Method of assessing risk of recurrent thrombotic events in patients with acute coronary syndrome

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.

1 ex, 1 tbl

 

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

The number of known ways to assess the risk of arterial thrombosis. Among them are the evaluation methods based on the determination of the number of erythrocytes 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 - Annex 1], the level of hematocrit H [Lowe G.D. Can haematological tests predict cardiovascular risk? Br. J. Haematol. 2006. Vol.133. PP. 239-240 - Annex 2].

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

There is a method of assessing the risk of thrombotic events in patients with cardiovascular disease, ischemic heart disease, diseases of the coronary and peripheral arteries. This method consists in the fact that taken blood samples add sodium citrate in the ratio of 9:1, put the sample into the receiver-analyzer instrument, run software measurement time of closure of the apertures of a special cassette, the which covered one of the inducers of platelet aggregation, get the desired value and interpret the results of the study patient. This method follows from the following domestic and foreign publications [Barkagan SS, Momot A.P. Diagnostics and controlled therapy of disorders of hemostasis. Moscow. 2001. - 43-44., Y.W. Chang, C.H. Liao, 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 - Annex 3;

Linnemann Century, Schwonberg J. Rechner A.R., H. Mani, 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 Century, Prochnow, S., H. Mani, 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. Application No. 4;

Christie D.J., Kottke-Marchant K., R.T. German 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 - Annex 5].

However, the method has drawbacks, which should include the use of citrate blood sample which is not subjected rekaltsifikatsii, which leads to non-physiological concentrations of calcium ions and distortion characteristics of coagulation link of the system of blood coagulation. Another disadvantage of the method described in all the above-mentioned publications, is that it is based on the use of the device, the principle of which is to study laminare is, and not turbulent blood flow characteristic of blood flow in the coronary arteries in patients with ACS. That is, reduces the accuracy and objectivity of the assessment of patients with ACS. In addition, the apparatus and consumables to him the road and not readily available for General use.

There is also known a method of estimating the risk of recurrence of thrombotic events, based on the analysis of biochemical parameters, such as inventor's certificate SU for the invention №1138122 priority from 05.02.1975, "a Method for predicting thromboembolic complications in patients with myocardial infarction". In this way determine the content of antiplasmin and urokinase activity of urine.

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

Also known "Method for diagnosis of incipient violations of the aggregation of platelets" [RU patent for the invention №2286580]. On a glass slide is applied to 0.02 ml of the standardized plasma of the subject and 0.02 ml of physiological inducers of aggregation, which are used ADP, collagen, thrombin and epinephrine. Determine the time aggregation for each inductor. Calculate the average time aggregation, when it is above the norm believe that the state of aggregation of platelets is in the nature of hemorragias the th thrombocytopathy, while values below normal - thrombophilic thrombocytopathy.

However, this method does not take into account indicators of coagulation hemostasis, which reduces the accuracy and objectivity of the evaluation of the patient, which is highly undesirable in the assessment of patients with ACS.

Also known "Method of determining the prognosis of unstable angina" [RU patent for the invention №2123700], which includes a multivariate discriminant analysis of thrombocyte stage of hemostasis and nature of the violations of the condition of the blood platelets. The prognosis of the disease is determined by the level of the three components contained in the platelets of the patient: cholesterol 64-76%, phosphatidylcholine 71-76%, sphingomyelin size 63-76%.

However, this method does not provide a specific estimate prognosis. The possibility of obtaining various parameters, keeping the values inside each of the above-mentioned patent No. 2133700 ranges and set as the number of combinations does not allow practitioners to determine what the Outlook is favorable or unfavorable is waiting for a specific patient, the subject in this way.

Also known "Method of predicting the course of acute myocardial infarction", [RU patent for the invention №2121691], including the study of biochemical parameters of blood of the patient in the dynamics and subsequent prediction of blagopriatnogo course of the disease. As the studied parameters using prothrombin index and the unfavorable course of the disease predict 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 recurrence of thrombotic events. This method is based on the study of 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 used device. That is, on the one hand, assessment is used exclusively coagulation component of the blood coagulation system and is not considered platelet mechanism of hemostasis. On the other hand, the specified parameter has a high variability and low reproducibility in different laboratories.

The closest analogue to the claimed invention is a Method of assessing the risk of adverse cardiac events in patients with acute coronary syndrome without ST-segment elevation" [RU patent for the invention №2312589], according to which the patient spend electrocardiographic study, angiographic examination and biochemical analysis of blood, and, given the presence of coronary artery stenosis more than 50% on the angiogram, the nalitch the e ST-segment depression in conjunction with a negative the T wave in the same lead ECG lead, the presence of arrhythmias, the level of troponin T in admission to the intensive care unit, determine the degree of risk of adverse coronary events in 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. require complex diagnostic procedures - koronaroangiografii, which is possible only in the presence of angiography and trained specialists, interventional cardiology, and expensive reagents and apparatus for determining the level of troponin, which limits the use of this method only highly specialized hospitals.

The task of the invention is to increase the objectivity of the method of estimating the risk of recurrence of thrombotic events in patients with ACS in its simplicity and lower cost estimates.

The essence of the invention is characterized by the fact that conducting a study of blood according to the following algorithm: taking venous blood and prepare samples of whole blood specimen, determine the level of hematocrit (N), the number of erythrocytes (E) and platelets (P), fix them, evaluate the values of the above indices; if they are placed in intervals ostanovivchish the values of these indicators for patients with ACS, prepare samples for the determination of time adenosintriphosphate-induced - ADP-induced coagulation (A) blood, which from whole blood prepared sample citrate blood in an amount of 0.4 ml and divide it into two samples of 0.2 ml, each of which contribute in a measuring cell, recalcification at 37°C for 2 minutes, then in each cell with the sample placed in a magnetic bead-beater, conduct the measurement is started and after three seconds injected 0.1 ml of ATP; after the formation of a clot record the time duration of the process for each sample separately, consider the arithmetic mean the obtained values ADP, scored the obtained values of each of these indicators:

Index1 point2 points3 points
A≥101 sec81-100 sec≤80 s
H45-48%--
E2,99-3·1012/l3,1-4,5·1012/sup> /l4,51-5,65·1012/l
P149-200·109/l201-449·109/l450-530·109/l
according to the total value of the indicators points∑=A+H+E+R
judge the risk of repeated thrombotic events: when 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. During the development of the proposed method of assessing the risk of recurrence of thrombotic events in patients with ACS due to study artificially simulated by the authors of the situation of the formation of a blood clot in the coronary arteries in vitro, the authors were able to find a parameter to evaluate the risk of recurrence of thrombotic events (ADP-induced clotting time of whole blood), which, according to the Applicant, up to the present time was not known. Unlike the currently used tools to assess the risk of recurrence of thrombotic events in similar clinical situations, this method is used throughout the world the following attributes technical solutions: the use of whole blood, standard coagulometer with magnetic bead-beater, ADP as the inductor of the blood clotting process. Until now, researchers used samples of blood plasma (platelet-poor), platelet or red blood cell suspension to determine the parameters of hemostasis, considered as markers of thrombosis. The use by the applicant of a sample of whole blood is the distinguishing feature of the invention, it is not obvious. ADP up to the present time actively used as the inducer of platelet aggregation techniques in the study of their functional activity. However, to initiate the processes of clot formation in whole blood in patients with ACS to study the risk of recurrence of thrombotic events ADP was not used. The use of magnetic bead-mixer coagulometer different types was due to the necessity of mixing for contact interaction of blood coagulation factors in aggregometry to create a certain shear stress on the surface of platelets that are necessary for their activation. While used in the inventive method, magnetic bead-stirrer provided modeling of turbulent flow of blood formed on the section of destabiliziruetsya plaques in the vessel of the patient with ACS. The advantages mentioned inventive techniques to more accurately with the given situation of thrombus formation in vitro. The combination of hematological parameters in conjunction with time ADP-induced blood coagulation has not been previously used to infer the degree of risk of blood clots. This is also the difference of the way. This ranking is called the set of four parameters selected by the severity of the changes each of them previously were not used because they were not known (revealed) before this application k intervals of values of the indicators developed and is typical for patients with ACS and included in the basis for conclusions on the assessment of the risk of recurrence of thrombotic events in this invention.

The way technically and technologically with the first manipulation until practically the last operation - processing of the data, since all manipulations: blood sampling, selection and preparation of samples, the effect of reagents on the sample, the incubation time, the order of steps in the present method are the signs of technically actions.

The inventive method is as follows. In patients with ACS in the first day of admission to hospital produce a sample of venous blood in two test tubes, 5 ml each. Blood from one tube is required to determine the default settings hematochrome: level of hematocrit, number of erythrocytes and platelet count any available staff by the way. D is I increase the speed it is desirable to use an automatic hematological analyzer. Record the measured values and evaluate the results of the three named parameters.

When evaluating the obtained values of H, E and R compare them with the corresponding values for the same characteristics obtained previously by the inventors as a result of research of all cardiac patients with ACS enrolled in the Saratov research Institute of cardiology. The inventors discovered as a result of long research, that for all these ACS patients without hematologic abnormalities, the values N, E, P are stacked in certain ranges of values; the hematocrit H 30-48%, erythrocytes E. 2,99-5,65·1012/l, platelets P 149-530·109/l If the patient with ACS values of H, E and R fall outside the established ranges of values, he should look for hematologic disease, such as anemia. This confirms the experience of research. Obtained by the authors for more information about the values N, E and P are new, important similar reference. Inside the ranges of values are established norm for patients with ACS. So, if the obtained values are plotted in intervals of current values for patients with ACS, the staff goes on to identify the last, the fourth parameter (If at least one of the first three options do not fit into the norm for this patient on the config method is not used and make him other blood tests to determine a more accurate perhaps adjusted diagnosis).

Then continue the way for those patients with ACS, to whom it is suitable. Whole blood from other tubes are stabilized by sodium citrate in the standard ratio of 9:1. Define the time of ADP-induced blood coagulation is as follows (the volume of blood sample, 0.4 ml of citrate blood; measuring time per patient ~5 minutes):

include warm up coagulometer in accordance with the instructions for its use;

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

divide the sample into two samples of 0.2 ml each;

make each measuring cuvette of 0.2 ml citrate whole blood;

then bring in a cuvette of 0.1 ml of the titrated solution l2;

include a timer to 120 seconds;

carry on 119th second cell in the measuring channel, 120-th second audio signal coagulometer placed in each of them a ball-mixer using the dispenser;

run measurements on 3-th second of which contribute to the cuvette of 0.1 ml, 0.2 mmol/l of a solution of ATP;

in the formation in a sample of blood coagulometer stops the timer and beeps;

record the time of the formation of a blood clot is telno for each sample;

calculate the arithmetic mean time ADP-induced clotting of blood for each patient, which is defined for the indicator for the end of the calculation;

put all four parameters, refer to the unit of measure in the score, and interpret the results.

If the ranking of parameters and calculation are given below.

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

The hematocrit (H): 45-48% - 1 point, 30-44%- 0 points,

The number of erythrocytes (E): 2,99-3·1012/l - 1 point. 3-4,5·1012/l - 2 points, 4,51-5,65·1012/l - 3 points.

The number of platelets (P): 450·109/l - 3 points, 201-449·109/l - 2 points, less than 200·109/l - 1 point.

For convenience and clarity, the calculation can use a table

Index0 points1 point2 points3 points
Time ADP-induced blood coagulation (A)-≥101 sec81-100 sec≤80 s
The hematocrit (H) 30-44%45-48%--
The number of erythrocytes (E)2,99-3·1012/l3,1-4,5·1012/l4,51-5,65·1012/l
The number of platelets (P)149-200·109/l201-449·109/l450-530·109/l

according to the total value of the indicators ∑=A+H+E+P in points judged on the risk of repeated thrombotic events: when the value of σ equal to 3-4 points - low risk; when ∑=5-6 points - average risk, when ∑=7-10 points - high risk.

In the ICU, Saratov research Institute of cardiology examined in accordance with the above-described method, the group of clinically healthy individuals and patients with acute coronary syndrome, including unstable angina, myocardial infarction, with the rise and without ST segment elevation. In patients with ACS test results were compared with the clinical outcome of the disease, death, complications, recurrence of the disease. Time ADP-induced blood coagulation in clinically healthy patients 120,81±45,57 seconds, for patients with ACS - 95,56±31,84 s is nd (differences are statistically significant: p level test Kruskall-Wallis equal 0,034). Application of the proposed method of assessing the risk of recurrence of thrombotic events for prediction of clinical outcome; the correct classification of the patient in 76.8% of cases. Clinical example: patient N., 54 years old, was admitted to the hospital with a diagnosis of CHD. Q-myocardial infarction of the anterior wall of the left ventricle, hypertension stage III. Time AFD induced coagulation 66 seconds, the number of platelets 293·109/l, the number of erythrocytes 5.2 x 1012/l, hematocrit 46%. The risk of recurrent thrombotic events is 9 points. Received standard therapy according to the recommendations of the Committee of experts of the all-Russian scientific society of cardiologists. After reaching a better state of the patient was discharged. Outpatient daily took the prescribed drugs. 20 days later, the patient was admitted with a diagnosis of Unstable angina. Conclusion about the risk of repeated thrombotic events were confirmed.

The above testifies to the adequacy of sufficient sensitivity and usefulness of the proposed method for objective assessment of the risk of recurrence of thrombotic events in patients with ACS.

Medico-social effect of the proposed method is more objective assessment of risk of recurrence of thrombotic events.

First proposed based on the study of the exact time AFD inducion the frame clotting whole nefraktsionirovannoi blood way of re-evaluation of thrombotic events in patients with ACS. In addition, the novelty of the claimed method is the use of specific these four indicators simultaneously in the same way to assess the risk of recurrence of thrombotic events in patients with ACS.

The economic effect of the proposed method in comparison with used, is to significantly reduce the costs of the research. For example, the average price of the cartridge PFA COLLAGEN/ADP CARTMG for ways analogues (see Appendix 1-5) on 20 measurements is 10817,3 rubles, that is, the cost only catridge to study for one patient is about 1081,73 rubles. While the cost of 1 bottle 5 ml of calcium chloride solution (0.025 M titrated solution l2) is 50 rubles, 1 bottle of ADP (ADP - adenosine diphosphate, lyophilized from 1.0 ml at a concentration of 0.2 mmol/l) - 110 rubles (NGOs "RENA"). The total cost of consumables for examination of a patient by the present method is 24 ruble. The cost of the research in a new way ten times lower only at the expense of consumables, not counting the expensive equipment.

The method allows prediction of the disease in the first hours of receipt of the patient in any medical institution, which has a laboratory equipped with coagulometer, a physician assistant which can handle data General analysis to the JVI and ADP-induced clotting time of blood.

The developed technology by the present method extends the Arsenal of tools to objectively monitor the condition of patients with ACS.

The method of estimating the risk of recurrence of thrombotic events in patients with acute coronary syndrome (ACS), characterized by the fact that conducting a study of blood, taking venous blood and prepare samples of whole blood specimen, determine the level of hematocrit (N), the number of erythrocytes (E) and platelets (P), fix them, evaluate the values of the above indices; if they are placed in the intervals of the steady-state values of these indicators for patients with ACS, prepare samples for the determination of time adenosintriphosphate-induced - ADP-induced blood coagulation (A), and from whole blood prepared sample citrate blood in an amount of 0.4 ml and divide it into two samples of 0.2 ml, each of which contribute in a measuring cell, recalcification at 37°C for 2 min, then in each cell with the sample placed in a magnetic bead-beater, conduct the measurement is started and after three seconds injected 0.1 ml of ATP; after the formation of a clot record the time duration of the process for each sample separately, consider the arithmetic mean of the obtained values And appreciate the points obtained values of each of these indicators:

Indicators1 point2 points3 points
And≥10181-100≤80
N45-48%--
E2,99-3·1012/l3,1-4,5·1012/l4,51-5,65·1012/l
P149-200·109/l201-449·109/l450-530·109/l

according to the total value of the indicators in the points Σ=A+H+E+P use to judge the risk of repeated thrombotic events when values: Σ=3-4 points - low risk, Σ=5-6 points - average risk, Σ=7-10 points - high risk.



 

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1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: venous blood filtration apparatus comprises a glass graduated cylinder 1 with an inlet 12 for venous blood supply and an outlet 13 for venous blood outflow, enclosed in a transparent plastic casing 2 with two tubes 3 and 4. The lower tube 4 is designed to deliver water heated to +36°C to the casing 2, while the upper tube 3 is used for air outlet from the casing 2. At the bottom of the glass graduated cylinder 1, there is a plastic grating 5 whereon a calibrated filter 7 of pore diametre equal to 5 mcm is fixed with a metal ring 6. The cylinder 1 accommodates an in-built piston 8 with a rod 9, and the piston has two channels 10 of different diametres on a junction of which there is a metal ball 11 for air discharge from the cylinder 1.

EFFECT: accelerated venous blood examination result with maintained precision of blood cancer cell detection, simplified designed of the related apparatus.

3 dwg

FIELD: veterinary science.

SUBSTANCE: method provides for blood test. In process of analysis quantity of platelets, fibrinogen and activated partial thromboplastin time. Conclusion on availability of liver failure is made when S<6.477, where S is determined using calculation formula.

EFFECT: method makes it possible to reliably diagnose liver failure in cows at early stage of disease origination.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: determined are degree of depression of consciousness, degree of depression of reflex activity and presence or absence of complications in the respiratory system. After that, total concentration of opioids in urine is determined by polarisation-immunofluorescent method and if its concenmtration is 15.84-19.04 mcg/ml, acute poisoning of light degree is diagnosed, if concentration is 19.05-38.40 mcg/ml, acute poisoning of medium degree is diagnosed, and if concentration is 38.41-57.70 mcg/ml - acute poisoning of severe degree is diagnosed.

EFFECT: increase of efficiency of diagnosing severity degree of poisoning.

3 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: erythrocytes are isolated, placed in aggregometre flask, lanthanum chloride is added to final concentration 400-600 mcM. Judgement about change in cytoskeleton condition is made basing on reduction of aggregation ability of erythrocytes.

EFFECT: method application makes it possible to determine condition of erythrocyte skeleton in a quick and simple way.

2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention relates to laboratory methods for blood analysis. Plasma is dropped in copper sulfate solution with density 1.023 g/cm3, not above, and time for drop falling on bottom of graduated cylinder with column height 243 mm is measured. The blood plasma density value is calculated by the formula:

wherein is the unknown blood plasma density (g/cm3); is copper sulfate solution density measured by areometer (g/cm3); t is average falling time of plasma drop in the copper sulfate solution (as seconds); 0.260130126 and 0.00290695 are correction coefficients. Temperature of plasma and copper sulfate solution is 20oC. Method is simple and suitable and allows carrying out analysis of small volumes of blood plasma and to reduce analysis time.

EFFECT: improved assay method.

2 ex

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