RU2700000C1 - Method for determining aspirin resistance - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for rapid assessment of aspirin resistance. That is ensured by ultrasonic oscillations with frequency of 150–160 kHz for a whole blood sample in volume of 25 mcl, placed in a measuring chamber before taking aspirin and 4 hours after taking it. Thereafter, echocoagulogram is recorded and in time intervals: 0.3 minutes, 2 minutes, 3 minutes, 5 minutes determine amplitude values, curve inclination angle, integral index, sector area. That is followed by analyzing the results, and if observing the results of the study after taking aspirin by more than 50 %, the absence of aspirin resistance in the patient is stated.

EFFECT: invention enables determining aspirin resistance in a patient using microvolumes of capillary blood.

1 cl, 8 dwg, 2 ex

Description

The invention relates to medicine, specifically to methods for determining aspirin resistance.

The use of antiplatelet therapy (AH) plays an important role in the prevention of thrombotic complications. The main representatives of hypertension are blockers of cyclooxygenase-1 (COX-1) - aspirin, cardiomagnyl, etc. and blockers of P ₂ Y ₁₂ platelet receptors - clopidogrel, ticlopedin, prazugrel, etc. Evaluation of the effectiveness of the therapy is an important link in the prevention of thrombotic complications.

Currently, the most common method in clinical practice for assessing aspirin resistance is the assessment of platelet aggregation activity before and after the appointment of an antiplatelet agent (after 2-3 and 5 days) - the gold standard in laboratory practice is the determination of platelet aggregation activity - the Bourd turbodimetric method, based on a study changes in the optical density of the plasma. Monitoring is carried out using an aggregometer, according to the dynamics of the decrease in platelet function of platelet-rich plasma of the patient while taking the drug (Born method) [1]. The prevalence of the phenomenon, aspirin resistance, according to many data reaches from 2% to 46% [5], this variability is due primarily to the lack of a generally accepted methodology for assessing this condition, wide variability and low reproducibility of the results of known tests. Platelet aggregation is examined in a sample of platelet-rich plasma (OTP) stabilized by some anticoagulant. Depending on the objectives of the study, it is possible to use various anticoagulants (Na citrate, acid citrate-dextrose solution, heparin hirudin, etc.). In addition, in research practice, platelets are sometimes isolated in an environment of a given composition. To study platelet aggregation. Platelet-rich plasma (OTP) and platelet-poor plasma (BTP) are prepared. Most often, a 130 mM sodium citrate solution is used as an anticoagulant. (This is a 3.2% solution of anhydrous sodium citrate).

Prepared for the study, the OTP is poured into 0.3 ml into cuvettes for an aggregometer and stored at room temperature until the start of the study.

A magnetic stirrer (stirring speed 800 rpm) is placed in a cuvette with a prepared OTP for research and a cuvette is placed in a cell of a laser platelet aggregation analyzer for preliminary incubation for 3 minutes at a temperature of 37 ° C, then the research process is started. After opening the recording window after 10 seconds, with the sound signal, add the necessary dose of the inducer (ADP, FAT, collagen, arachidonic acid) to the cuvette (without removing it from the working cell). When studying aggregation by the traditional Bourne method, it is customary to present the research results as a percentage, with 0% aggregation as the initial state of platelet-rich plasma (OTP), and 100% as platelet-poor plasma (BTP). [3.6].

To assess the effectiveness of the therapy, the above study is repeated after 2-3 and 5 days. According to the degree of change in the intensity of aggregation by 50% or more from the initial level, therapy is considered effective, and the state in which there is a change in platelet aggregation 20% less than the initial level is determined as aspirin resistance.

The main disadvantages of this method are:

- duration of the study

- complexity of sample preparation

- the impossibility of holding the patient at bedside - the variability of the results

- lack of information and reliability of the results in connection with the use of blood samples prepared for the study.

Closest to the proposed one chosen as a prototype is a method for determining aspirin resistance using low-frequency piezotromboelastography (NPTEG) [2] using the hardware-software complex ARP-01M Mednord (RS 2010/09767) [7], according to which to conduct a test, a test sample is taken from a cubital vein in the amount of 0.5 ml of venous blood, which is immediately transferred to a cuvette located in a thermostatically controlled cell of the device, where a resonator needle is mounted, mounted on a piezoelectric a viscous element, the viscosity chronometric characteristics of the blood are recorded, and according to the results of changes in t1 (the reaction period representing the time from the start of the study to the maximum decrease in amplitude - A1, in min) and ICK (intensity of the contact phase of coagulation), a conclusion is drawn on the effectiveness of the therapy - an increase in time t1 by 2 or more times, a decrease in KIC by 2 or more times, indicates the effectiveness of ongoing antiplatelet therapy and the absence of aspirin resistance in the patient [4].

The disadvantages of the NPTEG method are: - the need for highly qualified personnel;

- impossibility of assessment using capillary blood - the need for sampling

venous blood;

- high cost of the complex.

Thus, the known methods can determine aspirin resistance, but are complex, inaccessible to the patient, require qualified personnel.

A new technical task is the development of a reliable and affordable method for determining aspirin resistance at the patient’s bed (Point-of-care-test - GROWTH) using capillary blood.

A new technical result is an increase in accuracy, information content, accessibility and convenience of the method when using blood microvolumes for use in the GROWTH mode.

To achieve a new technical result in a method for determining aspirin resistance, which includes taking and placing a blood sample in a measuring chamber with subsequent exposure to it by a physical factor and recording blood characteristics, a whole blood sample in a volume of 25 μl is placed in the measuring chamber before aspirin is taken and after 4 hours after receiving it, they are exposed to ultrasonic vibrations with a frequency of 150-160 kHz, after which, an echocoagulogram is recorded and at time intervals T1 = 0.3 minutes, T2 = 2 minutes, T3 = 3 minutes, T4 (4) = 5 Inuit determine the magnitude of amplitude parameters A1, A2, A3, A4 (4), respectively, La curve angle between points on ehokoagulogramme A ₁ and A ₂ and the integral index: sector area S, bounded on one side of the segment of the curve between the points A1 and A4 ( 4) with the other abscissa axis, analyze the graphical results and, when the study indicators change after taking aspirin by more than 50%, determine the patient’s absence of aspirin resistance (or when the study indicators change after taking aspirin by less than 50%, the they show aspirin resistance in the patient).

The method is as follows: The method is implemented using the original device rapid assessment of the state of the hemostatic system (Fig. 1, Fig. 2).

In Fig 1. Cartridge device for rapid assessment of the state of the hemostatic system

1. Piezoceramic ultrasonic sensor (in section);

2. The measuring chamber;

3. Cartridge body;

4. External electrode;

4.1 Generator of ultrasonic vibrations; 4.2. The receiver of ultrasonic vibrations;

5. The internal electrode;

6. Contact group for switching;

7. Central contact;

8. Side contact.

In Figure 2. A device for rapid assessment of the state of the hemostatic system (general view)

1. Piezoceramic ultrasonic sensor (in section)

9. Display for displaying results;

10. USB output for charging / PC connection;

11. Connector for a cartridge;

12. Registration unit for rapid assessment of the state of the hemostatic system;

13. The keyboard.

When conducting research, use the standard procedure for taking blood. The ring finger of the patient should be slightly massaged before blood sampling. The upper phalanx of the finger is treated with cotton wool or gauze moistened with an antiseptic, then the finger is wiped with dry sterile material; perform a puncture, and the first few blood droplets are wiped dry with sterile. The next drop of blood is placed (due to the capillary effect), in the measuring chamber 2 of the sensor 1 of the device for rapid assessment of the state of the hemostasis system. After blood sampling, a sensor with a measuring chamber is placed in the cartridge of the device and the study is started. After the end of the study, the disposable sensor is disconnected from the device and disposed of (as waste class B).

This procedure is performed twice: before taking aspirin and 4 hours after taking it.

Measuring chamber 2 and electrodes 4.1. and 4.2 are located on the base of the cartridge case 3, moreover, the measuring chamber 2 is located inside the piezoelectric sensor 1 made in the form of a hollow cylinder, the external electrode 4 is located outside it, divided by symmetrical slots into two equal parts, one part of which 4.1 is a generator, and the second 4.2 - a receiver of ultrasonic vibrations, on the inner surface of the cylinder there is an inner electrode 5, the central 7 and side 8 contacts are built into the base of the cartridge 3 and fixed on its base.

α кривой между точками на эхокоагулограмме A₁ и А₂ и интегральный показатель: площадь сектора S, ограниченного с одной стороны отрезком кривой между точками А1 и А4 (4) с другой осью абсциссы, анализируют графические результаты и при изменении показателей исследования после приема аспирина более, чем на 50% определяют отсутствие у больного аспиринорезистентности (и при изменении показателей исследования после приема аспирина менее чем на 50% определяют аспиринорезистентность).An echocoagulogram is recorded and at intervals of time T1 = 0.3 minutes, T2 = 2 minutes, T3 = 3 minutes, T4 (4) = 5 minutes, the magnitude of the amplitude indicators A1, A2, A3, A4 (4) is determined, respectively, the angle of inclination

α curve between the points on the echocoagulogram A ₁ and A ₂ and an integral indicator: the area of sector S, limited on one side by the segment of the curve between points A1 and A4 (4) with the other abscissa axis, analyze the graphical results and when the study indicators change after taking aspirin more than 50% determine the absence of aspirin resistance in the patient (and when changing the research parameters after taking aspirin, aspirin resistance is determined by less than 50%).

The measurement results are recorded in the form of an echocoagulogram twice: before taking and four hours after taking aspirin, a second study.

After conducting 2 studies (before and after taking the drug), graphical results are analyzed and, when the results of the research indicators change after taking aspirin by more than 50%, the patient’s absence of aspirin resistance is determined, and when the research parameters change after taking less than 50% of aspirin aspirin resistance).

Clinical examples

Example 1. Conditionally healthy volunteer D. 28 years.

At the first stage, a voluntary written consent was obtained from the subject to participate in a scientific research.

To determine the initial state of platelet aggregation, blood was drawn from the cubital vein into a standard vacutainer (BD Vacutainer) to determine the Born aggregation study. Then, using a three-component syringe, blood is taken from the ulnar vein for low-frequency piezothromboelastography, and blood is taken from the finger for conducting an echocoagulogram.

The results of the study of platelet aggregation (Biola, Moscow) with ADP (5 μM): light transmission was 42% (Figure 3);

The study was carried out by means of low-frequency piezotromboelastography (NPTEG) [2] using the hardware-software complex ARP-01M “Mednord” (R.S. 2010/09767) [7]

NPTEG results - t1 = l, 4; KIC = 22.7 (Figure 4);

Further, the subject was prescribed aspirin (75 mg * 1 time / day)

A repeated study of NPTEG was carried out 4 hours after taking aspirin-t1 = 2.7; KIC = 11, 12 (Figure 4);

3 days after the start of aspirin administration, re-aggregation of platelets with ADP was performed: light transmission was 26% (Figure 3);

NPTEG data (an increase in t1 by a factor of 2 or more, a decrease in ICC by a factor of 2 or more) and aggregatometry data (according to Born) —a decrease in light transmission by more than 50%, indicate the effectiveness of antiplatelet therapy — the absence of aspirin resistance.

Additionally, a study was carried out according to the proposed method using a device for the rapid assessment of the state of hemostasis with registration of an echocoagulogram after exposure to ultrasonic vibrations on a blood sample of 150-160 KHz.

Echocoagulography results:

α=45°; S=431 (Фигура 5).Background values - A2 = 75; A3 = 105; A4 = 120;

α = 45 °; S = 431 (Figure 5).

α=12°; S=91 (Фигура 5).4 hours after taking aspirin A2 = 10; A3 = 20; A4 = 30;

α = 12 °; S = 91 (Figure 5).

Conclusion: Based on the results obtained, it can be concluded that aspirin is effective, i.e. the patient’s lack of aspirin resistance, which is confirmed by a decrease in the angle of inclination, a decrease in amplitude indicators, and a decrease in the value of the integral index of the area S by 2 or more times.

Example 2. Patient M. 30 years.

He was admitted to the hospital with a diagnosis of post-thrombophlebotic syndrome (PTFS), popliteal thrombosis.

To determine the initial state of platelet aggregation, blood was drawn from the cubital vein into a standard vacutainer (BD Vacutainer) to determine the Born aggregation study. Then, using a three-component syringe, blood is taken from the ulnar vein for low-frequency piezothromboelastography, and blood is taken from the finger for conducting an echocoagulogram.

The results of the study of platelet aggregation (Biola, Moscow) with ADP (5 μM): light transmission was 43% (Figure 6); NPTEG results - t1 = l, l; KIC = 15.45 (Figure 7).

Further, according to the prescription of the attending physician, the patient was prescribed aspirin at a dosage of 150 mg / 1 time per day.

4 hours after taking aspirin, a second study was conducted using NPTEG - t1 = l, 2; KIC = 15 (Figure 7);

3 days after the start of aspirin administration, re-aggregation of platelets with ADP was performed: light transmission was 38% (Figure 6);

The NPTEG data (an increase in t1 by less than a factor of 2, a decrease in ICC by a factor of less than two) and aggregatometry data (according to Born) - a decrease in light transmission of less than 50%, indicate the ineffectiveness of antiplatelet therapy - aspirin resistance.

Additionally, a study was carried out according to the proposed method using a device for the rapid assessment of the state of hemostasis with registration of an echocoagulogram after exposure to ultrasonic vibrations on a blood sample of 150-160 KHz.

Echocoagulography results:

α=40°; S=320 (Фигура 8). Через 4 часа после приема аспирина А2=100; А3=130; А4=150;

α=38°; S=260 (Фигура 8).Background values - A2 = 115; A3 = 145; A4 = 160;

α = 40 °; S = 320 (Figure 8). 4 hours after taking aspirin A2 = 100; A3 = 130; A4 = 150;

α = 38 °; S = 260 (Figure 8).

Conclusion: Based on the data of the studies, we can conclude that the action of aspirin is ineffective, that is, the patient has aspirin resistance, which corresponds to the research results obtained using the proposed method and is confirmed by the absence of statistically significant changes in the angle of inclination, amplitude indicators, and integral indicator area.

Figure 1. Cartridge device for rapid assessment of the state of the hemostatic system

1. Piezoceramic ultrasonic sensor (in section);

2. The measuring chamber;

3. Cartridge body;

4. External electrode;

4.1 Generator of ultrasonic vibrations; 4.2. The receiver of ultrasonic vibrations;

5. The internal electrode;

6. Contact group for switching;

7. Central contact;

8. Side contact.

Figure 2. Device for rapid assessment of the state of the hemostatic system (general view) 1. Piezoceramic ultrasonic sensor

9. Display for displaying results;

10. USB output for charging / PC connection;

11. Connector for a cartridge;

12. Registration unit for rapid assessment of the state of the hemostatic system;

13. The keyboard.

Figure 3. Aggregate of a conditionally healthy volunteer, according to Born (before and 5 days after taking aspirin). A - background study (before taking), B - study after taking aspirin.

Figure 4. Scaled low-frequency piezothromboelastogram, conditionally healthy volunteer, before and after the appointment of aspirin. A - background study (before taking), B - study after taking aspirin.

Figure 5. Echocoagulograms of a conditionally healthy volunteer, before and after prescribing aspirin. A - background study (before taking), B - study after taking aspirin.

Figure 6. Aggregate of patient M., according to Born (before and 5 days after taking aspirin). A - background study (before taking), B - study after taking aspirin.

Figure 7. Scaled low-frequency piezothromboelastogram, patient M., before and after the appointment of aspirin. A - background study (before taking), B - study after taking aspirin.

Figure 8. Echocoagulogram of patient M., before and after prescribing aspirin. A - background study (before taking), B - study after taking aspirin.

Information sources

1. Dolgov VV Laboratory diagnosis of hemostatic disorders / V.V. Dolgov, P.V. Svirin.- M.-Tver: Triad, 2005. -227 p.

2. Udut V.V., Tyutrin I.I. et al. Technology of low-frequency piezothromboelastography in monitoring antithrombotic therapy / V.V. Udut, I.I. Tyutrin et al. // Experimental and clinical pharmacology. - 2017. - No. 11. - FROM.

3. Gabbasov Z.A., Popov EG, Gavrilov I.Yu., Pozin E.Ya., Markosyan R.A. A new highly sensitive platelet aggregation assay. Laboratory Science, 1989, N10, p. 15-18

4. Soloviev MA, Udut VV, Tyutrin II, Karchagina OS Features of the pharmacodynamics of antiplatelet agents in the correction of thrombotic complications in polytrauma / Soloviev MA, Udut VV, Tyutrin I. AND. et al. // Medico-Biological and Socio-Psychological Problems of Safety in Emergency Situations, 2015.- No 1. C. 96-102.

5. Macchi L., Sorel N., Christiaens L. Aspirin resistance: definitions, mechanism, prevalence, and clinical significance. Curr Pharm Des. 2006; 12 (2): 251-258.

6. Born G.V.R. Quantitative investigation into the aggregation of blood platelets. J. Physiol. (Lond), 1962, p. 67P-68P.

7. Udut V.V., Kinzersky A.A. A method for evaluating platelet aggregation activity // Patent RU 2666945.

Claims (1)

A method for the rapid assessment of aspirin resistance by twice determining the viscosity characteristics of blood, including taking and placing a blood sample in a measuring chamber with subsequent exposure to it by a physical factor and recording blood characteristics, characterized in that a whole blood sample in a volume of 25 μl placed in the measuring chamber before taking aspirin and 4 hours after its administration, they are exposed to ultrasonic vibrations with a frequency of 150-160 kHz, after which an echocoagulogram is recorded at time intervals T 1 = 0.3 min, T2 = 2 min, T3 = 3 min, T4 (4) = 5 min determine the magnitude of the amplitude indicators A1, A2, A3, A4 (4), respectively, the angle of inclination of the curve between points A ₁ and A ₂ -

α and the integral indicator, the area of the S sector, limited on one side by a segment of the curve between points A1 and A4 (4) with the other abscissa axis, analyze the graphical results and, when the results of the study change after taking aspirin by more than 50%, determine the patient’s absence of aspirin resistance .

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