EA038901B1 - Method for prediction of thromboembolic complications in women with accompanying protrombin gene mutations [f2(20210)ga] - Google Patents

Abstract

The invention relates to medicine, in particular to laboratory diagnostics, and can be used for the prediction of thromboembolic complications in females with accompanying a prothrombin [F2(20210)GA] mutation by the determination of prothrombin activity for forming risk groups requiring anticoagulant preventive measures. A method is described for the prediction of thromboembolic complications in women-carriers of a prothrombin [F2(20210)GA] mutation, where the prothrombin activity is determined, and if its value is 174.8% and higher, a risk of the VTEC development is predicted. The claimed method provides high accuracy and high volume of information and allows of forming a risk group for the development of thrombotic events prior to the first clinical symptoms and prescribing personified preventive medication in order to reduce the risk of the development of pulmonary artery thromboembolia, and associated with it disablement and mortality.

Description

The invention relates to medicine, in particular to laboratory diagnostics, and can be used to predict venous thromboemolic complications (VTEC) in women, carriers of the prothrombin gene mutation [F2 (20210) GA], by determining the activity of prothrombin to form a risk group requiring anticoagulant prophylaxis.

Venous thromboembolic complications remain one of the important problems in clinical medicine. Despite significant advances in clinical practice and pharmacology, these diseases still remain the leading cause of death and disability in developed countries and represent a global medical and social problem.

Mutation of the prothrombin gene [F2 (20210) GA] occurs in 2-5% of the population, increasing the risk of developing venous thrombosis by 3-4 times. However, at present there are no precise and objective laboratory criteria for the selection of women with a mutation of the prothrombin gene [F2 (20210) GA] in the risk group for VTEC, who need personalized heparin prophylaxis to prevent thrombosis.

A known method for predicting the occurrence of venous thrombosis and thromboembolism of the pulmonary artery (Patent of Kazakhstan No. 17498, 14.07.2006). The essence of the invention lies in the fact that in the study of blood, DNA is isolated, an analysis of the polymorphism of the restriction length of DNA fragments is carried out, and when a mutation of factor 5 Leiden in the F5 gene and / or a mutation of factor F2 in the prothrombin gene is detected, the occurrence of venous thrombosis and thromboembolism of the pulmonary artery is predicted.

The disadvantage of this method is that it allows you to identify individuals with a hereditary predisposition to venous thrombosis, but does not predict the development of thrombosis in the selected cohort.

A known method for predicting thrombosis in a coronary stent during percutaneous coronary intervention in patients with ischemic heart disease (RF Patent No. 218.016.7535; 07.26.2018), which consists in the fact that the patient is determined by the content of creatine phosphokinase (CPK), prothrombin index (PTI), activated recalcification time (AVR). The obtained results of the survey are substituted into the mathematical model for predicting the risk of thrombosis in coronary stents: p = 1 / (1 + e ^P * ™ ^{0, 06} - ^{0, 076} * ^KF *

The disadvantage of this method is to determine the prognostic indicator for the development of thrombosis in a limited number of people suffering from coronary heart disease with planned stenting of the coronary vessels. This method makes it possible to predict the complication of an existing disease, and not to carry out the primary stratification of patients and possible prevention.

There is a method for predicting the development of thrombosis (USSR Patent No. 1634249; 03/15/1991), which consists in determining the ratio of the difference in the maximum amplitudes of the recorded thromboelastograms to the difference between the initial platelet count and the platelet count after repeated centrifugation. With an increase in this indicator by 1.5 times in relation to the control, the development of thrombosis is predicted in 71% of cases.

The disadvantage of this method is the use of expensive equipment, the complexity of the procedure.

The closest to the achieved technical result (prototype) is a method for predicting the development of deep vein thrombosis of the lower leg by determining the expression of a type 1 plasminogen activator inhibitor (PAI-1) (China; 2018) [Tang J., Zhu W., Mei X., Zhang Z . (2018) Plasminogen activator inhibitor-1: a risk factor for deep vein thrombosis after total hip arthroplasty. J. Orthop. Surg. Res., 13 (1): 8]. It was shown that with a median PAI-1 index of 32.1 ± 12.5ng / ml, the risk of venous thrombosis increases by 1.18 times (OR 1.18, 95% DI 1.04-1.29; p = 0.011).

The disadvantage of this method is its low accuracy.

The authors propose a simple, affordable method for predicting VTEC with high accuracy in women who are carriers of the prothrombin gene [F2 (20210) GA]. A method for predicting the development of thromboembolic complications in women with a mutation of the prothrombin gene [F2 (20210) GA] is to determine the activity of prothrombin, and if its value is 174.8% and higher, the risk of VTEC is predicted.

This method makes it possible to form a risk group for the development of thrombosis before the appearance of the first clinical symptoms and to prescribe personalized drug prophylaxis to reduce the risk of developing pulmonary embolism and associated disability and mortality.

The technical result of the proposed method is to improve the accuracy of predicting the development of thrombotic events in women, carriers of the mutation of the prothrombin gene [F2 (20210) GA], based on the prothrombin activity established by the authors in order to make a decision on anticoagulant prophylaxis.

The technical result is achieved by determining the activity of prothrombin (factor II) by using a substrate-deficient plasma.

The method can be carried out, for example, as follows:

Determine the clotting time of the test sample of platelet-poor plasma in the mixture, with

- 1 038901 containing factor II-deficient plasma, diluted test plasma and Tromborel S.

The quantitative determination of the activity of coagulation factor II is performed according to the graph of the dependence of the activity of factor II (in%) on the prothrombin coagulation time.

Equipment, materials, reagents

In accordance with the instructions for the set of reagents used, use an automatic or semi-automatic coagulometer;

Factor II deficient substrate plasma (catalog number 5190). Freeze-dried human blood plasma, the level of factor II in which is not higher than 1%;

Tromborel S (catalog number OUHP49). Freeze-dried thromboplast - calcium mixture from rabbit brain;

Saline 0.9% NaCl;

Control plasma (freeze-dried human blood plasma, pooled from 25 donors, with a known content of factor II);

Distilled water.

Preparation of analyzed samples

Blood for research is taken in the morning, on an empty stomach, from the cubital vein into 9 ml VACUETTE tubes containing 3.8% sodium citrate solution, the ratio of blood volume to sodium citrate is 9: 1. The blood is centrifuged at 3000-4000 rpm (1200 g) for 15 minutes, as a result, platelet-poor plasma is obtained, which is transferred to another tube, where it is stored until the study.

Centrifugation is usually carried out immediately after blood collection, and plasma collection for research is carried out immediately after centrifugation. Analysis of blood plasma that has clots, hemolysis and was obtained more than 2 hours ago is not allowed. Before analysis, dilute all test samples with 0.9% saline solution of NaCl 5 times (0.1 ml of sample + 0.4 ml of saline).

Preparation of reagents and analysis

1. Preparation of reagents for work

1.1. Dilution of Factor II Deficient Plasma

Add 1.0 ml of distilled water to a bottle with plasma deficient in factor II and dissolve the contents at room temperature (+18 - + 25 ° C) and gently shaking (avoiding the formation of foam) for 3 minutes. Before testing, keep the diluted plasma for at least 25-30 minutes at room temperature.

1.2. Preparation of the Tromborel S reagent

Add 2.0 ml of distilled water to one bottle with Tromborel S reagent. Shake the bottle and incubate at + 37 ° C (in a water bath) for 20 minutes. Stir the diluted reagent before each determination to avoid precipitation.

1.3. Dilution of Control Plasma

Add 1.0 ml of distilled water to the vial with control plasma and dissolve the contents at room temperature (+18 - + 25 ° C) for 3 minutes. Control plasma must be incubated at room temperature for 15 minutes prior to use.

1.4. The preparation of dilutions for constructing a calibration graph is presented in table. 1. Table 1. Scheme of dilutions of the calibration sample for constructing a control graph

Test tube, no. Saline 0.9% NaCl, ml Control plasma with certified value of factor II activity (100%) Mix and transfer to another tube 12 3 4 0.9 0.5 1.6 0.9 0.1 ml -II- - // - - // - ▼ AT AT А 1C), 5ml = LI = 0.4ml ^ k), 1ml ^ The resulting dilution 1:10 1:20 1: 100 1: 1000 Factor II activity one hundred % 50% 10% one%

1.5. Plotting a calibration curve

For each dilution of the control plasma, perform the determinations twice, mark the average result on the calibration curve. On the Y-axis, plot the clotting time of each diluted sample of the control plasma in seconds, on the X-axis — the corresponding value of the coagulation factor activity in percent,%. Draw a calibration graph through the obtained points, which in logarithmic coordinates should be a straight line in the range of factor II activity from 1 to 100%.

2. Analysis

2.1. Manual option:

2.1.1. To 0.1 ml of the test plasma diluted 5 times, taken in a test tube, add 0.1 ml perhaps

- 2,038901 day plasma deficient in factor II.

2.1.2. Incubate at + 37 ° C for 1 min.

2.1.3. Add 0.2 ml of diluted Tromborel S reagent at + 37 ° C and start the stopwatch. Register the clotting time.

2.1.4. Using the calibration curve, find the factor II activity. If the coagulation time of the tested plasma corresponds to a factor II activity of less than 100%, for example only 95%, the result read from the curve is multiplied by 0.95. In the case of the coagulation time, which corresponds to the content of the coagulation factor more than 100%, additional determinations are required using higher dilutions of the studied plasma (for example, 10 times). In this case, the result obtained in% must be multiplied by the factor corresponding to the dilution; for example, for a 10-fold dilution, the result is multiplied by 2.

2.2. Coagulometric option:

The study of blood plasma is carried out on coagulometers with a mechanical or optical (for example, Siemens BCS XP) principle of registration of results.

2.2.1. Select a program on the coagulometer to determine the activity of factor II by a one-stage clotting method;

2.2.2. Place the vials with the prepared reagents in the appropriate cells of the coagulometer;

2.2.3 Start the program for constructing a calibration straight line (for each new series of reagents);

2.2.4 Place control and test plasma samples into the coagulometer cuvette;

2.2.5 Start the measurement program;

2.2.6 Read the results.

Reading results

The activity of prothrombin (factor II) is expressed in international units (IU) or percentage, with 1 IU / ml corresponding to 100% activity. In healthy patients, without carriage of the prothrombin gene mutation, genotype [F2 (20210) GG], the activity of prothrombin (factor II) is 70-120% [Fickenscher K. Analysisbof individual coagulation factor. In: Thomas L, ed. Clinical Laboratory Diagnostics. Frankfurt: TH-Books Verlagsgesellschaft 1998: 607-9]. The reference intervals vary and depend on the population studied, the technique used, methods, equipment and reagent lot. Approbation of the proposed method for predicting thrombotic events when carrying a mutation of the prothrombin gene [F2 (20210) GA].

Approbation of the proposed method was carried out on 140 patients included in the database Carriers of the prothrombin mutation [F2 (G20210A)] in Russia (Certificate No. 2018621494 dated 09.19.2018) with the established carriage of the prothrombin gene mutation [F2 (20210) GA].

All patients underwent a study of the prothrombin activity indicator once every three months. In 32 (22.8% of 140) patients with dynamic observation, an episode of VTEC developed, while the median prothrombin activity indicator two weeks before thrombosis was 178.2% (95% CI 170.4-204.6%), which is significant more (p <0.0001) than in patients without a thrombotic event 148.8% (95% CI 145.3-156.9%).

The invention is illustrated by the figure, which depicts the ROC-curve, which determines the critical cutoff threshold of prothrombin activity (%) and built in the analysis of sensitivity and specificity in patients with the development of thrombosis when carrying a mutation of the prothrombin gene [F2 (20210) GA]. The area under the curve (AUC) for the prothrombin activity indicator of 174.8% is 0.904 (95CI 0.825-0.955) with a p-level p <0.0001. The predictor sensitivity is 81.8, the specificity is 91.5.

The results obtained indicate that the claimed method for predicting thromboembolic complications in women with the carriage of the mutation of the prothrombin gene [F2 (20210) GA], with prothrombin activity of 174.8%, ensures the forecast accuracy in 90.4% of cases (Table 2)

Clinical examples

1. Patient V.Yu.A., 28 years old. She was observed by a hematologist at the Altai branch of the Federal State Budgetary Institution National Medical Research Center of Hematology. She has a history of one premature birth at 3334 weeks, secondary infertility for 2 years. At the stage of pregnancy planning, examination revealed the carriage of the prothrombin gene mutation [F2 (20210) GA]. At the next dispensary observation, the activity of prothrombin was determined to be 198.2%. 3 days after the visit to the hematologist, the patient fell ill with ARVI of moderate severity with hectic fever, severe symptoms of intoxication. An episode of thrombosis occurred at the stage of recovery (8 days from the onset of the disease), with localization of a thrombus in the deep veins of the leg. The patient was admitted to the vascular surgery department.

2. Patient Ch.ON., 32 years old. She was observed in the center for the preservation and restoration of reproductive function of the Altai Regional Clinical Hospital with a diagnosis of primary infertility for 8 years. At the stage of examination, the carriage of the mutation of the prothrombin gene [F2 (20210) GA] was revealed, with prothrombin activity of 185.7%. In preparation for the protocol of assisted reproductive technologies, a study of the patency of the fallopian tubes (hysterosalpingography) was performed. On the second

- 3,038901 days after the procedure, she was hospitalized in the department of vascular surgery with acute thrombosis of the ilio-popliteal-femoral segment. During treatment, the patient was implanted with a cava filter

3. Patient G.I.G, 34 years old. She was observed in the city center for family planning and reproduction at a specialized appointment for miscarriage. A history of two non-developing pregnancies with a gestational age of 7-8 weeks. At the stage of pregnancy planning, examination revealed the carriage of a mutation in the prothrombin gene [F2 (20210) GA], with prothrombin activity of 187.2%. Without the consent of the hematologist, in order to regulate the menstrual cycle, the patient was prescribed estrogen-containing combined contraceptives. After taking 13 tablets, the patient was diagnosed with pulmonary embolism.

4. Patient M.N.V., 36 years old. She was observed in the center for the preservation and restoration of reproductive function of the Altai Regional Clinical Hospital with a diagnosis of secondary infertility for 5 years. At the stage of pregnancy planning, examination revealed the carriage of a mutation in the prothrombin gene [F2 (20210) GA], with prothrombin activity of 155.4%. According to the individual management plan, the patient underwent a gynecological operation - laparoscopy of endometrioid ovarian cysts. The postoperative period is favorable. Graduated compression hosiery was used as prevention of VTEC.

Thus, the claimed method for predicting VTEC in women with the carriage of the prothrombin gene mutation [F2 (20210) GA] by determining the activity of prothrombin has high accuracy, is highly informative and allows you to form a risk group for the development of VTEC for carrying out personalized drug prevention and therapy to reduce the risk of development pulmonary embolism

table 2

Indicator Number of examined Total women 140 true positive 32 false positive 2 true negative 103 false negative result 3 Total: the accuracy of the claimed method is 90.4% sensitivity of the claimed method - 81.8% specificity of the claimed method - 91.5%

CLAIM

Claims (1)

CLAIM A method for predicting the development of thromboembolic complications in women with the carriage of a mutation of the prothrombin gene [F2 (20210) GA], characterized in that the activity of prothrombin is determined and, if its value is 174.8% and higher, the risk of developing venous thromboembolic complications (VTEC) is predicted.