RU2530584C2 - Method for testing anticoagulant activity of blood - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to testing the anticoagulant activity of blood. Substance of the method consists in performing low-frequency piezothromboelastography and measuring blood viscosity characteristics: A0 is an initial blood state of aggregate, A1 is a value describing the maximum change of the blood state of aggregate at the stage of contact activation, A3 is a value describing the blood state of aggregate at the stage of the beginning of the polymerization process, t3 is the time to reach A3, A4 is a value describing the blood state of aggregate 10 min after the A3 value is achieved, t4 is always 10 minutes, as well as a clot polymerisation intensity (CPI); a coefficient of the anticoagulant activity of blood (AAB) is calculated by formula. If the AAB value falls within the range of 1.5-2, the normal anticoagulant activity is stated, while the AAB values less than 1.5 shows the decreased anticoagulant activity.

EFFECT: using the method enables monitoring the anticoagulant activity of blood both at the stage of diagnosing, and at any stage of therapy of haemostatic disorders with high accuracy and reliability by carrying out the integrated assessment of the anticoagulant activity of blood.

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Description

The invention relates to medicine and can be used to determine anticoagulant activity of blood.

The problem of assessing the state of anticoagulant blood activity is relevant, since the mechanisms underlying high vascular mortality in a number of serious diseases associated with endothelial damage remain unclear. Violations of hemostasis are observed at all stages of blood coagulation. An increase in blood coagulability due to depression of the anticoagulant link of the hemostatic system due to insufficient laboratory diagnostics, as a rule, remains unrecognized, and therefore, in the absence of timely correction, it can have a fatal outcome. It is well known that timely and adequate correction of the hemostasis system, including its anticoagulant component, prevents discirculatory disorders in vital organs. Therefore, the theme of the development is to propose a method for determining anticoagulant blood activity based on indicators of a low-frequency piezothromboelastogram (NPTG) of a patient.

If the vessel is damaged, blood should coagulate only at the site of damage. This is ensured by the anticoagulation system available in the blood. She, under normal conditions, plays a leading role in maintaining the liquid state of the blood. There are two types of anticoagulants: direct-acting anticoagulants (pre-existing) - self-synthesized (heparin, antithrombin III - AT-III, protein C, protein S, α ₂ -macroglobulin); indirect anticoagulants formed during blood coagulation, fibrinolysis and activation of other proteolytic systems (fibrin-antithrombin I, antithrombin IV, inhibitors of factors VIII, IX, etc.) [4].

Of all preexisting anticoagulants, the most versatile in action and activity of antithrombin III (AT-III). This is one of the α ₂ plasma globulins. Its concentration in blood plasma is about 240 mg / ml, and it makes up 75% of its anticoagulant activity. It inactivates thrombin and almost all other activated factors - XIIa, X1a, Xa, IXa. Another anticoagulant - heparin - is active only together with AT-III. Heparin promotes the fixation of AT-III on the surface of endothelial cells, which increases its activity by a factor of hundreds. In the liver, in the presence of vitamin K, protein C and protein S are synthesized. They reduce the activity of factors V and VIII. Endothelial cells of an intact vascular wall not only inhibit platelet adhesion, but also synthesize prostacyclin and heparin-like factor (the latter is also synthesized by mast cells of the connective tissue). Prostacyclin is a potent inhibitor of platelet aggregation. In the process of blood coagulation and fibrinolysis, many procoagulants and their metabolites acquire anticoagulant properties. The most powerful of them is fibrin, which adsorbs and inactivates the thrombin formed during coagulation, which also exhibits anticoagulant properties. Acting enzymatically on prothrombin, it cleaves the factor Xa inhibitor from the latter [1,2,5].

Closest to the proposed is a method of measuring the initial stages of blood coagulation using a low-frequency piezoelectric transducer using such blood parameters as:

A0 is the initial indicator of the state of aggregation of blood,

A1 is an indicator characterizing the maximum change in the state of aggregation of the test blood at the stage of contact activation,

A3 is an indicator characterizing the state of aggregation of blood at the beginning of the polymerization process,

t3 - time to reach A3, which are used in the calculation

coagulation drive intensity - an indicator characterizing the integrative effect of pro- and anticoagulation systems, which has the following form ICD = (A3-A0) / t3,

A4 - an indicator characterizing the state of aggregation of blood 10 minutes after reaching a value of A3,

t4 is always 10 minutes.

The intensity of bunch polymerization was determined as tgβ = (A4-A3) / (t4 is an indicator characterizing the rate of connection of monomer molecules) [3]. The disadvantage of this method is the lack of accuracy and information

A new technical result is an increase in the accuracy and information content of the method. To solve the problem in a method for determining the anticoagulant activity of blood, which consists in conducting low-frequency piezotromboelastography and measuring the viscosity characteristics of blood: A0 is the initial indicator of the state of aggregation of blood, A1 is an indicator characterizing the maximum change in the state of aggregation of the blood under study at the stage of contact activation, A3 is an indicator, characterizing the state of aggregation of blood at the beginning of the polymerization process, t3 - time to reach A3, A4 - indicator, characterization present state of aggregation of blood after 10 minutes after reaching the value A3, the indicator t4, always of 10 minutes, and the intensity of certain polymerization clot as tgβ = (A4-A3) / t4, blood anticoagulation activity coefficient KPA is calculated by the formula:

KPA = ICD / IPS, where

the ICD equation (coagulation drive intensity) has the form ICD = (A3-A1) / t3, and when the KPA value is within 1.5-2, normal anticoagulant activity is determined, and for values less than 1.5, the decrease in anticoagulant activity of blood is determined.

The method is as follows: Under standardization of the sampling process (with a 5 ml silicone syringe without applying a tourniquet from a cubital vein), 0.5 ml of whole unstabilized venous blood is taken for examination and placed in a cell of the ARP-01M Mednord device for 5-10 seconds "(Registration certificate No. ФСР 2010/09767 dated December 30, 2010). The study time is 60 ± 10 minutes. By the method of low-frequency piezotromboelastography using a hardware-software complex for clinical diagnostic studies of the rheological properties of blood ARP-01M Mednord, the following indicators are evaluated:

A0 is the initial indicator of the state of aggregation of blood (p.u.).

A1 is an indicator characterizing the maximum change in the state of aggregation of the test blood at the stage of contact activation (pu).

A3 is an indicator characterizing the state of aggregation of blood at the stage of the beginning of the polymerization process (pu).

t3 - time to reach A3 (min).

ICD - (A3-A1) / t3 — coagulation drive intensity — an indicator characterizing the integrative effect of pro- and anticoagulant systems on the clot formation rate (pu).

A4 is an indicator characterizing the state of aggregation of blood 10 minutes after reaching the value of A3 (min).

t4 = 10 min.

IPA is the intensity of bunch polymerization, defined as tgβ = (A4-A3) / (t4 is an indicator characterizing the rate of connection of monomer molecules).

Calculate the coefficient of anticoagulant activity of the CPA by the formula:

KPA = ICD / IPS

and when the KPA value is from 1.5 to 2, the norm is determined; when the KPA values are less than 1.5, a decrease in the anticoagulant activity of blood is determined.

The proposed method is based on the analysis of clinical trials in patients with preeclampsia against the background of correction of changes in the hemostatic system with direct anticoagulants. After signing the informed consent, in 15 patients with preeclampsia using low-frequency piezothromboelastography using a hardware-software complex for clinical diagnostic studies of blood rheological properties ARP-01M Mednord (Registration certificate No. ФСР 2010/09767 dated December 30, 2010). The complex is based on the principle of low-frequency piezometry and allows to study the rheological properties of whole blood, monitor and record the most minor changes in its state of aggregation, calculate the amplitude and chronometric constants, determine the intensity of the processes characterizing the main stages of blood coagulation, and obtain the results of standard rheological characteristics in the form graphs and tables of indicators characterizing the main stages of blood coagulation were determined with the following blood indicators (units of amplitude indicators are relative units (pu), time units are minutes):

"A0" is the initial indicator of the state of aggregation of blood.

“T0” is the start time of the study.

“A1” is an indicator characterizing the maximum change in the state of aggregation of the test blood at the stage of contact activation.

"T1" is an indicator reflecting the time to reach A1 (min).

“T2” is the time during which the amplitude A increases by 100 units.

"ICC" - the intensity of the contact phase of coagulation, is calculated empirically, by the ratio A0-A1 / t1.

"KTA" = (100 / t2-t1) is the thrombin activity constant, defined as the time elapsed from the end of the reaction period to an increase in the amplitude values by 100 relative units from the level of the minimum amplitude value for the reaction period.

“A3” is an indicator characterizing the state of aggregation of blood at the stage of the beginning of the polymerization process.

“T3” is the time to reach “A3”.

“ICD” - (A3-A1) / t3 — coagulation drive intensity — an indicator characterizing the integrative effect of pro- and anticoagulant systems on the rate of clot formation.

“A4” is an indicator characterizing the state of aggregation of blood 10 minutes after reaching the value “A3”.

“T4” is always 10 minutes.

“IPS” is the intensity of clot polymerization, defined as tgβ = (A4-A3) / t4 - an indicator that characterizes the rate of connection of monomeric molecules “side-by-side”, “end-to-end”, forming a fibrin network.

“A5” is an indicator reflecting the state of aggregation of blood in the final, stabilization stage of thrombus formation.

“T” is the time of clot formation (constant of total blood coagulation time).

"ITS" - the intensity of total blood coagulation, reflects the density of the fibrin-platelet structure of the clot. Empirically determined (A5-A1) / (T5-T1).

"IRLS" - the intensity of retraction and lysis of the clot. An indicator of spontaneous lysis of a clot.

When analyzing the obtained data (see Fig. 1) and knowing that the inclusion of the anticoagulation system in the coagulation cascade is delayed, the following indicators were chosen to determine the anticoagulation activity: "ICD" - the intensity of the coagulation drive - an indicator characterizing the integrative effect of and anticoagulant systems on the rate of clot formation; "IPS" - the intensity of the polymerization of the clot - an indicator that characterizes the speed of connection of monomeric molecules "side-by-side", "end-to-end", forming a network of fibrin (as the result of the action of coagulation and anticoagulation systems).

The coefficient of anticoagulant activity KPA = ICD / IPA was calculated, which reflects the total anticoagulant activity before and 6 hours after subcutaneous administration of 0.6 ml of fraksiparin. Statistical processing was performed on a personal computer using the SPSS 13.0 for Windows program with calculation of the median, lower and upper quartiles (Me [LQ; UQ]). The critical significance level p was taken equal to 0.05.

Indicators of a low-frequency piezothromboelastogram (NPTG) before and after administration of the drug were as follows (table 1, figure 1). After the introduction of the drug, aimed at increasing the anticoagulant activity of the blood, CPA statistically significantly (p <0.05) increased from 1.13 [1.07; 2.90] to 6.66 [4.13; 7.41], which suggests that the proposed method can be used with sufficient accuracy and reliability to determine the anticoagulant activity of blood

Example 1 (extract from the medical history): Patient P., 29 years old, gestational age 33 weeks was admitted to OGAUZ "Regional Perinatal Center" with complaints of an increase in edema within 3 weeks, an increase in blood pressure to 180/100 mm Hg The analysis showed moderate anemia, hypoproteinemia, hypercoagulation (IPI = 147%, Fibrinogen O. 5.82 g / l, APTT 30.2 s, AT III 79%, plasminogen 87%, D-dimer 6.92 mg / dL) . Under the conditions of standardization of the sampling process (with a 5 ml siliconized syringe without applying the tourniquet from the cubital vein), 0.5 ml of whole unstabilized venous blood was taken for examination and placed into the cuvette of the ARP-01M Mednord device for 5-10 seconds. Using indicators of the obtained NPTG ICD = 36.41 and IPA = 26.42, we calculated KPA = 36.41 / 26.42 = 1.37, which indicated the inhibition of anticoagulation activity, although laboratory determination of individual indicators of this system did not show a decrease below normal values (AT III 79%, plasminogen 87%). The patient was prescribed a direct anticoagulant fraxiparin 0.6 ml subcutaneously 1 time per day. Control tests the next day did not show significant changes in laboratory tests characterizing the individual components of the anticoagulation system (AT III 81%, plasminogen 86%), but the coefficient of anticoagulation activity, characterizing the total activity of the anticoagulation system, increased by more than 3 times (KPA = 5, 26). This allowed us to say that the application of the proposed method allowed us to prescribe adequate and timely therapy for the revealed condition with unfractionated heparin.

Example 2 (extract from the medical history): Patient B., 23 years old, gestational age 25 weeks was admitted to OGAUZ "Regional Perinatal Center" with complaints of drawing pains in the lower abdomen and scanty spotting from the genital tract. In the analyzes, hypoproteinemia, normocoagulation (IPI = 97%, Fibrinogen O. 3.92 g / l, APTT 35.7 s, AT III 92%, plasminogen 89%, D-dimer 4.9 mg / dL) were noted. Under the conditions of standardization of the sampling process (with a 5 ml siliconized syringe without applying the tourniquet from the cubital vein), 0.5 ml of whole unstabilized venous blood was taken for examination and placed into the cuvette of the ARP-01M Mednord device for 5-10 seconds. Using indicators of the obtained NPTG ICD = 49.21 and IPA = 26.42, we calculated KPA = 45.21 / 23.37 = 1.94, which indicated normal anticoagulant activity, as in laboratory tests (AT III 92%, plasminogen 89%). The patient was hospitalized with the threat of premature birth and was successfully treated with this condition by genipral. She did not need to correct hemostasis, during the hospitalization there were no thrombotic or hemorrhagic complications.

Thus, the clinical observations allow us to conclude that the proposed method makes it possible to diagnose changes in the anticoagulation system and carry out laboratory monitoring of the effectiveness of the therapy, which will undoubtedly reduce the frequency and severity of complications, including direct anticoagulants, which, as you know, requires careful monitoring.

Information sources

1. Barkagan Z.S. Diagnosis and controlled therapy of hemostasis disorders [Text]: monograph / Z.S. Barkagan, A.P. Momot. - M.: Newdiamed, 2008 .-- 292 p.

2. Barkagan Z.S. About new approaches to the monitoring of antithrombotic agents [Text] / Z. S. Barkagan, E.F. Kotovschikova, A.N. Shilova // Clinical Pharmacology and Therapy. - 2005. - T. 14, No. 3. - S. 51-53.

3. Tyutrin I.I. et al. Instrumental methods for assessing the functional state of the blood aggregate state regulation system (PACK) using low-frequency vibration piezoelectric hemocoagulography (NPHC) // Materials of the first international conference “Diagnosis, treatment and prevention of thrombosis and thromboembolism. Tomsk, 2011, S.11-17 (prototype).

4. Kishkun A.A. Clinical laboratory diagnostics: a training manual [Text] / A.A. Kishkun. - M .: GEOTAR-Media, 2010 .-- 976 p.

5. Momot A.P. Pathology of hemostasis. Principles and algorithms of clinical laboratory diagnostics [Text]: monograph. - SPb .: Form T, 2006. - 208 p.

6. Diagnosis, treatment and prevention of thrombosis and thromboembolism [Text]: collection of articles / Ed. Kairova G.T. - Tomsk, 2011 .-- 130 s.

Appendix: Table 1

Key indicators of NPTG before and after the administration of fraxiparin, Me [LQ; UQ], n = 15

Figure 1 Integrated NPTG of patients with preeclampsia (n = 15) before and after administration of fraxiparin.

Table 1 The method for determining anticoagulant activity of blood Indicator BEFORE after A0 164.00 [107.00; 291.00] 143.00 [102.00; 163.00] A1 116.00 [72.00; 181.00] 122.00 [94.00; 254.00] T1 1.20 [0.60; 1.40] * 3.10 [2.60; 3.90] KIC -123.33 [-130.00; -7.14] * -13.29 [-43.33; -10.00] A2 201.00 [168.00; 281.00] 163.00 [103.00; 243.00] T2 3.10 [2.90; 3.90] * 8.70 [7.60; 10.00] KTA 45.45 [32.26; 47.62] * 24.39 [16.03; 20.63] A3 387.00 [211.00; 534.00] 273.00 [149.00; 294.00] tk 5.40 [4.90; 7.30] * 19.90 [18.50; 21.70] ikd 49.81 [47.46; 72.80] * 22.66 [44.81; 66.29] A4 622.00 [586.00; 724.00] * 307.00 [371.00; 562.00] T4 15.40 [14.90; 17.30] * 29.90 [24.50; 32.70] KPA 1.13 [1.07; 2.90] * 6.66 [4.13; 7.41] IPS 21.70 [15.80; 21.70] * 3.40 [2.90; 7.90] A5 713.00 [692.00; 844.00] * 333.00 [388.00; 562.00] T5 31.00 [30.60; 43.70] 39.90 [26.70; 42.60] MA 597.00 [559.00; 702.00] * 331.00 [411.00; 538,001 ITS 19.26 [14.89; 20.24] 19.52 [16.15; 21.24] A6 722.00 [674.00; 836.00] 344.00 [384.00; 760.00] T6 41.00 [40.20; 51.20] 53.20 [44.70; 58.60] IRLS -1.51 [-1.89; 1.09] -0.15 [-0.17; 1.05] Note: * - p <0.05 - statistically significant differences before and after the administration of fraxiparin.

Claims (1)

A method for determining the anticoagulant activity of blood, which consists in conducting low-frequency piezotromboelastography and measuring the viscosity characteristics of blood: A0 is the initial indicator of the state of aggregation of blood, A1 is an indicator characterizing the maximum change in the state of blood of an investigated blood at the stage of contact activation, A3 is an indicator of the state of blood stage of the beginning of the polymerization process, t3 - time to reach A3, A4 - indicator characterizing the state of aggregation of blood 10 minutes after reaching a value of A3, an indicator of t4, always equal to 10 minutes, as well as the intensity of the polymerization of a clot (IPA), defined as tgβ = (A4-A3) / t4, characterized in that the coefficient of anticoagulant activity of blood KPA is calculated by the formula:
KPA = ICD / IPS, where
the ICD equation (coagulation drive intensity) has the form ICD = (A3-A1) / t3, and with a KPA value of 1.5-2, normal anticoagulant activity is determined, and for values less than 1.5, a decrease in anticoagulant activity of blood is determined.

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