RU2189590C2 - Method for detecting plasminogen - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics. SUBSTANCE: method deals with blood testing in case of disorders in hemostasis system. The method includes determination of the terms for fibrinous clot lysis at plasminogenic activation by streptokinase. Moreover, fibrin- monomer at 1.0-10.0 g/l concentration is used as a source of fibrinous clot development. The method enables to detect plasminogenic concentration according to the terms of fibrinous clot lysis despite fibrinogenic concentration in the plasma tested. EFFECT: higher efficiency and accuracy of detection. 1 dwg, 4 tbl

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 enzyme system that causes the breakdown of fibrinogen and fibrin is known as the fibrinolytic or plasmin system. Its main active principle is plasmin, a protease contained in plasma in the form of a proenzyme, plasminogen.

 A known method for determining plasminogen based on hydrolysis of the nitroamide bond of a chromogenic substrate with the formation of a colored substance (Momot A.P., Mamaev A.N., Barkagan Z.S., Nevedrova O.E., Makarov V.A., Vojushchina T. L ., Erin D.N. Method for determination of plasminogen and its diagnostic value. // Problems of Hematology, 1999. - 1. - P. 17-20).

 The disadvantage of this method is the complexity, high cost and the need to use special equipment for photometric analysis. In addition, amidolytic methods for the determination of enzymes, as is known, do not always coincide with the results of functional determinations, which may be due to the insufficient specificity of the chromogenic substrate.

 The closest to the achieved positive result (prototype) is a method for determining plasminogen by euglobulin lysis when activated by streptokinase (Barkagan Z.S., Momot A.P. Basics for the diagnosis of hemostasis, 1999. - P. 126-128).

 The prototype method is as follows.

 Pre-receive a platelet-poor control normal plasma and platelet-poor patient plasma.

 Stage 1. Obtaining euglobulin plasma fraction.

To obtain the euglobulin fraction, 0.15 ml of 1% acetic acid and 0.5 ml of the test plasma are added to 8.0 ml of distilled water. The mixture is maintained at a temperature of +2 ... + 8 ^o C for 30 minutes;
2 stage. Precipitation of the euglobulin fraction of plasma.

 Euglobulins are precipitated by centrifugation at 1500 rpm (240 g) for 7 minutes. The supernatant is drained, the tube is dried by tipping over onto filter paper.

 3 stage. Obtaining a fibrin clot of euglobulins and determining the time of its lysis.

The precipitate of euglobulins is diluted in 0.5 ml of Tris-HCl buffer (0.1 M, pH 7.4), the tube is installed in a water bath (+37 ^o C). Then, without removing the tube from the bath, add 0.1 ml of streptokinase solution (300 IU / ml) and 0.1 ml of thrombin solution (50 NIH units / ml), start the stopwatch and record the time (LIS) from the moment the solution was added calcium thrombin until the fibrin clot is completely dissolved.

 The control normal plasma is examined in a similar manner, the obtained result of the lysis time is designated as LIS-k.

где ЛИС-к - время лизиса эуглобулинов в контрольной нормальной плазме, ЛИС-и - то же в исследуемом образце.The plasminogen reserve index (RPI) is determined by the formula

where LIS-k is the time of lysis of euglobulins in the control normal plasma, LIS-i is the same in the test sample.

 Normal IRP is on average 100 ± 10%.

The disadvantages of the prototype method include:
1. The complexity, multi-stage and, associated with this, a significant expenditure of time during the study.

 2. The inability to quantify plasminogen.

 3. The dependence of the results not only on the concentration of plasminogen, but also on the concentration of fibrinogen in the patient’s plasma.

 The inventive method is devoid of these disadvantages: it is carried out in one step, which significantly reduces the study time, allows a quantitative assessment and does not depend on the concentration of fibrinogen in the studied plasma.

 The authors have developed a highly effective method for determining plasminogen, which allows determining the plasminogen concentration by the time of lysis of the fibrin clot, which does not depend on the concentration of fibrinogen in the studied plasma.

 A positive result of the proposed method is to increase the accuracy of determination of plasminogen. A positive result is achieved by using a fibrin monomer at a concentration of 1.0-10.0 g / l as a source of fibrin clot formation.

 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. Fibrin monomer from human blood plasma (freeze-dried), Tekhnologiya-Standart, Russia.

 3. Streptokinase (lyophilized), 1500 ME, Tekhnologiya-Standart, Russia.

 4. Tris-HCl buffer concentrated (20: 1), 1.0 M, pH 7.3-7.4, Technologiya-Standart, Russia.

 5. Kaolin (light fraction), a suspension with a concentration of 50 mg / ml of distilled water, the company "Technology Standard", Russia.

 6. Distilled water.

 7. Centrifuge OPN-8.

 8. Thermostat for the study of hemocoagulation (TPN).

 9. Stopwatch.

 10. Glass tubes with a volume of 20 ml.

 11. The cylinder glass measured on 100 ml.

Reagent preparation:
1. Dilution of the fibrin monomer: 2.0 ml of distilled water is added to the vial with the freeze-dried fibrin monomer and, with gentle swaying, the reagent is dissolved for 10 minutes.

 2. Dilution of streptokinase: 5.0 ml of distilled water is added to the vial with freeze-dried streptokinase and the reagent is dissolved with gentle swaying for 2 minutes.

 3. Preparation of a working buffer solution: A concentrated buffer in a volume of 1.0 ml and a suspension of kaolin in a volume of 2.0 ml are introduced into a graduated cylinder, and the total volume is adjusted to 100 ml with distilled water. The result is a buffer solution with the following composition: Tris-HCl 0.01 M, pH 7.3-7.4, containing kaolin at a concentration of 1.0 mg / ml.

 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 speed of 1000 rpm (200 g). The resulting platelet-rich plasma is re-centrifuged for 20 minutes at 4000 rpm (1200 g). 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. The plasminogen content in such a plasma pool is taken as 100% of the norm.

 The course of determination.

Add 0.1 ml of the test plasma to the test tube and incubate in a water bath for 1 min at +37 ^o C. Then, 0.1 ml of streptokinase solution, 0.8 ml of buffer (having a temperature of +37 ^o C) and 0.1 ml are successively added. fibrin monomer solution, immediately turn on the stopwatch and incubate in a water bath at +37 ^o C. Periodically removing the tube from the water bath (without shaking) and tilting it, note the time from the moment the fibrin monomer solution was added until the clot lysis was completed.

 The calibration curve is used to find the plasminogen content (in%).

 Construction of a calibration curve. To build a calibration curve, prepare a series of dilutions of the control normal plasma in accordance with table 1.

 With each of the dilutions of the control normal plasma pool, the lysis time is determined by the above procedure. All measurements are carried out three times, the average result is calculated. When constructing a calibration curve along the ordinate axis, the lysis time (in seconds) is postponed, and the abscissa axis shows the plasminogen level (in%) in accordance with the above dilutions. The calibration curve is shown in the drawing.

 Evaluation of the results. According to the results of a survey of healthy people (45 observations), the fibrin clot lysis time is 48-55 s (X = 51.2; σ = 1.65; m = 0.34). Moreover, the level of plasminogen (according to the calibration curve) ranges from 86 to 113% (X = 99.8%; m = 1.39%; σ = 6.84%).

 Testing of the proposed method.

 The testing of the proposed method was carried out on 25 patients with acute DIC-syndrome, including post-traumatic (8), septic (14), obstetric (3). Patient examination data are presented in table 2.

 It can be seen from it that the concentration of plasminogen in the plasma of patients with acute DIC syndrome, characterized by the consumption of plasminogen, naturally decreases.

 Assessment of the effect of fibrinogen concentration on the performance of the prototype method and the proposed method.

As you know, normal plasma fibrinogen concentration is 2.0-4.0 g / L. The pooled control normal plasma was heated at a temperature of +56 ^o C for 6 min, thus achieving the removal of fibrinogen. Standard amounts of dry fibrinogen preparation (Behringer, Germany) were added to this fibrinogen-free plasma in order to obtain a series of plasma concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 g / l (table 3).

 As can be seen from table 3, the measurement error of the prototype method under conditions of hypofibrinogenemia (comparison of results at a concentration of fibrinogen of 1.0 and 2.0 g / l) is + 28.4%, and with hyperfibrinogenemia (comparison of results at a concentration of fibrinogen 4, 0 and 7.0 g / l) equals - 26.1%.

 In comparison with this, the error in determining the plasminogen concentration by the proposed method for hypofibrinogenemia is + 4.6%, and for hyperfibrinogenemia (a comparison of the results with fibrinogen concentration of 4.0 and 7.0 g / l) is equal to 0.8%.

 The rationale for the selected concentration range of fibrin monomer to determine the concentration of plasminonogen in the proposed method.

 To select the required concentration of fibrin monomer in determining the concentration of plasminogen used samples of a solution of fibrin monomer with a concentration of from 0.5 to 12.0 g / l (table 4). It was found that the optimal concentration of fibrin monomer is in the range from 1.0 to 10.0 g / L. The decrease in the concentration of fibrin monomer below 1.0 g / l led to the fact that the clot did not form. On the other hand, the use of a fibrin monomer concentration in excess of 10.0 g / l led to the formation of an excessively dense clot, which practically did not lend itself to lysis.

 The specificity of the method and its reproducibility.

 Thus, the claimed method is highly accurate in determining the concentration of plasminogen. The pathological level of fibrinogen in the test plasma does not affect the indications of the proposed method.

 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 does not exceed 5%.

Claims (1)

 A method for determining plasminogen, including determining the lysis time of a fibrin clot upon plasminogen activation by streptokinase, characterized in that a fibrin monomer in a concentration of 1.0-10.0 g / l is used as a source of fibrin clot formation.

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