Devices, systems and methods for evaluation of hemostasis

DESCRIPTION

DEVICES, SYSTEMS AND METHODS FOR EVALUATING HEMOSTASIA

CROSS REFERENCE TO RELATED REQUESTS

This claim claims the benefit of the Interim Order in US 61 / 443,088, filed on February 15, 2011.

TECHNICAL FIELD

The present application relates to devices, systems and methods for assessing hemostasis in a subject by analyzing a test sample from the subject to determine one or more indices of hemostasis.

BACKGROUND

Hemostasis, the physiological control of bleeding, is a complex process that incorporates the vasculature, platelets, coagulation factors (FI-FXIII), fibrinolytic proteins and coagulation inhibitors. The interruption of hemostasis plays a central role in the onset of myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis and excessive bleeding. Consequently, in vitro diagnosis (IVD) is extremely necessary to quantify hemostatic dysfunction and to direct appropriate treatment. This need is particularly acute during cardiac surgeries that require extracorporeal circulation (CPB), in which post-surgical bleeding is a common complication that requires transfusion of blood products.

Existing IVDs include biochemical assessment criteria assays, platelet aggregation assays and viscoelastic clot measurement systems. Biochemical assessment criteria assays, such as prothrombin time (PT) and partial thromboplastin time (PTT), are widely used to assess coagulation. However, these tests measure only a part of the hemostatic process and operate in non-physiological conditions by incorporating only the function of the plasma. As a result of these limitations, complications such as postoperative bleeding often occur despite normal perioperative PT and PTT measurements.

activated clotting time (ACT) is an evaluation criterion assay applied most frequently to support CPB. This test applies a strong initiation of the (intrinsic) surface activation pathway to quantify heparinization. The limitations of TCA include its disregard for platelet function, lysis and coagulation kinetics, along with the use of large aliquots of whole blood (WB) (usually 2 mL) and moving mechanical parts. For these reasons, TCA is used for rapid assessment of heparinization and reversal of associated protamine, with limited utility for additional applications.

Platelets play a crucial role in the progression of clotting and suppression of arterial bleeding. In addition, modern cell-based hemostasis theory recognizes that platelets play a modulating role in coagulation. Platelet function is monitored clinically through both central laboratory and point of care (POC) tests, which use anticoagulated WB. Both approaches are limited in that they use platelet aggregation as a substitute for general platelet function. In addition, by disabling coagulation, these methods neglect the interaction between platelets and the coagulation cascade.

Techniques that monitor the WB's viscoelastic properties, such as thromboelastography (TEG) and rotational thromboelastomer (ROTEM), circumvent many of the limitations of biochemical assessment criteria tests and platelet aggregation tests by measuring the combined effects of all components of hemostasis. TEG has been shown to diagnose hyperfibrinolysis in bleeding patients, indicates transfusion requirements better than standard biochemical assays and reduces transfusion requirements during CPB when used with transfusion algorithms. Although these tests offer valuable clinical information, the devices are typically complex to operate and difficult to interpret. In addition, TEG applies relatively large shear stresses, which transgress the non-linear viscoelastic regime, thereby interrupting the formation of clots. For these reasons, TEG finds very limited utility as a POC test.

A novel ultrasound-based method to evaluate hemostatic function of whole blood, Clinica Chimica Acta 411 (2010) 106 to 113, Francesco Viola, F. William Mauldin Jr., Xiefan Lin-Schmidt, Doris M. Haverstick, Michael B. Lawrence, William F. Walker, discloses sonorometry, which can assess hemostasis function from a small blood sample. Sonorometry uses the phenomenon of the force of acoustic radiation to measure the dynamic changes in blood viscoelasticity during the formation and dissolution of the clot.

US No. 5,629,209 discloses an apparatus for detecting changes in the viscosity of a fluid, comprising a cartridge that has a fluid receiving / dispensing reservoir, one or more fluid receiving chambers and a conduit that allows fluid communication between the reservoir receiving / dispensing fluid and the fluid receiving chamber.

US2011034805 discloses methods, apparatus and systems for characterizing at least one physical property of blood, by generating a series of acoustic pulses and directing the series of pulses in the blood so that at least one of the pulses is of sufficiently high intensity to induce the physical displacement of blood. Acoustic pulses and / or optical waves reflected from the blood, or a flexible limb in contact with the blood that moves with the blood, are received and measured to estimate at least one characteristic of the physical displacement induced by it.

WO2011127436 discloses a system for displaying a plurality of hemostatic indices. The system includes a communication receiver configured to receive the hemostatic indexes and a graphical user interface (GUI) connected to the communication receiver and to display the hemostatic indexes simultaneously. Hemostatic indices are derived from a plurality of independent measurements, such as mechanical measurements determined using sonorometry systems and processes.

SUMMARY

Systems and methods for assessing hemostasis are provided. According to one aspect of the invention, a device for assessing hemostasis is provided as claimed in claim 1. In one embodiment, the reagents are selected from the group consisting of kaolin, celite, glass, abciximab, cytochalasin D, thrombin, tissue factor recombinant, reptilase, arachidonic acid (AA), adenosine diphosphate (ADP) and combinations thereof. Optionally, the reagents are lyophilized before interacting with the test samples.

The system comprises a transducer for transmitting ultrasound to one or more chambers and for receiving reflected sound from the chamber and the test sample in it. The system additionally comprises at least one processor configured to determine a hemostasis parameter of the received sound. The parameters are optionally selected from the group consisting of TC1, TC2, clot stiffness, clot formation rate (CFR), TL1 and TL2. 0 The processor is also optionally configured to determine an intrinsic pathway coagulation factor index, an extrinsic pathway coagulation factor index, a platelet index, a fibrinogen index and a fibrinolysis index value. The intrinsic and extrinsic coagulation factors are optionally combined to form an index of coagulation factors.

According to another aspect of the invention, a method is provided as claimed in claim 11. The parameters are optionally selected from the group consisting of TC1, TC2, clot stiffness, clot formation rate (CFR), TL1 and TL2. The methods disclosed may further include the determination of an intrinsic pathway coagulation factor index, an extrinsic pathway coagulation factor index, a platelet index, a fibrinogen index and a fibrinolysis index value. The intrinsic and extrinsic coagulation factors are optionally combined to form an index of coagulation factors. The reagents or combinations thereof are optionally lyophilized before being mixed with the blood.

These and other features and advantages of the present invention will become more readily apparent to those skilled in the art upon consideration of the following detailed description and accompanying drawings, which describe both the preferred and alternative embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA to G are schematic illustrations of an exemplary cartridge for assessing hemostasis.

Figure 2 is a schematic illustration of the biological fluid pathways of the exemplary cartridge of Figures IA to G.

Figure 3 is a schematic illustration of an exemplary processing system for use with the exemplary cartridge from Figures IA to G.

Figure 4 is a schematic illustration of a portion of a system for assessing hemostasis.

Figure 5 is a schematic illustration of a portion of a system for assessing hemostasis.

Figure 6A is a schematic illustration showing N acoustic pulses that are sent to a blood sample to generate a force. The resulting deformation can be estimated from the relative time delays between the N return echoes.

Figure 6B is a graph showing exemplary displacement curves generated within a blood sample. As the blood clots, reduced displacement is observed.

Figure 6C is a graph showing combined displacements to form relative rigidity graphs, which characterize the hemostatic process. The parameters described in the panel are estimated from parameters found by adjusting a sigmoidal curve.

Figure 7 is a flowchart that illustrates an exemplary method for estimating hemostasis parameters.

Figures 8A to D are schematic illustrations of an exemplary cartridge for assessing hemostasis.

Figures 9A to C are schematic illustrations of portions of a system for assessing hemostasis that includes pressure control mechanisms.

Figures 10A and 10B are schematic illustrations of an exemplary sample flow pattern for use with the devices and systems described and an exemplary cartridge for assessing hemostasis.

Figure 11 is a graph showing blood heating data inside an exemplary cartridge for assessing hemostasis.

Figures 12A to C are schematic illustrations of exemplary sound focusing mechanisms.

DETAILED DESCRIPTION

The present invention will be described more fully hereinafter with reference to specific embodiments of the invention. In fact, the invention can be embodied in many different forms and should not be interpreted as limited to the modalities presented in this document; instead, these modalities are provided so that this disclosure meets the applicable legal requirements.

As used in the specification and the appended claims, the singular forms one, one and o / a include the plural reference, unless the context clearly determines otherwise.

understand and variations thereof, as used herein, are used interchangeably with the term include and variations thereof and are open and non-limiting terms.

As used throughout the document, a subject is an individual. The subject may be a vertebrate, more specifically a mammal (for example, 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.

Figures IA to G illustrate an exemplary cartridge 100 for use in assessing hemostasis in a subject. The cartridge 100 includes a front surface 101 and a rear surface 126. Figure 1A shows a front view of the cartridge 100 and the corresponding front surface 101. 0 The cartridge includes an inlet 102, also referred to herein as an inlet or inlet port, as a nozzle, through which a biological sample of the subject can be introduced into the cartridge. Optionally, a blood sample from the subject is inserted into the cartridge at entry 102. Another biological sample that can be introduced for analysis is plasma. Inlet 102 is in fluid communication with channel 202, which is shown in Figure 2, and which directs the biological sample to other portions of the cartridge, as described in this document.

The cartridge additionally includes a port 106 for applying vacuum to the cartridge. When a vacuum is applied to port 106, the biological fluid introduced from inlet 102 to channel 202, the fluid is propelled along channel 202 towards port 106.

As shown in Figure 2, when moving between inlet 102 and port 106, biological fluid, or a portion thereof, moves along channel 202, to channel 204, channel 206 and along channels 208, 210, 212 and 214. Each of the channels 208, 210, 212 and 214 is in fluid communication with a test chamber, also referred to herein, for example, as a chamber, well or test well or the like. For example, as shown in Figure 2, channel 208 is in fluid communication with a test chamber 116, channel 210 is in fluid communication with a test chamber 114, channel 212 is in fluid communication with a test chamber 112 and channel 214 is in fluid communication with a test chamber 110.

Referring again to Figure 1, each test chamber comprises an open space 124 defined by a portion of the rear surface 126. Figure 1B shows a cross-sectional illustration through test chamber 116 taken along the line BB of Figure IA. Figure 1C shows a cross-sectional illustration taken along the line CC of Figure IA. Figure 1F shows an expanded view of the circled portion of Figure 1B. In addition, Figure ID shows a cross-sectional illustration along the line DD of Figure IA, which illustrates the open space of each of the four test chambers.

Each test chamber is configured to accept a quantity of the biological fluid in the open space. With reference to the test chamber 116, illustrated in detail in Figure 1F, a portion of the biological fluid introduced into the inlet 102 moves through channels 202, 204 and 214 and into the open space 124 of the test chamber 116.

The biological fluid can also leave each respective test chamber and continue along an outlet channel 130 towards port 106. Thus, the fluid introduced at inlet 102 flows under vacuum through the channels of the device and into the test chambers . From each test chamber (110, 112, 114, 116), biological fluid continues to flow along the outlet channels towards the vacuum.

Next to port 106, each outlet channel can direct the biological fluid flowing to a hydrophobic filter at location 222, 220, 218 and 216, respectively. The filters or filter prevent the movement of the biological fluid out of the cartridge 100 in port 106. Due to the volume of the channels and the test chamber being fixed, the vacuum can pull the biological fluid into the cartridge until the channels and each chamber test are filled with biological fluid.

The pressure can be controlled inside the cartridge 100 to, for example, manage the flow within the consumable 100 and to mitigate reliability problems related to the possible misuse of the user. To measure the properties of a target biological sample, such as a blood sample, a user of the hemostasis system optionally attaches a syringe filled with blood to the cartridge unit 100. There is a possibility that the user of the hemostasis system 300 (see Figure 3) could attempt to inject the contents of the syringe applied to the cartridge 100 manually, instead of allowing the device to automatically aspirate the sample. This action can lead to measurement or system errors. A pressure management device in the flow path of the consumable is used to prevent this action from the user.

Inadequate mixing of the biological sample with the reagents described in this document may result in variation in hemostasis measurements. Rapid aspiration of the blood sample is optionally used to provide increased mixing of the reagents with the biological sample, such as a blood sample. This is optionally achieved by creating a pressure differential between the cartridge and the aspiration mechanism of the hemostasis system.

In this regard, Figures 9A to C illustrate three exemplary configurations that can be used to control the pressure differential between the cartridge and the suction mechanism and, therefore, can be used to achieve the desired levels of mixing and reduce errors in the user.

Figure 9A schematically illustrates an exemplary system 900 for controlling the pressure in a cartridge 100. The cartridge includes four test chambers (110, 112, 114 and 116). Each test chamber optionally includes a reagent and the operation of the system causes a biological sample to enter one or more test chambers. The exemplary system 900 includes a bidirectional pump 908 that operates to aspirate a biological sample, such as a blood sample. For example, a blood sample can be aspirated into the cartridge from a 902 sample container. Pump 908 is in fluid communication with cartridge 100 and therefore pump activation can be used to move the biological sample through cartridge 100. A pressure transducer 904 is in communication with the pump that measures the gauge pressure sucked by pump 908. A solenoid-operated valve 906 operates to block the flow downstream of the pump, allowing the pressure gauge to increase. The solenoid can be selectively actuated to quickly expose the pressure gradient to the cartridge. The sample can progress through the cartridge and is optionally collected in a 910 sample container.

Figure 9B schematically illustrates another exemplary system 920 for controlling the pressure in a cartridge 100. The cartridge includes four test chambers (110, 112, 114 and 116). Each test chamber optionally includes a reagent and the operation of the system causes a biological sample to enter one or more test chambers. The exemplary system 920 includes a bidirectional pump 908 that operates to aspirate a biological sample, such as a blood sample. For example, a blood sample can be aspirated into the cartridge from a 902 sample container. Pump 908 is in fluid communication with cartridge 100 and therefore pump activation can be used to move the biological sample through of cartridge 100. A pressure activated membrane 912 is positioned upstream or downstream of cartridge 100 of pump 908. The membrane 912 is configured to burst at a predetermined cartridge gauge pressure, thereby controlling the pressure at which the sample is sucked through the cartridge. The sample can progress through the cartridge and is optionally collected in a 910 sample container.

Figure 9C schematically illustrates another example system 930 for controlling the pressure in a cartridge 100. The cartridge includes four test chambers (110, 112, 114 and 116). Each test chamber optionally includes a reagent and the operation of the system causes a biological sample to enter one or more test chambers. The exemplary system 930 includes a bidirectional pump 908 that operates to aspirate a biological sample, such as a blood sample. For example, a blood sample can be aspirated into the cartridge from a 902 sample container. Pump 908 is in fluid communication with cartridge 100 and therefore pump activation can be used to move the biological sample through cartridge 100. A 916 closed-loop actuated valve contains an internal pressure control mechanism and is used to block the flow downstream of the pump, allowing the gauge pressure to rise to a valve pressure set point. Once the gauge pressure set point is reached, valve 916 is actuated, thereby exposing the cartridge to a desired pressure gradient. The sample can progress through the cartridge and is optionally collected in a 910 sample container.

The sample level in each chamber can also be monitored. For example, as shown in Figures 8A to 8D, the fluid level in each chamber can be monitored optically. Figure 8A is a schematic illustration of an exemplary consumable cartridge placed in an exemplary hemostasis assessment system. Figure 8B is a schematic illustration of a cross-section taken along the BB line of Figure 8A. Figure 8C is an expanded schematic illustration of the circled portion of Figure 8B. Figure 8D is a schematic illustration of an exemplary consumable cartridge.

It can be indicated if a desired level has been reached in a given chamber, by an LED or other visual indicator. Employing a single beam of light from an 802 LED emitter that reflects from the chamber into a target blood detection reservoir 224, which is then detected by a detector 800, can optionally be used to monitor the fluid level of the chamber optically.

For example, blood entering a test chamber reduces the reflection of light from an 802 transmitter located next to detector 800 and aimed at the test chamber. A dual-beam approach can be used in which two sources of different wavelengths are reflected from the test chamber. The blood has a deep red color that can be differentiated by comparing the reflection of the wavelength of red with that of another color.

The difference in intensity of the red light reflected by itself is sufficient to determine when the blood entered the chamber. The intensity of the red light reflected from the test chamber that contains blood was about half that of the well that contains air and about two thirds of that of the well that contains water.

To control the temperature of the biological sample entering the test chambers, the cartridge 100 may comprise a heat exchanger in communication with channel 204. The heat exchanger can be used to maintain, raise or lower the temperature of the biological fluid prior to analysis in each test chamber. Optionally, the temperature of the biological fluid for analysis in each test chamber is the same, so that the common portion of the channel system, as shown in Figure 2, is subjected to temperature manipulation by the heat exchanger. Optionally, in modes not pictured, the temperature of the biological fluid entering each test chamber can be controlled separately.

For example, to heat the biological fluid, it can be passed through channel 204 through a polystyrene labyrinth held against a copper block. The copper block can be thin (for example, less than 2 mm) and sized slightly larger than the labyrinth to minimize thermal mass. A thermistor can be embedded in the block so that a control circuit can maintain a defined stable temperature in the block. A heater is used which optionally comprises two Watlow® serpentine sheet heating elements (St. Louis, MO) joined to a flexible kapton plastic substrate, and the interface between the block and the heater may be a thin layer of heatsink compound. silicone heat.

Various flow rates, for example, up to and including 5.99 ml / min or 6.0 ml / min can be used, and the input power to the heater can optionally be varied between 8 and 16 Watts. Blood or other biological fluid can be heated in the cartridge from room temperature (approximately 20 ° C) to 37 ° C at a nominal flow rate of 6 mL / min, which is fast enough to fill the cartridge in 20 seconds. The surface area of ​​the labyrinth used was less than 8 cm2.

Physiologically, the coagulation process is highly dependent on the temperature at which it occurs. Under normal conditions, coagulation occurs at body temperature (37 ° C), which is ideal for the proper enzymatic action of coagulation factors in the cascade.

Blood can be heated from its inlet temperature, which varies between 18 ° C and 37 ° C, to an arbitrary or desired temperature, such as body temperature, 37 ° C, passing through a serpentine channel next to a heater block. To perform heating in a short time on a short trajectory, the block can be heated to almost 60 ° C when the incoming blood is at the lower end of its temperature range. The blood temperature can also be measured and the heating block can optionally be adjusted to a temperature ranging from 40 ° C to 58 ° C.

To measure the temperature, a sensor can be incorporated in the system 300 (Figure 5) or in the cartridge. Optionally, a thermistor or thermocouple is placed in physical contact with the cartridge or blood and an IR thermometer is pointed at the cartridge or blood. In both cases, the cartridge can incorporate a small well through which the incoming blood passes, instead of having direct contact with the blood. When the cartridge material (polystyrene) is thin and the blood is kept moving through the well, then the greater thermal capacity of the blood ensures that the temperature of the well wall is close to that of the blood. Optionally, a window that allows the passage of IR is used. The window may comprise a thin layer (e.g., 20 µm or less) of polyethylene or polystyrene.

Temperature changes can occur in the body due to fever or in hospital environments, such as the emergency department (SE) or the operating room (OR). Trauma patients arriving at the ER are treated with large volumes of intravenous saline solution, which reduces body temperature to up to 17 ° C. In SC, patients undergoing cardiopulmonary bypass surgery (CPB) have their entire blood volume passing through a lung-heart machine, which also lowers the blood temperature and can adversely affect clotting. In addition, if there is a time lag between the time of blood collection and the measurement, the blood temperature will have time to change.

Styron® 666 polystyrene (Styron Inc. Berwyn, PA) and microfluidic heat exchanger channel 204 allow a blood sample to be heated by a copper block outside the cartridge that is maintained at constant 37 ° C. When a sample enters the cartridge at temperatures substantially below 37 ° C, it is optionally desirable to use a modified cartridge to allow faster heating of the biological sample. For example, in a model that simulates changes in temperature over time when blood enters the polystyrene cartridge at 17 ° C, Styron® 666 has been found to reduce the ability to heat blood and blood leaving the heat exchanger. heat did not reach 37 ° C. These shortcomings of the Styron® 666 are due to its relatively low thermal conductivity. When faster or more efficient heating of the biological sample is desired than is possible with Styron® 666, the cartridge may include materials with greater thermal conductivity than Styron® 666. For example, a thermally conductive polymer (E1201®) from Cool Polymers Inc. (North Kingstown, RI) with improved thermal conductivity properties can be used. This polymer can form a portion of the cartridge between the heating block and channel 204. By using this polymer in a portion of the cartridge between the heating block and the sample, the sample can be heated more efficiently. For example, Figure 11 shows that in a cartridge that comprises this material, the blood entering the heat exchanger at 17 ° C reaches 37 ° C in 15 seconds.

Cartridges optionally include both materials, EI 201® and Styron® 666, to improve heat transfer to the sample with EI 201® on the heated side while maintaining flow visibility on the other side of the consumable with Styron® 666 Another alternative is to use EI 201® as an insert that fits over the copper heater and on a chassis made from Styron® 666. This is done optionally by overmoulding the separate parts into a single part or by attaching the EI 201® to the Styron® chassis by means such as laser, ultrasonic or RE welding. Changing the geometry of the EI 201® insert to fit the larger chassis like a puzzle piece can further improve the assembly of the separate parts and help seal the microfluidic flow chambers.

It may also be desirable to cool the biological fluid in the cartridge. In this example, and similar to when heating is desired, the cartridge can include materials with higher thermal conductivity than Styron® 666. For example, the thermally conductive polymer (E1201®), described above, with improved thermal conductivity properties can be used. This polymer can form a portion of the cartridge between a cooling device, such as a Peltier cooling device, and channel 204. With the use of this polymer in a portion of the cartridge between the cooling device and the sample, the sample can be cooled with efficiency.

Each test chamber comprises one or more reagents useful in the analysis of one or more hemostasis indices. Optionally, the reagents are lyophilized. Optionally, one or more reagents of the lyophilized microsphere type are used. For example, the lyophilized microsphere can be a LyoSphere® produced by BioLyph (Minnetonka, MN). A self-contained lyophilized microsphere is a format that allows compatible coexistence of immunochemical and clinical chemistry reagents that require two or three components that are incompatible as liquids due to their pH level or reaction to each other. Because these lyophilized microspheres are stable and non-reactive, the chemicals can be packaged together in the same test chamber.

To produce lyophilized reagents, a lyophilizer device can be used. For example, the reagent for a given test chamber can be frozen to solidify all of its water molecules. Once frozen, the product is placed in a vacuum and gradually heated without melting the product. This process, called sublimation, turns the ice directly into water vapor, without first going through the liquid state. The water vapor released by the product in the sublimation phase condenses like ice in a collector, known as a condenser, inside the vacuum chamber of the freeze dryer. Optionally, the lyophilized product contains 3% or less of its original moisture content. The lyophilized product, which can be a pellet, can then be positioned in each test chamber. Once placed in a test chamber, the test chamber can be sealed to prevent unwanted rehydration of the product.

To locate the lyophilized reagents in the test chambers, the components can first be lyophilized and then the resulting lyophilized product can be placed in the test chambers. With the use of UV curing epoxy glue or a welding process (such as ultrasonic or RE welding), the lens assembly is sealed over each of the test chambers. The assembled cartridge can be sealed in a vapor proof barrier (for example, a bag) and the vapor barrier can be sealed to preserve the dehydrated nature of the product in the test chambers. When ready for use, the cartridge can be removed from the pouch or vapor barrier and placed in an analysis system 300, which is described in more detail below.

Antistatic treatment of plastic cartridges is optionally used with lyophilized reagents. Lyophilized reagents are inherently devoid of water, which gives them significant electrical insulation.

Materials that are electrical insulators accumulate static charge more readily than materials that act as electrical conductors. This can create problems with process control when assembling the cartridges and loading the reagents. Since the cartridges are optionally produced from an electrical insulating material (polystyrene, for example), it is unlikely to dissipate an accumulated static charge within the lyophilized reagents. As a result, lyophilized reagents can statically adhere to the inner walls of the consumable. In order to prevent this from occurring, three techniques are optionally implemented to remove static build-up.

Air ionization is a method that passes ionized air directed over a target material to neutralize the residual static charge on the material's surface. Directing ionized air into one or more cartridge test chambers and / or reagents during the assembly process improves the manufacturing capacity by reducing the adhesion of the reagent microsphere to the cartridge test chambers.

A second method implements the construction of the cartridge using a plastic material that exhibits significantly more conductivity than standard injection molding materials. PermaStat® RTF plastics (Winona, MA) are an example of these materials. The use of this material for the cartridge reduces the adhesion of lyophilized reagents to the walls of the cartridge's test chamber.

Third, antistatic liquid sprays are used to temporarily create a dust-free coating on optical lenses and equipment. These sprays reduce the static charge on the target surface and are useful for static reduction during the cartridge assembly process.

When lyophilized reagents are exposed to the fluid sample, they can generate foam that floats on the sample surface in the test chambers. As shown in Figures 10A and B, consumable cartridge 1002 optionally comprises a fluidic circuit 202 that delivers the sample from an external container, such as a syringe or vacutainer, to one or more test chambers (110, 112, 114, 116) where measurements are performed.

Figure 10A shows an exemplary fluidic circuit that can be implanted in a consumable cartridge 1002. This circuit includes an input port 102, a channel 202, four test chambers (110, 112, 114, 116), a filter 1004 and a port outlet 1006. The biological sample can be distributed inside the chamber by applying vacuum to the outlet port, with the filter allowing air to escape, but which interrupts the fluid. Several different reagents can be placed inside the test chamber, for example, as described throughout this document. In order to generate accurate measurements, the reagents are mixed in the sample before the test is started. For example, the ultrasound emitted in the test chambers can be used to mix the reagents with the sample, as described below.

As shown in Figures 10A and 10B, to improve foam mixing, a sample of biological fluid can flow through channel 202, which enters the test chamber on the side at a tangent to the chamber. In addition, changing the channel diameter from large to small increases the flow speed (flow conservation) at the entrance to the test chamber. This high flow rate, in collaboration with gravity, helps to generate a rotational recirculation flow pattern that improves the mixing and dispersion of the reagent with the sample. As the flow enters laterally, it causes any foam formed to be pulled into the flow stream and pushed under the surface.

Figure 10B shows a flow pattern implanted in a consumable cartridge designed for injection molding. The fluidic circuit was repeated four times in order to deliver the sample and mix reagents in four different test chambers.

The circuit shown in Figure 10B also includes a serpentine heat exchanger to adjust the temperature of the inlet sample to a desired level.

The reagents are mixed with the sample before the assay is started. The mixing of the reagents can be carried out using passive and / or active mechanisms. Passive methods include, for example, the use of serpentine channels and built-in barriers to create flow turbulence. Active methods include, for example, magnetic microspheres, pressure disturbance and artificial eyelashes. 0 Consumable cartridge contains a lens that focuses the ultrasound energy within the sample, which can be used to generate current and mix. The lens, also referred to herein as a lens set or sound focusing set, is designed using a soft material, such as a thermoplastic elastomer 134, in conjunction with a rigid substrate 132, such as polystyrene. This combination provides a dry ultrasound coupling that does not require the use of any fluid or gel coupler. Note that the same lens and ultrasound controller used for hemostasis measurement can be used in this case to provide mixing. The increase in acoustic energy for the mixture can be delivered, for example, by increasing the pulse length, pulse amplitude or pulse repetition frequency.

The mixture can also be supplied by a variable magnetic field applied by a series of coils placed outside a test chamber or each test chamber. A magnetic microsphere or small magnetic stirrer can be placed inside a test chamber and when the fluid sample enters the chamber, the current through the coils can be modulated to generate a variable magnetic field. This generates movement of the magnetic microsphere or magnetic stirrer, which in turn generates the mixing of the sample with the reagent.

Exposure of blood to surface proteins, as in the case of collagen or von Willebrand factor (vWF) in damaged blood vessel walls, is an essential part of the clotting process. These proteins not only contribute to the coagulation cascade, but also modulate several steps that lead to clot formation and hemostasis.

Although exposure to these proteins is essential to the coagulation cascade, standard point-of-care (POC) coagulation devices and assays do not take this interaction into account. Optionally, the test well (or wells) and / or channel (or channels) of a consumable cartridge, such as those described in this document, are coated with such surface proteins for the measurement of coagulation within a medical device.

POC.

The use of surface protein coatings includes collagen, vWF, fibronectin and any other molecule that modulates coagulation, such as fibrinogen and thrombin. A layer of protein on a substrate (glass, polystyrene, polypropylene) creates binding sites that allow for the mediation of receptor-ligand interactions between the substrate and other biological materials, such as blood, in a way that improves the evaluation of coagulation or provides new information of test.

The internal surfaces of a consumable cartridge can be coated using, for example: (1) a layer of these proteins by covalent bonding using ligand molecules, (2) covalent bonding using photochemistry or (3) adsorption of simple protein. Binding molecules, such as streptavidin or avidin and biotin, can be used for this purpose. With the binding molecules, the surface of any internal portion of the cartridge that will be exposed to the biological sample is biotinylated (coated with a layer of biotin) using commercially available biotin that is conjugated to a reactive group that binds nonspecifically and covalently with the substrate. A solution with a high concentration of streptavidin or avidin, which has a high affinity for biotin, is added to create a layer of biotin bound to streptavidin / avidin. The addition of biotinylated protein (collagen, vWF, fibronectin, thrombin, fibrinogen) then creates a layer of protein attached to the surface of the test well that specifically affects coagulation through interactions with plasma proteins and platelets.

The adsorption of proteins can be carried out by filling the wells with a highly concentrated protein solution. Adsorption on the plastic surface occurs almost immediately, depending on temperature, pH, surface charges, surface morphology and chemical composition. The solution can then be removed and the surface air dried. Brushing a highly concentrated protein solution on the surface of the wells or immersing the wells in this solution will serve the same purpose.

The concentration of molecules in the solutions used for the coating, whether using binding proteins or adsorption, can be changed to modulate the amount of protein that binds to the substrate and thus modulate the effects on the coagulation cascade in a way that relevant to physiology and hemostasis.

Referring again to Figure 1F, to seal each test chamber, for example, test chamber 116, a lens assembly 131 includes a rigid substrate 132 and a coupling 134 that can be positioned at the rear end of each test chamber. Each coupling 134 comprises an elastomeric material. Optionally, the elastomeric material is a thermoplastic elastomer (TPE). Exemplary elastomeric materials optionally include Dynaflex D3202, Versaflex OM 9-802CL, Maxelast S4740, RTF 6035. Optionally, the coupler is overmolded on the rigid substrate.

Between each coupling 134 and the open space of each test chamber there is a rigid substrate 132. The rigid substrate and the coupler form an interface that focuses on the ultrasound transmitted (for example, set of lenses) by an ultrasonic transducer in the open space of the chamber and in any biological fluid and / or reagents in the chamber. The rigid substrate of the lens may comprise a material that allows sound to pass through and that can act to focus ultrasound at some level within the space. Optionally, the rigid substrate comprises styrene, such as, for example, Styrene® 666.

lens set can be glued or welded to the surface 101 to fix the lens in place in an orientation that allows the desired sound focusing. Alternatively, the lens assembly is optionally manufactured in conjunction with the surface 101. In that regard, the rigid substrate 132 can be molded with the surface 101 and the coupler 134 can be overmoulded on the rigid substrate. A wide variety of materials can be used to build the device. For example, plastics can be used for single-use disposable cartridges.

Each test chamber (116, 114, 112 and 110) can have a set of lenses positioned over the wide opening in the open space of each chamber. In this way, each camera can be interrogated separately by focused ultrasound.

When placed in the analysis system 300, coupler 134 can be placed in acoustic communication with a transducer to deliver ultrasound through the lens assembly and into a test chamber. Optionally, an intermediate layer of an acoustically permeable material is positioned between an ultrasonic transducer and the coupler. For example, an intermediate layer or block of Rexolite® can be used. The intermediate layer can be forced against the coupler and can be in acoustic contact with the transducer.

sound generated by a transducer passes through the middle layer, through the coupler, through the rigid substrate and is focused within the biological and reagent sample in the test chamber. Part of the sound directed to the chamber comes into contact with the distal internal surface 111 of the test chamber, which is defined by surface 126. Optionally, the surface is polystyrene. The inner distal surface has a known geometry and is positioned at a known distance from the ultrasound source. The distal inner 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 the baseline and during the process of clot formation and clot dissolution. These measures can be used, for example, to estimate the subject's hematocrit along with hemostasis indexes. 0 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.

Figure 12A illustrates an exemplary geometry for a parabolic mirror that can be used to focus the sound in one or more test chambers, where f (x, y) is the shape of the focusing reflector, z is the height of the reflector above of the active element at the origin and (xf, yf, Zf) is the coordinate of the focal point. The focusing reflector is defined by a curve that is equidistant from the point of emission in the active acoustic element and the focal point. This can be expressed as:

d = - xf -í V, -yV + - /ÍA.y (1)

Where d is the total distance from the face of the acoustic source to the focus. If the distance is defined from the origin to the reflector as zo, the total length of the trajectory is:

<img file="PT2676136T_D0001.tif" />

reflector shape can be determined by solving for f (x, y) as follows:

<img file="PT2676136T_D0002.tif" />

<img file="PT2676136T_D0003.tif" />

7 'ύι ·? /: V, y) - Ω ú' J t -2y / ba)

27/67) -2 &lt;(. Yy) - (7 -tV -r (7 - tf f A - /

<img file="PT2676136T_D0004.tif" />

<img file="PT2676136T_D0005.tif" />

If z is defined, then equation 2 above can be evaluated and replaced in equation 10 above to produce an equation for the reflector surface. The reflector is a parabolic section. Exemplary parameters are optionally an 8 mm orifice with a 16 mm focus on the side, a 4 mm range and a displacement between the mirror and the 0.5 mm orifice. A diagram of this geometry is shown in Figure 12B. This geometry is useful when the focusing mirror is placed inside the system. The mirror can also be placed inside the cartridge. In this case, the focus is optionally moved closer in the axial dimension, but additionally in the lateral dimension, as shown in Figure 12C.

The cartridge 100 can be positioned in the pocket 302 of an analysis system 300. As shown in Figure 4, the pocket includes an actuator system 402 for pressing the intermediate layer, such as Rexolite®, which is acoustically coupled to a transducer in contact with coupling 134. In this way, the pouch holds the cartridge securely in place and in an orientation so that the ultrasound can be focused on each test chamber.

Figure 5 shows additional aspects of the cartridge 100 positioned in the analysis system. The cartridge is positioned so that the intermediate layer 504 is pushed into the coupler 134, which is in communication with the rigid substrate 132 of the lens assembly 131. The ultrasonic generating means 502, which includes at least one ultrasonic transducer, is positioned so that the ultrasound is transmitted through the intermediate layer, set of lenses and to the test chamber.

At least a portion of the sound is reflected by the biological sample positioned in the same chamber, and a portion of the sound transmitted to the chamber can also be reflected from the distal surface of chamber 111. The reflected ultrasound can be received by the ultrasonic transducer and transmitted to the system for processing. Thus, the cartridge and the analysis system 300 can be in communication so that data and other operational or processing signals can be communicated between the cartridge and the analysis system.

A suitable analysis system 300 can therefore comprise one or more processing devices. The processing of the disclosed methods, devices and systems can be performed by software components. Thus, the disclosed systems, devices and methods, including the analysis system 300, can be described in the general context of computer executable instructions, such as program modules, which are executed by one or more computers or other devices. Generally, program modules comprise computer code routines, programs, objects, components, data structures, etc., that perform particular tasks or implement particular abstract data types. For example, program modules can be used to cause ultrasound transmission that has desired transmission parameters and to receive and process ultrasound to assess the hemostasis rates of a subject sample. The software can also be used to control the heating of the biological sample using the heat exchanger and to monitor and indicate the filling level of a given chamber. The processor can also be used to run algorithms, to determine hemostatic and hematocrit indices. In some examples, the software can be used to remove the determined hematocrit from certain hemostatic indexes. The hemostatic indices and determined hematocytes can be displayed to a medical professional or medical agent for the purpose of making medical decisions for a subject.

Thus, a person skilled in the art will recognize that the systems, devices and methods disclosed in this document can be deployed by means of a general purpose computing device in the form of a computer. The computer, or portions thereof, may be located in the analysis system 300. Computer components may, however, include, without limitation, one or more processors or processing units, a system memory and a system bus that couples various system components, including the processor to the system memory. In the case of multiple processing units, the system can use parallel computing.

The computer typically comprises a variety of computer-readable media. Exemplary readable media can be any available medium that is accessible by the computer and comprises, for example, and in a non-limiting way, both volatile and non-volatile media, removable and non-removable media. System memory comprises a computer-readable medium in the form of volatile memory, such as random access memory (RAM), and / or non-volatile memory, such as read-only memory (ROM). System memory usually contains data, such as data and / or program modules, such as the operating system and software that are immediately accessible and / or currently operated by the processing unit.

In another aspect, the computer may also comprise other removable / non-removable, volatile / non-volatile computer storage media. For example, a mass storage device, which can provide non-volatile storage of computer code, computer-readable instructions, data structures, program modules and other data to the computer. For example, and in a non-limiting way, a mass storage device can be a hard disk, a removable magnetic disk, a removable optical disk, magnetic cassettes or other magnetic storage devices, flash memory cards, CD-ROM, disks digital versatile (DVD) or other optical storage, random access memories (RAM), read-only memories (ROM), electrically erasable programmable read-only memory (EEPROM) and the like.

Optionally, any number of program modules can be stored on the mass storage device, including, for example, an operating system and software. Each of the operating system and the software, or some combination thereof, can comprise elements of the programming and the software. The data can also be stored on the mass storage device. The data can be stored in any one or more databases known in the art. Examples of such databases include DB2®, Microsoft® Access, Microsoft® SQL Server, Oracle®, mySQL, PostgreSQL and the like. Databases can be centralized or distributed across multiple systems.

In another aspect, the user can enter commands and information on the computer using an input device. Examples of such input devices include, but are not limited to, a keyboard, pointing device (e.g., a mouse), a touch screen, a scanner and the like. These and other input devices can be connected to the processing unit via a human-machine interface that is coupled to the system bus, but can be connected via another interface and bus structures, such as a parallel port, game port, an IEEE 1394 port (also known as a Firewire port), a serial port, or a universal serial bus (USB).

In yet another aspect, a display device 304, such as a touchscreen, can also be connected to the system bus via an interface, such as a video adapter. It is contemplated that the computer may have more than one video adapter and the computer may have more than one video device. For example, a display device can be a monitor, an LCD (liquid crystal display) or a projector.

Any of the disclosed methods can be performed by computer-readable instructions embedded in a computer-readable medium. The computer-readable medium can be any available medium that can be accessed by a computer. By way of example and without limitation, the computer-readable medium may comprise a computer storage medium and a communication medium. The computer storage medium comprises volatile and non-volatile, removable and non-removable media implanted in any method or technology for storing information, such as computer-readable instructions, data structures, program modules or other data.

Example 1

The reagents in each test chamber, also referred to as the test well, can include all reagents necessary to assess one or more hemostasis indices.

Optionally, the cartridge is a single-use disposable cartridge with pre-loaded lyophilized reagents. The cartridge can be used with a subject's whole blood. The cartridge or assay components include the following for fresh whole blood samples. Four separate wells containing lyophilized reagents to which 1.6 ml of fresh whole blood is added. Each test well uses approximately 300 mL of fresh whole blood along with the following reagents:

Table 1:

<td>Test Well 1</td><td>Test Well 2</td><td>Test Well 3</td><td>Test Well 4</td>

<td>0.15 mg of kaolin</td><td>0.15 mg of kaolin</td><td>0.3 U of thrombin</td><td>recombinant tissue factor</td>

<td>earplugs and stabilizer es</td><td>earplugs and stabilizer es</td><td>earplugs and stabilizer es</td><td>earplugs and stabilizer es</td>

<td>0 pL of abciximab 2 mg / mL</td><td>12 pL of abciximab 2 mg / mL</td><td>12 pL of abciximab 2 mg / mL</td><td>0 pL of abciximab 2 mg / mL</td>

The systems and methods of the devices use the phenomenon of the force of acoustic radiation to measure changes in the mechanical properties (for example, stiffness) of a blood sample during the processes of coagulation and fibrinolysis. These changes are representative of the role of the four main components of hemostasis: (i) plasma clotting factors, (ii) platelets, (iii) fibrinogen and (iv) plasma fibrinolytic factors. The basic approach is shown in Figures 6A to C.

A series of N-focused ultrasound pulses are sent to a blood sample at short AT intervals (AT is in the order of microseconds), as shown schematically in panel A. Each pulse generates a small, localized force within the blood as it moves. acoustic energy is absorbed and reflected during propagation. This force, which is concentrated around the focus of the ultrasound beam, induces a small displacement within the blood sample that depends on the local mechanical properties. These displacements are of the order of 40 micrometers or less at the focus of the ultrasound beam.

Each pulse also returns an echo as a portion of its energy is reflected from within the blood sample. As the sample moves slightly from one pulse transmission to the next, the length of the path between the fixed ultrasound emitter and any region within the target increases with the pulse number. This change in the trajectory length can be estimated from the differences in the arrival times of the echoes in the same region. The combination of these delays forms a time shift curve that contains combined information about the sample's viscoelastic properties. These time shift curves are shown in Figure 6B. These time displacement curves are measured every 6 seconds to fully characterize the coagulation and fibrinolysis dynamics, which represents the entire hemostatic process.

When the blood sample is in a viscous fluid state, the application of acoustic force generates large displacements. Once coagulation is activated and fibrinogen is crosslinked in fibrin filaments, the sample behaves like a viscoelastic solid and the induced displacement reduces as the sample's stiffness increases. The interaction of platelets and fibrin mesh also further reduces the displacements induced as the clot stiffness increases. As the clot progresses to the fibrinolysis phase, the fibrin mesh is dissolved by fibrinolytic enzymes and the sample returns to the viscous fluid, which exhibits increasing displacements.

The evolution of the magnitude of the displacements induced over time is therefore directly related to changes in the mechanical properties of the blood sample during hemostasis. A curve obtained with this method is shown in Figure 6. Functional data, which highlight the role of clotting factors, platelets, fibrinogen and fibrinolysis, can be extracted from the curve, as labeled in Figure 6.

The force of acoustic radiation results from the momentum transfer that occurs when a propagating acoustic wave is absorbed or reflected. This body force acts in the direction of the propagating wave and can be approximated by the following expression:

where [m-1] is the acoustic attenuation coefficient, c [m / s] is the speed of sound, I (t) [W / m²] is the instantaneous intensity of the ultrasound beam, PII is the integral of the pulse intensity, AT [s] is the time interval between successive transmissions of the ultrasound pulse and <> indicates an average amount of time.

The acoustic energy used by the instrument to generate the force of the acoustic radiation is comparable to the acoustic energy normally used for common medical ultrasound procedures, such as color Doppler imaging. The estimated maximum acoustic intensity is of the order of 2.5 W / cm² (mean time), which results in a blood sample temperature increase of 0.01 ° C for each measurement set (performed approximately every 6 seconds).

Since the blood sample changes rapidly from viscous fluid to viscoelastic solid during coagulation and back to viscous fluid after clot lysis, the applied acoustic radiation force is changed adaptively to induce displacements above the noise threshold, but below levels that could induce mechanical disruption (typically below 40 micrometers).

The magnitude of the force is adjusted to follow changes in the mechanical properties of the blood sample, varying the time interval ΔΤ between successive pulses, as shown in equation 1. The maximum displacement induced during the (ml) th acquisition is used to determine whether the force should be increased or decreased for the mth acquisition, based on predetermined threshold values. This adaptive process allows the characterization of five orders of magnitude in stiffness without generating high tension within the blood sample that could alter the dynamics of coagulation and fibrinolysis.

As shown in equation (1), the applied acoustic radiation force changes as a function of acoustic attenuation and the speed of sound, both of which change as a function of coagulation. The system uses echoes that return from inside the cartridge to estimate changes in these parameters and to normalize the force of acoustic radiation.

The force of acoustic radiation is generated with the use of conventional piezoelectric materials that act as acoustic emitters and receivers. These materials deform when a stress is applied to them and, conversely, generate a stress when they are deformed. Similar to optics, an acoustic lens can be placed in front of the piezoelectric material to focus the acoustic energy on a single focal point.

In the exemplary systems, methods and devices, piezoelectric discs that have an active diameter of 7.5 mm are used. The acoustic lens is provided by the curved shape of the disposable cartridge. Four discs are placed side by side to send sound through the four test wells in a disposable cartridge. The vibration frequency of these piezoelectric discs is centered at 10 MHz, well within the frequency range used in conventional ultrasound imaging.

The ultrasound echo signals that return to the blood sample transducers are first filtered to remove electronic noise, digitized and further processed in a processor built into the system. A flowchart of the data analysis steps performed by the system is shown in Figure 7, in which a test starts at block 700. Ultrasound pulses are transmitted to a target sample in a test well at 702. The echoes are received, filtered and digitized in 704. After a short 706 wait, steps 702 to 704 can be repeated. A time delay estimate is applied at 708 and a curve fit at 710. The system then determines whether sufficient data has been acquired to estimate the desired hemostasis indices at 712. If there is sufficient data to estimate a hemostasis index, the hemostasis index is estimated at 714 and displayed at 716. If at 712 it is determined that insufficient data has been acquired to estimate a hemostasis index, the system determines whether the test should be stopped at 718 and, if so, an output summary is generated at 722. If the test should continue, after a long wait 770, one or more steps 702 to 770 are optionally repeated.

Estimated time delay

Once a set of N pulses is sent to the blood sample and feedback echoes are obtained, the time delay estimate (TDE) is performed to estimate a local time shift curve, similar to the one shown in Figure 6B . TDE involves measuring the relative time change from one received echo to the next; the known value of the speed of sound in the blood allows the conversion of time changes into displacements. TDE is performed around the focus of the ultrasound beam. This process is repeated every 6 seconds (arbitrary fixed waiting) to obtain time displacement curves throughout the coagulation and fibrinolysis process.

A variety of ready-to-use algorithms are available to perform this operation. TDE is a common signal processing step in fields of application ranging from RADAR, SONAR and medical ultrasound imaging (Doppler).

Curve adjustment

The viscoelastic properties of the blood sample during hemostasis are modeled using a modified model consisting of the well-known mechanical Voigt-Kelvin model with the addition of inertia. Although dynamic changes in blood viscoelasticity during hemostasis are certainly complex, the modified Voigt-Kelvin model is simple and robust and has been well validated in the past.

Each time shift curve is fitted to the characteristic equation of the modified Voigt-Kelvin model to estimate a variety of parameters related to the sample's viscoelastic properties. These parameters include relative elasticity, relative viscosity, time constant and maximum displacement. The mathematical expression of the equation of motion for the modified Voigt-Kelvin model is

<img file="PT2676136T_D0006.tif" />

where ξ, is the damping ratio, ω is the natural frequency and s is the static sensitivity.

Among the parameters obtained by adjusting the curve, the system uses the magnitude of the displacement estimated in 1 second as a qualitative measure of the sample stiffness. When the blood is in a viscous fluid state, the displacement in 1 second is high. As the blood coagulates, this displacement decreases in proportion to the generation of the fibrin mesh and the activity of the platelets. The value increases again during the fibrinolysis process.

Estimate hemostatic function indices

The displacement values ​​obtained in 1 second for each data acquisition are compiled to form a curve that shows the relative stiffness as a function of time (Figure 6C). This curve, shown earlier, completely characterizes hemostasis and can be further processed to estimate direct indices of hemostatic function.

Hemostasis indices are calculated by fitting a sigmoidal curve to the stiffness-time curve (Figure 6C) and evaluating the first derivative of the curve. The times to coagulate TC1 and TC2 are calculated based on a threshold value of the derived curve (20% of the minimum value) and are indicative of the initial and final phase of fibrin polymerization. The CFR coagulation slope is the maximum of the derived curve and is indicative of the rate of fibrin polymerization. The stiffness S is estimated from the stiffness curve 3 minutes after TC2. S depends on platelet function and the final stiffness of the fibrin network. Identical methods and indices are calculated for the fibrinolytic process. In particular, the times TL1 and TL2 can be defined to represent the initial and final phases of the fibrinolytic process and the consequent dissolution of the fibrin network (time for lysis).

A summary of the parameters generated for each test chamber is shown in table 2:

<td>Parameter ro</td><td>Information provided</td><td>Dependent on</td>

<td>TCi, TC₂</td><td>Measurement of initial and final fibrin formation</td><td>Fibrinogen function and other clotting factors</td>

<td>s</td><td>Fibrin and platelet activity</td><td>Function of the fibrin network and platelet aggregation</td>

<td>CFR</td><td>Fibrin polymerization rate</td><td>Fibrinogen function and other clotting factors</td>

<td>TLi, tl₂</td><td>Clot dissolution process</td><td>Protein function plasma fibrinolytics</td>

In order to isolate the four main components of hemostasis, four measurements are performed in parallel inside the disposable cartridge using a combination of agonists and antagonists in each of the four wells. The measurements in each well are combined to form hemostasis indices, as shown in table 3:

Output Method index of factors of coagulation (Intrinsic Via) Time to coagulate TCi in the final well index of factors of coagulation (Extrinsic Route) Time to clot TCi in well na4 Platelet index Differential stiffness S between non-oil well 2 Fibrinogen index Rigidity S in well na3 Output Method Fibrinolysis index Time to smooth TL1 in well n £ 4