RU2058031C1 - Method for determining activation of blood plasma coagulation by cell membrane enzymes - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves determining recalcification time in the mixture of the sample under study with substrate plasma. The recalcification time is determined in substrate plasma having cells of the sample under study. The sample coagulation activity is estimated from calibration curve built using relationship between substrate plasma recalcification time and membrane coagulation activity. EFFECT: enhanced accuracy of method. 2 dwg, 1 tbl

Description

 The invention relates to medicine, in particular to a blood test, and can be used for laboratory diagnosis of bleeding disorders.

 As is known, modified fragments of cell membranes circulate in the blood, which, in contrast to damaged cell membranes, have coagulating activity. This background activity is determined under physiological conditions. According to modern concepts, many types of bleeding disorders (DIC, thrombosis, embolism, etc.) are accompanied by excessive accumulation in the bloodstream of coagulatively active fragments of cell membranes that trigger blood coagulation both by external and internal mechanisms. The search for reliable methods for determining the membrane activation of blood coagulation would create the basis for obtaining new data on the pathogenesis of coagulopathy, new criteria for laboratory diagnosis and monitoring of the treatment of these diseases.

 Known methods for determining membrane activation of blood coagulation (MASK) have low accuracy and are little suitable for practical purposes. In this regard, the development of a method for determining MASK is an important scientific and practical task of coagulology.

 A known method for determining circulating in plasma membrane cells by determining the activity of a specific marker of 5'-nucleotidase. A change in the content of this enzyme indicates a possible role of membrane fragments in the development of a hypercoagulable state. The main disadvantage of this method is the one-sided assessment of the number of membranes (in the equivalent of 5'-nucleotidase) without taking into account their coagulation activity.

 This disadvantage is largely eliminated in the closest in technical solution to the method for determining the activity of blood plasma thromboplastin. It should be noted that the term "thromboplastin" refers to coagulation-active cell membranes.

100 (%)
Недостатком способа-прототипа является то, что при оценке результатов не учитывается исходный (обусловленный плазменными факторами свертывания) коагуляционный потенциал исследуемой плазмы, который варьирует на практике у больных в широком диапазоне. Это приводит к ошибке измерения МАСК. Влияние коагуляционного потенциала плазмы крови на результаты определения МАСК (%) при нормальной (100%) коагуляционной активности мембран представлено в таблице.The prototype method is as follows. A mixture of platelet-poor plasma is obtained from 5 healthy donors. The mixture is centrifuged with an acceleration of 52,000 g for 60 minutes, resulting in a substrate plasma, freed from cell membranes. Samples of the platelet-poor plasma of the test and healthy donor are prepared. The clotting time (recalcification) in a mixture of equal volumes of substrate and donor platelet-poor plasma (T ₁ ) is determined. Following this, the coagulation (recalcification) time of a mixture of equal volumes of substrate and plasma studied by platelets (T ₂ ) is determined. The hemocoagulating activity of cell membranes circulating in plasma is determined by the formula
A

one hundred (%)
The disadvantage of the prototype method is that when evaluating the results, the initial (due to plasma coagulation factors) coagulation potential of the studied plasma, which varies in practice in patients in a wide range, is not taken into account. This leads to an MASK measurement error. The influence of the coagulation potential of blood plasma on the results of determination of MASK (%) with normal (100%) coagulation activity of the membranes is presented in the table.

 As can be seen from the table, if, under normal plasma coagulation, the MASK indices for the compared methods were practically the same, then in the study of blood plasma samples of patients with hypercoagulation, the prototype method falsely underestimated the results by an average of 36% (p <0.001).

 In the study of blood plasma with hypocoagulation (for example, in patients with hemophilia or in the hypocoagulation phase of DIC), the results of determining MASK, on the contrary, were falsely overestimated, by an average of 66% (p <0.001). Thus, the error of the prototype method is from 36% (with hypercoagulation) to 66% (with hypocoagulation).

 The technical result of the invention is to increase the accuracy of determining membrane activation of blood plasma coagulation (MASK) by determining the recalcification time in a substrate plasma containing the membranes of the test sample, and their coagulation activity is evaluated by a calibration curve constructed from the dependence of the time of recalcification of substrate plasma on the coagulation activity of the membranes.

 If the membranes of the studied plasma sample are deposited by ultracentrifugation or on a filter with a certain pore diameter (0.2-0.4 μm) (the pore diameter of the filter is determined by the size (more than 0.04 μm) of membranes with coagulation activity), it becomes possible to wash them from the plasma on the filter and introduce into the composition of the standard substrate plasma, devoid of autologous membranes. Thus, the time of recalcification of the substrate plasma will depend on the activity of the membranes introduced into it. The calibration curve of the dependence of the time of recalcification of the substrate plasma on the number of coagulation-active membranes introduced into it allows us to evaluate the activity of the membranes of the studied sample of blood plasma by the claimed method. This excludes the influence of the coagulation potential of the studied plasma sample on the results of the determination of MASK (see table).

 In FIG. 1 shows a diagram of a filtering device and the stages of its use; figure 2 calibration chart for determining membrane activation of blood coagulation (MASK).

 The inventive method is as follows.

Reagents and equipment:
Sodium citrate, trisubstituted, 3.8% solution;
calcium chloride, 0.025 M solution;
a suspension of kaolin, is prepared at the rate of 5.0 mg of kaolin in 1 ml of medinal-HCl buffer (pH 7.4);
donor plasma, standard, lyophilized (used for the production of substrate plasma) produced by the Kirov Research Institute of Hygiene and Epidemiology;
membrane nylon microporous filters (TU 15-16-3-85) manufactured by Hiu Kalur (Estonia) with a pore size of 0.2 μm. Filters with a similar pore size from Nucleopore, Millipore;
a compressor for creating excess air pressure from 0 to 2.0 kgf / cm ² , for example, "Installation of UK 25-1.6M";
centrifuge;
a filtering device that allows filtering in the forward and reverse direction (see figure 1).

 Preparation of substrate and test blood plasma.

 1. Preparation of substrate plasma.

On the day of the study, lyophilized plasma produced by the Kirov Research Institute of Hygiene and Epidemiology is diluted with distilled water to its volume prior to drying. For research, 3-10 ml of substrate plasma is needed. It should be noted that autologous membranes of substrate plasma at this stage are included in its composition, but they are discarded during work with the filtering device (see the course of determination);
2. Preparation of the test plasma sample.

 With a silicone needle, blood is drawn from a vein into a test tube containing 3.8% sodium citrate. Blood is mixed with an anticoagulant in a ratio of 9: 1 (9 ml of blood and 1 ml of anticoagulant) and centrifuged at 1500 rpm (180 g) for 5 minutes. The resulting platelet rich plasma was transferred to another tube and centrifuged again at 3000 rpm (700 g) for 20 minutes. The result is a platelet-poor plasma that is used for analysis.

 The course of determination.

In section A of the plasma filtering device (FIG. 1, step 1), 1.0 ml of the test plasma is introduced and filtered under a pressure of 0.8-1.0 kg / cm ² . Filtered plasma is discarded. To wash the membrane deposited on the filter from plasma residues in the same (forward) direction and under the same pressure, 1.0 ml of the medial buffer is passed. Further (Fig. 1, stage 2), a substrate plasma in a volume of 1.0 ml is passed under pressure in a reverse direction through a filtering device, it is collected in a test tube and used for research (determination of recalcification time).

 As a result of this operation, the membranes of the studied sample deposited on the filter and washed from the remnants of the studied plasma are transferred to the substrate plasma, and the autologous membranes of the substrate plasma remain on the filter.

The time of recalcification of substrate plasma is carried out with standardization by factor XIII contact with kaolin (see Clinical and laboratory methods in hematology. Edited by V. G. Mikhailov and G. A. Alekseev. Tashkent, Medicine. 1986). For this, 0.1 ml of substrate plasma taken in a test tube is mixed with 0.1 ml of a kaolin suspension. The tube was placed in a water bath (37 ° ^C) and after 3 minutes to the above mixture was added 0.1 ml of calcium chloride solution, immediately start the stopwatch and note the time of coagulation in seconds (up to the appearance of fibrin strands). From the calibration curve (figure 2), knowing the time of recalcification of the substrate plasma, find the MASK of the studied plasma sample (%).

 Construction of a calibration curve.

 A mixture of equal volumes of platelet-poor plasma from 5 healthy donors is prepared. The content (coagulation activity) of the membranes in this plasma mixture is taken as 100%. Then, by filtering in the forward and backward direction by varying the volume of the studied (donor) plasma with the same volume (1.0 ml) of substrate plasma, we obtain substrate plasma samples with different contents ( activity) membranes. So, when filtering during stage 1 (Fig. 1) 0.25 ml, 0.5 ml, 1.0 ml, 2.0 ml and 4.0 ml of the studied donor plasma and washout of membranes obtained from these plasma volumes, 1 ml of substrate plasma (stage 2), prepare samples of substrate plasma having membrane activity of 25, 50, 100, 200 and 400%, respectively. Determination of recalcification time in the listed samples allows you to build a curve of the interdependence of these indicators.

 An example of constructing such a curve in logarithmic coordinates is presented in FIG. 2 (used donor standard plasma of the Kirov NIIIGiPK, series 030492).

 It should be noted that a single construction of a calibration graph is sufficient when working with the same batch of lyophilized dried standard plasma over its shelf life.

 Methods for preparing plasma with various coagulation potential, having normal (100%) coagulation activity of membranes.

 1. Prepare several samples of platelet-poor plasma from healthy donors and patients. Each sample is placed in a filtering device and passed under pressure through the filter in the forward direction in accordance with step 1 (Fig. 1), while achieving removal of autologous membranes. Filtered samples determine the recalcification time and take samples, the analysis of which is 180-195 s (normocoagulation in healthy donors), less than 165 s (hypercoagulation shift) and more than 210 s (hypocoagulation shift).

 2. Prepare a mixture of equal volumes of platelet-poor plasma from 5 healthy donors. The content (coagulation activity) of the membranes in this mixture is taken as 100%. The mixture in a volume of 1.0 ml is placed in a filtering device and passed under pressure through a clean filter in the forward direction (Fig. 1, step 1). Filtered plasma is discarded, and the filter (after washing by passing through it 1.0 ml of the medinal buffer) is used as a source of 100% membrane activity for a given plasma volume of 1.0 ml. To introduce the obtained membranes into a plasma with different coagulation potential, pre-filtered plasma samples with hyper-, hypo-, and normocoagulation (in a volume of 1.0 ml) are passed under pressure through a filtering device in the opposite direction (Fig. 1, step 2). The result is plasma samples with hyper-, hypo-, and normocoagulation potential and normal (100%) in all cases coagulation of the membranes (see table).

 In healthy donors, MASK according to the claimed method varies in the range from 86 to 112% on average 99.5 ± 4.8%. In patients with pathology of the hemostatic system, MASK increases (with thrombosis and DIC), or, conversely, decreases (with hemophilia, primary antiphospholipid syndrome, etc.) in comparison with the norm.

PRI me R 1. Patient N. 27 years. Diagnosis: 38 weeks pregnancy, premature placental abruption, DIC. Membrane activation of blood coagulation was 270%
PRI me R 2. Patient G. 68 years. Diagnosis: condition after surgery for prostate adenoma. Membrane activation of blood coagulation was 185%
PRI me R 3. Patient T. 19 years. Diagnosis: 8 weeks pregnancy, primary antiphospholipid syndrome. Membrane plasma coagulation activity was 16%
The determination of MASK using the proposed method, as well as using a well-known prototype, is carried out by assessing the coagulation effect of the membranes of the test sample on a substrate plasma lacking autologous membranes. However, the inventive method for determining MASK by cleaning the membranes of the test sample from plasma impurities can improve the accuracy of the method (see table) by 36% (with hypercoagulation) and 66% (with hypocoagulation), which is achieved by eliminating the influence of the coagulation potential of the plasma impurity under study sample.

 In addition, it should be noted that the optimal procedure for obtaining substrate plasma purified from autologous membranes is the filtration procedure in comparison with ultracentrifugation, since the latter requires sophisticated equipment and additional room, and the time required to obtain substrate plasma is 3-5 min, respectively and 1.5-2 hours

 The proposed method is simple, standardized (through the use of reference freeze-dried plasma of healthy donors), performed in a short period of time (10-15 minutes) and can be used in laboratories of scientific and practical medical institutions of various types.

Claims (1)

 METHOD FOR DETERMINING BLOOD PLASMA CLOSING ACTIVATION FROM CELL MEMBRANE FRAGMENTS, including determining the recalcification time in a mixture of a test sample with a substrate plasma, characterized in that the recalcification time is determined in a substrate plasma containing fragments of cell membranes of a test sample, and their according to the time of recalcification of substrate plasma on the coagulation activity of fragments of cell membranes.

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