RU2419800C1 - Method of assessing risk of recurrent thrombotic events in patients with acute coronary syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: blood is examined. A hematocrit level (H), erythrocyte count (E), thrombocyte count (T) are determined. Said parametres are evaluated. In the event if they keep within the determined limits for the patients with acute coronary syndrome (ACS), then adenosine phosphate induced (ADP-induced) clotting time test samples are prepared. Citrated blood sample 0.4 ml is prepared of whole blood and divided on two samples 0.2 ml. Each of these samples is introduced in a measuring cell, recalcified at temperature 37°C for 2 minutes. Then a magnetic ball mixer is placed in each cell. The measurement is activated, and in three seconds the ADP solution 0.1 ml is introduced. After a clotting reaction, a process time duration is recorded separately for each sample. An arithmetical mean of the derived values is calculated (A). The derived values of each of said parameters are scored. Total score Σ=A+H+E+P shows the risk of recurrent thrombotic events. If Σ=4 points, the low risk is observed; the value Σ=5-6 points shows the medium risk, while Σ=7-10 points - the high risk.

EFFECT: method provides more objective risk evaluation of recurrent thrombotic events in the patients with ACS with its simplicity and low cost.

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Description

The invention relates to practical medicine, clinical laboratory diagnostics and experimental medicine. The method is designed to assess the thrombogenic risk of patients with acute coronary syndrome (ACS) and allows you to clarify the picture of the course and outcome of the disease - myocardial infarction with and without ST segment elevation, unstable angina, having a common pathogenesis, etc.

A number of methods for assessing the risk of arterial thrombosis are known. Among them, we can distinguish evaluation methods based on determining the number of red blood cells E [Sagesaka T. Influence of red blood cell concentration on the initiation time of blood coagulation: risk of thrombus formation by hemoconcentration. Clinical Hemorheology and Microcirculation. 2004. Vol. 31. PP 243-249 - Appendix No. 1], the level of hematocrit H [Lowe G.D. Can haematological tests predict cardiovascular risk? Br. J. Haematol. 2006. Vol. 133. PP 239-240 - Appendix No. 2].

However, an assessment of only one or two blood parameters cannot comprehensively show the picture of the patient's condition.

A known method for assessing the risk of thrombotic events in patients with cardiovascular diseases, with coronary heart disease, diseases of the coronary and peripheral arteries. This method consists in adding sodium citrate in a ratio of 9: 1 to the taken blood samples, placing the samples in the receiver-analyzer of the device, running programs for measuring the closing time of the apertures of a special cassette, which is coated with one of the platelet aggregation inducers, obtaining the desired values and interpreting according to them the results of the study of the patient. This method follows from a number of the following domestic and foreign publications [Barkagan Z.S., Momot A.P. Diagnosis and controlled therapy of hemostasis disorders. Moscow. 2001. - C 43-44., Chang Y.W., Liao C.H., Day Y.J. Platelet function analyzer (PFA-100) offers higher sensitivity and specificity than thromboelastography (TEG) in detection of platelet dysfunction. Acta Anaesthesiol Taiwan. 2009. Sep. Vol. 47 (3). PP 110-7 - Appendix No. 3;

Linnemann B., Schwonberg J., Rechner A.R., Mani H., Lindhoff-Last E. Assessment of clopidogrel non-response by the PFA-100 (R) system using the new test cartridge INNOVANCE (R) PFA P2Y. Ann Hematol. 2010. Jan 5., Linnemann B., Prochnow S., Mani H., Schwonberg J., Lindhoff-Last E. Variability of non-response to aspirin in patients with peripheral arterial occlusive disease during long-term follow-up. Ann Hematol. 2009. Oct. Vol. 88 (10). PP. 979-88. - Appendix No. 4;

Christie D.J., Kottke-Marchant K., German R.T. Hypersensitivity of platelets to adenosine diphosphate in patients with stable cardiovascular disease predicts major adverse events despite antiplatelet therapy. Platelets. 2008. Mar. Vol.19 (2). PP 104-10 - Appendix No. 5].

However, the method has disadvantages, which include the use of a citrated blood sample, which is not subjected to recalcification, which leads to a non-physiological concentration of calcium ions and a distortion of the characteristics of the coagulation unit of the thrombus formation system. Another disadvantage of the method described in all of the above publications is that it is based on the use of a device whose principle of operation is to study the laminar rather than turbulent blood flow characteristic of blood flow in the coronary arteries in patients with ACS. That is, the accuracy and objectivity of assessing the condition of a patient with ACS is reduced. In addition, the device and its consumables are expensive and inaccessible for widespread use.

There is also a method for assessing the risk of recurring thrombotic events, based on an analysis of biochemical parameters, for example, SU certificate for invention No. 1138122 with priority dated 02/05/1975, “A method for predicting thromboembolic complications in patients with myocardial infarction”. In this method, the content of antiplasmin and urokinase activity of urine is determined.

However, the method does not correspond to the modern level of development of medicine and has mainly historical significance.

Also known is a "Method for diagnosing incipient violations of platelet aggregation function" [RU patent on invention No. 2286580]. On a glass slide, 0.02 ml of standardized plasma of the examined and 0.02 ml of a solution of physiological aggregation inducers are applied, which are used as ADP, collagen, thrombin and adrenaline. The aggregation time for each inductor is determined. The average aggregation time is calculated, when it is above the norm, it is believed that the state of platelet aggregation function is hemorrhagic thrombocytopathy, and if it is below normal, it is thrombophilic thrombocytopathy.

However, this method does not take into account the indicators of the coagulation link of hemostasis, which reduces the objectivity and accuracy of assessing the patient's condition, which is extremely undesirable in assessing the condition for patients with ACS.

Also known is the "Method for determining the prognosis of unstable angina pectoris" [RU patent for the invention No. 2133700], including a multivariate discriminant analysis of the state of platelet link of the hemostatic system and the nature of violations of the state of the blood platelets. The prognosis of the disease is determined by the level of the three components contained in the patient’s platelets: cholesterol 64-76%, phosphatidylcholine 71-76%, sphingomyelin 63-76%.

However, this method does not provide a specific assessment of the forecast. The possibility of obtaining different parameters that fit into the values of each of the ranges mentioned in the patent No. 2133700 and the combination in the form of the number of combinations does not allow practical doctors to determine which prognosis is favorable or unfavorable for a particular patient being examined by this method.

Also known is a "Method for predicting the course of acute myocardial infarction", [RU patent for the invention No. 2121691], including the study of biochemical parameters of the patient’s blood in dynamics with subsequent 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, only the coagulation link of the blood coagulation system is used and the platelet mechanism of hemostasis is not taken into account. On the other hand, this parameter has high variability and low reproducibility in the conditions of various laboratories.

The closest analogue to the claimed invention is a "Method for assessing the risk of adverse cardiac events in patients with acute coronary syndrome without ST segment elevation" [RU patent for invention No. 2312589], according to which the patient undergoes an electrocardiographic study, angiographic study and biochemical blood test, and , taking into account the presence of coronary artery stenosis of more than 50% on the angiogram, the presence of depression of the ST segment in combination with a negative T wave in one lead of the ECG lead , The presence of cardiac arrhythmias, troponin T levels on admission to the intensive care unit, determine the degree of risk of adverse coronary events according to a mathematical formula.

The main disadvantage of this method is the inability to assess the risk of adverse events in patients with ACS with myocardial infarction with ST segment elevation. In addition, the method is invasive, i.e. requires a complex diagnostic procedure - coronary angiography, which is possible only with an angiograph and trained specialists in invasive cardiology, as well as expensive reagents and apparatus for determining the level of troponin, which limits the use of this method only to highly specialized hospitals.

The objective of the invention is to increase the objectivity of the method for assessing the risk of repeated thrombotic events in patients with ACS with its simplicity and with a decrease in the cost of evaluation.

The essence of the invention is characterized by the fact that they conduct a blood test according to the following algorithm: venous blood is sampled and whole blood samples are prepared, the hematocrit (H) level, the number of red blood cells (E) and platelets (P) are determined, they are fixed, and the values of these indicators are evaluated ; in cases where they fit into the intervals of steady-state values of these indicators for patients with ACS, samples are prepared to determine the time of adenosine phosphate-induced - ADP-induced coagulation (A) of blood, for which a sample of citrated blood in the amount of 0.4 ml is prepared from whole blood and divide it into two 0.2 ml samples, each of which is introduced into the measuring cell, recalcified at 37 ° C for 2 minutes, then a magnetic stirring ball is placed in each sample cell, the measurement is started and after three seconds s inject 0.1 ml of ADP; after the formation of a clot, the time of the process duration for each sample is recorded separately, the arithmetic mean of the obtained ADP values is considered, and the obtained values of each of the indicated indicators are evaluated in points:

Indicator 1 point 2 points 3 points A ≥101 sec 81-100 sec ≤80 sec H 45-48% - - E 2,99-3 · 10 ¹² / l 3.1-4.5 · 10 ¹² / l 4.51-5.65 · 10 ¹² / l P 149-20010 ⁹ / l 201-44910 ⁹ / L 450-53010 ⁹ / L by the total value of indicators in points бал = A + H + E + P judge the risk of recurring thrombotic events: at values ∑ = 3-4 points - low risk,
∑ = 5-6 points - average risk,
∑ = 7-10 points - high risk.

The technical result of the claimed invention and the differences of the proposed method. When developing the proposed method for assessing the risk of recurring thrombotic events in patients with ACS by studying the in vitro formation of a thrombus in a coronary artery artificially simulated by the authors, the authors were able to select a parameter for assessing the risk of recurring thrombotic events (ADP-induced whole blood clotting time), which, according to the Applicant , until now was not known. Unlike currently used tools for assessing the risk of recurring thrombotic events in similar clinical situations, this method uses the following attributes of a technical solution: the use of whole blood, a standard coagulometer with a magnetic stirring ball, and ADP as an inducer of the blood coagulation process. Until now, researchers have used plasma samples (platelet-poor), platelet or erythrocyte suspension to determine the parameters of the hemostatic system, considered as markers of thrombosis. The applicant's use of a whole blood sample is a feature of the invention, it is not obvious. ADP is still actively used as an inducer of platelet aggregation in methods for studying their functional activity. However, to initiate the processes of thrombosis in whole blood in patients with ACS in order to study the risk of repeated thrombotic events, ADP was not used. The use of a magnetic stirring ball in various types of coagulometers was due to the need for mixing for contact interaction of blood coagulation factors in aggregometers to create a certain shear stress on the platelet surface necessary for their activation. While used in the claimed method, a magnetic stirrer ball provided modeling of turbulent blood flows generated on the site of a destabilizing plaque in the patient's vessel with ACS. The advantages of the claimed inventive methods more accurately create a situation of thrombosis in vitro. The combination of hematological parameters together with the time of ADP-induced blood coagulation was not previously used to conclude the degree of risk of thrombosis. This is also the difference between the method. A similar ranking of the named set of four selected parameters by the degree of severity of changes in each of them was also not previously used, since k intervals of values of indicators that have developed and are characteristic for patients with ACS and which form the basis of the conclusions about the assessment were not known (were not identified) prior to this application the risk of recurring thrombotic events in this invention.

The method 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 present method are signs of a technically feasible action.

The inventive method is as follows. In patients with ACS, on the first day of admission to the hospital, a venous blood sample is taken in two test tubes of 5 ml each. Blood from a single tube is required to determine the standard parameters of the hematogram: hematocrit level, red blood cell count and platelet count in any way that is available to staff. To increase the speed, it is desirable to use an automatic hematological analyzer. The obtained values are recorded and the obtained results of the three named parameters are evaluated.

When assessing the obtained values of H, E and P, they are compared with the corresponding values of the same characteristics obtained earlier by the inventors as a result of the study of all cardiological patients with ACS who entered the Saratov Research Institute of Cardiology. The inventors found, as a result of lengthy studies, that for all these patients with ACS who do not have hematological pathologies, the values of H, E, P fit into well-defined ranges of values; hematocrit H 30-48%, red blood cells E 2.99-5.65 · 10 ¹² / l, platelets P 149-530 · 10 ⁹ / l. If in a patient with ACS the values of H, E and P fall outside the established ranges of values, he should look for a hematological disease, for example, anemia. This confirms the research experience. The additional information obtained by the authors about the values of H, E and P is new, significant, similar to the reference. Values within the ranges are the established norm for patients with ACS. So, if the obtained parameter values fit into the intervals of prevailing values for patients with ACS, the staff proceeds to determine the last, fourth, parameter (If, however, at least one of the first three parameters does not fit the norm, this method is not used for this patient , and they do other blood tests for him to establish a more accurate, possibly corrected diagnosis).

Then continue the method for those patients with ACS, to whom it is suitable. Whole blood from another tube is stabilized with sodium citrate in a standard 9: 1 ratio. The determination of the time of ADP induced blood coagulation is carried out as follows (blood sample volume 0.4 ml of citrated blood; measurement time per patient ~ 5 minutes):

turn on and warm up the coagulometer in accordance with the instructions for its use;

prepare and warm the used reagents to 37 ° C in the appropriate cells of the coagulometer;

divide a blood sample into two samples of 0.2 ml each;

0.2 ml of citrated whole blood is added to each measuring cell;

then add 0.1 ml of titrated CaCl ₂ solution to the cuvette;

turn on the timer for 120 seconds;

at the 119th second, the cuvette is transferred to the measuring channel; at the 120th second, according to the sound signal of the coagulometer, a stirring ball is placed in each of them using a dispenser;

start the measurement, at the 3rd second of which 0.1 ml of 0.2 mmol / L ADP solution is introduced into the cuvettes;

when a clot forms in the sample, the coagulometer stops the timer and emits an audible signal;

fix the time of formation of a blood clot separately for each sample;

calculate the arithmetic average time of ADP-induced blood coagulation for each patient, which is taken as a determined indicator for the final calculation;

add up all four parameters reduced to the unit of measurement in points and interpret the results.

At the same time, the ranking of the parameters and the calculation are carried out taking into account the following.

Time ADP-induced blood coagulation (A): less than 80 seconds - 3 points, 81 - 100 seconds - 2 points, 101 seconds and above - 1 point;

Hematocrit (N): 45-48% - 1 point, 30-44% - 0 points,

The number of red blood cells (E): 2,99-3 · 10 ¹² / l - 1 point. 3-4.5 · 10 ¹² / l - 2 points, 4.51-5.65 · 10 ¹² / l - 3 points.

The number of platelets (R): more than 450 · 10 ⁹ / l - 3 points, 201-449 · 10 ⁹ / l - 2 points, less than 200 · 10 ⁹ / l - 1 point.

For convenience and clarity of calculation, you can use the table

Indicator 0 points 1 point 2 points 3 points ADP induced coagulation time (A) - ≥101 sec 81-100 sec ≤80 sec Hematocrit (N) 30-44% 45-48% - - The number of red blood cells (E) 2,99-3 · 10 ¹² / l 3.1-4.5 · 10 ¹² / l 4.51-5.65 · 10 ¹² / l Platelet Count (P) 149-20010 ⁹ / l 201-44910 ⁹ / L 450-53010 ⁹ / L

by the total value of the indicators ∑ = A + H + E + P in points, the risk of repeated thrombotic events is judged: with a value of рав equal to 3-4 points - low risk; with ∑ = 5-6 points - medium risk, with ∑ = 7-10 points - high risk.

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 time of ADP-induced blood coagulation in clinically healthy patients is 120.81 ± 45.57 seconds, for patients with ACS - 95.56 ± 31.84 seconds (the differences are statistically significant: the p level of the Kruskell-Wallis test is 0.034). The use of the proposed method for assessing the risk of recurring thrombotic events to predict clinical outcomes; correct patient classification in 76.8% of cases. Clinical example: patient N., 54 years old, was admitted to the hospital with a diagnosis of coronary heart disease. Q-myocardial infarction of the anterior wall of the left ventricle, arterial hypertension stage III. The AFD time of induced blood coagulation is 66 seconds, the platelet count is 293 · 10 ⁹ / L, the number of red blood cells is 5.2 · 10 ¹² / L, and the hematocrit is 46%. The risk of recurring thrombotic events is 9 points. Standard therapy was carried out according to the recommendations of the Committee of Experts of the All-Russian Scientific Society of Cardiology. After achieving improvement, the patient was discharged. I took prescribed medications on an outpatient basis daily. After 20 days, the patient was admitted with a diagnosis of unstable angina. The conclusion about the risk of recurring thrombotic events was confirmed.

The above indicates the adequacy, sufficient sensitivity and usefulness of the proposed method for an objective assessment of the risk of repeated thrombotic events in patients with ACS.

The medical and social effect of the proposed method consists in a more objective assessment of the risk of repeated thrombotic events.

For the first time, a method for evaluating recurring thrombotic events in patients with ACS, based on the study of the exact time of AFD-induced coagulation of whole nefraciopyirovoy blood, has been proposed. In addition, the novelty of the claimed method is the use of specific four of these indicators simultaneously in one way of assessing the risk of repeated thrombotic events in patients with ACS.

The economic effect of the proposed method, compared with used, is a significant reduction in research costs. For example, the average price of a PFA COLLAGEN / ADP CARTMG cartridge for analogue methods (see Appendix 1-5) for 20 measurements is 10,817.3 rubles, that is, the cost of only a cartridge for research for one patient is about 1081.73 rubles. While the cost of 1 bottle of 5 ml of calcium chloride (0.025 M titrated solution of CaCl ₂ ) is 50 rubles, 1 bottle of ADP (ADP - adenosine diphosphate, freeze-dried from 1.0 ml at a concentration of 0.2 mmol / l) - 110 rubles (NGO "RENAM"). The total cost of consumables for examining one patient by the present method is 24 rubles. The cost of research in a new way is tens of times lower only due to consumables, not including expensive equipment.

The method makes it possible to predict the course of the disease in the first hours of the patient’s admission to any medical institution where there is a laboratory equipped with a coagulometer, the laboratory doctor of which can process the data of a general blood test and ADP-induced blood coagulation time.

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 assessing the risk of repeated thrombotic events in patients with acute coronary syndrome (ACS), characterized in that they conduct a blood test, collect venous blood and prepare samples of whole blood, determine the level of hematocrit (H), the number of red blood cells (E) and platelets (P ), fix them, evaluate the values of these indicators; in cases where they fit into the intervals of steady-state values of these indicators for patients with ACS, samples are prepared to determine the time of adenosine phosphate-induced - ADP-induced blood coagulation (A), for which a citrate blood sample of 0.4 ml is prepared from whole blood and divide it into two samples of 0.2 ml, each of which is introduced into the measuring cell, recalcified at 37 ° C for 2 minutes, then a magnetic stirring ball is placed in each sample cell, the measurement is started and after three seconds lead, 0.1 ml of ADP solution; after the formation of a clot, the time of the process duration for each sample is recorded separately, the arithmetic average of the obtained values of A is considered, the obtained values of each of the mentioned indicators are evaluated in points:
Indicators 1 point 2 points 3 points BUT ≥101 s 81-100 s ≤80 s N 45-48% - - E 2,99-3 · 10 ¹² / l 3.1-4.5 · 10 ¹² / l 4.51-5.65 · 10 ¹² / l R 149-20010 ⁹ / l 201-44910 ⁹ / L 450-53010 ⁹ / L
by the total value of indicators in points Σ = A + H + E + P, the risk of repeated thrombotic events is judged - with values: Σ = 3-4 points - low risk, Σ = 5-6 points - medium risk, Σ = 7-10 points - high risk.