Method of determining platelet resistance to acetylsalicylic acid

FIELD: chemistry.

SUBSTANCE: invention relates to method of determining platelet resistance to acetylsalicylic acid (ASA) by impedance analysis of aggregation function of platelets in vivo, in which aggregation activity is analyses after incubation of biological material sample with ASA with application of aggregation inducer, and aggregation of platelets is induced by collagen in optimal concentration 2 mg/ml, and simultaneously with measurement of impedance determination of dynamics of platelet granules release is performed by luminescent method, where before carrying out aggregation samples are calibrated by means of adenosine triphosphate (ATP) standard, value of aggregation amplitude are determined in Ohms by obtained aggregatograms and obtained values are given points: values ≤6 correspond to 0 points, values 7-9 correspond to 1 point, values 10-12 correspond to 2 points, values >12 correspond to 3 points; after that, intensity of ATP release from platelet granules is determined in nmoles and obtained values are given points: values <0.5 correspond to 0 points, values 0.5-1.0 correspond to 1 point, values 1.0-1.5 correspond to 2 points, values 1.5 correspond to 3 points, and then resistance index (RI) is calculated by formula, value of IR parameter points to presence of aspirin-resistance of platelets.

EFFECT: carrying out fast complex diagnostics of platelet resistance to ASA with estimation of functional state of platelet granules before beginning treatment and possibility of preventing development of undesirable thrombotic or hemorrhagic complications.

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The invention relates to the problem of clinical and preventive personalized medicine - definition individual characteristics reaction platelet drugs, namely preventive methods in vitro diagnostics resistance of platelets to acetylsalicylic acid (ASA).

In connection with certain preventive effectiveness for cardiovascular diseases and affordability ASC is the most commonly prescribed antiplatelet drug [Volkov, C. I., rabuka centuries, Zapravlena O. E., fine A. I. Diagnosis of resistance to aspirin in patients with ischemic heart disease. RBM. Cardiol. zhurn., January 6, 2006 [online]. Found in the Internet: <URL: http://rq1.net.ua/cardio_j/2006/3/IMAG306/volkkov.gif>]. However, it is known that to achieve adequate reduction of platelet aggregation and prevent re-thrombosis and the occurrence of cardiovascular events not all patients taking ASA [Ashikhmin, J. I. the Phenomenon of resistance to the antithrombotic action of aspirin and ways to overcome it / I. I. Ashikhmin O. M. Drapkin // Russian medical news, 2008. - So 13. No. 2]. According to some authors, this phenomenon is called "resistance" to aspirin, distributed among the population with a frequency of from 5 to 48% [Hankey, G. Aspirin resistance / G. Hankey, J. Eikelboom // Lancet, 2006 - Vol.367]. About the Noah of the most important issues occurs when you attempt in any way to measure (evaluate) the degree of inhibition of platelet function after exposure to pharmacological agents is that none of the existing laboratory tests does not include the evaluation of all the versatility of platelet function [Bugaenko, Century Century Modern principles of antiplatelet therapy in patients with ischemic heart disease. Part 2. Tolerability and possible side effects / centuries Bugaenko // heart journal, 2009. No. 5]. This in vitro diagnostic efficiency ASC does not evaluate mechanisms to reduce the function of platelets, which may be due to defects such an important structural and functional component of platelets as granules [Vorobyev A. I. Manual of Hematology / A. I. Vorobiev. - M.: Medicine, 1985]. Impaired functioning of these specific organelles of platelets is the pathogenetic basis of a number of diseases in which the receiving ASA is contraindicated. It is known that in certain pathological conditions of blood there is inadequate response of platelets to acetylsalicylic acid, expressed in occurrence of hemorrhage [Avram, S., Lupu, A. Abnormalities of platelet aggregation in chronic myeloproliferative disorders / S. Avram, A. Lupu // J. Cell. Mol. Med., 2001 - Vol 5]. Use ASC causes an increased risk of gastrointestinal bleeding and hemorrhage what is a mini stroke [J. He, Whelton, P. K. Aspirin and risk of haemorrhagic stroke: a meta-analysis of randomized controlled trials // JAMA. 1998. Vol.280. P. 1930-1935]. In this regard, it is highly important to develop diagnostic tests conducted before administration of the drug by the patient to forecast individual reactivity of platelets to the action of the ASC, including the assessment of secretory activity.

There is a method of definition of aspirin resistance by measurement of light transmission in induced platelet aggregation in blood plasma [RF Patent №2413953C1, G01N 33/86 (2006.01). Publ. 10.03.2011. Bull. No. 7]. The method allows to quantify the amplitude of the aggregation process in vitro. To study the antiaggregatory properties in the test tube with platelet-rich plasma was pre-made solution of ASC and incubated. Then made an inductor - ADP and recorded the amplitude of the aggregation. The results were compared with the parameters of the aggregation caused by the inductor without prior exposure of the ASC with the calculation of the coefficient of inhibition of aggregation (KIA).

Diagnostic value of this method is limited to the obvious drawbacks: 1) the duration, complexity and lack of standardization of the process of preparing platelet-rich plasma; 2) during centrifugation of the blood platelets are exposed to significant mechanical stress to the e significantly alters their functional viability; 3) at the stage of sample preparation disturb the natural environment of platelets, which does not take into account the effects of other blood cells, modulation of platelet function; 4) increases the likelihood of a significant reduction in the concentration of labile mediators to the time of measurement. All of this allows us to create optimal standard conditions necessary to ensure the full realization of the functions of platelets after stimulation of the inductor. A significant drawback of optical aggregatometry is the inability to examine the blood of patients with severe hyperlipidemia, common in cardiology practice. In addition, the above measurement method skips 67% of patients with a defect in the accumulation of granules and the threat of hemorrhagic complications during therapy [Research society Clinical hemostaseology". Diagnostics [online]. Found in the Internet: <URL:http://www.hemostas.EN/society/diagnostics/ecomeds/index.shtml>].

Known another method for diagnosis of aspirin resistance, which is the study of the functional activity of platelets before and after incubation of the specimen with acetylsalicylic acid, while aggregation activity of platelets is determined in whole blood impedance method to calculate the inhibitory capacity of ASC in percent [atent of the Russian Federation No. 2379684 C2, G01N 33/48 (2006.01). Publ. 20.01.2010. (prototype)]. This method allows to predict the resistance to the ASC before therapy, but does not provide information about the state of the granular unit of platelets, which are important for their functioning in the hemostatic system.

The technical result of the invention consists in carrying out a quick, comprehensive diagnosis of resistance of platelets to aspirin with the assessment of the functional status of granules of platelets prior to treatment and possible prevention of adverse thrombotic or hemorrhagic complications.

The technical result is achieved in that in the method for determining the resistance of platelets to acetylsalicylic acid (ASA) by impedance studies of the aggregation of platelets in vitro, which investigate aggregation activity after incubation of the specimen with an ASC using aggregation inductor new is that the aggregation of platelets induce collagen in optimal concentration of 2 mg/ml and simultaneously with the measurement of impedance spend the determination of release granules of platelets by the luminescence method, where prior to the aggregation of the samples are calibrated using standard adenosine triphosphate (ATP), on the obtained agregatogramme determine the values of the amplitude and is regali in ohms and assign the obtained values of the points: values < 6 correspond to 0 points, the values of 7-9 correspond to 1 point, the values of 10-12 correspond to 2 points, the values of >12 correspond to 3 points, then determines the rate of ATP release from granules of platelets in nolah and assign the obtained values of the points: values <0,5 0 correspond to points, values of 0.5-1.0 correspond to 1 point, the values of 1.0 to 1.5 corresponds to 2 points, the values of >1.5 to correspond to 3 points, and then calculate the index resistivity (ER) by the formula:

IL=Band+Bl,

where Bandand Bl- the degree of sensitivity to aspirin points in the impedance and fluorescence tests, respectively, with the value of the index IL more than 4 indicates the presence of userinitiated platelets.

The proposed method differs from the prototype in that the definition of aspirin resistance is carried out in vitro by studying the functional activity of platelets only after incubation of the specimen with an ASC, while aggregation activity of platelets and their granules is determined in whole blood complex impedance-fluorescent method that allows to obtain additional diagnostic parameter is the degree of influence of ASA on the release of the contents of platelet granules and allows for a more objective approach to the assessment of individual sensitivity is lnasty patient to the drug ASC.

Thus, the above distinctive features of the prototype characteristics allow to draw a conclusion on the conformity of the proposed technical solution the criterion of "novelty". The features distinguishing the claimed technical solution to the prototype, not identified in other technical solutions and, therefore, provide the claimed solution according to the criterion of "inventive step".

The invention is illustrated using graphic materials:

In Fig. 1 shows curves of collagen-induced aggregation and release curves granules of platelets in whole blood up to (1-2) and after (3-4) incubation of samples with ASA for individuals who are sensitive to aspirin.

In Fig. 2 presents curves of collagen-induced aggregation and release curves granules of platelets in whole blood up to (1-2) and after (3-4) incubation of samples with ASA for the individual, resistant to aspirin (1 - the original curve aggregation, 2 - the original curve of the intensity of the release pellets, 3 - curve aggregation after incubation with ASA, 4 - curve intensity release pellets after incubation of blood samples with ASA).

The method is as follows.

Blood samples obtained from the cubital vein and stabilize a 3.8% solution of sodium citrate in a volume ratio of 9:1. During the study blood remain in closed plastic probescope room temperature in accordance with accepted safety precautions when working with blood cells.

Analysis of the functional activity of platelets is carried out not earlier than 15 minutes and no later than 3 hours after receipt of the blood using a Lumi-aggregometer at 37°C and stirring the contents of a ditch with the mixer rotation speed of 1000 rpm, the Ratio of the volume of blood samples and the inductor is 100:1.

Prior to the aggregation of fluorescent method samples are calibrated using standard ATP. When this is set individually for the patient gain (in %) response of luminescence. Further registers the intensity of the release of ADP from granules of platelets (in nmol) with simultaneous recording of impedance parameters induced platelet aggregation in the same cell.

To induce aggregation, use a solution of collagen at a final concentration of 2.0 mg/ml, shipped ready to use.

Study of the functional activity of platelets each patient in the sample after pre-incubation of blood with ASA.

The incubation is conducted for 15 minutes at 37°C. in a pilot sample of whole blood add 40 ál 3,36 mm solution of ASC.

Processing agregators is generally characteristic points of the curves of aggregation to determine the maximum amplitude of rise of impedance in ohms to 5 minutes from the bearing into the measuring cell of the inductor - collagen and determination of the luminescence intensity when ATP release from granules of platelets in nolah. Identifying the source of low release granules may indicate pathology of platelets and contraindications to the appointment of the ASC. In addition to routine evaluation settings agregatogramme calculated the resistance index (IR). This parameter represents the amount of points characterizing the degree of sensitivity of the patient impedance test and fluorescent test for the maximum amplitudes of aggregation and release of granules, and is calculated by the formula: IL=Band+Blwhere Bandand Bl- the degree of sensitivity to aspirin points in the impedance and fluorescence test, respectively. This parameter determines the final degree of individual sensitivity to aspirin and includes a comprehensive assessment of the inhibitory effect of ASA on the accumulation of ATP, the intensity of the release granules, the dynamics of the membrane potential of platelets and agregation-adhesive. IL allows you to determine the individual sensitivity of the patient to ask in absolute units of the scale that can serve as a criterion to identify individuals with high and low response to the drug. This will allow you to assign therapy with aspirin, a reasonable timely is th predictive laboratory evaluation, and to avoid undesirable consequences of inadequate treatment.

Examples of the obtained agregators with individual assessment of aspirin resistance is shown in Fig. 1-2.

The degree of resistance is determined in accordance with the proposed scale:

Table 1 presents the results of a survey of 13 clinically healthy people, conducted on the basis of the Krasnoyarsk branch of the fgbi Hematological scientific center of the Russian Ministry of health. The proposed method was determined by the magnitude of the IL. In samples of ten patients, the value of this indicator was below 4. Three people according to the test results IL was equal to or more than 4.

Because healthy people who didn't ask, is the IR source without conducting incubation with the ACU were equal to or greater than 4, the level values of IL was adopted as the criterion of resistance. After the patient was admitted per os single dose ASA in the amount of 125 mg/day, the next day the newly defined metrics aggregation in a sample of fresh blood without pre-incubations. Three patients revealed by the results of preliminary testing with incubation in vitro IL was again more than 4, as before taking the drug. This confirms that the proposed solution allows to identify patients with resistance to ant is the platelet effect of aspirin. In contrast, among individuals with indicator IL for less than 4, a single receiving 125 mg ASA caused a significant inhibition of indicators aggregation, indicating their sensitivity to this drug.

The result shows a high diagnostic capabilities of the proposed method and the clinical usefulness of preventive determine antithrombotic actions ASC in vitro complex impedance-luminescent technique.

td align="center"> 6
Table 1
The results of the research degree of resistance to aspirin
№ p/pIn the test, IN VITROIn the test, IN VIVOForecast
The amplitude, ΩRelease granules, nmolBandBlILIL
120,3000sensitive
240,66011sensitive
340,64011sensitive
460,92011sensitive
570,34101sensitive
690,69112sensitive
761,08/td> 022sensitive
8100,921121sensitive
9100,751121sensitive
1061,7033sensitive
11160,583146resistant
12130,73314resistant
13162,243366resistant

The method for determining the resistance of platelets to acetylsalicylic acid (ASA) by impedance studies of the aggregation of platelets in vitro, which investigate aggregation activity after incubation of the specimen with an ASC using aggregation inductor, characterized in that the aggregation of platelets induce collagen in optimal concentration of 2 mg/ml and simultaneously with the measurement of impedance spend the determination of release granules of platelets by the luminescence method, where prior to the aggregation of the samples are calibrated using standard adenosine triphosphate (ATP), resulting agregatogramme determine the amplitude value aggregation in ohms and assign the obtained values of the points: values ≤6 correspond to 0 points, the values of 7-9 correspond to 1 point, the values of 10-12 correspond to 2 points, the values of >12 correspond to 3 points, then determines the rate of ATP release from granules of platelets in nolah, and assign the obtained values of the Alla: value < 0,5 0 correspond to points, values of 0.5-1.0 correspond to 1 point, the values of 1.0 to 1.5 corresponds to 2 points, the values of >1.5 to correspond to 3 points, and then calculate the index resistivity (ER) by the formula:
IL=Band+Bl,
where Bandand Bl- the degree of sensitivity to aspirin points in the impedance and fluorescence tests, respectively, with the value of the index IL more than 4 indicates the presence of userinitiated platelets.



 

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