Method of determining quantity of fibrin-monomers in blood

FIELD: medicine.

SUBSTANCE: quantity of fibrin-monomers, dissolved in 0.5 N sodium hydroxide, is determined spectrophotometrically with application of ethanol test. Claimed method of quantitative determination of fibrin-monomers in blood makes it possible to reveal pathological process in organism with 95% reliability.

EFFECT: increase of determination accuracy.

2 tbl, 4 ex

 

The invention relates to medicine, namely to biochemical method for determining the concentration of components of the coagulation and products of their transformation in biological fluids.

In many diseases and pathological conditions (hemorrhagic and ischemic strokes, atherosclerosis, myocardial infarction, surgical trauma, cesarean section, and so on) there is a need for quantitative determination of hybridmonolith and degradation products of fibrin in plasma, however, to date only apply qualitative methods of determining the presence of hybridmonolith in plasma.

There is a method of identifying hybridmonolith in plasma for the formation of gelatinous masses under the influence of 50% ethanol [Lychev VG Diagnosis and treatment of disseminated intravascular coagulation. - M.: Medicine, 1993. - 160 S.]. Releasetest plasma 10 min after addition of 50% ethanol is considered as a sign of the presence of hybridmonolith. If form coarse particles, then the result is evaluated as slabopolozhitelnym or negative. The method does not allow to determine the concentration of hybridmonolith.

Angomont et al. proposed method for the determination of hybridmonolith and degradation products of fibrin on education in the plasma grains (procoagulant) fibrin after you add the possible solution phenanthroline [Moment A.N., Lakomov VA, Barkagan SS Clinical and laboratory diagnosis. 1996. No. 4. - P.17-20]. The main disadvantages of the method are its subjectivity and the presence of false positive results that arise, in particular, as a result of insufficient mixing of blood and citrate or storage of plasma for more than 1 hour, which leads to inactivation of coagulation factors.

The present invention is to develop an objective method for determining the number of hybridmonolith in plasma.

The technical result is simple, does not require high material costs, based on spectrophotometric determination of hybridmonolith.

The technical result is achieved in that the precipitate plasma homogenized in 0.5 N sodium hydroxide spend spectrophotometric analysis of suspension and the number of hybridmonolith determined by phrogram.

The positive effect of the proposed method achieve due to the quantitative determination of hybridmonolith spectrophotometrically. Comparative characteristics of the proposed method and the method of the prototype are presented in table 1.

The proposed method is as follows.

From the cubital vein blood sample in a plastic test tube containing 3.8% solution of sodium citrate 3-substituted (sodium citrate). The ratio of the volume of the RH blood and citrate - 9:1 (4.5 ml blood and 0.5 ml of sodium citrate). The contents centrifuged at 4000 rpm for 10 min (2000g). The result is a platelet-poor plasma, then 0.4 ml of blood plasma is added 0.15 ml of 50% ethanol and centrifugum at 3500 rpm for 15 minutes Received centrifugal (sludge) wash of 0.14 M solution of sodium chloride and centrifugum twice at 3.500 rpm for 10 min. the precipitate homogenizers in 7 ml of 0.5 n solution of sodium hydroxide within 1-2 min and placed in a quartz cuvette for spectrophotometry at 280 nm transmittance and phrogram determine the number of hybridmonolith. Fluctuations in the extinction from 0.1 to 0.8 indicate concentrations hybridmonolith in plasma.

Examples of the practical use of the proposed method

Example 1

In the neurological Department Bureau No. 2 Tyumen entered patient Shcherbakova A.I., 68 years old with the diagnosis of Ischemic stroke" (moderate severity).

Patient blood samples were taken from the cubital vein into a syringe with a stabilizing solution (3,8% aq sodium citrate) in the ratio 1:9. A sample of 5 ml was placed in a centrifuge tube and centrifuged with acceleration 2000g for 10 min, then 0.4 ml of plasma was transferred to another tube and was added 0.15 ml of 50% ethanol. Included stopwatch and after 10 min was noted by the appearance of grains of procoagulant, then centrifuge holds Aravali at 3500 rpm 15 minutes Received centrifugal (sludge) were washed of 0.14 M sodium chloride solution and twice centrifuged at 3500 rpm (acceleration 1500g) for 10 min Decanted removed. The precipitate homogenized in 7 ml of 0.5 n solution of sodium hydroxide within 1-2 min and were placed in a quartz cuvette for spectrophotometry at 280 nm transmittance and phrogram determine the number of hybridmonolith. Concentration of hybridmonolith in plasma were judged by the magnitude of the refraction of light. Registered transmittance - 0,41.

Example 2

Sick men E.N., 46 years was delivered to the Department of reanimacii design Bureau No. 2 Tyumen SMP machine in an emergency. Clinically diagnosed with "Ischemic stroke" (high severity).

From the cubital vein blood samples were taken in a plastic test tube containing 3.8% solution of sodium citrate 3-substituted (sodium citrate). The ratio of the volume of blood and sodium citrate to 9:1 (4.5 ml blood and 0.5 ml of sodium citrate). The contents were centrifuged at 4000 rpm for 10 min (2000g). The result made a club platelet-poor plasma, then 0.4 ml of plasma was added 0.15 ml of 50% ethanol and centrifuged at 3500 rpm for 15 minutes Received centrifugal (sludge) were washed of 0.14 M sodium chloride solution, and then centrifuged twice at 3.500 rpm for 10 min. the precipitate homogenized in 7 ml 05 N. solution of sodium hydroxide within 1-2 min and were placed in a quartz cuvette for spectrophotometry at 280 nm transmittance and phrogram determined the number of hybridmonolith. The transmittance is 0.76, indicating that the concentration of hybridmonolith in plasma.

To quantify the content of hybridmonolith in the blood plasma used phrogram, which is in units of activity reflects Concentratio of hybridmonolith, corresponding to 6.0 EA.

Example 3

In the neurological Department Bureau No. 2 Tyumen entered patient Lagutina L.A., 48 years with a diagnosis of Ischemic stroke" (mild severity).

From the cubital vein blood samples were taken in a plastic test tube containing 3.8% solution of sodium citrate 3-substituted (sodium citrate). The ratio of the volume of blood and sodium citrate to 9:1 (4.5 ml blood and 0.5 ml of sodium citrate). The contents were centrifuged at 4000 rpm for 10 min (2000g). As a result he received platelet-poor plasma. Then 0.4 ml of plasma was added 0.15 ml of 50% ethanol and centrifuged at 3500 rpm for 15 minutes Received centrifugal (sludge) were washed of 0.14 M sodium chloride solution and centrifuged twice at 3500 rpm for 10 min. the precipitate homogenized in 7 ml of 0.5 n solution of sodium hydroxide within 1-2 minutes Received) suspension was placed in a quartz Kyu the etu for spectrophotometry. Used a wavelength of 280 nm.

To quantify the content of hybridmonolith in the blood plasma used phrogram, which reflects the light transmission in units of activity. Light transmission rate amounted to 0.27, and according to phrogram - 2,2 EA.

Example 4

Patient eagles E.A., 34 years, was delivered to the Department of reanimacii design Bureau No. 2 Tyumen SMP machine in an emergency. Clinical diagnosis of Ischemic stroke high severity".

Patient blood samples were taken from the cubital vein into a syringe with a stabilizing solution (3,8% aq sodium citrate) in the ratio 1:9. A sample of 5 ml was placed in a centrifuge tube and centrifuged with acceleration 2000g for 10 min, then 0.4 ml of plasma was transferred to another tube and was added 0.15 ml of 50% ethanol. Included stopwatch and after 10 min note the appearance of grains of procoagulant, then centrifuged at 3500 rpm for 15 minutes Received centrifugal (sludge) were washed of 0.14 M sodium chloride solution and twice centrifuged at 3500 rpm (acceleration 1500g) for 10 min Decanted removed. The precipitate homogenized in 7 ml of 0.5 n solution of sodium hydroxide within 1-2 min and were placed in a quartz cuvette for spectrophotometry at 280 nm transmittance and phrogram determined the number of hybridmonolith. On the concentration of the emission of hybridmonolith in plasma judged also by the magnitude of the refraction of light. Registered transmittance - 0,79.

The examples illustrate the applicability of this method for the quantitative determination of hybridmonolith in plasma on the basis of spectrophotometric analysis and results phrogram. Altogether there were 20 studies with blood plasma from patients undergoing treatment in the regional clinical hospital №2 Tyumen (table 2). The positive effect of applying the proposed method in comparison with the prototype presented in table 2. The proposed method allows to detect the pathological process in the body with a certainty of 95%.

The method of quantitative determination of hybridmonolith in the blood

Table 1
Comparative characteristics determine hybridmonolith blood on the proposed method and prototype
SignsThe proposed methodPrototype method
1. Ispolzovanie reagents for research50% solution of ethanolsolution phenanthroline
2. Processing plasma for research Repeated centrifugationNot done
3. Resolution methodQuantitative determination of hybridmonolithQualitative determination of hybridmonolith
4. Objectivity of researchSpectrophotometric determination of hybridmonolithVisual definition of research results
5. Determining the severity of the pathological process in order to assign specific treatmentThe possibility of using the method to assign specific treatmentSpecific treatment is not provided

The method of quantitative determination of hybridmonolith in the blood

Table 2
Fluctuations in the readings of the optical density in patients with ischemic stroke
The sequence numberThe indicators light transmissionEA per ml
10.31 3.1
20.4424.2
30.4524.3
40.434.2
50.696.1
60.4114.1
70.2682.5
80.7616.5
90.3153.1
100.3613.4
110.5725.3
120.7036.4
130.5685.2
140.5755.3
150.5215.1
160.3453.2
170.3953.9
180.4614.5
190.4624.5
200.7947.0

Method of determining hybridmonolith in the blood, including the use of an ethanol test, characterized in that after treatment of plasma with ethanol, centrifugation and washing the precipitate with sodium chloride re-centrifugation, the precipitate homogenized with 0.5 N sodium hydroxide within 1-2 minutes, and the result take into account on the basis of spectrophotometric analysis at 280 nm transmittance and the number of hybridmonolith determined by ferrogram.



 

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