SU1146002A1 - Method of quantitative determination of fibrinogens in blood plasma - Google Patents

Abstract

A METHOD FOR QUANTITATIVE DETERMINATION OF FIBRINOGEN IN THE BLOOD PLASMA, including the closure of the clotting of the coagulation with plasma fibrinogen, followed by a quantitative determination of coagulated fibrinogen, which in order to increase the accuracy of the method, is determined by the failure of the bearers, and in the rest of the screen, it is defined as foldable and uncharacterized, with the help of the method, which can be used to reduce the accuracy of the method, which means that the volume of the coagulated fibrinogen should be reduced, and in the case of reproducibility of the accuracy of the method, it is determined that the volume of the coagulated fibrinogen should be reduced. polysculate efy (Echis multisguamatus).

Description

This invention relates to medicine, in particular to the laboratory diagnosis of bleeding disorders.

A known method for determining plasma fibrinogen concentration by salting out ammonium sulfate followed by grinding the coagulum by a photocalorimetric or spectrophotometric method 1.

However, this method does not provide a selective determination of fibrinogen due to the partial precipitation of other plasma proteins.

The closest to the invention in its technical essence and the achieved result is a method for quantitative determination of plasma fibrinogen content, including fibrinogen coagulation by the natural enzyme thrombin, which has a specific effect on fibrinogen and Not coagulates other proteins, with the next photo calorimetric determination of Circle Circuit .

The disadvantages of this method are the impossibility of determining the TOI part of fibrinogen, which has lost the ability to coagulate with thrombin, as well as the difficulty of collecting without loss) weakly consolidated clots formed in the syndrome of disseminated intravascular coagulation of blood and other types of pathology.

The purpose of the invention is to increase the accuracy of the method.

This goal is achieved by the fact that according to the method of quantitative determination of fibrinogen in plasma, including coagulation of plasma fibrinogen with a coagulating agent followed by a quantitative determination of coagulated fibrinogen, both coagulable and non-clotted thrombin fibrinogens are used as a co-scientist asparagistur is determined by a body builder who is coagulated.

(Echis raultisguamatus).

-.- iExample 1. KO, 2 ml of citrate plasma was added 4.8 ml of medaline buffer (pH 7.4), the mixture was incubated for 5 min, then 0.1 ml of working solution iiiHoroscale syrup (1-1 (J-5. -10 mg / ml) mix the contents of the tube with a stick and incubate for 10 min.

at. Then, the clot formed in the tube is removed, washed in cold NaCl solution, dried, pressed on filter paper, and transferred to a tube with 0.2 ml of a 0.1 M solution of NaOH. The tube is placed for 10 minutes in a boiling water bath until the clot is dissolved, then 6 ml of a 12.5% solution and 1 ml of 0.1 M CuSO solution are introduced into the test tube, stirred for 10 minutes, 1 ml of Folina solution is added. Chikalto, diluted 1: 2 with distilled water and after 30 minutes. The optical density of the solution was determined on a photoelectric calorimeter with a red filter. According to the scion of dilutions of fibrinogen, the optical density indices translate the amount of fibrinogen, g / l.

The poison of the snake epha multi-scaly (Echis multisguamatus), has a hemocoagulating effect, differs from thrombin by the following properties: its action is not blocked by heparin and antithrombin III-heparin complex, it coagulates the so-called blocked or thrombin-resistant fibrinogen, i.e. fibrinogen incorporated into fibrin-monomeric complexes.

Example 2. Comparative determination of the concentration of fibrinogen by thrombin and the house of polysplate effa when blocking the coagulation of heparino

Studies on mixed normal plasma (source of fibrinogen).

Reagents: mixed normal plasma with 3.5 g / l fibrinogen; source of antithrombin Ш-defibrinated plasma (plasma, incubated at 56 ° С and 5 min); thrombin solution with an activity of 20-5 s; working solution yes; dilution of heparin 0.1-1.0 u / ml. one

A parallel determination of fibrinogen concentration is carried out by known and proposed methods at various levels of heparinization of the plasma samples tested.

The results of the studies are presented in Table. TO

From tab. 1, it can be seen that, against the background of heparinization, the amount of fibrinogen thrombin contracted progressively decreases, starting with a heparin concentration of 0.3-0.4 units / ml, and at 0.6 units / ml it is not determined due to complete blocking of coagulation . AT

by contrast, when used as a coagulating agent on Echis muktiseuamatus. heparinization does not affect the quantitative determination of fibrinogen in those doses that completely block natural coagulation.

Thus, the proposed method allows to determine the concentration of fibrinogen in the plasma, even with very intensive heparinization of patients, giving complete non-clotting of blood.

Studies on purified fibrinogen solution.

Reagents: a solution of purified fibrinogen containing 94% of the coagulated protein (according to Lowry) at a concentration of 4.0 g / l; source of antithrombin Ш-defibrinated plasma; thrombin solution, with activity; working solution and efy; dilution of heparin 0.1-1.0 u / ml.

The parallel determination of fibrinogen concentration is performed by known and proposed methods at different levels of heparinization (Table 2).

From tab. 2 that when coagulation with thrombin, heparin causes a decrease in the amount of detectable fibrinogen, while using yes, it reveals all fibrinogen introduced into the system (4.0 g / l). I

Example 3. Comparative

determination of the fibrinogen content in coagulation with thrombin (in a known manner) and the house of the snake Echis multisguamatus (in the proposed method) in patients with the syndrome of disseminated intravascular coagulation of blood (DIC).

In these patients, more or less pronounced blocking of fibrinogen in the fibrin-monomer complexes was detected, which was established by determining the degradation products

plasma fibrinogen, and according to positive results, ethanol-log and protamine-sulfate tests. Contraceptive studies were performed on the plasma of healthy people. In the two experimental groups, there were 35 patients, and in the control group, 20 healthy people. The first experimental group consisted of patients with DIC-syndroms in the hypocoagulation phase (20 people), in the second experimental group (15 people), patients with DIC-syndromes with complete blood clotting.

The results of the research are presented in table. 3

From tab. 3, it can be seen that in healthy people, approximately the same amount of fibrinogen is determined during coagulation with thrombin (a known method) and house of an epha (the proposed method). In contrast, in patients with the help of yes, the amount of fibrinogen was significantly higher than during thrombin coagulation. After coagulation with thrombin and removal of the formed clots with the additional administration of Echis multisguamatus, a second clot of thrombin-resistant fibrinogen was formed. In different cases, the amount of such blocked fibrinogen averaged 1.27-0.78 g / l, i.e. reached significant values. In the study of patients in the phase of complete incoagulability by thrombin, plasma coagulation was not possible, and q epha caused the formation of a dense consolidated fibrin clot. Conducting the ethanol test after coagulation with thrombin revealed fibrin-monomer complexes in patients with DIC in 80% of cases. In contrast, after the coagulation of the house of etha, the ethanol test is always negative, which indicates precipitation of blocked fibrinogen and fibrin-monomeric complexes with this coagulating agent.

EXAMPLE 4 Practical use of the proposed method for determining the fibrinogen content in the blood plasma of patients.

Sick K.N., 25 years. Diagnosis: pregnancy 29 weeks, intrauterine fetal death, DIC, uterine bleeding with unsubstituted blood.

The results of a laboratory study of the hemostatic system: autocoagulant test for 10 minutes 35 seconds: kaolin-kefaline clotting time 87 seconds (control 45 seconds); prothrombin time 42 s (control 17); thrombin time 73 s (control 20 s); ethanol test is positive.

The fibrinogen content during coagulation with thrombin was 0.70 g / l.

The conclusion was made about severe hypofibrinogenemia. The definition of house efa showed 2.5 g / l fibrinogen. Thus, using yes you see

There was a significant amount of fibrinogen, not thrombin-convertible), and therefore it was concluded that there was no true hypofibrinogenemia and the presence of a large amount of blocked fibrinogen in plasma, the amount of which was estimated at 2.5 g / l - 0.7 g / l 1 , 8 g / l. The ethanol test after coagulation with thrombin remained positive, and after coagulation the house of Epha became negative. This allowed us to prescribe adequate treatment.

Patient K.S., 23g. Diagnosis; condition after surgery for pulmonary artery branch aneurysm, disseminated intravascular coagulation syndrome, massive bleeding from postoperative suture, and body sync.

The results of a laboratory study of the hemostatic system: autocoagulation test for 10 minutes - 17 seconds; kaolin-kefalin coagulation time of 50 s (control 45 s); prothrombin time 25 s (control 18 s); thrombin time 23 s (control 15 s); ethanol test is positive; when the clotting of thrombin formed a poor-quality fine-grained clot, which could not be squeezed and dried, the house of epha was determined to be 3.5 g / l fibrinoGen. It was concluded that massive intravascular coagulation of blood with the formation of fibrin-monomer complexes and blocking of fibrinogen in them. A60026

Patient RA, 45 years old. Diagnosis: osteo-undifferentiated leukemia, DIC, hemorrhage in the brain with hemiparesis, gastric hemorrhage. 5 Sin Ki on the body, gross hematuria.

I

The results of the laboratory study of the hemostatic system: the autocoagulant test for the 10th minute does not have curvature; kaolin-kefalin coagulation time - no coagulation (control 50 s); protorombin time 75 s (control 18 s); thrombin time 110 s (control 15 s);

15 ethanol test positive; fibrinogen at coagulation with thrombin was 0.5 g / l, while coagulation was 4.5 g / l at home. The amount of blocked fibrinogen was

20. 4.5-0.5 4.0 g / l. Prescribed treatment corresponding to the phase of DIC.

The proposed method provides an increase in the accuracy of determination of plasma fibrinogen concentration due to coagulation of both graft and thrombin-non-clotting (thrombin-resistant) fibrinogen, which is important in the diagnosis of massive thrombosis, disseminated intravascular coagulation - blood, differentiation of these conditions, and of these conditions, it is important to diagnose massive thrombosis, disseminated intravascular coagulation. which is very important for diagnosis and treatment.

Thrombin coagulated 3,5 3,5 3,0 2,2 1,3 Multiple cochlear house 3.5 3.5 3.5 3.5 3.5 No coagulation 3.5 3.5 3.5 3.5 3.5

Claims (1)

METHOD FOR QUANTITATIVE DETERMINATION OF FIBRINOGEN IN BLOOD PLASMA, including coagulation of plasma fibrinogen with a coagulating agent, followed by quantitative determination of coagulated fibrinogen, characterized in that, in order to improve the accuracy of the method, both coagulable and non-coagulable coagulation are used, and they use ephas (Echis multisguamatus).