KR20230149730A - Thromboelastography test system and method - Google Patents

Abstract

The present invention relates to a thromboelastography test device and method capable of continuously performing thrombus formation and thrombolysis tests, wherein the thromboelastography test device includes a sample reservoir into which a blood sample is injected and stored; a sample preprocessing unit that is in fluid communication with the sample storage unit and preprocesses the blood sample introduced from the sample storage unit by mixing it with a reagent; a sample inspector communicating with the sample preprocessing unit and monitoring movement of the blood sample; A pressure supply unit connected to the sample test tube and providing pressure to the sample test tube so that a certain amount of blood sample is introduced from the sample pretreatment unit into the sample test tube, wherein the blood sample moves within the sample test tube by the pressure. It is characterized by acquiring one or more parameter data by monitoring the periodic movement characteristics of the blood sample.

Description

Thromboelastography test system and method}

The present invention relates to a thromboelastography test device and method for examining the process of blood clot formation and blood clot dissolution. More specifically, the present invention relates to a thromboelastography test device and method that examines the process of blood clot formation and blood clot dissolution. More specifically, the present invention relates to the viscoelastic properties where blood coagulation occurs when a small amount of blood sample passes through a microfluidic tube. A thromboelastography test that can continuously perform thrombus formation and thrombolysis tests using the characteristics of blood flow stopping due to changes and the blood flow occurring again through the naturally occurring thrombolysis process. It relates to devices and methods.

Blood is in liquid form and flows throughout the body through blood vessels, supplying oxygen and nutrients. However, when a wound occurs inside a blood vessel, the blood begins to clot, which can cause a rapid blood clot at the site of the wound. . Also, when these thrombi block the vascular passages, serious diseases such as myocardial infarction or stroke may occur. In some people, the blood vessel flow blocked by the thrombus may be effectively re-opened according to its own lysis ability. This type of case may be overlooked without the patient being aware of it, but if the blockage caused by the blood clot continues, immediate surgery is required.

Therefore, accurate measurement of the ability of a patient's blood to coagulate and subsequently dissolve in a timely and effective manner has become very important information in general and medical procedures. In addition, accurate detection of abnormal hemostasis is treated as clinically very important information by allowing appropriate treatment to be prepared in advance for patients suffering from blood coagulation disorders.

Hemostasis is the result of a very complex and complex biochemical chain reaction that changes blood from a liquid state to a gel state. A blood clot is accompanied by changes in rheological and mechanical properties that occur when liquid blood gels, and through this, information such as the strength and formation time of the blood clot can be obtained, and the hemostatic properties can be determined.

For example, if the adhesion strength of the blood clot that has begun to form through the coagulation mechanism can resist the shear stress of the circulating blood flow, the blood clot can attach to the damaged blood vessel wall and stop bleeding.

In another example, if there is no damage to the blood vessel wall, a blood clot may form in the stenosis of the blood vessel and occlude the blood vessel. At this time, when the thrombus that begins to form overcomes the shear force of blood flow, the thrombus grows at a very rapid rate and blocks the blood vessel, causing heart attack, ischemic stroke, and pulmonary embolism (depending on the location of the blockage caused by the thrombus). Pulmonary Embolism (PE), or Deep Vein Thrombosis (DVT).

A typical thromboelastography test device provides hemostatic properties of a blood sample by applying periodic rotational oscillation or linear oscillation from the outside and monitoring the blood's response to it. In other words, the thromboelasticity test device records and analyzes the viscoelastic characteristics of blood samples that react during the coagulation and dissolution process over time to derive hemostasis characteristic information. For this purpose, the thromboelastometry test device includes a container for receiving the blood sample, a shaker or exciter for periodically oscillating or vibrating the blood sample, and an exciter of the blood sample. It usually includes a sensor to measure the amplitude results.

A conventional thromboelastography (TEG) system consists of a set of cups and pins spaced 1 mm apart. Kaolin activated blood is placed in a cup that vibrates by 4°45 at a frequency of 0.1 Hz. One of the cups or pins is fixed and the other rotates. TEG, including rotational thromboelastometry (ROTEM) and the TEG 5000 analysis system, can provide comprehensive information on all stages of hemostasis in oscillating dynamic environments. Additionally, TEG uses parameters such as R-time, K-time, α-angle, and maximum amplitude (MA) to determine initial fibrin formation, fibrin-platelet plug formation, clot formation, and Blood clot lysis can be analyzed. Additionally, TEG analysis can measure fibrinolysis by monitoring the decrease in amplitude indicated by Ly30. Due to these characteristics, TEG has demonstrated excellent performance in a variety of clinical applications such as coagulopathy diagnosis, transfusion therapeutic guidance, liver transplantation, and heart surgery.

Recently, several new point-of-care TEG devices have been introduced. The TEG6s system (Haemonetics, Braintree, MA), consisting of a vibrated test cell and a meniscus monitoring system, measures the resonant frequency of suspended blood droplets with applied vibration. By tracking the resonance frequency along with the coagulation process, TEG equivalent parameters were derived using the mapping function. Optical thromboelastography (O-TEG), which consists of a laser, an optical lens, and a CMOS camera, measures transient laser speckle fluctuations in a few drops of blood. The decrease in speckle fluctuation signal due to coagulation is converted to the viscoelastic modulus of blood (viscoelastic mod uLus). In sonic estimation of elasticity via resonance (SEER) sonorheometry, which consists of an ultrasound transducer and a probe, the acoustic deformation of a developing clot measures the viscoelastic properties and functional properties of the clot. Used to extract scale.

However, existing or improved TEG systems do not fully integrate the in vivo hemodynamic shear environments that play a critical role in the coagulation process. In fact, the intravascular flow environment where thrombosis occurs has different flow characteristics from the existing cup-and-pin-type oscillatory flow. First, intravascular flow can be characterized as pipe flow, which generates a radially variable shear-rate distribution. Under normal physiological flow conditions, wall shear rate increases from about 10 s ^-1 in veins to about 2000 s ^-1 in the smallest arteries. In fact, shear rate is closely related to hemostatic behavior. Second, oscillatory shear is a dominant flow characteristic that influences lesion progression patterns and plaque vulnerability in patients with coronary artery disease. For oscillatory flow, bidirectional (or oscillatory) shear flow was significantly more associated with thrombotic events near atherosclerotic areas than unidirectional shear flow.

KR 10-2016-0011622 A1

Therefore, the present invention was devised to solve the above problem, and the purpose of the present invention is to stop blood flow due to a change in the viscoelastic properties where blood coagulation occurs, or to allow blood flow to resume due to the blood clot dissolution process, etc. To provide a thromboelasticity testing device and method that can continuously perform thrombus formation and thrombolysis tests using .

Other objects and advantages of the present invention will be explained below and will be learned by practice of the present invention. Additionally, the objects and advantages of the present invention can be realized by the means and combinations indicated in the claims.

In order to achieve the above object, the thromboelastography test device according to the present invention includes a sample storage unit into which a blood sample is injected and stored; a sample preprocessing unit that is in fluid communication with the sample storage unit and preprocesses the blood sample introduced from the sample storage unit by mixing it with a reagent; a sample inspector communicating with the sample preprocessing unit and monitoring movement of the blood sample; and a pressure supply unit connected to the sample test tube to provide oscillating pressure, and obtaining one or more parameter data by monitoring periodic movement characteristics of the blood sample as it moves within the sample test tube due to pressure. It is characterized by

Additionally, the method for testing thromboelastosis according to the present invention includes the steps of injecting a blood sample into a sample storage unit; Preprocessing the blood sample introduced from the sample storage unit by mixing it with a reagent in a sample preprocessing unit; providing a oscillating pressure to the sample test tube by a pressure supply unit so that a certain amount of blood sample is introduced from the sample pretreatment unit into the sample test tube; and acquiring one or more parameter data by monitoring periodic movement characteristics of the blood sample as it moves within the sample test tube due to pressure.

In one embodiment, the periodic movement characteristics of the blood sample can be measured by monitoring the moving distance of the blood sample in the sample test tube, or by monitoring the fluctuation of the oscillation pressure in the sample test tube supplied from the pressure supply unit. .

In one embodiment, the periodic movement characteristics of the blood sample are measured using an image sensor that measures the round-trip movement distance of the blood sample, or a pressure measurement unit that measures the variation of the oscillation pressure in the sample test tube supplied from the pressure supply unit. It can be measured.

In one embodiment, the pressure supply unit may be connected to one end of the sample storage unit, and the pressure measurement unit may be connected to one end of the sample test tube.

In one embodiment, the pressure supply unit may be connected to one end of the sample test tube, and the pressure measurement unit may be connected to one end of the sample storage unit.

In one embodiment, a three-way valve may be provided between the sample storage unit and the sample test tube to change the flow direction to measure the supply of a blood sample to the sample test tube or a change in oscillation pressure in the sample test tube. there is.

In one embodiment, a plurality of sample preprocessing units are connected to one end of the sample storage unit, one end of a sample inspector is connected to each of the plurality of sample preprocessing units, and a main shaking pressure is provided to the other end of the sample storage unit. The pressure supply unit may be connected to the other end of the sample test tube, and each auxiliary pressure supply unit may be connected to provide auxiliary swing pressure.

In one embodiment, the sample storage unit is composed of a double cylindrical structure (annulus) including a central cylindrical tube located at the center, and only the blood sample filled in the central cylindrical tube flows into the sample test tube. It can be.

In one embodiment, the blood sample flowing into the sample test tube may be branched to one or more sample preprocessing units to perform one or more tests simultaneously.

In one embodiment, the volume of the blood sample flowing into each sample test tube may range from 5 uL to 200 uL.

In one embodiment, the sample preprocessing unit and the sample test tube are each provided in one or more plurality, and different reagents may be supplied to each sample preprocessing unit.

In one embodiment, the reagent may be coated on the sample pretreatment unit or the inner wall of the sample test tube.

In one embodiment, the reagent is calcium chloride (CaCl ₂ ), kaolin, tissue factor, heparinase, thrombin, adenosine diphosphate (ADP), arachidonic acid (AA), It may include any one or more of reptilase, blood coagulation factor 13 (Factor XIIIa), prostaglandin E1, epinephrine, and combinations thereof.

In one embodiment, the sample pretreatment unit may include a stirrer that is rotated using a non-contact method of magnetic force.

In one embodiment, when the reagent is coated on the inner wall of the sample test tube, after the blood sample is placed in the test area of the sample test tube, the blood sample is mixed with the reagent by the reciprocating flow generated within the sample test tube. You can.

In one embodiment, the inner wall of the sample pretreatment unit may form a hydrophobic surface.

In one embodiment, when the blood sample is located in the examination area of the sample examiner, the pressure supply unit may repeatedly operate according to a predetermined operation cycle of one cycle.

In one embodiment, the waveform generated by the pressure supply unit is a sinusoidal wave, and the operating cycle time may range from 1 second to 60 seconds.

In one embodiment, the shear rate applied to the blood sample by the pressure generated by the pressure supply unit may range from 0.1 to 60 s ^-1 .

In one embodiment, the hydraulic diameter of the sample tube may range from 0.1 mm to 4 mm.

In one embodiment, for the purpose of activating platelets, the shear rate applied to the blood sample by the pressure generated by the pressure supply unit may be at least 500 s ^-1 or more, and the shear force may be at least 1 Pa or more.

In one embodiment, the pressure supply unit includes a linear driving unit that moves linearly; a piston that performs linear reciprocating motion by being driven by the linear driving unit; The piston may include a cylinder installed to enable linear reciprocating motion.

In one embodiment, a control valve that controls the transmission and release of driving pressure may be installed between the sample test tube and the pressure supply unit.

In one embodiment, the parameter data is obtained by applying an input pressure (Input Magnitude, IMp) of oscillating pressure to the blood sample and monitoring the pressure as the anticoagulated blood sample moves back and forth according to the input pressure, where the blood clot R-time (min), which is the reaction time for formation and is defined as the time for the change in oscillating pressure to reach 1/40 of the input pressure; K-time (min), which is the thrombus formation time required for a thrombus to form and is defined as the time for the change in oscillating pressure to reach 1/4 of the input pressure; The α-angle (°), which is the slope of the straight line connecting the curves of the R-time and the K-time, with the rate of thrombus formation; MA _P (Pa), which is the maximum amplitude indicating the maximum value of the change in oscillation pressure; Coagulation strength (MAp/Imp) obtained by dividing the maximum amplitude by the input pressure; tMA(min), which is the time required to reach the MA _P ; and Ly(au), which is the clot lysis (lysis) after a certain period of time from the MA _P , and the MA _P (Pa) value is calculated by referring to the correlation with the MA value for the known TEG 5000 system. (mm) can be converted to a value.

In one embodiment, the measurement time of the thrombus solubility (ly) is determined according to the value of the shear rate applied to the blood sample by the pressure generated by the pressure supply unit, and is measured 1 to 30 minutes after the MA _P. You can.

As described above, according to the present invention, it is possible to comprehensively test blood clot formation and dissolution-related tests at once.

In particular, the present invention has the characteristic of best simulating the formation of thrombus in in-vivo vascular flow in an in vivo environment, and can simultaneously measure the ability for blood coagulation and thrombus solubility according to the patient's blood. It has features that can be

In addition, according to the present invention, a thromboelastography test, including thrombus formation and thrombolysis mechanisms, can be performed under various conditions in a relatively very short time (<10 minutes) while minimizing measurement error.

1 is a configuration diagram of a thromboelasticity testing device according to a first embodiment of the present invention;
Figure 2 is a diagram showing an example of the configuration of a sample storage unit according to the present invention;
Figure 3 is a diagram showing an example of the configuration of a sample inspector according to the present invention;
Figure 4 is a configuration diagram of a thromboelasticity testing device according to a second embodiment of the present invention;
Figure 5 is a configuration diagram of a thromboelasticity testing device according to a third embodiment of the present invention.
6 and 7 are diagrams for explaining a configuration for measuring fluctuations in shaking pressure in an embodiment of the present invention;
8 to 17 are drawings for explaining various test examples of the present invention.
Figure 18 shows a microfluidic TEG system according to one embodiment of the invention developed for thromboelastographic analysis.
Figure 19 is a diagram showing signal changes over time in anticoagulated blood and re-calcified blood.
Figure 20(a) is a diagram showing parameter definitions of the microfluidic TEG system according to the present invention from typical thromboelastography (TEG).
Figure 21(a) is a diagram showing the effect of platelet dysfunction on the results of the existing TEG 5000 and the microfluidic TEG system according to the present invention.

Specific details of other embodiments are included in the detailed description and drawings.

The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. In the description below, when a part is connected to another part, this is not only the case when it is directly connected. This also includes cases where they are connected through other media in between. In addition, in the drawings, parts unrelated to the present invention are omitted to clarify the description of the present invention, and similar parts are given the same reference numerals throughout the specification.

In the present invention, a small amount of blood sample (5 to 50 uL) is placed in a sample test tube that simulates a blood vessel, and then pressure (positive or negative pressure) vibration is applied and released to the sample test tube.

Before the blood coagulation process begins, the amplitude of the blood transport distance appears regularly due to periodic pressure fluctuations. However, after the blood coagulation process begins, the viscoelastic properties of the blood become prominent and change to a gelled state, and the amplitude of the blood transfer distance gradually decreases and sometimes stops. Afterwards, the stopped blood may begin to gradually increase the amplitude of the transfer distance again, and in some cases, the amplitude of the transfer distance may completely return to its original state. Therefore, measurement of changes in the amplitude of the sample's transport distance during the thrombus and lysis process under pressure can provide information on hemostasis or intravascular occlusion in intravascular blood flow.

As such, in the present invention, a method of measuring the distance of blood movement or fluctuation using the characteristics of blood flow stopping due to changes in viscoelastic properties where blood coagulation occurs or the characteristics of blood flow occurring again due to the blood clot dissolution process, etc. Thrombogenesis and thrombolysis tests can be performed continuously by measuring pressure fluctuations.

Hereinafter, the present invention will be described with reference to the attached drawings.

Figure 1 is a configuration diagram of a thromboelastometry test device according to a first embodiment of the present invention, Figure 2 is a diagram showing a configuration example of a sample storage unit according to the present invention, and Figure 3 is a configuration of a sample test tube according to the present invention. It is a diagram showing an example, FIG. 4 is a configuration diagram of a thromboelastometry test device according to a second embodiment of the present invention, and FIG. 5 is a configuration diagram of a thromboelastometry test device according to a third embodiment of the present invention. FIGS. 6 and 7 are diagrams for explaining a configuration for measuring fluctuations in shaking pressure in an embodiment of the present invention, and FIGS. 8 to 17 are diagrams for explaining various test examples of the present invention.

Figure 18 shows a microfluidic TEG system according to an embodiment of the present invention developed for thromboelastographic analysis, Figure 18(a) shows a syringe pump providing sinusoidal input pressure, a test tube, and a coagulation process. A schematic diagram of a microfluidic TEG system consisting of a camera or pressure sensor that monitors the oscillatory motion of blood, Figure 18(b) shows pressure over time for recalcified blood, and Figure 18(c) shows the initial It represents the pressure converted from pressure amplitude to pressure difference.

Figure 19 shows signal changes over time for anticoagulated blood and re-calcified blood, where Figure 19(a) is the pressure measured for anticoagulated blood, and Figure 19(b) is the pressure measured for anticoagulated blood. ) is the pressure conversion over time for anticoagulated blood, Figure 19(c) is the pressure over time for blood recalcified by adding kaolin, and Figure 19(d) is the pressure over time for recalcified blood. is the conversion pressure according to .

Figure 20(a) shows the parameter definition of the microfluidic TEG system according to the present invention from a typical thromboelastography (TEG), and Figure 20(b) shows the MA _p and TEG 5000 of the microfluidic TEG system according to the present invention. It represents the correlation between the MAs.

Figure 21(a) shows the effect of platelet dysfunction on the results of the existing TEG 5000 and the microfluidic TEG system according to the present invention, and Figure 21(b) shows the MA of the microfluidic TEG system according to the present invention. 21(c) shows the effect of antiplatelet concentration on various blood, such as whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma. This is a thromboelastogram of (platelet-poor plasma: PPP), and Figure 21(d) shows the variation of MA for various blood types.

Referring to FIG. 1, the thromboelastography test device according to the first embodiment of the present invention includes a sample storage unit 110, a sample preprocessing unit 111, a sample test tube 131, and a pressure supply unit 140. It consists of:

Blood samples can be injected and stored in the sample storage unit 110.

The sample storage unit 110 is made in the shape of a roughly circular chamber. The size of the storage unit 110 may be manufactured in various sizes depending on the purpose of use. In addition, the sample storage unit 110 is preferably manufactured to be optically transparent so that it can be easily observed from the outside.

As shown in FIG. 2, the sample storage unit 110 is preferably configured as a double cylindrical structure (annulus) including a central cylindrical tube 110a located at the center. At this time, the central cylindrical tube 110a is connected to fluid communication with the sample test tube 131, and therefore only the blood sample filled in the central cylindrical tube 110a flows into the sample test tube 131. may flow into.

The sample preprocessing unit 111 is in fluid communication with the sample storage unit 110 and can preprocess the blood sample introduced from the sample storage unit 110 by mixing it with a reagent.

The sample pretreatment unit 111 provides calcium chloride (CaCl ₂ ) at an appropriate concentration to blood to induce blood coagulation, and various reagents can be additionally provided as needed. Representative reagents include kaolin, tissue factor, heparinase, thrombin, ADP (adenosine diphosphate), AA (arachidonic acid), reptilase, and blood coagulation factor 13. (Factor XIIIa) and combinations thereof may be included.

Additionally, other reagents may include prostaglandin E1 (PGE1) and epinephrine.

Kaolin is one of the substances studied as a blood coagulation promoting substance and is known to have an excellent effect in promoting coagulation in the blood.

Tissue factor is a factor that plays a very important role in the blood coagulation cascade and is a key protein that maintains vascular homeostasis.

Heparinase is an enzyme that degrades heparin.

For example, the reagent may be coated on the sample pretreatment unit 111 or the inner wall of the sample test tube 131. This can ensure that the reagent is effectively mixed with the blood sample when the blood sample flows into the sample pretreatment unit 111 or the sample test tube 131.

The sample preprocessing unit 111 may include a stirrer 121 inside. The shape of the stirrer 121 may be either a circular rod or a circular plate shape.

For example, the stirrer 121 is a stirrer made of a thin metal material that is magnetized by the magnetic force of a separately installed stirring induction device (not shown) and rotated by a non-contact method of magnetic force that can be influenced without mechanical connection. You can.

When the reagent is coated on the inner wall of the sample test tube 131, after the blood sample is located in the test area of the sample test tube 131, the blood sample is moved by the reciprocating flow generated within the sample test tube 131. Can be mixed with reagents.

Additionally, the inner wall of the sample pretreatment unit 111 may form a hydrophobic surface, thereby preventing contaminants such as bacteria from attaching to the inner wall of the sample pretreatment unit 111.

If the inner wall of the sample pretreatment unit 111 is not a hydrophobic surface, blood clots may adhere to the inner wall of the sample pretreatment unit 111, which may lead to difficulties in hemostasis testing.

The sample inspector 131 is in communication with the sample preprocessing unit 111 and is for monitoring the movement of the blood sample.

The hydraulic diameter of the sample test tube 131 may range from 0.1 mm to 4 mm, preferably from 0.2 mm to 2 mm. The sample test tube 131 can be manufactured in various shapes such as squares and polygons rather than circles, and the hydraulic diameter can be determined by taking this into consideration.

The pressure supply unit 140 is connected to the sample test tube 131 and provides a constant pressure to the sample test tube 131 so that a certain amount of blood sample is introduced from the sample preprocessing unit 111 to the sample test tube 131. You can.

Here, the pressure supply unit 140 may be a device that simultaneously includes the function of fluctuation pressure. That is, the pressure supply unit 140 can provide a oscillating pressure with a oscillating pressure. At this time, the form of the oscillating pressure may include one of a sine wave, a sawtooth wave, a triangle wave, a square wave, or an arbitrary waveform.

Referring to FIG. 3, a rubber stopper 132 may be provided at the end of the sample test tube 131 through which the injection needle 145 connected to the control valve 144 can penetrate. Accordingly, the sample test tube 131 can easily communicate with the external pressure supply unit 140 through the injection needle 145. The control valve 144 functions to control the transmission and release of driving pressure from the pressure supply unit 140, as will be described later.

Preferably, the volume of the blood sample flowing into the sample test tube 131 may be determined based on the volume of the central cylindrical tube 110a of the sample storage unit 110.

For example, the volume of the blood sample flowing into the sample test tube 131 may range from 5 uL to 200 uL.

The pressure supply unit 140 includes a linear driving unit 141 that moves linearly, a piston 142 that performs a linear reciprocating motion by driving the linear driving unit 141, and the piston is installed so that the piston can perform a linear reciprocating motion. It may include a cylinder 143.

Here, the linear drive unit 141 may be configured as a crank device that converts rotational motion into linear reciprocating motion.

Preferably, when a blood sample is located in the inspection area of the sample test tube 131, the pressure supply unit 140 may repeatedly operate according to a predetermined operation cycle of one cycle.

For example, one cycle of the operation cycle includes any one of a Withdraw - Forward (Infusion) cycle, a Forward - Backward cycle, a Backward - Stop - Forward - Stop cycle, and a Forward - Stop - Backward - Stop cycle. can do. At this time, the time taken for one cycle may range from 1 second to 60 seconds.

Here, the reverse cycle may be, for example, the piston 142 moving from left to right by driving the linear drive unit 141 as seen in the drawing. On the contrary, the forward cycle may mean that the piston 142 moves from right to left by driving the linear drive unit 141 when viewed in the drawing. Of course, the reverse is also possible.

The shear rate applied to the blood sample by the pressure generated by the pressure supply unit 140 may range from 0.1 to 60 s ^-1 .

In particular, when the purpose is to activate platelets, the shear rate of the pressure generated by the pressure supply unit 140 is at least 500 s ^-1 or more, and in this case, the shear force may be 1 Pa or more, preferably 8 Pa or more.

Additionally, a control valve 144 that controls the transmission and release of driving pressure may be installed between the sample test tube 131 and the pressure supply unit 140.

Meanwhile, the linear driving unit 141 is connected to a control unit (not shown) so that the driving can be controlled.

Therefore, in the present invention, a method of monitoring the moving distance of the blood sample flowing by the pressure of the pressure supply unit 140 within the sample test tube 131 or measuring the fluctuation of the oscillation pressure in the sample test tube 131 You can obtain one or more parameter data. As a result, in the present invention, thromboelastography tests, including thrombus formation and thrombolysis mechanisms, can be performed in a short period of time while minimizing measurement errors under various conditions.

Here, the parameter data is obtained by applying an input pressure (Input Magnitude, IMp) of the oscillating pressure to the blood sample and monitoring the pressure when the anticoagulated blood sample moves back and forth according to the input pressure, which is a reaction for thrombus formation. R-time (min), defined as the time for the change in oscillating pressure to reach 1/40 of the input pressure; K-time (min), which is the thrombus formation time required for a thrombus to form and is defined as the time for the change in oscillation pressure to reach 1/4 of the input pressure; The thrombus formation rate, α-angle (°), which is the slope of the straight line connecting the curves of the R-time and the K-time, and MA _P (Pa), which is the maximum amplitude representing the maximum value of the change in oscillation pressure. ); Coagulation strength (MAp/Imp) obtained by dividing the maximum amplitude by the input pressure; tMA(min), which is the time required to reach the MA _P ; and Ly(au), which is the clot lysis (lysis) after a certain period of time from the MA _P , and the MA _P (Pa) value is calculated by referring to the correlation with the MA value for the known TEG 5000 system. (mm) can be converted to a value.

Referring to FIG. 4, a thromboelastometry test device according to the second embodiment of the present invention is shown, and compared to the thromboelastometry test device according to the first embodiment, the only difference is in the arrangement of the sample test tube 131. and all remaining components are the same.

Specifically, in the thromboelasticity testing device according to the first embodiment of the present invention, the sample test tube 131 is arranged in a horizontal direction with respect to the ground surface, whereas in the thromboelasticity testing device according to the second embodiment of the present invention, The difference is that the sample inspector 131 is arranged in a U-shape in a vertical direction with respect to the ground surface. Accordingly, the blood sample in the sample test tube 131 may move up and down around the virtual horizontal line of the U-shaped portion in the test area when the pressure of the pressure supply unit 140 is applied. Here, the vertical direction can be understood as a direction parallel to the direction in which gravity acts and perpendicular to the ground surface.

Referring to Figure 5, a thromboelastography test device according to a third embodiment of the present invention is shown.

As shown in FIG. 5, in the thromboelastography test device according to the third embodiment of the present invention, a plurality of sample preprocessing units 111 and 112 are connected to one end of one sample storage unit 110, and the plurality of sample preprocessing units 111 and 112 are connected to one end of the sample storage unit 110. One end of the sample test tubes 131 and 132 may be connected to the sample preprocessing units 111 and 112, respectively. At this time, different reagents can be supplied to each sample preprocessing unit 111 and 112, and thus more than one test can be performed simultaneously.

In addition, a pressure supply unit 140 is connected to the other end of the sample storage unit 110 to provide the main shaking pressure, and a pressure supply unit 140 is connected to the other end of the sample test tubes 131 and 132 to provide auxiliary shaking pressure. An auxiliary pressure supply unit 170 may be connected. At this time, the pressure measuring unit 170 and the auxiliary pressure supply unit 180 may be respectively connected to the sample test tubes 131 and 132 through one connection port 138.

When multiple sample test tubes 131 and 132 are included, it becomes difficult to control the position of the blood sample in each sample test tube (131 and 132) using only one pressure supply unit 140. Therefore, in order to solve this problem, in this embodiment, in addition to the main pressure supply unit 140, a separate auxiliary pressure supply unit 180 is provided.

The auxiliary pressure supply unit 180 may be configured as, for example, a syringe pump. Here, the syringe pump is equipped with a syringe containing air to auxiliary control the position of the blood sample in each sample tube (131, 132), and pushes or suctions a certain amount of air at a constant speed to provide auxiliary flow pressure to each sample. Refers to the pump provided to the inspector (131, 132).

In Figure 5, one sample storage unit 110, a plurality of sample preprocessing units 111 and 112, a plurality of sample test tubes 131 and 132, and a plurality of connection ports 138 perform a thromboelastography test. It constitutes a thromboelastography test chip, and the pressure supply part 140, the pressure measurement part 170, and the auxiliary pressure supply part 180 can be viewed as a mechanism that can be connected separately from the thromboelastancy test chip 100. .

The inside of the sample preprocessing unit 111 may include a stirrer 121, and the sample preprocessing unit 112 may include a stirrer 122.

As shown in FIG. 5, it is exemplified that a plurality of sample preprocessing units 111 and 112 are provided, but of course, a plurality of sample storage units 110 may be provided.

Figures 6 and 7 are diagrams for explaining a configuration for measuring fluctuations in shaking pressure in an embodiment of the present invention.

In an embodiment of the present invention, as a result of measuring the fluctuation of the shaking pressure between the sample test tube and the pressure supply unit, the pressure signal representing the fluctuation of the shaking pressure was detected to be very complex. In consideration of this, the present inventor provided a three-way valve between the sample storage unit and the sample inspection tube to maintain the pressure constant, so that the pressure signal representing the fluctuation of the oscillating pressure was also constantly detected.

Referring to FIG. 6A, as a configuration for measuring fluctuations in oscillation pressure in an embodiment of the present invention, between the sample storage unit 110 and the sample test tube 131, a blood sample is placed in the sample test tube 131. A 3-way valve 150 may be provided to change the flow direction to measure the fluctuation of the oscillation pressure in the supply and sample inspection tube 131. At this time, the 3-way valve 150 may operate in cooperation with the pressure supply unit 140, and its operation may be controlled by a control unit (not shown). Here, the pressure supply part 140 is connected to one end of the sample test tube 131, and a pressure sensing part (PS) is connected to one end of the sample storage part 110. The pressure sensing part (PS) Measures the periodic movement characteristics of the blood sample by monitoring changes in air pressure in the sample test tube 131.

Referring to FIG. 6B, the sample storage unit 110 is connected to the first port 150a of the 3-way valve 150, and the sample inspector is connected to the second port 150b of the 3-way valve 150. 131 may be connected, and the pressure measuring unit 150 may be connected to the third port 150c of the 3-way valve 150. At this time, a pressure supply unit 140 may be connected to the other end of the sample inspection tube 131 through a control valve 144.

In this configuration, during the first operation of the 3-way valve 150, a certain amount of blood sample S is measured by opening the second port 150b of the 3-way valve 150, and the measured blood sample ( Since S) moves to the inspection position of the sample inspector 131 (see the direction of arrow 1), the blood sample S is supplied to the sample inspector 131. At this time, in order to measure a certain amount of blood sample S, an optical sensor 3 capable of measuring the movement of the blood sample S may be additionally provided.

In addition, the 3-way valve 150 opens the third port 150c during the second operation, thereby enabling the pressure measurement unit 150 to measure the fluctuation of the oscillation pressure in the sample inspection tube 131 (arrow) (see direction in 2).

Referring to FIG. 7A, as another configuration for measuring fluctuations in oscillation pressure in an embodiment of the present invention, between the sample storage unit 110 and the sample test tube 131, a blood sample to the sample test tube 131 is provided. A three-way valve 160 may be provided to change the flow direction to measure fluctuations in the oscillation pressure in the supply or sample tube 131. At this time, the 3-way valve 160 may operate in cooperation with the pressure supply unit 140, and its operation may be controlled by a control unit (not shown). Here, the pressure supply unit 140 is connected to one end of the sample storage unit 110, and the pressure measurement unit (PS) is connected to one end of the sample test tube 131.

Referring to FIG. 7b, a sample test tube 131 is connected to the second port 160b of the 3-way valve 160, and a pressure supply unit 140 is connected to the third port 160c of the 3-way valve 160. can be connected. At this time, the third port 160c of the 3-way valve 160 and the pressure supply unit 140 may be connected to each other through a connection pipe 162 of a certain length, and a pressure measurement unit (PS) at the other end of the sample inspection tube 131. ) can be connected.

In this configuration, during the first operation of the 3-way valve 160, a certain amount of blood sample S is measured within the connection pipe 152 by opening the third port 160c of the 3-way valve 160. It can be (see direction of arrow 4). At this time, in order to measure a certain amount of blood sample S within the connection pipe 152, an optical sensor 3 capable of measuring the movement of the blood sample S may be additionally provided.

In addition, during the second operation of the 3-way valve 160, the second port 160b of the 3-way valve 160 is opened, so that a certain amount of blood sample S measured in the connection pipe 152 is transferred to the sample examiner. Since it moves to the test position of (131), the blood sample (S) is supplied to the sample test tube (131). At this time, it is possible to measure the fluctuation of the oscillating pressure in the sample inspection tube 131 by the pressure measuring unit 150 connected to the other end of the sample inspection tube 131 (see the direction of arrow 5).

[Test Example 1]

Figure 8 shows Test Example 1 of the present invention.

In Test Example 1, after a certain amount (10 uL) of blood sample is introduced into the sample test tube 131, the pressure supply unit 140 operates in one cycle, for example, withdraw - stop - battery in units of 5 seconds. (infuse) It operates repeatedly at intervals of 15 seconds in total. Here, the waveform generated by the pressure supply unit 140 is a sinusoidal wave, and the operating cycle time may range from 1 second to 60 seconds.

Here, the total length of the sample test tube 131 was approximately 160 mm. At this time, the length of the entrance area (L _ent ) where the blood sample (S) is introduced was 20 mm, and the length of the test area (L _Test ) where the test was performed was 30 mm. The blood sample S was assumed to move, for example, in steps of 2 mm.

The total length of the sample test tube 131 may be preferably 70 mm to 90 mm, and most preferably 80 mm.

Although the sample preprocessing unit 111 is not shown in Test Example 1, it should be understood that the sample preprocessing unit 111 is included when reagents are mixed.

Before the blood coagulation process begins, the amplitude of the blood sample's transport distance appears regularly due to periodic pressure fluctuations.

However, after the blood coagulation process begins, the viscoelastic properties of the blood become prominent and change to a gelled state, and the amplitude of the blood transfer distance gradually decreases and sometimes stops. Afterwards, the stopped blood may begin to gradually increase the amplitude of the transfer distance again, and in some cases, the amplitude of the transfer distance may completely return to its original state. Therefore, measurement of changes in the amplitude of the sample's transport distance during the thrombus and lysis process under pressure can provide information on hemostasis or intravascular occlusion in intravascular blood flow.

Referring to FIG. 9A, it can be seen that in the case where there is no blood coagulation, the blood flow periodically moves a certain distance during one operating cycle.

Referring to FIG. 9B, when blood coagulation begins (an appropriate concentration of calcium is provided to the blood to induce blood coagulation), blood flow gradually stops, and the periodic blood movement distance decreases and sometimes reaches a value of 0. In other words, the periodic blood movement distance gradually decreases and then stops.

Referring to FIGS. 10A and 10B, it can be seen that when blood coagulation and dissolution occurs, the amplitude of the transfer distance of the stopped blood begins to gradually increase again. In other words, when the thrombolytic mechanism acts, the stopped blood flow has the characteristic of periodically moving again.

The change in the decrease in blood movement distance associated with blood coagulation and blood dissolution as described above can be analyzed in the form of thrombus elasticity as shown in FIG. 9. This thromboelastography is very similar to existing thromboelastography.

Figure 11 shows one or more parameter data that can be obtained in the present invention.

One or more parameter data that can be obtained in the present invention include initial thrombus reaction time (R-time), thrombus formation time (K-time), thrombus formation rate (α-angle), and maximum amplitude of maximum thrombus strength. MA) and thrombus solubility (Ly30).

This one or more parameter data includes all equivalent parameter characteristics as in thromboelastography according to the prior art.

Therefore, in the present invention, the blood flow is stopped due to changes in the viscoelastic properties where blood coagulation occurs, or the blood flow is re-generated due to the thrombolysis process, to measure the distance the blood moves to determine the formation of blood clots. and thrombolysis tests can be performed continuously.

[Test Example 2]

Figure 12 shows Test Example 2 of the present invention.

In Test Example 2, after a certain amount (10 uL) of blood sample is introduced into the sample test tube 131, the pressure supply unit 140 operates for one cycle, for example, withdraw in 5 second units (0.12 ml/ min) - Infuse (0.12 ml/min) cycle operates repeatedly at a total time interval of 10 seconds.

In Test Example 2, as in Test Example 1, the decrease in blood movement distance associated with blood coagulation and blood dissolution could be analyzed in the form of thrombus elasticity as shown in FIG. 11.

[Test Example 3]

Figure 13 shows Test Example 3 of the present invention.

In Test Example 3, after a certain amount (25 uL) of blood sample is introduced into the sample test tube 131, the pressure supply unit 140 operates for one cycle, for example, backward in units of 5 seconds (0.3 ml/min) - Stop - Forward (0.3 ml/min) - Stop cycle operates repeatedly at a total time interval of 20 seconds.

In Test Example 3, as in Test Example 1, the decrease in blood movement distance associated with blood coagulation and blood dissolution could be analyzed in the form of thrombus elasticity as shown in FIG. 11.

[Test Example 4]

Figure 14 shows Test Example 4 of the present invention.

In Test Example 4, the sample test tube 131 is arranged in a U-shape in the vertical direction with respect to the ground surface, and therefore, when periodic pressure is provided, the blood sample in the sample test tube 131 is moved by gravity (g) in the test area. It showed a pattern of moving up and down around the virtual horizon (H) of the U-shaped part.

In Test Example 4, as in Test Example 1, the decrease in blood movement distance associated with blood coagulation and blood dissolution could be analyzed in the form of thrombus elasticity as shown in FIG. 15.

[Test Example 5]

Figure 16 shows Test Example 5 of the present invention.

In Test Examples 1 to 4, the periodic movement characteristics of the blood sample were measured by monitoring the movement distance of the blood sample in the sample test tube 131, but in Test Example 5, the periodic movement characteristics of the blood sample were measured through the pressure measuring unit (PS). The difference is measured by monitoring changes in air pressure in the sample tube 131.

In Test Example 5, as in Test Examples 1 to 4, the decrease in blood movement distance associated with blood coagulation and blood dissolution could be analyzed in the form of thrombus elasticity as shown in FIG. 17.

[Implementation example]

The present invention proposes an innovative microfluidic thromboelastography (abbreviated as μ-TEG) system that mimics the in vivo hemodynamic environment in a circular tube. By adopting a sinusoidal input pressure, the blood sample moving back and forth in the tube was reproduced and the monitored pressure was converted to TEG equivalent parameters. Therefore, we demonstrated a novel μ-TEG system to rapidly assess blood coagulation properties using a simple microfluidic tube system.

samples and materials

This invention was approved by the Human Use Ethics Committee of Korea University Guro Hospital (IRB No. 2020GR0559). Blood samples were collected via venipuncture in sodium citrate and K2EDTA tubes (BD Vacutainer Systems, Franklin Lakes, NJ, USA). All tests were completed within 4 hours to minimize the risk of blood's time-dependent coagulation properties. Complete blood counts were measured using an automated hematology analyzer (Beckman Co uLter, Miami, FL, USA). Before μ-TEG analysis, 1 mL of citrated blood was added to a vial (40 μL) of kaolin solution. 20 μL calcium chloride solution (0.2M) was added to another vial, and then 340 μL of kaolin mixed blood was added. The mixture of blood samples was mixed gently and 40 μL of the mixture was immediately transferred to a test tube for μ-TEG measurements. Each sample was tested simultaneously using the TEG 5000.

Identification of shear rate

Shear rate is an important factor in blood coagulation. In fact, shear rate is a biophysical agent that causes platelet activation and aggregation, and high shear rates are highly correlated with increased thrombin generation. Additionally, arterial thrombus formation under high shear rates has been extensively studied. Normal vascular flow in a vein can be simplified to steady laminar blood flow in a circular tube with a parabolic velocity profile. In this configuration, the maximum shear rate was observed at the wall (known as wall shear rate, γ _wall ), which was defined as 8 V _avg /d. When applying a syringe pump speed of 0.6 mL/min, the average velocity of the in vitro blood sample was approximately 0.75 mm/s. Considering this, the wall shear rate near the pipe wall was 4.6 s ^-1 , which is frequently observed in venous flow. Additionally, the shear rate of μ-TEG can be easily varied using syringe pump speed, stroke volume, tube diameter, and cyclic time. The shear rate of TEG 5000 rotating at 4°45 for 5 seconds is approximately 0.03 s ^-1 , which is a shear flow close to zero that is rarely observed in circulatory flow in the human body.

Experimental setup and operating principle

Figure 18 shows a microfluidic system according to an embodiment of the present invention developed for thromboelastographic analysis, Figure 18(a) shows a syringe pump providing sinusoidal input pressure, a test tube, and a blood clotting process. A schematic diagram of a μ-TEG system consisting of a camera or pressure sensor that monitors the oscillatory motion of Indicates the pressure converted to pressure difference. The system consists of a reciprocating syringe pump, dead volume, straight silicone tubing, pressure transducer, and valve. The weighed blood sample (40 μL) was aspirated into a silicone test tube (d = 1.3 mm, L = 80 mm) using a syringe pump (Legato 200, KD Scientific, SeouL, Korea) and pumped into the test tube with a syringe pump. was located at a specific location. Next, the other end of the test tube was connected to a pressure transducer. The blood sample was periodically moved back and forth through the circular tube when oscillating air pressure was generated by a reciprocating syringe pump. The typical period was 10 seconds (f = 0.1 Hz), which was the same as that of the TEG 5000. The oscillating distance of the blood sample ranged from 1 to 20 mm and varied depending on the stroke volume of the syringe pump.

With the appropriate combination of dead volume and stroke volume of the syringe pump, a sawtooth wave pressure was created. In this experiment, a dead volume of 2 ml and a stroke volume of 50 μL were carefully selected through optimization experiments. Syringe pump speeds ranged from 0.5 to 3.0 mL/min for backward and forward modes. Accordingly, various operating conditions, such as “stop-and-go” operation, were established. For example, a combination of ramp modes from 0.5 mL/min to 1.2 mL/min for 5 seconds each for reverse and forward modes. Then, a sinusoidal pressure wave profile was obtained. The average shear rate for this test ranged from 1.2 to 24.0 s ^-1 .

When an input sinusoidal pressure was applied to the blood sample, the pressure across the blood sample created a phase lag related to fluid properties that are not considered in the present invention. In particular, depending on the location of the pressure sensor, completely different signals may be obtained. When the pressure sensor was positioned between the syringe pump and the blood sample, the measured pressure was superimposed on the original input pressure generated by the syringe pump and the pressure wave reflected from the blood sample. Pressure wave reflection, frequently observed in the human vascular system, causes excessive aortic pressure elevation and increases the risk of cardiovascular disease. Using superimposed pressure signals, it is extremely difficult to evaluate the viscoelastic properties of clotted blood. Therefore, in the present invention, a pressure sensor is placed across the blood from a syringe pump as shown in Figure 18(a).

Examination of various material tubes (silicone, PolyTetraFluoroEthylene (PTFE), and Tygon tubes) showed that hydrophobic materials tend to exhibit relatively better repeatability than hydrophilic materials. This may be due to the increase in surface tension during solidification. Therefore, a silicone tube was chosen as the test tube for the μ-TEG system. As shown in Figure 18(b), the oscillatory pressure signal was monitored for 20 minutes. The initial pressure amplitude was stable and had a constant magnitude until full-fledged solidification characteristics appeared. While the solidification cascade progresses gradually, the oscillatory pressure amplitude tends to decrease. The pressure difference reduced from the initial amplitude, defined as the converted pressure, is again displayed in Figure 18(c). The converted pressure signal shows a typical thromboelastographic form with an initial value of zero (0) and then gradually increasing.

result

Monitoring pressure fluctuations

First, pressure changes over time were measured to evaluate the viscoelastic properties of coagulated blood. When a sinusoidal input pressure was applied to the blood sample, the anticoagulated blood sample began to move back and forth according to the input pressure, and the pressure was monitored (FIG. 19(a)). Due to calcium ion deficiency, the blood samples did not clot and did not exhibit time-dependent characteristics associated with blood clotting. Therefore, in the current μ-TEG system, the anticoagulated blood sample continuously oscillates back and forth, and the pressure amplitude exhibits a constant pressure amplitude. The converted pressure, defined as the pressure difference from the initial pressure amplitude, yielded a constant zero value over time for the anticoagulant blood sample, as shown in Figure 19(b). Similar results were observed with water and mineral oil.

When the anticoagulant blood sample was recalcified, the oscillatory pressure amplitude signal tended to change with time, as shown in Figure 19(c). The pressure amplitude remained constant for the first few minutes and then decreased noticeably from a certain moment known as R-time. R-time represents the time at which blood clot or fibrin formation begins after recalcification by adding kaolin. Normal R-time values range from 4 to 10 minutes. Within 2 to 3 minutes after the pressure amplitude decreased, the pressure amplitude reached a minimum value and then began to slowly increase again. When converting these results, the converted pressure change was very similar to the existing TEG results, as shown in Figure 19(d). When fibrin formation began, the converted pressure yielded values close to zero. Blood also tends to stick to the tube walls, resulting in a rapid increase in converted pressure. The increased pressure reached a maximum value and then decreased slightly.

Definition of μ-TEG parameters

Similar to conventional TEG, the present invention developed parameters as shown in Figure 20(a) and Table 1. These parameters can be obtained by applying an input pressure (Input Magnitude, IMp) of oscillating pressure to the blood sample and monitoring the pressure when the anticoagulated blood sample moves back and forth according to the input pressure.

The “R-time” of the μ-TEG system according to the invention represents the first indicator of thrombus formation. R-time stands for “reaction time” and refers to the time it takes for blood to begin to clot. The R-time of the μ-TEG system is defined as the time for the change in oscillating pressure to reach 1/40 of the input pressure. Considering the sensor's internal jittering signal, the cutoff amplitude for R-time can be significantly different from the noise signal by a value of more than three times the standard deviation of the jittering noise. R-time represents fibrin formation in the early stages of blood coagulation, corresponding to the International normalized ratio (INR) and Partial Thromboplastin Time (PTT). Increased R-time was correlated with deficiencies in coagulation factors, the action of anticoagulant agents, and hypofibrinogenemia.

Similarly, “K-time” was defined as the time for the change in oscillation pressure to reach 1/4 of the input pressure. K-time refers to the speed at which a blood clot forms and clots. Shortened K-time indicates rapid thrombus formation with fibrin polymerization and platelet aggregation. The slope of the straight line in the μ-TEG curve connecting time R and time K is defined as the alpha (α)-angle. This angle, like K-time, represents the speed at which the blood clot forms and clots. K-time and α-angle reflect thrombus dynamics as well as thrombus formation rate.

The maximum amplitude (MA) of the pressure change is defined as the maximum amplitude (MA _p ), which represents the maximum thrombus strength, resulting in maximum fibrin-platelet interaction. Since the units of MA _p are different from those of TEG 5000, the MA _p value was converted to MA using the correlation obtained in Figure 20(b). Additionally, tMA represents the time required to reach MA. After calculating the peak value of the pressure amplitude, the final step begins with fibrinolytic-induced dissolution of fibrin-platelet bonds.

On the other hand, when the pressure amplitude decreases to baseline, fibrinolysis can be observed. The percentage reduction from MA for a certain period of time after the peak was defined as solubility (lysis) (Ly) after a certain period of time. Most existing equipment uses 30 as the standard constant time. However, in the present invention, fibrinolysis progresses quickly, so the same amount of fibrinolysis can be observed within 3 to 15 minutes instead of 30 minutes.

Additionally, coagulation strength (MAp/Imp) was defined as the value obtained by dividing the maximum amplitude by the input pressure.

Table 1 shows the analysis performance of μ-TEG through eight repeated measurements.

Tests
(n = 8) R
(min) K
(min) Angle α
(degree) M.A.
(mm) tMA (min) Ly30 (au) μ-TEG 6.4 ± 0.31
(4.8%) 0.7 ± 0.1
(13.9%) 78.2 ± 3.6
(5.0%) 55.2± 2.5
(4.8%) 10.7 ± 1.51
(14.2%) 3.6 ± 0.5 ^{Note 1)}
(13.8%) TEG 5000 6.9±0.2
(3.2%) 2.7 ± 0.4
(16.3%) 55.0 ± 4.0
(7.4%) 53.2 ± 3.8
(7.2%) 23.5 ± 2.3
(9.6%) 3.8 ± 0.8
(20.7%) RV(%) 7.2%↓ 74.1%↓ 42.2%↑ 3.7%↑ 54.5%↓ 6.1%↓

Note 1) In the case of μ-TEG of the present invention, Ly30 is replaced with the amount measured for 5 minutes. As a key characteristic of the present invention, in the present invention, tMA is reached very quickly, and then fibrinolysis is also accelerated, so that the amount achieved in the existing 30 minutes can be obtained in about 3-10 minutes. In the present invention, the solubility in about 5 minutes is the same as the solubility in 30 minutes in existing equipment.

Analytical performance of μ-TEG

Repeated measurements were performed eight times per sample using the μ-TEG system, and the measured results were analyzed using μ-TEG parameters and are shown in Table 1. Additionally, μ-TEG measurements were compared with those of TEG5000. First, the R-time of μ-TEG was 6.4 minutes, which was 0.5 minutes shorter than that of TEG5000, showing a relative variation (RV) of 7.2%. The R-times of both devices showed excellent repeatability. The coefficient of variation (CV) for R-time was less than 5%. For K-time, μ-TEG showed relatively short values (0.7 min) compared to TEG5000 (2.7 min), resulting in a clear RV (74.1%). The coefficient of variation (CV) for K-time was slightly larger at 13.9% for μ-TEG and 16.3% for TEG5000. This may be because the standard deviations are similar in size and K-time is shorter than R-time. Reduced K-time has a direct effect on the α-angle since its definition is highly dependent on K-time. The μ-TEG system produced a much steeper angle (78.2°) than the TEG5000 (53.2°), while both devices showed excellent repeatability for the α-angle (4.4% and 6.0%, respectively). Considering K-time and α angle, μ-TEG shows a faster fibrin polymerization rate than TEG 5000. The reason may be the higher shear rate compared to TEG 5000. This will be explained later.

The MA _p unit of μ-TEG is pressure (Pa), and the MA of TEG5000 is amplitude length (mm). Therefore, the MA values of these two devices cannot be directly compared. Therefore, MA _p of μ-TEG was converted to MA of TEG 5000 using a correlation curve as shown in Figure 20(b). The converted MA of μ-TEG in mm has almost the same value as TEG 5000 (RV < 3.7%). Meanwhile, μ-TEG quickly reached MA at 10.7 minutes, which was 13 minutes shorter than TEG5000 (23.5 minutes). This characteristic of the short tMA of μ-TEG is thought to be due to the shear rate associated with the coagulation process. For Ly30, both devices showed significantly greater variation across replicates (13.0% and 20.7%, respectively). Ly30 of TEG5000 and Ly5 of μ-TEG were 3.8% and 3.6%, respectively.

Detection of platelet function deficiency

The effect of platelet dysfunction on thromboelastogram was investigated using various concentrations of antiplatelets. To produce platelet dysfunction, we employed 2-methylthio-AMP (2MeSAMP), which inhibits platelet function through a P2Y12-dependent mechanism both in vitro and in vivo. As shown in Figure 21(a), the thromboelastogram immediately increases due to ADP-induced platelet activation. As the concentration of 2MeSAMP increases, the maximum amplitude (MA) of μ-TEG decreases, as shown in Figure 21(b). Therefore, platelet inhibition appears to have a direct effect on coagulation strength. Additionally, we show thromboelastograms for whole blood, platelet-rich plasma (PRP), and platelet-poor plasma (PPP) in Figures 21(c) and 21(d). was compared. PRP samples showed a delayed increase in thromboelastogram (i.e., prolonged R-time) with reduced MA compared to whole blood samples. Additionally, the R-time of the PPP sample was more prolonged and the MA was decreased compared to the PRP sample. These results suggest that red blood cells and platelets each independently affect the coagulation process and outcome.

In general, TEG provides a more comprehensive assessment of coagulation, including coagulation factors, anticoagulants (heparin), platelet function, and thrombolysis, which cannot be measured using traditional coagulation assays. However, it has recently been reported that conventional TEG is not sensitive enough to detect platelet dysfunction even when specific platelet mapping tests are performed. This drawback is mainly due to the extensive thrombin generation of standard activated viscoelastic tests, which bypasses other pathways and activates platelets through protease activated receptors. In addition, existing TEG cannot separate the roles of platelet count and function, but appears to reflect count more than function.

Argument

Although the clinical utility of TEG has recently rapidly increased and is being applied to various clinical fields, TEG measurement technology has not significantly departed from the technology proposed 60 years ago. Of course, new attempts are being made by introducing optical or ultrasonic measurement principles along with innovative information about coagulation. However, this technology does not reflect the shear flow field of existing TEG, so the fibrinolysis phenomenon is not easily observed. Therefore, we proposed a novel microfluidic TEG system that provides an in-vivo mimicking flow field. Therefore, in this microfluidic TEG system, the oscillatory flow occurring in the tube was adopted by applying a sinusoidal pressure profile. This system reflects the oscillatory velocity field of the pulsating lumen as well as the non-uniform shear rate characteristics of vascular flow. In addition, an in-vivo mixing mechanism was proposed in which coagulation initiators such as CaCl ₂ and kaolin are injected and mixed into a vibrating blood sample. Finally, the viscoelastic coagulation phenomenon was observed by measuring the air pressure between the blood sample and the pressure transducer.

The converted pressure signal tends to generate a conventional thromboelastogram, and TEG parameters such as R-time, K-time, α-angle, MA and Ly30 have been successfully analyzed. Above all, this microfluidic TEG system has dramatically improved several fatal shortcomings of existing TEG systems. First, the time required to acquire MA (tMA), a key parameter, is about 10 minutes for this system, compared to about 23 minutes for conventional TEG. Since it is very important to determine coagulation characteristics in a short period of time in emergency situations, the shortened testing time of the μ-TEG system is expected to be used as a great advantage. The characteristics of the μ-TEG system can be attributed to the shear rate (2 to 20 s ^-1 ), which is much larger than that of conventional TEG (0.03 s ^-1 ). This shear rate environment may better simulate an arterial thrombus environment where high shear rates act relative to vascular flow until blocked by thrombosis.

In previous studies, shear rates (15 to 100 s ^-1 ) were found to play a more important role in the rapid formation of fibrin and fibrin polymerization than zero shear rate (Tippe, A., Muller-Mohnssen, H Thromb Res. 1993, 72, 379-388). These results were reported using viscosity measurements during solidification at various shear rates (Ranucci, M., Laddomada, T., Ranucci, M., Baryshnikova, E., Physiol. Rep. 2014, 2, e12065 ). As the shear rate increased, the time-to-gel point (TGP) shortened significantly. Recent studies have further investigated the influence of shear rate gradients on the ability of platelets to adhere to fibrinogen (Receveur N, Nechipurenko D, Knapp Y, Yakusheva A, Maurer E, Denis CV, Lanza F, Panteleev M, Gachet C, Mangin PH.. Haematologica. 2020, 105, 105, 2471-2483). In fact, shear-induced platelet adhesion and platelet-fibrin crosslinking affect K-time and tMA. Therefore, elevated shear rate accelerated fibrin formation and cross-linking between fibrin polymers, resulting in shortened K-time and tMA in μ-TEG measurements.

Shear rate also affected clot strength and fibrinolysis. Previous studies have reported that shear rates promote the production of plasminogen and tissue plasminogen activator (tPA) near the formed thrombus, which in turn enhances fibrinolysis (Li, M., Hotaling, N.A., Ku, D.N., Forest, C.R., PLOS ONE. 2014, 9, e82493). tPA catalyzes the conversion of plasminogen to plasmin, which can break down fibrin clots. Elevated tPA induced by shear rate results in rapid and large thrombolysis. Therefore, the significant increase in Ly30 of μ-TEG may be due to the shear rate applied to blood. However, excessive shear can form hard clots that are more resistant to fibrinolysis.

conclusion

A new microfluidic TEG system has been introduced to evaluate the viscoelastic properties of blood clots at the point-of-care. Simple reciprocating blood flow in the tube successfully produces the characteristics of conventional thromboelastography. The main features of the μ-TEG system are the simple structure of the test tube, non-contact measurement, and linear reciprocating shear mechanism. In particular, testing at the shear rate of tube flow not only reproduces the in vivo hemodynamic environment but also shortens extended testing times. This approach makes it possible to identify the causes of cardiac events that previously could not be diagnosed using existing equipment. Additionally, because the method of the present invention is based on a precise pressure monitoring system for blood coagulation, the reproducibility of the present system is as good as that of the existing TEG system. In conclusion, this microfluidic TEG system can be practically implemented for screening testing in a clinical setting.

Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. must be interpreted.

110: sample storage unit
111: Sample preprocessing unit
131: Sample Inspector
140: pressure supply unit
141: linear driving unit
142: piston
143: cylinder
144: control valve
PS: Pressure measuring unit
170: Auxiliary pressure supply unit

Claims (50)

a sample storage unit where blood samples are injected and stored;
a sample preprocessing unit that is in fluid communication with the sample storage unit and preprocesses the blood sample introduced from the sample storage unit by mixing it with a reagent;
a sample inspector communicating with the sample preprocessing unit and monitoring movement of the blood sample; and
It includes a pressure supply unit connected to the sample test tube to provide a oscillating pressure,
A thromboelastometry test device, characterized in that one or more parameter data is acquired by monitoring periodic movement characteristics of the blood sample as it moves within the sample test tube due to pressure. According to paragraph 1,
The cyclical movement characteristics of the blood sample are measured by monitoring the moving distance of the blood sample in the sample test tube, or by monitoring the fluctuation of the oscillation pressure in the sample test tube supplied from the pressure supply unit. test device. According to paragraph 2,
The periodic movement characteristics of the blood sample are measured using an image sensor that measures the round-trip movement distance of the blood sample, or a pressure measurement unit that measures fluctuations in the oscillating pressure in the sample test tube supplied from the pressure supply unit. A thromboelastography testing device. According to paragraph 2,
A thromboelastometry test device characterized in that the pressure supply unit is connected to one end of the sample storage unit, and the pressure measurement unit is connected to one end of the sample test tube. According to paragraph 2,
A thromboelastometry test device characterized in that the pressure supply unit is connected to one end of the sample test tube, and the pressure measurement unit is connected to one end of the sample storage unit. According to clause 4 or 5,
Thromboelasticity, characterized in that a three-way valve is provided between the sample storage unit and the sample test tube to change the flow direction to measure the supply of the blood sample to the sample test tube or the change in oscillation pressure in the sample test tube. Road test device. According to paragraph 1,
A plurality of sample preprocessing units are connected to one end of one sample storage unit,
One end of a sample inspector is connected to each of the plurality of sample preprocessing units,
A pressure supply unit is connected to the other end of the sample storage unit to provide main swing pressure,
A thromboelastometry test device, characterized in that each auxiliary pressure supply unit is connected to the other end of the sample test tube to provide auxiliary oscillating pressure. According to paragraph 1,
The sample storage unit is composed of a double cylindrical structure (annulus) including a central cylindrical tube located at the center, and only the blood sample filled in the central cylindrical tube flows into the sample, characterized in that a certain volume of blood sample flows into the sample test tube. Thromboelasticity testing device. According to paragraph 1,
A blood sample flowing into the sample test tube is branched into one or more sample preprocessing units to perform one or more tests simultaneously. According to clause 9,
A thromboelasticity test device, characterized in that the volume of the blood sample flowing into each sample test tube is in the range of 5 uL to 200 uL. According to paragraph 1,
A thromboelasticity test device, characterized in that the sample preprocessing unit and the sample test tube are each provided in one or more plurality, and different reagents are supplied to each sample preprocessing unit. According to clause 11,
The reagent is a thromboelasticity test device, characterized in that the sample pretreatment unit or the inner wall of the sample test tube is coated. According to clause 11,
The reagents include calcium chloride (CaCl ₂ ), kaolin, tissue factor, heparinase, thrombin, adenosine diphosphate (ADP), arachidonic acid (AA), and reptilase. , Blood coagulation factor 13 (Factor According to clause 11,
The sample pretreatment unit is a thromboelasticity test device characterized in that it includes a stirrer rotated by a non-contact method of magnetic force. According to clause 12,
When the reagent is coated on the inner wall of the sample test tube, after the blood sample is placed in the test area of the sample test tube, the blood sample is mixed with the reagent by the reciprocating flow generated within the sample test tube. Elasticity testing device. According to clause 11,
A thromboelasticity test device, characterized in that the inner wall of the sample pretreatment unit forms a hydrophobic surface. According to paragraph 1,
When the blood sample is located in the inspection area of the sample examiner, the pressure supply unit repeatedly operates according to a predetermined operation cycle of one cycle. According to clause 17,
The waveform generated by the pressure supply unit is a sinusoidal wave, and the operation cycle time is in the range of 1 second to 60 seconds. According to clause 17,
A thromboelasticity testing device, characterized in that the shear rate applied to the blood sample by the pressure generated by the pressure supply unit is in the range of 0.1 to 60 s ^-1 . According to paragraph 1,
A thromboelasticity testing device, characterized in that the hydraulic diameter of the sample test tube is in the range of 0.1 mm to 4 mm. According to clause 20,
For the purpose of activating platelets, a shear rate applied to the blood sample by the pressure generated by the pressure supply unit is at least 500 s ^-1 or more, and the shear force is at least 1 Pa or more. According to paragraph 1,
The pressure supply unit,
a linear driving unit that moves linearly;
a piston that performs linear reciprocating motion by being driven by the linear driving unit;
A thromboelasticity testing device comprising a cylinder on which the piston is installed to enable linear reciprocating motion. According to paragraph 1,
A control valve for controlling the transmission and release of driving pressure is installed between the sample test tube and the pressure supply unit. According to paragraph 1,
The parameter data is,
An input pressure (Input Magnitude, IMp) of oscillating pressure is applied to the blood sample, and it is obtained by monitoring the pressure when the anticoagulated blood sample moves back and forth according to the input pressure.
R-time (min), which is the reaction time for thrombus formation and is defined as the time for the change in oscillation pressure to reach 1/40 of the input pressure;
K-time (min), which is the thrombus formation time required for a thrombus to form and is defined as the time for the change in oscillating pressure to reach 1/4 of the input pressure;
The α-angle (°), which is the slope of the straight line connecting the curves of the R-time and the K-time, with the rate of thrombus formation;
MA _P (Pa), which is the maximum amplitude indicating the maximum value of the change in oscillation pressure;
Coagulation strength (MAp/Imp) obtained by dividing the maximum amplitude by the input pressure;
tMA(min), which is the time required to reach the MA _P ; and
Contains at least one of Ly(au), which is the clot lysis (lysis) after a certain period of time from the MA _P ,
The MA _P (Pa) value is converted to a MA (mm) value by referring to the correlation with the MA value for the known TEG 5000 system. According to clause 24,
The measurement time of the clot solubility (ly) is determined depending on the value of the shear rate applied to the blood sample by the pressure generated by the pressure supply unit, and is measured 1 to 30 minutes after the MA _P. Elasticity testing device. Injecting a blood sample into a sample reservoir;
Preprocessing the blood sample introduced from the sample storage unit by mixing it with a reagent in a sample preprocessing unit;
providing a oscillating pressure to the sample test tube by a pressure supply unit so that a certain amount of blood sample is introduced from the sample pretreatment unit into the sample test tube; and
A method for testing thromboelastography, comprising the step of acquiring one or more parameter data by monitoring periodic movement characteristics of the blood sample as it moves within the sample tube due to pressure. According to clause 26,
The cyclical movement characteristics of the blood sample are measured by monitoring the moving distance of the blood sample in the sample test tube, or by monitoring the fluctuation of the oscillation pressure in the sample test tube supplied from the pressure supply unit. How to test. According to clause 27,
The periodic movement characteristics of the blood sample are measured using an image sensor that measures the round-trip movement distance of the blood sample, or a pressure measurement unit that measures fluctuations in the oscillating pressure in the sample test tube supplied from the pressure supply unit. How to test thromboelasticity. According to clause 27,
A method for testing thromboelasticity, characterized in that the pressure supply unit is connected to one end of the sample storage unit, and the pressure measurement unit is connected to one end of the sample test tube. According to clause 27,
A method for testing thromboelasticity, characterized in that the pressure supply unit is connected to one end of the sample test tube, and the pressure measurement unit is connected to one end of the sample storage unit. According to claim 29 or 30,
Thromboelasticity, characterized in that a three-way valve is provided between the sample storage unit and the sample test tube to change the flow direction to measure the supply of the blood sample to the sample test tube or the change in oscillation pressure in the sample test tube. How to test too. According to clause 26,
A plurality of sample preprocessing units are connected to one end of one sample storage unit,
One end of a sample inspector is connected to each of the plurality of sample preprocessing units,
A pressure supply unit is connected to the other end of the sample storage unit to provide main swing pressure,
A method for testing thromboelasticity, characterized in that each auxiliary pressure supply unit is connected to the other end of the sample test tube to provide auxiliary oscillation pressure. According to clause 26,
The sample storage unit is composed of a double cylindrical structure including a central cylindrical tube located at the center, and only the blood sample filled in the central cylindrical tube flows in, so that a certain volume of blood sample flows into the sample test tube. How to test. According to clause 26,
A blood sample flowing into the sample test tube is branched into one or more sample preprocessing units to perform one or more tests simultaneously. According to clause 34,
A method for testing thromboelasticity, characterized in that the volume of the blood sample flowing into the sample test tube is in the range of 5 uL to 200 uL. According to clause 26,
A method for testing thromboelasticity, characterized in that the sample preprocessing unit and the sample test tube are each provided in one or more plurality, and different reagents are supplied to each sample preprocessing unit. According to clause 36,
A method for testing thromboelasticity, characterized in that the reagent is coated on the sample pretreatment unit or the inner wall of the sample test tube. According to clause 36,
The reagents include calcium chloride (CaCl ₂ ), kaolin, tissue factor, heparinase, thrombin, adenosine diphosphate (ADP), arachidonic acid (AA), and reptilase. , Blood coagulation factor 13 (Factor According to clause 36,
A method for testing thromboelasticity, wherein the sample pretreatment unit includes a stirrer rotated by a non-contact method of magnetic force. According to clause 37,
When the reagent is coated on the inner wall of the sample test tube, after the blood sample is placed in the test area of the sample test tube, the blood sample is mixed with the reagent by the reciprocating flow generated within the sample test tube. Elasticity test method. According to clause 36,
A method for testing thromboelasticity, characterized in that the inner wall of the sample pretreatment unit forms a hydrophobic surface. According to clause 26,
When the blood sample is located in the inspection area of the sample examiner, the pressure supply unit repeatedly operates according to a predetermined operation cycle of one cycle. According to clause 42,
A thromboelasticity test method, characterized in that the waveform generated by the pressure supply unit is a sinusoidal wave, and the operation cycle time is in the range of 1 second to 60 seconds. According to clause 42,
A method for testing thromboelasticity, characterized in that the shear rate applied to the blood sample by the pressure generated by the pressure supply unit is in the range of 0.1 to 60 s ^-1 . According to clause 26,
A method for testing thromboelasticity, characterized in that the hydraulic diameter of the sample test tube is in the range of 0.1 mm to 4 mm. According to clause 42,
For the purpose of activating platelets, a shear rate applied to the blood sample by the pressure generated by the pressure supply unit is at least 500 s ^-1 or more, and the shear force is at least 1 Pa or more. According to clause 42,
The pressure supply unit,
a linear driving unit that moves linearly;
a piston that performs linear reciprocating motion by being driven by the linear driving unit;
A thromboelasticity test method comprising a cylinder on which the piston is installed to enable linear reciprocating motion. According to clause 26,
A thromboelasticity test method, characterized in that a control valve for controlling the transmission and release of driving pressure is installed between the sample test tube and the pressure supply unit. According to clause 26,
An input pressure (Input Magnitude, IMp) of oscillating pressure is applied to the blood sample, and it is obtained by monitoring the pressure when the anticoagulated blood sample moves back and forth according to the input pressure.
R-time (min), which is the reaction time for thrombus formation and is defined as the time for the change in oscillation pressure to reach 1/40 of the input pressure;
K-time (min), which is the thrombus formation time required for a thrombus to form and is defined as the time for the change in oscillating pressure to reach 1/4 of the input pressure;
The α-angle (°), which is the slope of the straight line connecting the curves of the R-time and the K-time, with the rate of thrombus formation;
MA _P (Pa), which is the maximum amplitude indicating the maximum value of the change in oscillation pressure;
Coagulation strength (MAp/Imp) obtained by dividing the maximum amplitude by the input pressure;
tMA(min), which is the time required to reach the MA _P ; and
Contains at least one of Ly(au), which is the clot lysis (lysis) after a certain period of time from the MA _P ,
The MA _P (Pa) value is converted to a MA (mm) value by referring to the correlation with the MA value for the known TEG 5000 system. According to clause 49,
The measurement time of the clot solubility (ly) is determined depending on the value of the shear rate applied to the blood sample by the pressure generated by the pressure supply unit, and is measured 1 to 30 minutes after the MA _P. Elasticity test method.