SU1508169A1 - Method of determining activity of antithrombin-iii in blood plasma - Google Patents

Abstract

The invention relates to medicine, in particular, to hemocoagulation, and can be used in the diagnosis of disorders of hemostasis. The aim of the invention is to improve the accuracy of the method. For this, the plasma to be tested is mixed with a stabilizer. Then, platelets are precipitated from it by centrifugation. Platelet-depleted plasma is defibrinated by heating to 56 ° C in a water bath. Then, the test plasma is added to a solution of thrombin heated in a water bath at 37 ° C for 2 minutes, the mixture is incubated. Fibrinogen is heated and a mixture of thrombin and plasma is introduced into it, immediately noting the time of the start of the reaction, the time for the formation of a fibrin clot is determined, and a sample is additionally carried out with the addition of 0.01-0.02 ml of 1% protamine sulfate solution or 1.0- 1.5 mg per 1 ml of a mixture of polybrene. A calibration curve is constructed for both samples, and the antithrombin-3 activity is calculated taking into account the additional sample data. The method allows to more accurately determine the activity of antithrombin-3, in addition, it is simple and reproducible in any biochemical laboratory.

Description

j

(20 A231374 / 28-14

(22) 04/17/87

(46) .89. Bul Number 34

(71) Moscow Medical Dental Institute. ON. Semashko

(72) V.N. Aleksandrov, G.N. Bud Kova, IG Bobrinsk, T. And. Taranova

and lf Karmolin

(53) .07 (088.8)

(56) Hensen A., Loeliger E.A. Anti-thrombin III: Coagulation. Stuttgart, 1963. In Abildgaard U.5 Gravem K., Godal H. Thromoos, Diathes, Haemorrh., -1970, v 24, N 1/2, p. 224.

(54) METHOD FOR DETERMINING THE ACTIVITY OF ANTI-THROMBIN -111 IN BLOOD PLASMA

(57) The invention relates to medicine, in particular to hemocoagulation, and can be used in the diagnosis of hemostasis. The aim of the invention is to improve the accuracy of the method. For this study

the plasma is mixed with a stabilizer. Then, platelets are precipitated from it by centrifugation. Platelet-depleted plasma is defibrinated by heating to a water bath. Then, the test plasma is added to a solution of thrombin heated in a water bath at 37 ° C for 2 min, and the mixture is incubated. Fibrinogen is heated and a mixture of thrombin and plasma is introduced into it, immediately noting the time of the start of the reaction, the time of formation of a fibrin clot is determined, and a sample is additionally carried out with the addition of 0.01-0.02 ml of 1% protaminesulfate solution or 1.0- 1.5 mg per 1 ml of a mixture of polybrene. A calibration curve is constructed for both samples and the antithrombin-111 activity is calculated taking into account the additional sample data. The method allows to more accurately determine the activity of antithrombin-111, in addition, it is simple and reproducible in any biochemical laboratory.

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The invention relates to medicine, namely to hemocoagulation, and can be used in clinical diagnostic and research laboratories x in the diagnosis of disorders of hemostasis.

The purpose of the invention is to improve the accuracy of the method by conducting an additional sample with the addition of protamine sulfate or polybrene to the defibrinated platelet-poor plasma and determining the time of formation

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clot in the presence of thrombin and fibrinogen (to represent the activity of antithrombin-111 as a percentage, a calibration curve is constructed).

The method is carried out as follows.

The investigated plasma is mixed with a stabilizer in the usual ratio. Then platelets are precipitated from it by centrifugation. The plasma depleted with thromboocytes is defibrinated. De31508

fibrination is performed by heating to a water bath.

Solutions for the standard mixture: a solution of thrombin with an activity of 15-16 seconds (i.e. in an amount of ml, capable of reducing -0.3 mp of a 0.4% solution of commercial fibrinogen); fibrinogen solution 0,; 4 in Michaelis buffer, pH 7s8,

The course of determination.

Sample I. The thrombin solution is incubated at 37 ° C and the test plasma is added to it. The mixture is then transferred with B solution-fibrinogen; warmed up to 37 ° C, the formation time of the fi phy rin in seconds is noticed.

Sample II. The same mixture of thrombin with the plasma under study is introduced into a solution of fibrinogen and a solution of npOTai-ssulfate or polybrene is added. The time of fibrin clot formation in seconds is noted.

A calibration curve was constructed for the activity of antithrombin-III and the rate of formation of a fibrin clot in a mixture of thrombin – donor plasma fibrinogen, and the donor plasma is analyzed as whole and assessed by 2.4 VS 8 times, respectively, taking the activity of antktrombin-111 as 1008 25 and 12; 5%. An additional calibration curve is added; protamysulfate or polybrene is added to all dilutions. These curves are odd als; 1Y and in the exact preparation of the buffer may not fail,

Estrapoliru from the time of formation of a clot in the first sample by the percentage of activity in the first curve, and the time of formation of a clot in the second sample by the percentage of activity in the second curve. Indicators of the second sample - the activity of antithrombin-111,

Example 1, Patient D., 43 years old, was diagnosed with a head injury, took blood (the first 0.5 ml was drained) and mixed it in a plastic tube with a 358% solution of sodium citrate in a ratio of ... 9: 1. Centrifuged at 1500 rpm for 5 min. Sucked platelet-rich plasma and re-centrifuged at 4000 rpm for 20 minutes. The platelet-poor plasma was sucked away and heated in a water bath with (this parameter is strictly observed) for 5 minutes. Centrifuged

10 min at 4000 rpm The plasma supernatant was aspirated for analysis.

The course of determination.

Sample 1, A plastic tube was collected 0.8 sh rast. Jurassic tiombin, heated in a water bath at 37 ° C for 2 min, and 0.2 1-40 plasma was added to it. Incubated at for 3 min. 0.3 fibrinogen solution was heated for 1 min and a mixture of thrombin and plasma was added to it immediately noting the time of the onset of the reaction. Determined that the formation of cryciKa fibrin was 75 s. According to the calibration curve, antigronbin activities were found - 11 1 0%.

Sample 2. In a plastic tube, 0.8 ml of the thrombinj solution was added and heated in a water bath for 2 minutes and O ,, 2 ml was added to it. test plasma mixed with OjOl ml of 1% com: a mercury solution of protamine sulfate and incubated at 37 ° C for 1 min. Incubated at 37 s for 3 min. 0s3 ml of fibrinogen solution was heated for 1 M-IH at 37 ° C and added to it a mixture of thrombin to plasma immediately marking the time; According to the kaish calibration curve: and the activity of T1-Trom6in-T II ant1 with protamine sulphate - 63%, with M; that the clotting time was 42 seconds. Sample 3. A plastic tube was taken up with 0.8 ml of thrombin solution, heated in a water bath at 37 ° C for 2 minutes, and 0.2 1-1L of the test plasma, previously mixed with polybrep, was added in a ratio of i mg per ml and incubated at 37 ° C for 1 min. Inkubirov, PI at 37 ° C for 3. 0.3 ml of fibrinogen solution was heated for 1 min at 37 ° C and a mixture of thrombin and plasma was added to it, immediately noting the time of the onset of the reaction. The fibrin clot formation time was determined to 41 seconds. According to the calibration curve, the antithrombin 111 activity with a polybamy was found - 63%,

To extrapolate the time of fibrin clot formation in antithrombin-III activity, a calibration curve is constructed,

For the time being, the time of formation of a fibrin clot in a thrombin mixture is determined - a complete mixture of dasm 10 health

out donor - fibrinogen: 60 s. In the same way, the clotting time of a clot in a mixture of thrombin, plasma of donors diluted 2 times, fibrinogen is determined: 35 seconds, then the time of clot formation in a mixture5 of donor containing gshasmu, diluted 4 times, 27 seconds, and the mixture of donor plasmas is diluted 8 times and determine the time of formation of the NIN clot - 23 s. Build a curve of fibrin clot formation time versus antithrombin-111 activity, conventionally taking the activity of antithrombin-III in a whole mixture of donor plasmas for 100%, diluted 2 times - 50%, - 4 times - 25% , 8 times - for.

Thus, the determination of the activity of antithrombin-III according to the prototype method (sample 1) showed the result (110%), At the same time, the severity of the patient's condition, the presence of DIC-syndrome accompanied by hypercoagulation of blood and consumption of antithrombin -Sh - anti-coagulation factor, objectively indicated a decrease in the activity of antithrombin-III.

The analysis of the proposed method (samples 2 and 3) showed that the activity of antithrombin-III in this patient is reduced to 63%. Adding protamine sulfate or polybrene to the samples allowed suppressing the activity of endogenous heparin (whose presence slowed down the rate of formation of a fibrin clot due to the formation of fibrinogen complexes — heparin or thrombin — heparin, and consequently, overestimated the calculated antithrombin-111 activity) the actual value of the activity of antithrombin-111.

Example 2. Patient H., 32 years old, took blood (the first 0.5 ml was drained) and mixed it in a plastic tube with 3.8% Hbw sodium citrate solution in a 9: 1 ratio. Centrifuged at 1500 rpm for 5 min. Sucked platelet-rich plasma and again centrifuged at 4000 rpm for 20 minutes. The platelet-poor plasma was sucked away and heated in a water bath at 56 ° C (this parameter should be strictly observed) for 5 minutes. Centrifuged for 1 min at 4000 rpm. The plasma supernatant was aspirated for analysis.

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The course of determination.

Sample 1. A 0.8 ml solution of thrombin was collected into a plastic tube. heated in a water bath at 37 ° C for 2 minutes and 0.2 ml of the plasma under investigation was added to it. Incubated at 37 ° C for 3 min. 0.3 ml of a solution of fibrinogen was heated for 1 min at 37 ° C and a mixture of thrombin and plasma was introduced into it, immediately noting the time at which the reaction began. The fibrin clot formation time was determined to be 81 s. The antithrombin-111 activity was found by the calibration curve - 142%.

Sample 2. 0.8 ml of the thrombin solution was collected in a plastic tube, heated in a water bath at 37 ° C for 2 minutes, and 0.2 ml of the test plasma, previously mixed with protamine sulfate, was added to it in an amount of 0.02 ml 1% a commercial solution and incubated at 37 ° C for min. Incubated at 37 ° C for 3 min. 0.3 ml of a solution of fibrinogen was heated for 1 min at 37 ° C and a mixture of thrombin and plasma was introduced into it, immediately noting the time at which the reaction began. The fibrin clot formation time was determined to 37 seconds. The calibration curve found the activity of antithrombin-111 with protamine sulfate - 41%.

Sample 3. A plastic tube was filled with 0.8 ml of thrombin solution, heated in a water bath at 37 ° C for 2 minutes, and 0.2 ml of the test plasma, previously mixed with polybrene, was added to it in a ratio of 1.5 mg i ml and incubated at 37 ° C for 1 min. Incubated at 37 ° C for 3 min. 0.3 ml of a solution of fibrinogen was heated for 1 min at 37 ° C and a mixture of thrombin and plasma was introduced into it, immediately noting the time at which the reaction began. The fibrin clot formation time was determined to 36 seconds. The calibration curve found the activity of antithrombin-111 with polybrene - 41%.

Thus, when determining the activity of antithrombin-111 according to the method of the prototype, the activity value was 110% in example 1, 142% in example 2, which was falsely overestimated due to the presence of elevated concentrations of endogenous heparin in the plasma of patients plasma heparin was confirmed by Soulier, 1959). .

The definition of the proposed method showed that the total antithrombin activity in Example 1 was 110%, the activity of antithrombin 111 itself was 63%.

In Example 2, the total antithrombin activity was 142%, the activity of antithrombin-111 itself was A1%. These indicators correspond to the severity of the studied resuscitation patients.

Clearly traced1 .:::;: shots falsely overestimated test results in the determination by the method prototype. When determining the proposed method, it is possible to simultaneously simultaneously determine the activity of the actual antithrombin-111o.

Example 3: The course of determination is the same as in Example 2. But 0.03 ml of 1% protamine sulfate solution was added to sample 2. Fibrin clot was not formed. In sample 3, 2 mg of polybrene was added. Fibrin clot not formed,

Thus, an increase in the dose of protamine sulfate or polybrene more than proposed is an overdose, and no fibrin clot is formed. The dose of 1 ml sulfate less than 0.0 ml or polybrene less than 1 mg in the sample does not completely inactivate the action of heparin and gives falsely high figures for the activity of the product under investigation.

Editor M. Kelemesh

Compiled by T. Kovaleva

Tehred M. Morgental Proofreader A. Obruchar

Order 5536/48

Circulation 789

 State Committee for Inventions and Discoveries under the State Committee on Science and Technology of the USSR I 13035s Moscow; Zh-355 Raushsk nab. D. 4/5

The invention allows to increase the accuracy of determining the activity of antithrombin-111 compared with the prototype (see table).

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As can be seen from the table, the error (-m) is ± 20% more than ± 3.1% 9 and the variance of sigma (b) is 8.9 more than K8 according to the prototype method than according to the application of the corresponding method, Therefore, the proposed method is more precise than the prototype method.

The method is simple and reproducible B of any biochemical laboratory.

Claims (1)

Invention Formula The method of determining plasma antithrombin-111 activity by incubating thrombin with platelet-poor plasma-defibrated plasma of adding the mixture to fibrinogen, determining the formation of a clot and calculating antithrombin-111 activity, characterized in that 5 to improve the accuracy of the method, additionally T sample with the addition of 0.01-0.02 ml per 1% solution of protamine sulfate or J, 0-15 5 mg per i ml of a mixture of polybrene j and the calculation of the activity of antithrombin-III is carried out taking into account the data of this sample. Subscription