RU2722280C1 - Method for prediction of thromboembolic complications in women accompanying protrombin gene mutations [f2(20210)ga] - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and concerns a method for prediction of thromboembolic complications (VTEC) in females accompanying a prothrombin [F2(20210)GA] mutation, where prothrombin activity is determined and if its value is 174.8 % and higher, a risk of developing VTEC is predicted, wherein prothrombin activity is expressed in international units (IU) or percentage, wherein 1 IU/ml corresponds to 100 % activity.

EFFECT: invention provides higher accuracy of prediction of thrombotic events in females, carriers of mutation of prothrombin gene [F2(20210)GA].

1 cl, 4 ex, 1 dwg, 2 tbl

Description

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

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

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

A known method for predicting the occurrence of venous thrombosis and pulmonary embolism (Patent of Kazakhstan No. 17498, 07/14/2006). The essence of the invention lies in the fact that DNA is isolated during a blood test, an analysis of the restriction length polymorphism of DNA fragments is carried out, and when a factor 5 Leiden mutation in the F5 gene and / or factor F2 mutation in the prothrombin gene is detected, the occurrence of venous thrombosis and pulmonary embolism is predicted.

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

.A known method for predicting thrombosis in a coronary stent with percutaneous coronary intervention in patients with coronary heart disease (RF Patent No. 218.016.7535; 07.26.2018), which consists in the fact that the content of creatine phosphosokinase (CPK), prothrombin index (IPT), is determined in a patient, Recalcification Time (ABP). The results of the examination are substituted into the mathematical model for predicting the risk of developing thrombosis in the coronary stent:

.

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

A known 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 platelet count after repeated centrifugation. With an increase of 1.5 times in relation to the control, 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 technical result achieved (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 score of 32.1 ± 12.5ng / ml, the risk of venous thrombosis increases 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 VTEO, which is highly accurate in women carriers of the prothrombin gene mutation [F2 (20210) GA]. A method for predicting the development of thromboembolic complications (VTEO) in women with a prothrombin [F2 (20210) GA] gene mutation, characterized in that the prothrombin activity is determined, and with its value of 174.8% or higher, the risk of developing VTEO is predicted, while the activity prothrombin is expressed in international units (ME) or percent, with 1 IU / ml corresponding to 100% activity.

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

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

The technical result is achieved by the fact that the activity of prothrombin (factor II) is determined by using a plasma deficient in the substrate.

The method can be carried out, for example, as follows: The clotting time of the test sample of platelet-poor plasma in a mixture containing factor II deficient plasma, diluted test plasma and Trsborel S reagent is determined. 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 clotting time. Equipment, materials, reagents

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

- Substrate plasma with factor II deficiency (catalog number 5190). Lyophilized dried human blood plasma, the level of factor II in which is not higher than 1%;

- Thromborel S (catalog number OUHP49). Lyophilized dried thromboplasty - a rabbit brain calcium mixture;

- Saline solution of 0.9% NaCl;

- Control plasma (freeze-dried human blood plasma with known Factor II content pulsed from 25 donors);

- Distilled water.

Sample Preparation

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

Centrifugation is usually carried out immediately after blood sampling, and the selection of plasma for research - immediately after centrifugation. An analysis of blood plasma having clots, hemolysis and obtained more than 2 hours ago is not allowed. Before analysis, dilute all test samples with 0.9% saline 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

In a bottle with a plasma deficient in factor II, add 1.0 ml of distilled water and dissolve the contents at room temperature (+ 18 ... + 25 ° C) and gently swaying (avoiding the formation of foam) for 3 minutes. Dilute the plasma before the study to withstand at least 25-30 minutes at room temperature.

1.2. Preparation of reagent Thromborel S

In one bottle with Thromborel S reagent, add 2.0 ml of distilled water. Shake the bottle and stand at + 37 ° C (in a water bath) for 20 minutes. Stir the diluted reagent before each determination to prevent precipitation.

1.3. Dilution of control plasma

In a bottle with a control plasma, add 1.0 ml of distilled water and dissolve the contents at room temperature (+ 18 ... + 25 ° C) for 3 minutes. Before use, the control plasma should be kept at room temperature for 15 minutes.

1.4. Preparation of dilutions for constructing a calibration graph is presented in table 1.

1.5. Building a calibration curve

For each dilution of the control plasma, perform the determination twice, mark the average result on the calibration curve. On the Y axis, set the clotting time of each diluted control plasma sample in seconds, on the X axis, the corresponding value of the coagulation factor activity in percent,%. Through the obtained points, draw a calibration graph, which in bilogarithmic 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 a 5-fold diluted test plasma taken in a test tube, add 0.1 ml of diluted factor II deficient plasma.

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

2.1.3. Add 0.2 ml of diluted reagent Thromborel S, having a temperature of + 37 ° C, and turn on the stopwatch. Record clotting time.

2.1.4. Using the calibration curve, find the activity of factor II. If the coagulation time of the test 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 a coagulation time that corresponds to a coagulation factor content of 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% should be multiplied by a coefficient corresponding to the dilution; for example, to dilute 10 times, 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 recording results.

2.2.1. On the coagulometer, select a program for determining the activity of factor II using the one-step clotting method;

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

2.2.3 Run the program for constructing the calibration line (for each new series of reagents);

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

2.2.5 Run the measurement program;

2.2.6 Read the results. Reading results

The activity of prothrombin (factor II) is expressed in international units (ME) or in percent, with 1 IU / ml corresponding to 100% activity. In healthy patients without prothrombin gene mutation carriers, the genotype [F2 (20210) GG], prothrombin (factor II) activity 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 studied population, used equipment, methods, equipment and batch of reagents. Testing of the proposed method for predicting thrombotic events in the carriage of a prothrombin gene mutation [F2 (20210) GA].

The proposed method was tested on 140 patients included in the database “Prothrombin [F2 (G20210A)] mutation carriers in the Russian Federation” database (Certificate No. 2018621494 of 09/19/2018) with established prothrombin [F2 (20210) GA] gene mutation carriers. All patients underwent a study of the prothrombin activity indicator once every three months. 32 (22.8% of 140) patients under dynamic observation developed an episode of VTE, while the median of prothrombin activity 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 in figure 1, which depicts an ROC curve that defines the critical threshold for prothrombin activity cutoff (%) and is constructed by analyzing sensitivity and specificity in patients with thrombosis with prothrombin gene mutation [F2 (20210) GA]. The area under the curve (AUC) of the 174.8% prothrombin activity index is 0.904 (95CI 0.825-0.955) with a significance level (p-level) p <0.0001. The predictor sensitivity is 81.8, the specificity is 91.5.

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

Clinical examples.

1. Patient V.YU.A., 28 years old. It was observed by a hematologist in the Altai branch of the Federal State Budgetary Institution National Medical Research Center for Hematology. A history of some preterm birth at 33-34 weeks, secondary infertility 2 years. At the stage of pregnancy planning, examination revealed the carriage of a prothrombin gene mutation [F2 (20210) GA]. At the next follow-up, prothrombin activity was determined to be 198.2%. 3 days after a visit to a hematologist, the patient fell ill with moderate ARVI with hectic fever, severe symptoms of intoxication. The episode of thrombosis was realized at the stage of recovery (8 days from the onset of the disease), with the localization of the thrombus in the deep veins of the leg. The patient was hospitalized in the department of vascular surgery.

2. Patient Ch.O.N., 32 years old. It was observed at the center for preserving and restoring the reproductive function of the Altai Regional Clinical Hospital with a diagnosis of primary infertility for 8 years. At the stage of examination, a prothrombin [F2 (20210) GA] gene mutation was detected, with a 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 day after the procedure, she was hospitalized in the department of vascular surgery with acute thrombosis of the iliac-popliteal femoral segment. During treatment, the patient was implanted with a cava filter

3. Patient G.I.G., 34 years old. It was observed in the city center for family planning and reproduction at a specialized reception for miscarriage. A history of two non-developing pregnancies with a gestational age of 7-8 weeks. At the stage of pregnancy planning during the examination, a prothrombin [F2 (20210) GA] gene mutation was detected by carriage, with a prothrombin activity of 187.2%. Without coordination with a 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. It was observed at the center for preserving and restoring the 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 a prothrombin gene mutation [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. As prevention of VTEO, graduated compression knitwear was used.

Thus, the claimed method for predicting VTEO in women with a prothrombin [F2 (20210) GA] gene mutation is highly accurate in determining prothrombin activity, is highly informative and allows one to form a risk group for the development of VTEO for conducting personalized drug prophylaxis and therapy to reduce the risk of development pulmonary embolism

Claims (1)

A method for predicting the development of thromboembolic complications (VTEO) in women with a prothrombin [F2 (20210) GA] gene mutation, characterized in that prothrombin activity is determined and, with its value of 174.8% or higher, the risk of developing VTEC is predicted, while prothrombin activity is expressed in international units (ME) or percent, with 1 IU / ml corresponding to 100% activity.

Images