a corresponding fill channel 11e that is in fluid communication with a corresponding filter chamber 12 (shown in duplicates (“×4”) in FIG. 5B with a “+” symbol) with a filter therein. The filter chamber 12 in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a channel 12a (see FIG. 8) in the second plane of fluid pathways of the cartridge 100. The channel 12a is in fluid communication with a pressure port 22g (see FIG. 8).

(64) In some configurations, when more than one sample chamber 11 are implemented on the cartridge 100, the channel 12a (see FIG. 8) is in fluid communication with all of the sample chambers 11 (see FIG. 5B) via corresponding fill channels 11e and filter chambers 12. Accordingly, channel 12a acts as a manifold for applying negative pressure to all of the sample chambers 11 via a single pressure port 22g. Therefore, a separate pressure port is beneficially not needed for filling each of the sample chambers 11. FIGS. 6C and 6D show a front perspective view and a back perspective view of FIGS. 5A, 5B, and 8 with additional labels corresponding to the description of this section.

(65) Heating Chamber Vent Pathway

(66) Referring to FIGS. 5A, 5B, and 8, the heating chamber 6 comprises a vent pathway 31a, 31b, 31c along the first side 6c of the heating chamber for venting the heating chamber 6 as the sample chambers 11 are filled. The vent pathway 31 (not shown) includes the fluid pathway through conduit elements 31a-31d. Channels 31a-31b terminate at one end in the heating chamber 6 along the first side 6c and terminate at the other end at a filter chamber 31c with a filter therein. Accordingly, channels 31a-b provide a fluid pathway between the heating chamber 6 and the filter chamber 31c. The filter chamber 31c (see FIG. 5A) in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a channel 31d (see FIG. 8) in the second plane of fluid pathways of the cartridge 100, which in turn is in fluid communication with a vent port 22c (see FIG. 8). As discussed in more detail below, cartridge 100 couples with the instrument via the vent port 22c for the instrument to provide atmospheric pressure to vent the heating chamber 6. FIGS. 6C and 6D also show labels corresponding to the description of this section.

(67) Sample Chamber Vent Pathway

(68) Referring to FIGS. 5A, 5B, and 8 each of the sample chambers 11 includes a vent pathway 18 that terminates at a first end in a corresponding sample chamber 11. A second end of the vent pathway 18 in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a vent manifold 18 in the second plan of fluid pathways of the cartridge 100. When more than one sample chamber 11 is present, all of the vent pathways 18 (see FIG. 5B) of the sample chambers are in fluid communication with the vent manifold 18a (see FIG. 8). The vent manifold 18a (see FIG. 8) in the second plane of fluid pathways of the cartridge 100 is further in fluid communication with a first end of a channel 18b (see FIG. 5B) in the first plane of fluid pathways. A second end of channel 18b (see FIG. 5B) is in fluid communication with a filter chamber 18c (see FIG. 5B) with a filter therein. Filter chamber 18c (see FIG. 5B) in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a vent port 22e (see FIG. 8). FIGS. 6C and 6D also show labels corresponding to the description of this section.

(69) Sample Aliquot (Metering) Chambers Fill

(70) During operation, once the sample is at, or near, the desired temperature, the sample is aliquoted (or metered) into one or more independent sample chambers 11 (see FIG. 5B). Referring to FIG. 5B unless indicated otherwise, in various embodiments, the sample chambers are filled by applying a negative pressure at pressure port 22g (see FIG. 8) via a pump in the instrument while venting the heating chamber 6 by way of the vent port 22c (see FIG. 8). Each filter chamber 12 has a filter therein that will clog when the corresponding sample chamber 11 is filled and will trigger the pressure sensor of the instrument to turn the pump off, similar to filling the heating chamber 6. As discussed above, alternative filling detection techniques could be used. In various embodiments, all the sample chambers 11 are controlled by a single valve and fluidic pathway in the instrument via pressure port 22g (see FIG. 8). The cutoff pressure will not trigger until all sample chambers' filters clog in the corresponding filter chambers 12. The sample chambers 11 are used to separate samples into independent functional channels, aliquot a known volume of sample, and stage the sample for mixing with reagents. While the sample chambers 11 are filled, pressure ports 22b, 22d, 22f, and 22h (see FIG. 8) as well as vent port 22e (see FIG. 8) are closed by the instrument so as to prevent fluid from leaking past the location 19, arranged below the sample chambers 11. Once the sample chambers 11 have been filled, the vent port 22e (see FIG. 8) is opened to atmospheric pressure so that the one or more sample chambers 11 can be fluidically separated from each other and the heating chamber 6. Vent port 22e (see FIG. 8) remains open to atmosphere during sample mixing as discussed below. FIGS. 6C and 6D also show labels corresponding to the description of this section.

(71) Mixing and Testing Pathway

(72) Referring to FIG. 5B unless indicated otherwise, each of the sample chambers 11 is in fluid communication with a corresponding one or more reagent pocket 14 (see FIG. 5A) configured to house at least one lyophilized bead comprising a reagent. As shown in FIGS. 5A and 5B, two reagent pockets 14 (shown as 14a and 14b in FIG. 5A for one of the test channels) are provided. In other embodiments, a single reagent pocket is used for each testing channel. In yet other embodiments, more than two reagent pockets 14 are used for each testing channel. The reagent pockets 14 are in turn in fluid communication with a serpentine channel 13 (see FIG. 5A, and shown with a duplication symbol (“×4”). Each of the serpentine channel 13 has a first end in fluid communication with the reagent pockets and a second end that terminates at an optical detection zone 15. As discussed below, the instrument (i.e., measurement system) may optically interrogate the optical detection zone 15 of the cartridge 100 to facilitate the control of a pump that facilitates mixing of the individual aliquots with the corresponding reagent(s). Each of the serpentine channel 13 (see FIG. 5A) is in fluid communication with a test chamber 16 (see FIG. 5B, with duplication symbol “×4”) which in turn is in fluid communication with a filter chamber 17, with a filter therein. The filter chamber 17 in the first plane of fluid pathways of the cartridge 100 is in fluid communication with a first end of a corresponding one of fluid channels 17a-17d (see FIG. 8) in the second plane of fluid pathways of the cartridge 100. A second end of each of the fluid communication channels 17a-17d (see FIG. 8) are in fluid communication with a corresponding pressure port 22b, 22d, 22f, and 22h (see FIG. 8). The cartridge 100 couples with instrument via the pressure ports 22b, 22d, 22f, and 22h (see FIG. 8) to supply positive and negative pressure to facilitate mixing and testing of the sample as described below. FIGS. 6E and 6F also show a front perspective view and a back perspective view of FIGS. 5A, 5B, and 8 with additional labels corresponding to the description of this section.

(73) Sample Mixing

(74) Referring to FIG. 5B unless indicated otherwise, each individual aliquot in the sample chambers 11 is pulled into a separate reservoir or channel (a serpentine channel pathway 13 (see FIG. 5A) in various embodiments) and brought into contact with the channel specific reagent, located in one (or both) of the two reagent pockets 14 (see FIG. 5A). Specifically, a pump in the instrument applies negative pressure to pressure ports 22b, 22d, 22f, and 22h (see FIG. 8) to draw the sample through the reagent pockets 14 (see FIG. 5A) and serpentine channel pathway 13 (see FIG. 5A). The reagents and sample are kept separate from each other during sample chamber 11 filling so as to avoid reagents floating on the blood and getting trapped in the filter in filter chambers 12, as well as to ensure fluidic isolation of the one or more sample chamber 11 from the heating chamber 6. To this end, precise time of when the test sample is in contact with the reagents can be measured thereby facilitating accurate and precise clot time measurements (e.g., from when mixing begins). In addition, the reagents and test samples are kept separated from one another, in some embodiments, until all channels are metered to prevent undesired siphoning of test samples from other channels or from the unprocessed sample in the fluidic pathways. The sample is aspirated through the serpentine channel 13 (see FIG. 5A) until it triggers an optical sensor in the instrument (detection zone is top of serpentine channel near the optical detection zone 15 (see FIG. 5A)) which closes off the channel from the pump. Mixing in the serpentine channels or zones, or region, therein can be controlled by one or more independent valves and pathways that allow individual channel control. Alternative sensor techniques could be utilized: pressure, pass through optical sensors, ultrasound detection, time, volumetric control, etc. Once all the channels' optical sensors trigger, the pump reverses and applies positive pressure to pressure ports 22b, 22d, 22f, and 22h (see FIG. 8). The positive pressure pushes the biological sample, such as blood, down the serpentine path 13 (see FIG. 5A) for a designated time, or until a second set of optical sensors in the instrument is tripped (in an alternative embodiment). This process, to pull the sample up the serpentine path 13 (see FIG. 5A) to the optical detection zone where it is detected by an optical sensor of the instrument and push back for a given time, repeats until full sample mixing is achieved. FIGS. 6E and 6F also show labels corresponding to the description of this section.

(75) Other sensors (e.g., impedance sensors), pressure sensor, and etc., may be used. Alternatively, additional sensors may be used to detect both ends of the optical detection zone. Alternate pathway geometries, obstructions to create turbulence, cycle numbers, and cycle speed are all design alternatives that can be used with varying test types to achieve optimal results. In alternative embodiments, mixing could be achieved with one or more ferromagnetic beads or bars placed within the cartridge and controlled by the instrument.

(76) Test Chamber Filling

(77) Referring to FIGS. 5A, 5B, and 8, one or more testing chambers 16 are filled after mixing is completed. Using one or more independent valves and pathways, each testing chamber 16 is filled with a sample via the application of pressure perturbations (negative and/or positive pressure) at pressure ports 22b, 22d, 22f, and 22h. Specifically, the instrument pump will apply negative pressure to pressure ports 22b, 22d, 22f, and 22h until all of the filters are clogged in the one or more filter chambers and cause a pressure spike to cause the instrument to turn the pump off, similar to filling the heating chamber 6. As discussed above, alternative filling detection techniques could be used. The testing chambers 16 have design features such as the ridges 24a which prevent the formation of bubbles in the testing chambers 16 during filling. Once filled, the instrument begins viscoelastic testing of the sample. FIGS. 6E and 6F also show labels corresponding to the description of this section.

(78) In some embodiments, the cartridge apparatus includes, at least, four independent fluidic circuits configured with different sets of reagents for measurements (and/or sample preparation) to be performed in parallel. The measurements are performed per channel of the, at least, four channels of the cartridge. The measurement, in some embodiments, include viscoelastic properties such as a sample shear modulus. The measurement, in another embodiment, includes other properties such as viscosity, elastic modulus, or any other mechanical property of the sample, or combinations thereof.

(79) Table 1 provides an example set of reagents and measurement parameters for use in an example cartridge apparatus (e.g., apparatus 100, among others). As shown in Table 1, Channel #1 in the example cartridge apparatus is interrogated to measure clot time of the test sample in the presence of kaolin, which is an activator of the intrinsic pathway of coagulation. As shown in Table 1, Channel #2 is interrogated to measure clot time of the test sample in the presence of kaolin and in further presence of heparinase I, which is a neutralizer of the anticoagulant heparin. As shown in Table 1, Channel #3 is interrogated to measure overall clot stiffness of the test sample in the presence of i) thromboplastin, which is an activator of the extrinsic pathway of coagulation, and ii) polybrene, which is a neutralizer of the anticoagulant heparin. As shown in Table 1, Channel #4 is interrogated to measure clot stiffness of the test sample with the same reagents as channel #3, but with the addition of abciximab (e.g., Clotinab® and/or ReoPro®), which is an inhibitor of platelet aggregation/contraction. As shown in Table 1, when the assay is configured to operate with citrated whole blood samples, calcium is added to all the reagent formulations.

(80) TABLE-US-00001 TABLE 1 Reagents utilized in a preferred embodiment Measurement Channel # Reagents (units) 1 Kaolin, calcium, buffers and stabilizers Clot time (Seconds) 2 Kaolin, heparinase I, calcium, buffers and Clot time stabilizers (Seconds) 3 Thromboplastin, polybrene, calcium, Clot stiffness buffers and stabilizers (hecto Pascals) 4 Thromboplastin, polybrene, abciximab Clot stiffness (and/or cytochalasin D), calcium, (hecto Pascals) buffers and stabilizers

(81) Table 2 provides an additional example set of reagents and measurements for use in an example cartridge apparatus (e.g., apparatus 100, among others). As shown in Table 2, channel #2 includes an extrinsic pathway activator with inhibition of fibrinolysis by tranexamic acid (TXA). In addition to the measurements previously presented in Table 1, channels #2, channel #3, and channel #4 are interrogated to also measure clot stiffness changes, which, for example, can be related to the fibrinolytic process. In some embodiments, other channels can include reagents that inhibit fibrinolysis and can also be interrogated to measure clot stiffness changes. For example, channel #4 could also include TXA or other fibrinolysis inhibitor in order to measure clot stiffness in the absence of fibrinolysis.

(82) TABLE-US-00002 TABLE 2 Reagents utilized in a preferred embodiment Measurement Channel # Reagents (units) 1 Kaolin, calcium, buffers and stabilizers Clot time (Seconds) 2 Thromboplastin, polybrene, calcium, Clot stiffness tranexamic acid, buffers and stabilizers (hectoPascals) and Clot stiffness change 3 Thromboplastin, polybrene, calcium, Clot stiffness (hecto buffers and stabilizers Pascals) and clot stiffness change 4 Thromboplastin, polybrene, abciximab Clot stiffness (hecto (and/or cytochalasin D), calcium, Pascals) and clot buffers and stabilizers stiffness change

(83) In some embodiments, clot time and clot stiffness are measured by analyzing a shear modulus (clot stiffness) versus time curve that is generated within each measurement channel in the cartridge. FIG. 13 shows an example shear module versus time curve, in accordance with an illustrative embodiment. Clot time may be determined by identifying when the clot stiffness meets or exceeds a threshold value, or when the first or higher derivative of such property being measured meets or exceeds a threshold value, or at the point of maximum acceleration in the rate of clot stiffness, or some combination of the above methods. Clot stiffness may be estimated by the clot stiffness at a fixed time after clot time, or the maximum overall clot stiffness measured within some time limit, or the clot stiffness at the point of maximum rate of change in clot stiffness, or some combination of the above methods. Similar methods can also be applied to measure the effects of fibrinolysis (i.e., clot dissolution) and the corresponding reduction in clot stiffness. In some embodiments, clot stiffness changes can be calculated as percentage drop in clot stiffness over a fixed time window, as a rate of change of clot stiffness over time, as area under or over the clot stiffness vs time curve within a predefined time window, as the time required to achieve a predefined drop in clot stiffness, or combinations thereof. Similar curves and similar measurements to those just described can be formed by plotting the Young's modulus, viscosity, or other viscoelastic property of the sample being measured.

(84) TABLE-US-00003 TABLE 3 Parameters reported from measurement of the preferred embodiments discussed in relation to Table 1. Hemostatic Index Units Description Measurement Clot Time Minutes Clot time in citrated Clot time measured from (min) whole blood channel #1 with kaolin activation (intrinsic pathway) Heparinase Minutes Clot time in citrated Clot time measured from Clot Time (min) whole blood with channel #2 with kaolin heparin activation and heparinase I neutralization Clot hecto Stiffness of the Clot stiffness measured Stiffness Pascals whole blood clot from channel #3 with (hPa) thromboplastin activation (extrinsic pathway) and polybrene Fibrinogen hecto Contribution of Clot stiffness measured Contribution Pascals functional from channel#4 with (hPa) fibrinogen to thromboplastin activation, clot stiffness polybrene, and abciximab Clot Time Unit Assessment of Calculated ratio of clot Ratio less residual heparin time values from anticoagulation channels #1 and #2 Platelet hecto Contribution of Calculated from subtraction Contribution Pascals platelet activity to of the clot stiffness values (hPa) clot stiffness from channels #3 and #4

(85) A person of ordinary skills in the art should recognize that clot time and clot stiffness can be estimated using a number of methodologies and criteria. Clot times and clot stiffness values obtained from the, at least, four channels/measurements may be combined to provide, at least, six parameters can depict a functional status of the patient's hemostatic system. The indexes are summarized in Table 3. Relationship between results (clot time, clot stiffness, clot stiffness change, etc.) from different channels may be verified to be within expected ranges as additional quality control checks to verify instrument, cartridge, and sample function.

(86) In other embodiments, other reagents can be used and other hemostatic indexes or output parameters can be obtained such as in the case of a fibrinolytic index, indexes corresponding to the functionality of anti-platelet treatments, indexes corresponding to the functionality of anti-coagulation treatments, etc.

(87) For example, one or more fibrinolysis indexes could be formed using the clot stiffness changes measured in any of the channels presented in Table 2, but preferably channels #3 and #4. Alternatively, a fibrinolysis index could be formed by differential combination of the clot stiffness changes measured in channels #2 and channel #3 presented in Table 2. Such combination could be in the form of a ratio, a difference, or combinations thereof. One of the benefit of using a combination of clot stiffness changes measured with and without an anti-fibrinoltyic reagent is the ability to mitigate the interfering effects of non-fibrinolysis driven reductions in clot stiffness values. In some embodiments, TXA or other fibrinolysis inhibitor reagent can be included in both channel #2 and channel #4 of the example cartridge of Table 2. With such modifications the parameters Clot Stiffness, Platelet Contribution, and Fibrinogen Contribution could be derived without the influence of fibrinolysis by combination of the clot stiffness measurements obtained in channel #2 and channel #4.

(88) As discussed above, an example user interface is described in commonly assigned U.S. Pub. No. 2011/0252352 to Viola et al., which is incorporated by reference herein in its entirety. The example user interface may be used to display the measured hemostatic indexes as discussed in relation to Table 4, among other parameters.

(89) TABLE-US-00004 TABLE 4 Parameters reported from measurement of the preferred embodiments discussed in relation to Table 2. Hemostatic index Units Description Measurement Clot Time Minutes Clot time in Clot time measured from (min) citrated whole channel #1 with kaolin blood activation (intrinsic pathway) Clot hecto Stiffness of Clot stiffness measured Stiffness Pascals the whole from channel #3 with (hPa) blood clot thromboplastin activation (extrinsic pathway) and polybrene Fibrinogen hecto Contribution of Clot stiffness measured from Contribution Pascals functional channel #4 with (hPa) fibrinogen to thromboplastin activation, clot stiffness polybrene, and abciximab Platelet hecto Contribution of Calculated from subtraction of Contribution Pascals platelet activity the clot stiffness values from (hPa) to clot stiffness channels #3 and #4 Clot % or Clot stiffness Changes (% or rate of change) Stiffness hPa/sec change over in clot stiffness measured Change or sec time from channels #2 and #3 Clot % or Differential rate Differential comparison of Reduction hPa/sec, of clot stiffness clot stiffness change measured Differential or no changes with in channels #2 and #3. units and without anti-fibrinolytic

(90) As noted before, in various embodiments, the testing chambers 16 are shaped to facilitate ultrasound testing of viscoelastic properties of the sample, but alternative geometries can also be implemented to facilitate other types of testing. Such an ultrasound testing system is described in commonly assigned U.S. Pat. No. 9,726,647 and U.S. Pub. No. 2016/0139159, both of which are hereby incorporated by reference in their entirety. Ultrasound transducers in the measuring system connect with the testing chambers 16 of the cartridge 100 via compliant and deformable elastomers 21 which are affixed to a testing block 21d on the cartridge 100.

(91) Example elastomeric materials optionally include, Dynaflex D3202, Versaflex OM 9-802CL, Maxelast 54740, RTP 6035, Versaflex CL2003X, among others. Referring now to FIG. 9 unless indicated otherwise, the testing block 21d is aligned with the testing chambers 16 (see FIG. 5B) via alignment slots 23 and 24 on the cartridge 100. Referring still to FIG. 9, the elastomers 21 may be affixed to the testing block 21d via a flange 21a on the elastomers 21. The flange 21a may have a plurality of alignment holes 21b that may receive corresponding alignment pegs (not shown) from the testing block 21d. The soft elastomers 21 may also each include a lens 21c that focuses ultrasound energy within the sample at the testing chambers 16.

(92) FIG. 16 shows a photograph of an exemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9 for use in a disposable system, in accordance with an illustrative embodiment. FIG. 17 shows a front view of the exemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9, in accordance with an illustrative embodiment. FIG. 18 shows a front view of the exemplary cartridge of FIGS. 1, 2, 3, 4, 5A, 5B, 7, 8, and 9, in accordance with an illustrative embodiments.

(93) As described in U.S. Pat. No. 9,272,280, which is incorporated by reference herein in its entirety, in various embodiments, the consumable cartridge contains a lens assembly that focuses ultrasound energy within the sample that can be used to generate streaming and mixing. The lens assembly, or sound focusing assembly, is designed using a soft material, such as a thermoplastic elastomer 134 (previously referred to as 21), in conjunction with a rigid substrate 132 (e.g., formed of testing block 21d), such as polystyrene as shown in FIGS. 10, 11, and 12. This combination provides a dry ultrasound coupling that does not require the use of any fluid or gel couplant. Note that the same lens and ultrasound driver used for hemostasis measurement can be used in this matter to provide mixing. Increasing acoustic energy for mixing can be delivered by, for example, increasing pulse length, pulse amplitude or pulse repetition frequency.

(94) Referring now to FIG. 10, a top cross-sectional view of the testing chamber 116 (referred to previously as testing chamber 16) is shown. To seal each test chamber, e.g. test chamber 116, a lens assembly 131 includes a rigid substrate 132 and a couplant 134 that can be positioned at the back end of each test chamber.

(95) Referring still to FIG. 10, each couplant 134 comprises an elastomeric material. Optionally, the elastomeric material is a thermoplastic elastomer (TPE). Example elastomeric materials optionally include, Dynaflex D3202, Versaflex OM 9-802CL, Maxelast 54740, RTP 6035, Versaflex CL2003X, among others. Optionally the couplant is over-molded to the rigid substrate. Optionally the couplant is mechanically anchored to the rigid substrate.

(96) Referring still to FIG. 10, between each couplant 134 and the open space of each test chamber is a rigid substrate 132. The rigid substrate and the couplant form an interface that focuses ultrasound transmitted (e.g. lens assembly) by an ultrasonic transducer into the chamber's open space and onto any biological fluid and/or reagents in the chamber. The rigid substrate of the lens can comprise a material which allows sound to pass and that can act to focus ultrasound at some level within the space. Optionally, the rigid substrate comprises a styrene.

(97) Referring now to FIG. 11, The lens assembly may be glued or welded to the surface 101 of the testing block 21d (shown in FIG. 11 as element 132) to secure the lens in place in an orientation that allows the desired focusing of sound. Alternatively, the lens assembly is optionally manufactured together with the surface 101 of the testing block 21d. In this regard, the rigid substrate 132 can be molded with the surface 101 of the testing block 21d and the couplant 134 can be overmolded or mechanically anchored on the rigid substrate. A wide variety of materials can be used to construct the device. For example, plastics can be used for single use, disposable cartridges.

(98) Referring still to FIG. 11, each of the test chambers 116 can have a lens assembly positioned over the large opening of each chamber's open space. In this way, each chamber can be separately interrogated by focused ultrasound.

(99) Referring still to FIG. 11, when placed in the instrument, the couplant 134 can be placed in acoustic communication with a transducer for supplying ultrasound through the lens assembly and into a test chamber 116. Optionally, an intermediate layer of an acoustically permeable material is positioned between an ultrasonic transducer and the couplant. For example, and intermediate layer or block of Rexolite® or TPX® can be used. The intermediate layer can be forced against the couplant and can be in acoustic contact with the transducer.

(100) Referring still to FIG. 11, sound generated by a transducer passes through the intermediate layer, through the couplant, through the rigid substrate, and is focused within the biological sample, such as blood, and reagent in the test chamber. Some of the sound directed into chamber contacts the distal interior surface 111 of the test chamber, which is defined by the surface 126. Optionally, the surface is polystyrene. The distal interior surface has a known geometry and is positioned at a known distance from the ultrasound source. The distal interior surface 111 is used as a calibrated reflector, which is used to estimate the speed of sound and attenuation of sound in a test chamber at base line and during the process of clot formation and clot dissolution. These measurements can be used, for example, to estimate hematocrit of the subject along with the indexes of hemostasis. The sound generated by the transducer can be focused within the biological sample in a test chamber using a parabolic mirror that is coupled to the biological sample using an elastomer.

(101) Other example cartridge apparatus and measurement system, and methods thereof, are described in U.S. Pat. No. 9,031,701; U.S. Provisional Appl. No. 61/443,084; U.S. Pat. Nos. 9,272,280; 9,410,971; U.S. Provisional Appl. No. 61/443,088; U.S. Publication No. 2011/0252352; published PCT Publication No. WO2011/127436; U.S. Publication No. 2012/0294767; U.S. Pat. Nos. 7,892,188; 8,740,818; and U.S. Publication No. 2016/0274067, each of which is incorporated by reference herein in its entirety.

(102) As noted, the cartridge and features described herein can be modified for use with other types of measurement systems such as thromboelastography-based systems, thromboelastometry-based systems, optical-based systems, fluorescence-based systems, colorimetric-based systems, aggregometry-based systems, resonance-based system, and an electrical impedance-based system, among others.

(103) Many modifications and other embodiments of the invention set forth herein will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

(104) As used in the claims, the term “first”, “second”, and “third” are provided merely as labels and do not intended to connote a sequence.