RU2486523C1 - Method for estimating clinical effectiveness of antiaggregant therapy in patients with acute coronary syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: taken venous blood is separated into two samples. The first sample is stabilised with a solution of sodium citrate, the second one - with ethylene diamine tetraacetate. The first sample of whole blood is added with adenosine diphosphate as an aggregation inducer and tested for a peak amplitude of thrombocyte aggregation and a peak amplitude of adenosine triphosphate release profile by impedance method. The second sample is used to measure a fraction of thrombocytes and a fraction of blood corpuscles. It is followed by calculating a thrombocyte aggregation potential index by formula $$I=\frac{L_{\max }\cdot PTC}{{\Omega }_{\max }\cdot HTC}\cdot 100\%;$$

wherein L_max is the peak amplitude of adenosine triphosphate release profile, Ω_max is the peak amplitude of thrombocyte aggregation, PCT is the fraction of thrombocytes, HTC is the fraction of blood corpuscles. If the value I is less than 0.5%, the low clinical effectiveness of the antiaggregant therapy is stated, and the value I being 1.5-2.5% shows the high effectiveness thereof.

EFFECT: improving the objective estimation of the clinical effectiveness of the antiaggregant therapy in the patients with acute coronary syndrome, and providing an opportunity for predicting the clinical course of the disease.

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Description

The invention relates to practical medicine and clinical laboratory diagnostics. The method is intended to build a short-term forecast of the effectiveness of combination antiplatelet therapy in patients with acute coronary syndrome (ACS) and allows to assess the individual risk of recurring thrombotic events (myocardial infarction with and without ST segment elevation, unstable angina) in patients undergoing antiplatelet therapy according to about acute coronary syndrome.

There are a number of methods for evaluating the effectiveness of antiplatelet therapy, based on the assessment of parameters of the termination of blood flow in conditions of high shear stress in vitro by the formation of platelet plugs (PFA-100); study of platelet adhesion under shear stress (IMPACT cone and plate analyzer); the study of changes on the surface of a platelet associated with its activation (flow cytometry, immunochemical markers, for example P-selectin); study of platelet release reaction (determination of thromboxane B2 in serum, its degradation products in urine, indirect determination of arachidonic acid-induced formation of thromboxane A2) [Hezard N, Tessier-Marteau A, Macchi L., Cardiovasc. Hematol. Disord Drug Targets, 2010; 10 (3): 224-33; Gorlinger K, Jambor C, Dirkmann D, Dusse F, Hanke A, Adamzik M, Hartmann M, Philipp S, Weber AA, Rahe-Meyer N. Herz. 2008 Jun; 33 (4): 297-305.].

However, the described methods are complex and time-consuming to use, have high variability of the estimated parameters, low sensitivity, require special preparation of the test blood sample and an experienced operator, and are expensive to use.

A known method for predicting the course of acute myocardial infarction (see RF patent No. 2121691, class IPC G01N 33/86, publ. 10.11.1998), including the study of biochemical parameters of the patient’s blood in dynamics, followed by prediction of the adverse course of the disease. The prothrombin index is used as the studied indicator, and an adverse course of the disease is predicted when the index decreases from the third to the fourth day from the onset of the disease.

The disadvantage of this method is the low sensitivity and low specificity for assessing the risk of repeated thrombotic events. This method is based on the study of the prothrombin index, the value of which is determined by the functional activity and the number of coagulation factors - II, VII, V and X and the sensitivity of the thromboplastin used. That is, on the one hand, the assessment exclusively uses the coagulation link of the blood coagulation system and does not take into account the platelet mechanism of hemostasis, the effect on which is the essence of antiplatelet therapy aimed at preventing the development of repeated thrombotic events. On the other hand, this parameter has high variability and low reproducibility in the conditions of various laboratories.

There is also a method of assessing the risk of recurring thrombotic events in patients with acute coronary syndrome (see RF patent No. 2419800 according to class IPC G01N 33/86, publ. 05.27.2011), which consists in determining the level of hematocrit, the number of red blood cells, platelets, determining the time of adenosine phosphate -induced-ADP-induced blood coagulation, scoring.

However, the method does not take into account a number of functional features of platelets, in particular, the characteristic of the reaction of platelet release, their volume fraction in whole blood. The described method is an integral assessment of the state of the hemostatic system and cannot adequately assess the risk of recurring thrombotic events in patients with thrombocytopathies, including drugs.

Also known is the "Method for the diagnosis of aspirin resistance in patients with coronary heart disease" (see RF patent for the invention No. 2348041, class IPC G01N 33/68, published on 02.27.2009), for which prior to treatment with aspirin in platelets of peripheral blood of patients is determined activity of reactions of NAD- and NADP-dependent dehydrogenases. Then, the platelet cofactor exchange coefficient (CCOT) is calculated according to the formula CCOT = IOR / IVN, where IOR is the ratio of the activity levels of NADP- and NADPH-dependent glutamate dehydrogenase reactions, and IVN is the ratio of the activity level of the NADH-dependent glutamate dehydrogenase reaction to the activity level of the NAD-dependent lactate dehydrogenase. And when the value of CCOT is higher than 0.3, aspirin resistance is diagnosed, and if the value of CCOT is equal to or less than 0.3, sensitivity to aspirin is diagnosed. The method allows the diagnosis of aspirin resistance of patients prior to treatment with aspirin and provides high accuracy of the prognosis.

However, the implementation of this method requires significant material costs and is extremely laborious.

Closest to the claimed is a method for evaluating the effectiveness of antiplatelet therapy in the treatment of cardiovascular diseases (see RF patent No. 2379684, class IPC G01N 33/48, publ. 10.06.2009), which consists in determining the aggregation activity of platelets in whole blood by the impedance method , determining the amplitudes of the aggregation process before and after incubation in vitro with acetylsalicylic acid.

However, this method of evaluating the effectiveness of antiplatelet therapy is not objective enough, since the disaggregation effect of acetylsalicylic acid after its incubation in vitro is evaluated, which does not take into account the effect of its deacylation in the gastrointestinal tract.

On the other hand, the proposed method is based on the analysis of the integral parameter of platelet aggregation ability and does not take into account the contribution of the release reaction (the state of platelet granules and its cell membrane), as well as the aggregate-suspension state of the blood, which may ultimately lead to incomplete consideration of the parameters leading to the risk of recurring thrombotic events in patients undergoing antiplatelet therapy.

The objective of the invention is to increase the objectivity of evaluating the effectiveness of antiplatelet therapy in patients with acute coronary syndrome due to the possibility of a comprehensive consideration of the parameters of the aggregate-suspension state of the blood and the aggregation ability of platelets, including the release reaction of adenosine triphosphate with technical simplicity and reducing the cost of the method.

To solve the problem in a method for evaluating the clinical efficacy of antiplatelet therapy in patients with acute coronary syndrome, which consists in taking venous blood, stabilizing it, determining platelet aggregation activity by the impedance method, determining platelet aggregation amplitudes, according to the invention, venous blood is divided into two whole blood samples, one of which they are stabilized with sodium citrate solution, and the second with ethylene diamine tetraacetate, an inductor is introduced into the first whole blood sample platelet aggregation adenosine diphosphate, determine in it the maximum amplitude of platelet aggregation and the maximum amplitude of the adenosine triphosphate release curve, in the second sample determine the fraction of the volume occupied by platelets, and the proportion of blood volume on the formed elements, determine the platelet aggregation index at a blood count value less than 0 and a value of less than 0 , 5% judge the low effectiveness of antiplatelet therapy, and with a value of the index of 1.5-2.5% - about its high effectiveness, while the platelet a regatsionnogo capacity of whole blood is determined by the formula

$$I=\frac{L_{\max }\cdot PTC}{{\Omega }_{\max }\cdot HTC}\cdot \mathrm{one\; hundred}\%$$

where L _max - the maximum amplitude of the release curve of adenosine triphosphate,

Ω _max - the maximum amplitude of platelet aggregation,

PCT - the proportion of whole blood volume occupied by platelets,

NTS - the proportion of blood volume attributable to the shaped elements.

Sources of patent and scientific and technical information known to the authors do not describe a method for evaluating the effectiveness of antiplatelet therapy, which allows an objective and high probability to assess the risk of repeated thrombotic events in patients with acute coronary syndrome due to the possibility of a comprehensive assessment of the parameters of the aggregate-suspension state of blood and aggregation ability platelets, including adenosine triphosphate release reactions.

The impedance and lumi method of aggregation of whole blood are known (see, for example, W. Heptner, et. Al. Thromb. Haemostas. 58, 1676 (1987); P. Sathiropas, et. Al. Thromb. Res. 51, 55 ( 1988)), which allows you to evaluate the aggregation activity of platelets in a sample of diluted whole blood in response to the introduction of aggregation inducers, including the quantitative parameters of the adenyl nucleotide release reaction (ATP, nmol). However, the parameters determined by this method have high variability, which is determined by the features of taking a blood sample, its type (venous or arterial), the age of the patient, the presence of a particular type of thrombophilia and / or thrombocytopathy, background and / or concomitant pathology, the spectrum of drug therapy used, including antithrombotic, concentration, time and method of introducing an aggregation inducer, etc.

For them, there are no standardized assessment parameters, their predictive power has not been practically studied.

In the claimed method, by studying the artificially simulated situation of the formation of a blood clot in the coronary arteries in vitro, the authors were able to select a parameter for assessing the risk of repeated thrombotic events (platelet aggregation potential index of whole blood), which, according to the Applicant, has not yet been known.

The index of platelet aggregation potential of whole blood with its subsequent ranking for predicting the effectiveness of antiplatelet therapy was also not previously used, since the intervals of values characteristic of patients with acute coronary syndrome indicators reflecting the aggregate ratio were not known and were not identified (before the present application for the invention) -suspension state of the blood and the state of the reaction of platelet release, which form the basis of the conclusions about the risk assessment of repeated thrombotic events herein.

In addition, the use of whole blood samples, adenosine diphosphate as an inducer of platelet aggregation, the absence of a preanalytical preparation of samples allows optimizing the cost of implementing the method, which generally increases its manufacturability and allows evaluating the effectiveness of antiplatelet therapy for a larger number of patients.

The method claimed by the authors is technical and technological from the first manipulation to, practically, the last operation — processing the obtained data, since all manipulations: blood sampling, selection and preparation of samples, exposure to reagents on samples, incubation time, procedure in the inventive method are signs of a technically carried out action .

The foregoing allows us to conclude that there is a criterion of "inventive step" in the claimed invention.

The method is as follows.

Venous blood is sampled, it is divided into two samples, one of which is stabilized with a 3.8% solution of trisubstituted sodium citrate in a standard ratio of 9: 1 and the aggregation activity of platelets in whole diluted blood is immediately evaluated by impedance measurement with parallel determination of the dynamics of the ATP release reaction by lumiaggregation as follows.

1. Turn on and warm up the aggregometer to the state of maintaining a stable temperature in the cells 37ºС in accordance with the instructions for its use.

2. Prepare and warm the used reagents to 37 ° C in the appropriate cells of the aggregometer.

3. When using a multi-channel aggregometer, it is possible to conduct studies in duplicates to increase the accuracy of the study.

4. Add 0.45 ml of saline and a magnetic stirrer to each measuring cuvette, warm for 5 minutes.

5. Add 0.45 ml of the patient’s whole blood to the cuvette, followed by warming for 5 minutes, after which 0.1 ml of the phosphor (CHRONOLOG LUME) is introduced by bolus.

6. After adjusting the device according to the standard ATP solution and the level of basic impedance, according to the instructions for use of the aggregometer, the ADP working solution with a final concentration of 10 μL is introduced in a bolus manner.

7. Aggregate pattern and ATP release curve are recorded for 5 minutes (maximally, depending on the time the determined parameters reach the plateau).

8. Determine the maximum amplitude of the aggregate and the response curve of the release (using standard software).

The second part of the whole blood sample is stabilized with ethyleneamine tetraacetate (potassium EDTA) in a standard ratio of 1.2-2 mg per 1 ml of whole blood and is used for hematological examination using hematological analyzers - flow-type counters (in our case, ABXMicros). Thrombocrit (PCT) is determined - the proportion of the volume of whole blood occupied by platelets, and hematocrit (HCT) - the proportion of the volume of blood attributable to the formed elements.

In this case, the use of stabilizers of whole blood in this embodiment is crucial. Sodium citrate is the most gentle stabilizer in relation to cell membranes. It binds calcium ions of blood plasma, thereby breaking the cascade of its coagulation, which largely determine the kinetics and severity of thrombosis, and also minimally complex with coagulation proteins, therefore it is the stabilizer of choice in coagulological blood tests.

The use of EDTA is crucial for cytomorphometry and hematological analyzes. For the use of blood stabilized with EDTA, practically all hematological counters used in clinical laboratories have been calibrated.

The effectiveness and prognosis of antiplatelet therapy are assessed by the platelet aggregation potential index of whole blood using the following parameters: Ω _max is the maximum amplitude of platelet aggregation determined by the impedance method, L _max is the maximum amplitude of the ATP release curve, PCT platelet count, and NTS hematocrit (in shares).

In this case, the calculation of the platelet aggregation potential index of whole blood is carried out according to the following formula:

$$I=\frac{L_{\max }\cdot PTC}{{\Omega }_{\max }\cdot HTC}\cdot \mathrm{one\; hundred}\%$$

This formula was obtained by analyzing the results of a study of the dynamics of platelet aggregation activity by impedance and lumiaggregometry in patients with ACS (n = 174) undergoing standard antiplatelet therapy. The observation period was 12 months. Based on the forces of information and correlation of all the estimated parameters of aggregatograms and clinical outcomes at the end of the observation period, as well as the pathophysiological essence of the blood coagulation process, a platelet aggregation potential index of whole blood was derived, which is the ratio of the total amount of ATP released to the initial stages of aggregation "Prevalence" of platelet aggregation in the conditional volume of whole blood, expressed as a percentage.

Then the ranking and evaluation of the specified index is carried out according to the following scheme, given for clarity in the table.

I values The risk of recurring thrombotic events in the context of ongoing antiplatelet therapy The need for additional diagnostic steps 0.00-0.50 Tall Verification of the etiology of thrombophilia 0.51-1.50 Average No 1.5-2.5 Low No > 2.5 Vague Assessment of platelet aggregation ability using other aggregation inducers

The risk of recurring thrombotic events in the context of antiplatelet therapy was evaluated as a quantitative description of the “danger” of recurring thrombotic events (this is the ratio of the number of adverse effects (the sum of deaths, the development of repeated non-fatal thrombotic events (n) to their possible number over a certain period (N) R = N (t) / Q (f), where N (t) is a quantitative indicator of the frequency of adverse events per unit time t; Q (f) is the number of risk objects (patients included in the study), p those with a particular risk factor f).

In the intensive care unit of the Saratov Research Institute of Cardiology, a group of clinically healthy individuals and a group of patients with acute coronary syndrome, including unstable angina pectoris, myocardial infarction with and without ST segment elevation, were examined in accordance with the method described above. In patients with ACS, the test results were compared with the clinical outcome of the disease - death, the development of complications, relapses of the disease. The maximum amplitude of impedancemetry for clinically healthy patients ranged from 6 to 18 Ohms with a maximum amount of released ATP from 0.38 to 1.1 nmol, which corresponded to the declared reference intervals when using ADP aggregation as an inductor in a final concentration of 10 μmol. The maximum degree of ADP induced platelet aggregation ranged from 0 to 21 Ohms in patients with NS and from 0 to 17 Ohms in patients with MI, all patients were on standard antiplatelet therapy with a maximum amount of released ATP from 0.0 to 1.71 nmol. Differences in these parameters among patients without and with repeated thrombotic events did not reach the degree of statistical significance, which is due to the high degree of data dispersion and the nonequilibrium proportion of patients in the studied groups. The authors' index of platelet aggregation potential of whole blood revealed statistically significant differences in the group of patients with repeated thrombotic events and without them (ManWhitney p level = 0.041), and a negative moderate strength correlation between the rank of outcome and the used index of platelet aggregation potential of whole blood was revealed ( Kendall τ 0.69, plevel 0.002), while the variability of the index within each group did not exceed 17.2%. The use of the proposed method for predicting the effectiveness of antiplatelet therapy leads (based on the studied sample of patients with ACS) to the correct classification of patients in 83.5% of cases.

Clinical example 1. Patient S., 65 years old, was admitted to the hospital with a diagnosis of coronary heart disease, left ventricular posterior wall Q myocardial infarction, stage III arterial hypertension, complication: Killip class 1 acute heart failure. Standard therapy was carried out according to the recommendations of the Committee of Experts of the All-Russian Scientific Society of Cardiology - double antiplatelet therapy, Xa factor inhibitors, statins, ACE inhibitors, beta-blockers. According to Doppler echocardiography, hypokinesis zones were detected, the ejection fraction was preserved (61%), there were no signs of stagnation at rest, and diastolic dysfunction was revealed. Biochemical indicators and indicators of the general analysis of blood are within normal limits. Ω _max - 7 Ohms, L _max - 0.01 nmol, PCT - 0.180 and NTS - 0.456. The platelet aggregation potential index of the patient’s whole blood was 0.056 (high risk). According to coronarography, occlusion of the right coronary artery, hemodynamically insignificant stenosis of the anterior descending artery was revealed. The right coronary artery was stented with a drug-coated stent with a good angiographic effect - complete restoration of blood flow in a heart attack-related artery. 3 days after stenting, against the background of antithrombotic treatment continued in accordance with the recommendations, the condition suddenly worsened - a second anginal attack developed, against which the ST segment elevation was recorded in the anterolateral leads. The patient died 1.5 hours after the onset of a pain attack in the clinical picture of cardiogenic shock.

Clinical example 2. Patient A., 63 years old. He was admitted to the hospital with Q-myocardial infarction of the anterior wall of the left ventricle. A history of arterial hypertension, myocardial infarction, exertional angina III FC, chronic heart failure 3 tbsp., III FC, type II diabetes mellitus. The course of the disease was complicated by the development of acute class III heart failure by Killip, pneumonia, and left-sided hydrothorax. He received standard therapy according to the recommendations of the Committee of Experts of the All-Russian Scientific Society of Cardiology - double antiplatelet therapy, X-factor inhibitors, statins, ACE inhibitors, beta-blockers, diuretics inside and intravenously, inotropic drugs, insulin. Doppler echogardiography revealed areas of hypo- and akinesia, aneurysm of the apex of the left ventricle, signs of decompensation of blood circulation in both circles, the ejection fraction of the left ventricle 40%. Complete blood count without pathology. Upon receipt, biochemical signs of decompensation of diabetes. Coronarography was not performed. Biochemical indicators and indicators of the general analysis of blood are within normal limits. Ω _max - 5 Ohms, L _max - 0.16 nmol, PCT - 0.201 and NTS - 0.455. The platelet aggregation potential index of the patient’s whole blood was 1.413 (medium risk).

Against the background of the treatment, the manifestations of chronic heart failure decreased - exercise tolerance (at discharge at level II FC) increased, edema of the lower extremities, signs of stagnation in the small circle and hydrothorax (according to repeated x-ray examination) disappeared, and no angina attacks occurred, according to biochemical analysis achieved compensation of metabolic processes. The patient was discharged under the supervision of a cardiologist at the place of residence, for 11 months - the absence of repeated thrombotic events.

Clinical example 3. Patient C., 31 years old. He was admitted to the hospital with Q myocardial infarction of the anterior wall of the left ventricle, complication: acute heart failure grade 1 according to Killip. Previously, there were no diseases of the cardiovascular system. Standard therapy was carried out according to the recommendations of the Committee of Experts of the All-Russian Scientific Society of Cardiology - thrombolytic therapy, double antiplatelet therapy, X-factor inhibitors, statins, ACE inhibitors, beta-blockers. According to Doppler echocardiography, zones of hypo- and akinesis were detected, the ejection fraction of the left ventricle was 48%, there were no signs of stagnation at rest. Biochemical indicators and indicators of the general analysis of blood are within normal limits. Ω _max - 14 Ohms, L _max - 0.65 nmol, PCT - 0.175 and NTS - 0.371. The platelet aggregation potential index of the patient’s whole blood was 2.19 (low risk). According to coronarography, an occlusion of the anterior descending coronary artery in the proximal segment was revealed. An anterior descending coronary artery was stented with a drug-coated stent with a good angiographic effect - complete restoration of blood flow in a heart attack-related artery. The course of the disease without complications. Discharged under the supervision of a cardiologist at the place of residence.

The above indicates the adequacy, sufficient sensitivity and usefulness of the proposed method for predicting the clinical effectiveness of antiplatelet therapy in patients with ACS.

The medical and social effect of the proposed method consists in a pathogenetically substantiated highly sensitive risk assessment of repeated thrombotic events, i.e. clinical efficacy of antiplatelet therapy in patients with ACS.

For the first time, a method for predicting the clinical effectiveness of antiplatelet therapy in patients with ACS, based on a comprehensive assessment of the parameters of the aggregate-suspension state of the blood and the aggregation ability of platelets, is proposed. In addition, the novelty of the claimed method is the use of specific four of these indicators simultaneously in one method for predicting the clinical effectiveness of antiplatelet therapy in patients with ACS.

The economic effect of the proposed method compared with the currently used is on the one hand to reduce research costs. For example, the average price of a PFA COLLAGEN / ADP CARTRIG cartridge for analogous methods for 20 measurements (two patients) is 10,817.3 rubles, that is, the cost of only a cartridge for examining one patient is about 1,081.73 rubles. While the cost of reagents for conducting a study of 1 patient is about 270.75 rubles, which is several times cheaper due to consumables. When compared with whole blood aggregometry with an impedance and lumimetric method, the advantage lies in obtaining additional information about the condition of patients with ACS with similar personnel, time and resource costs.

The method makes it possible to predict the course of the disease already in the first hours of the patient’s admission to any medical institution where there is a laboratory equipped with an aggregometer (impedance and lumiaggregation) and a hematological counter, and a laboratory doctor who can process data from a general blood test and studies of platelet aggregation activity .

The developed technology according to the claimed method expands the arsenal of means of objective monitoring of the condition of patients with ACS.

Claims (1)

A method for evaluating the clinical efficacy of antiplatelet therapy in patients with acute coronary syndrome, which consists in taking venous blood, stabilizing it, determining platelet aggregation activity by the impedance method, determining platelet aggregation amplitudes, characterized in that the venous blood is divided into two whole blood samples, one of which is stabilized a solution of sodium citrate, and the second - ethylenediaminetetraacetate, in the first sample of whole blood make as an inducer of platelet aggregation adenosindi osfate, determine in it the maximum amplitude of platelet aggregation and the maximum amplitude of the adenosine triphosphate release curve, in the second sample, the fraction of the volume occupied by platelets and the fraction of the blood volume falling on the formed elements are determined, the index of platelet aggregation potential of the blood is determined, and if the index value is less than 0.5% judge the low effectiveness of antiplatelet therapy, and with a value of the index of 1.5-2.5% - about its high efficiency, while the platelet aggregation potential index of the whole cr ovi are determined by the formula:
$$I=\frac{L_{\max }\cdot PTC}{{\Omega }_{\max }\cdot HTC}\cdot \mathrm{one\; hundred}\%$$

where L _max - the maximum amplitude of the release curve of adenosine triphosphate,
Ω _max - the maximum amplitude of platelet aggregation,
PCT - the proportion of whole blood volume occupied by platelets,
NTS - the proportion of blood volume attributable to the shaped elements.