RU2366955C2 - Method for determination of fibrin monomer self-assembly time - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly laboratory diagnostics. There is disclosed method for determination of fibrin monomer self-assembly time. The method can be used for blood analysis in disturbed haemostasis system. It involves determination of coagulation time with thrombin with urea added in concentration 180 g/l. Thrombin applied therefor is characterised with activity 4-6 seconds. The offered method allows estimating both delayed and accelerated fibrin monomer self-assembly in relevant typical clinical situations.

EFFECT: higher accuracy of determination fibrin monomer self-assembly time.

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Description

The invention relates to medicine, in particular to laboratory diagnostics, and can be used for blood testing for disorders in the hemostatic system.

The blood coagulation system is characterized as a cascade of enzymatic reactions with the transformation at the final stage of fibrinogen coagulation under the influence of thrombin into fibrin. The final stage of coagulation includes a number of enzymatic and non-enzymatic reactions. At first, thrombin cleaves two peptides A and two peptides B from the α and β chains of fibrinogen. The molecular weight of each of these peptides is about 4 kDa. The remaining main molecule with mol. weighing about 300 kDa (fibrin monomer) when interacting with similar fibrin monomers forms a fibrin dimer, then a tetramer, octamer, etc., resulting in the formation of a large-molecular complex, referred to as “soluble fibrin” or “soluble fibrin-monomer complexes ". Having reached a certain critical mass (about 12000-15000 kDa), they coagulate, turning into fibers and clots of fibrin. This fibrin is unstable and readily soluble in 5M urea or 1% acetic acid, but it is immediately additionally fixed by a fibrin-stabilizing factor (factor XIIIa), which forms disulfide bonds between the γ chains of fibrin monomers.

Elongation of time at the final stage of coagulation, associated with defects in the separation of fibrinopeptides A and / or B from fibrinogen and the interaction of fibrin monomers with each other (self-assembly of fibrin monomer), are characteristic of congenital or acquired dysphibrinogenemia, in which hemorrhagic syndrome is often observed. On the other hand, the acceleration of the self-assembly time of the fibrin monomer may be one of the causes of the hypercoagulation syndrome leading to intravascular coagulation.

A known method for determining activated partial thromboplastin time (APTT), based on measuring the coagulation time of human blood plasma during its recalcification under standardized contact and phospholipid coagulation activation (Barkagan ZS, Momot AP Diagnosis and controlled therapy of hemostasis disorders. Edition 2nd supplemented. - M.: “Newdiamed”, 2001. - P.79-81).

The disadvantage of this method is the effect on APTT of deficiency of coagulation factors (XII, XI, X, IX, VIII, V, II), the presence of their inhibitors and the presence in the blood plasma of direct anticoagulants (heparin, etc.).

In addition, the known method is slightly sensitive to violations of the self-assembly of fibrin monomer and is rarely indicative in this regard.

A known method for determining the duration of fibrin self-assembly in plasma, including treating fibrinogen with thrombin directly in the plasma in the presence of urea, adding a buffer with low ionic strength and subsequent determination of fibrin self-assembly time (copyright certificate SU 1210094, published 07.02.1986, abstract and description of the invention )

The fraction of the duration of fibrin self-assembly in the total duration of fibrin formation is determined.

Reagents

1. Urea, 40% in 0.02M acetate buffer with a pH of 5.2

2. A solution of thrombin 0.1%, 1% in 0.14 M sodium chloride

3. Borate buffer 0.0075M, pH 7.6

Material for research. Citrated blood plasma.

The course of determination. 0.2 ml of the test plasma add 0.2 ml of a 40% urea solution and 0.2 ml of a 0.1% thrombin solution. Mix and leave in a water bath at + 37 ° C for 10 minutes.

After exposure, 0.1 ml of the mixture is transferred to a test tube where 0.3 ml of borate buffer is pre-poured. At the time of application, the stopwatch is turned on, turning it off when a clot appears, recording the duration of fibrin self-assembly. Stopwatch reading, e.g. 16.3 s.

0.3 ml of borate buffer is added to the test tube in a water bath at + 37 ° C, 0.033 ml of a 40% solution of urea and 0.033 ml of the test plasma are added, everything is mixed. 0.033 ml of a 1% thrombin solution is added to the mixture, the stopwatch is immediately turned on, and the moment of clot formation is noted, recording the total duration of fibrin formation. Stopwatch readings, e.g. 58.7 s.

Reading the results. Calculation of the fraction of self-assembly in the total duration of fibrin formation in the studied plasma: (16.3: 58.7) × 100 = 27.7%.

The disadvantages of the method for determining the duration of self-assembly of fibrin in plasma include:

1. The inability to use thrombin, a standard activity.

2. The inability to conduct coagulometric studies.

3. The complexity and duration of the test.

4. Low accuracy of the method.

The inventive method is devoid of these disadvantages: the test is easily performed on coagulometers of various designs using standard thrombin activity, has higher accuracy in assessing both the slowdown and shortening of the self-assembly time in the plasma of patients with various types of hemostasis disorders.

The authors have developed a highly effective and high-precision method for determining the self-assembly time of a fibrin monomer, which allows one to determine both the elongation and shortening of the self-assembly time of a fibrin monomer.

The technical result of the proposed method is to increase the accuracy of determining the time of self-assembly of fibrin monomer.

The technical result is achieved by the fact that urea is introduced into the reaction mixture at a concentration of 150-250 g / l and thrombin with an activity of 40-60 NIH / ml or 4-6 s is used.

The method is as follows.

Reagents and equipment:

1. Sodium citrate, trisubstituted, 3.8% solution, obtained by dissolving 3.8 g of sodium citrate in 100 ml of distilled water.

2. Human thrombin (freeze-dried), 500 NIH, Technologiya-Standart, Russia.

3. Buffer Tris-Hcl concentrated (20: 1), 1.0 M, 5 ml, pH 7.3-7.4, Tekhnologiya-Standard, Russia.

4. Urea, osch, firm "Merck", Germany.

5. Physiological (0.9%) sodium chloride solution.

6. Distilled water.

7. Centrifuge OPN-8.

8. Coagulometer Minilab-701 firm Unimed, Russia.

9. Glass tubes with a volume of 20 ml.

10. The cylinder is glass, measured, on 100 ml.

Reagent preparation.

1. Thrombin dilution: 10.0 ml of physiological sodium chloride solution is added to the bottle with freeze-dried thrombin and, with gentle swaying, the reagent is dissolved within 5 minutes.

2. Preparation of a working buffer solution containing urea: a concentrated buffer in a volume of 2.0 ml and a portion of urea (18 g) are introduced into a graduated cylinder and the total volume is adjusted with distilled water to 100 ml. The result is a working buffer solution of the following composition - Tris-HCl 0.01 M, pH 7.3-7.4, containing urea at a concentration of 180 g / L.

Preparation of the studied plasma of the patient and the pool of the control normal blood plasma (taken from healthy people).

Blood is obtained from the cubital vein by gravity through a needle into a plastic tube containing a 0.1 M sodium citrate solution (blood to citrate ratio 9: 1). Blood is centrifuged for 5 min at a rotation speed of 1000 rpm./min (200g). The resulting platelet-rich plasma is re-centrifuged for 20 minutes at 4000 rpm. (1200g). The resulting platelet-poor plasma is used for the study.

To prepare a pool of control normal plasma, samples of a platelet-poor plasma from 8-10 healthy people are mixed in equal proportions.

Definition Progress

0.1 ml of the studied plasma is introduced into the coagulometer cuvette and incubated in a temperature-controlled cell for 1 min at + 37 ° C. Then, 0.05 ml of working buffer solution containing urea at a concentration of 180 g / l was added and incubated there at + 37 ° C for 1 min. The cuvette is transferred to the measuring channel, then 0.05 ml of thrombin solution is added, the measuring channel is immediately launched and the clotting time is recorded at the final stage.

Evaluation of the results. According to the results of a survey of healthy people (42 observations), the coagulation time in the final stage is 24.5-37.8 s (X = 30.7; σ = 3.01; m = 0.46).

An elongation of the fibrin monomer self-assembly time above the indicated upper limit indicates congenital or acquired dysfibrinogenemia, or the inhibitory effect of fibrin monomer self-assembly inhibitors (products of fibrin degradation - D-dimer, paraproteins). Shortening the self-assembly time of the fibrin monomer indicates the presence of a thrombogenic shift at the final stage of coagulation.

Testing of the proposed method.

The testing of the proposed method was carried out on 25 patients with lengthening of the coagulation time at the final stage (group I), including hemorrhagic mesenchymal dysplasias (14), with von Willebrand disease (7), cirrhosis of the liver (4), as well as 27 patients (II group) with a shortening of the self-assembly time of the fibrin monomer in hypercoagulable conditions, namely with hematogenous thrombophilia (11), severe gestosis of the last trimester of pregnancy (9), and septic conditions (7). The results of the examination of patients with disorders of the final stage of coagulation by the proposed method are presented in table 1. From the table it follows that the proposed method allows to evaluate both the slowdown and the acceleration of self-assembly of fibrin monomer in the corresponding standard clinical situations.

It should be noted that the claimed method has significant differences from the known SU 1210094, namely the use of thrombin standardized for coagulation activity, a significant reduction in analysis time, the possibility of coagulometric studies, and improving the accuracy of the results.

In particular:

1. Improving the accuracy of the claimed method is achieved using thrombin standard activity of 4-6 s (or 40-60 NIH / ml).

The activity of the thrombin used (0.1% or 0.1 g / 100 ml) described in the prototype method cannot be standardized, since thrombin is an enzyme whose activity is difficult to compare with its weight in different batches of this reagent from various manufacturers.

To clarify the choice of the range of activity of the thrombin used, we present table 2, which shows the effect of thrombin activity on the result in the present method. It provides average data on the results of a 10-fold repetition of the self-assembly time of the fibrin monomer. As can be seen from table 2, the optimal activity of thrombin in the present method is the range from 40 to 60 NIH / ml Lower thrombin activity (less than 40 NIH / ml) gives erratic results and lengthens the study time. The use of thrombin activity above 60 NIH / ml is not economically feasible.

2. In addition, in the claimed method, the time required to perform the analysis is 2.5-3 minutes, while the prototype method requires 15-17 minutes to complete, i.e. 5.8 times less on average.

3. It is customary to evaluate the coagulation of blood or plasma obtained from it using coagulometers. Evaluation of the results in the inventive method can be carried out on a coagulometer of any design. At the same time, the determination of coagulation time in the prototype method is carried out only manually and is not captured coagulometrically due to the low concentration of fibrinogen in the mixture being evaluated ("tiny" coagulation). The latter is due to the high, 12 times, dilution of plasma during the analysis by the prototype method. In the inventive method, the plasma during the analysis is diluted 2 times. A multiple of the dilutions reduces the amount of coagulation substrate required for thrombin - fibrinogen.

4. When performing the proposed method for determining the time of self-assembly of fibrin monomer, the accuracy of determinations is increased in comparison with the prototype method. Table 3 shows that the coefficient of variation of the results in the claimed method is more than 3 times lower than the same indicator in the prototype method, and is 4.4%. The calculation of the coefficient of variation of the results obtained is presented in the proposed method coagulometrically and the prototype method manually.

The rationale for the required range of urea concentration to assess the final stage of coagulation in the proposed method.

To select the required urea concentration when determining the self-assembly time of the fibrin monomer, we used samples of a urea buffer solution with a concentration of 50 to 350 g / l (table 4). It was established that its optimal concentrations lie in the range from 150.0 to 250.0 g / l. A decrease in urea concentration below 150.0 g / l leads to the fact that the clot formation time is shortened to 17.1-19.0 s or less and does not significantly differ in patients of different groups. On the other hand, the use of urea concentrations in excess of 250.0 g / l leads to a significant lengthening of clotting time and a decrease in reproducibility of the method. To determine the optimal range of urea concentration in the test system, the ratio C = K / B was used to determine the self-assembly time of the fibrin monomer

where C is the ratio;

K is the time of self-assembly of the fibrin monomer in the control normal pooled plasma;

B - the time of self-assembly of fibrin monomer in the patient’s plasma.

From the studies shown in Table 3, it can be observed that the required range of urea concentrations in the test system is 150-250 g / l, since it is in this range of urea concentrations that the ratios remain unchanged and have diagnostic value.

To assess the reproducibility of the proposed method used the coefficient of variation CV (%). The coefficient of variation of the method during the study on different days of the study does not exceed 8.5%.

Thus, the claimed method for determining the time of self-assembly of fibrin monomer is highly accurate and allows you to diagnose congenital or acquired dysphibrinogenemia, in which hemorrhagic syndrome is often observed, as well as identify hypercoagulable syndrome leading to intravascular coagulation.

The method for determining the self-assembly time of fibrin monomer
Table 1 I group II group Patient FM self-assembly time (difference from the normal indicator,%) Patient FM self-assembly time (difference from the normal indicator,%) 1. N.M. 38.4 (+ 25.1%) 1. U.M. 22.4 (-27.0%) 2. Z.V. 39.5 (+ 28.7%) 2. Z.A. 24.6 (-19.9%) 3. N.G. 45.2 (+ 47.2%) 3. K.R. 20.6 (-32.9%) 4. A.I. 39.6 (+ 29.0%) 4. A.I. 17.1 (-44.3%) 5. T.I. 46.7 (+ 52.1%) 5. V.O. 14.7 (-52.1%) 6. I.G. 41.4 (+ 34.9%) 6. L.G. 25.3 (-17.6%) 7. I.A. 44.6 (+ 45.3%) 7. I.D. 24.9 (-18.9%) 8. T.S. 39.8 (+ 29.6%) 8. H.S. 22.4 (-27.0%) 9. N.V. 38.9 (+ 26.7%) 9.R.D. 20.3 (-33.9%) 10. O.A. 43.0 (+ 40.1%) 10. R.A. 21.6 (-29.6%) 11. E.S. 47.0 (+ 53.1%) 11. A.S. 22.3 (-27.4%) 12.O.O. 56.7 (+ 84.7%) 12. L.L. 21.8 (-29.0%) 13. A.P. 49.4 (+ 60.9%) 13. A.T. 24.6 (-19.0%) 14. E.R. 39.4 (+ 28.3%) 14. I.R. 17.7 (-42.4%) 15. V.I. 44.5 (+ 45.0%) 15. V.I. 19.6 (-36.2%) 16. A.N. 46.3 (+ 50.8%) 16.O.L. 15.9 (-48.2%) 17. G.K. 48.2 (+ 57.0%) 17. D.A. 24.6 (-19.9%) 18. E.A. 41.3 (+ 34.5%) 18. E.A. 22.6 (-26.4%) 19. T.A. 40.3 (+ 31.3%) 19. S.A. 23.0 (-25.1%) 20. V.V. 48.2 (+ 57.0%) 20. V.R. 19.1 (-37.8%) 21. Yu.N. 42.7 (+ 39.1%) 21. D.N. 20.3 (-33.9%) 22. A.K. 38.3 (+ 24.8%) 22. D.K. 20.3 (-33.9%) 23.T.G. 39.4 (+ 28.3%) 23. B.G. 21.4 (-30.3%) 24. S.S. 42.4 (+ 38.1%) 24. B.S. 23.5 (-23.5%) 25. A.D. 49.4 (+ 60.9%) 25. R.L. 16.5 (-46.3%) 26. S.P. 18.7 (-39.1%) 27. A.A. 24.3 (-20.3%) I group II group The average result for the I group of patients (X ± m) 43.6 ± 0.9 The average result for the II group of patients (X ± m) 21.1 ± 0.6 Regulatory indicators based on a survey of 42 healthy donors The average control result (X ± m) 30.7 ± 0.5 The average control result (X ± m) 30.7 ± 0.5

METHOD FOR DETERMINING THE SELF-ASSEMBLY TIME OF FIBRIN MONOMER
table 2 Thrombin concentration, NIH / ml twenty thirty 40 fifty 60 70 80 Time in s in the claimed method 77.2 ± 9.6 46.5 ± 5.9 32.7 ± 0.9 31.3 ± 0.6 29.1 ± 0.7 28.2 ± 0.7 28.8 ± 0.6 the effect of thrombin activity on the result of the present method

METHOD FOR DETERMINING THE SELF-ASSEMBLY TIME OF FIBRIN MONOMER
Table 3 Coefficient of variation of the fibrin monomer self-assembly time estimate According to the claimed method, with by the prototype method,% 32,0 22 33.0 27 30.5 23 32.8 31 31.9 23 35.7 22 33.5 27 32.6 29th 31.8 thirty 34.2 33 Coefficient of variation - 4.39% Coefficient of variation - 15.09% coefficient of variation of the results in the present method and in the prototype method

The method for determining the self-assembly time of fibrin monomer
Table 4 Urea concentrations in the test system, g / l fifty one hundred 150 200 250 300 350 Control normal pooled plasma (K) Fibrin monomer self-assembly time, s 5.9 18.5 26,4 46,4 60.3 More than 120 More than 120 Patient N.'s plasma, with prolongation of the self-assembly time of fibrin monomer (B ₁ ) Fibrin monomer self-assembly time, s 6.3 19.0 34.5 59.2 79.5 More than 120 More than 120 The ratio of the time of self-assembly of fibrin monomer (K / B ₁ ), s 0.94 0.97 0.76 0.79 0.76 Not determined Not determined Plasma of patient B. with shortening of the time of self-assembly of fibrin monomer (B ₂ ) Fibrin monomer self-assembly time, s 5.8 17.1 20,0 35,4 46.0 More than 120 More than 120 The ratio of the time of self-assembly of fibrin monomer (K / B ₂ ), s 1,02 1,08 1.32 1.31 1.31 Not determined Not determined

Claims (1)

A method for determining the self-assembly time of a fibrin monomer, including determining the clotting time using thrombin and urea, characterized in that the clotting time is evaluated on a coagulometer with successive mixing of 0.1 ml of the test plasma, 0.05 ml of urea solution in a concentration of 180 g / l, incubation at a temperature of 37 ° C for 1 min and adding 0.05 ml of a thrombin solution, activity of 4-6 s or 40-60 NIH / ml.