RU2732388C1 - Method for determining fibrinogen and functionality evaluation thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to laboratory diagnostics and can be used to determine fibrinogen and evaluate its functionality. Method for determining fibrinogen (FG) during recalcification of citrate plasma and its functionality involves activating a coagulation contact path in polystyrene 96-well flat-bottomed immunoassay plates by mixing citrated blood plasma with calcium chloride followed by photometric recording of coagulation, wherein to intensify fibrinogen coagulation, osmolarity is reduced in the test sample by adding 50 mcl of distilled water to 150 mcl of the test system, 50 mcl of VBS buffer is added in the control sample instead of water, coagulation reaction is started by addition of 50 mcl 25 mM CaCl₂ into sample, thoroughly mixed, optical density of samples is measured, then incubated for 120 min at 37 °C, determination of plasma coagulation is carried out photometrically by turbidity change of samples at wave length of 450 nm with measurement intervals of 0 and 120 minutes, in initial plasma content of FG is determined by Klaus method, calculating an individual coefficient for transfer of changes in optical density in the sample during coagulation as a relation of FG by Klaus to change in the optical density of the sample (ΔA₄₅₀), average coefficient and fibrinogen content are calculated by formula: FG (g/l) = ΔA₄₅₀ × 4.9, where: ΔA₄₅₀ is change of optical density of the sample during plasma coagulation; 4.9 is the average value of the coefficient for transforming the optical density of the sample in plasma coagulation in g/l of fibrinogen; the fibrinogen dysfunctions are determined as the difference between the amounts of fibrinogen determined by the given method and method.

EFFECT: invention provides a simple, informative and specific test for determining fibrinogen and its functionality and enables wide screening and detecting people prone to thrombosis.

1 cl, 4 tbl, 2 dwg

Description

Fibrinogen is the main blood plasma glycoprotein required for blood clotting. The amount of fibrinogen present in plasma is directly proportional to the blood's ability to form blood clots. Fibrinogen is transformed into fibrin during thrombus formation, so fibrinogen deficiency will result in insufficient fibrin formation and impaired blood clotting.

The lack of fibrinogen in the blood plasma can be caused by a decrease in the formation of fibrinogen, due to hepatic cell failure or congenital hypofibrinogenemia. Disseminated intravascular coagulation of blood, which is a pathological condition, leads to an increased consumption of fibrinogen, which is suitable for use in normal thrombus formation. Obviously, a lack of fibrinogen can lead to bleeding.

An excess of fibrinogen in the blood also represents a pathological condition that is usually associated with inflammatory processes. The high content of fibrinogen in the blood can contribute to the occurrence of hypercoagulation, which is undesirable for the body.

It was found that an increase in the concentration of fibrinogen in the blood is also a risk factor for the occurrence of cardiovascular diseases. From the above, it is obvious that monitoring the content of fibrinogen in the blood is an informative test for assessing the state of human health, and can be useful as a marker for detecting the pre-thrombotic state in the human body before vascular catastrophes.

In clinical practice, the most widely used determination of fibrinogen (FG) according to Clauss, based on the study of the time of clot formation when an excess concentration of thrombin is added to a citrated plasma diluted by 10-20 times. The test is performed on special coagulometers, while the logarithm of the clot formation time is inversely proportional to the logarithm of the fibrinogen concentration [Dolgov V.V., Sviridov P.V. Laboratory diagnostics of hemostasis disorders. - M. - Tver: LLC "Publishing house" Triada "2005: 227 p.].

The main disadvantages of the method for determining FG according to Clauss is the sensitivity of the results to hypo-, dis- and hyperfibrinogenemia, as well as to fibrin degradation products, which affect the process of fibrin monomer polymerization and can cause both falsely low and falsely increased results [Polevod O A.A., Galstyan G.M., Berkovsky A.L., Sergeeva E.V. Method for determining functional fibrinogen // RF Patent No. 2669796],

Currently, there are numerous data on the important pathogenetic role of lipid peroxidation, which is an integral component of oxidative stress, in the initiation and development of many acute and chronic diseases. However, active oxygen metabolites (AKM), along with the oxidative modification of lipids, also cause oxidative modification of proteins, leading to pathological changes in their properties and functions, to fragmentation and aggregation. Considering that oxidative modification of proteins plays an important role in atherosclerotic processes, oxidized FG is of interest in the study of the pathology of the cardiovascular system. Since an increased level of FG is a diagnostically and prognostically significant marker of not only atherosclerosis, but also many other chronic inflammatory diseases, and its oxidative modification even more significantly potentiates disorders of the hemostasis system associated with endothelial dysfunction, which ultimately leads to impaired platelet and erythrocyte aggregation and to increased secretion of cytokines [Roitman EV, Azizova OA, Morozov YA, Aseichev AV. Influence of oxidized fibrinogen on the blood coagulation system // Bulletin expert. Biology and Medicine, 2004, 138, 5: 467-469.].

There are also known methods for determining the oxidative modification of blood plasma fibrinogen [Ragino Yu.I., Baum VA, Polonskaya Ya.V. RF patent No. 2298189. Published on April 27, 2007. Bul, no. 12; Shvachko A.G., Piryazev A.P., Azizova O.A., Sergienko V.I., Bykova A.A. A method for determining the oxidative modification of blood plasma fibrinogen by the content of carbonyl groups in a fibrin clot // RF Patent No. 2595806]. The essence of both methods is the preliminary preparation of a fibrin clot according to Rutberg, determining its mass. Then, equal volumes of saline solution and 20% trichloroacetic acid (TCA) are added to the clot to denature and precipitate FG. Next, the oxidative modification of fibrin with 2,4-dinitrophenylhydrazine (2,4-DNPH) is determined, as described in the work of Dubinina et al., 1995 [Dubinina EE, Burmistrov C.O., Khodov DA, Porotov G.E. Oxidative modification of human blood serum proteins, a method for its determination // Questions of medicinal chemistry, 1995, 41.1: 24-26.].

The disadvantage of the described methods is the need to isolate the fibrin clot, and incubation for 10 minutes is not sufficient for coagulation of oxidized fibrinogen. The method is difficult to carry out in the context of routine research.

A known method for determining the functionality of fibrinogen, including the study of whole blood using thromboelastography (TEG). Heparinized blood and batroxobin are introduced into the cuvette for TEG, after which the test is carried out, the parameter of the maximum amplitude on the thromboelastogram is determined, and the concentration of functional fibrinogen is calculated by the formula [Polevodova O.A., Galstyan G.M., Berkovsky A.L., Sergeeva E .IN. Method for determining functional fibrinogen // RF Patent No. 2669796].

The main drawback of determining the functionality of FG is the use of a special enzyme isolated from the venom of the rattlesnake Boothrops atrox, batroxobin, which is a non-physiological enzyme for human FG, as well as low productivity and high cost of analysis using a special device, a thromboelastograph. The listed disadvantages will not allow widespread use of this method for assessing fibrinogen functionality.

The closest technical solution to the claimed method is a method for determining FG during recalcification of citrate plasma and assessing its functionality [RF Patent No. 2703541, G01N 33/86, publ. 21.10.2019 Bul. No. 30], which consists in the recalcification of blood plasma with calcium chloride, followed by registration of the degree of coagulation on a photometer for enzyme immunoassay. In order to improve the accuracy of the test, polyethylene glycol with a molecular weight of 4000 is used as an activator of fibrin-stabilizing factor. The reaction is started in parallel 2 series by adding 25 mM calcium chloride solution to a sample containing citrate plasma, PEG-3350 and veronal buffer (VBS) with pH 7 , 4, mix thoroughly, measure the optical density of the samples, then incubate for 60 min at 37 ° C. In the first series of experiments, the protein content in the fibrin clot is determined by the standard method using a biuret reagent; in the second series of the experiment, the determination of plasma coagulation is carried out photometrically by the change in the turbidity of the samples at a wavelength of 450 nm with measurement intervals of 0 and 60 minutes. The calculation of the content of fibrinogen is carried out according to the formula: FG (g / l) = ΔA ₄₅₀ × 5.12, where: ΔA ₄₅₀ is the change in the optical density of the sample in the second series, during plasma coagulation; 5.12 - coefficient for converting changes in the optical density of the sample during plasma coagulation in g / l, which is the ratio of the protein in the fibrin clot to the change in optical density in the sample during plasma coagulation. The functionality of fibrinogen is calculated as the difference between the amount of fibrinogen determined by this method and by the Clauss method, calculating the difference in% to the protein in the fibrin clot. Fibrinogen functionality is defined as impaired with a difference of more than 10%.

The disadvantage of this method is the use of an additional chemical reagent (polyethylene glycol) to activate the fibrin stabilizing factor.

The objective of the present invention is to expand the arsenal of laboratory tests for determining fibrinogen and its functionality, as well as to reduce the cost by eliminating PEG from the test.

The technical result of the claimed invention consists in increasing the productivity, the availability of the test for routine research, increasing the accuracy due to the complete coagulation of plasma fibrinogen during the recalcification of citrated human blood plasma under conditions of hypoosmolarity.

The technical result is achieved by the fact that to increase productivity, the determination of FG analysis is carried out in 96-well flat-bottomed immunological plates and turbidimetric determination of coagulation using a photometer for enzyme immunoassay. High accuracy of determination of FG is ensured by a decrease in osmolarity by adding distilled water to the sample, which enhances the coagulation of fibrinogen during recalcification of citrated plasma.

The invention is illustrated by the figures:

FIG. 1. Correlation analysis of FG according to Clauss and the proposed method. Designations: X-axis - Clauss fibrinogen; Y-axis - fibrinogen according to the developed method

FIG. 2. Correlation analysis of FG according to Clauss and the proposed method without 3 samples of citrated plasma influencing the emissions from the linear trend of FG data. Designations: X-axis - Clauss fibrinogen; Y-axis - fibrinogen according to the developed method.

The method is carried out as follows: a standard blood sampling is carried out in a solution of 3.8% sodium citrate in a ratio of 9: 1, platelet-depleted plasma is prepared by centrifugation at 3000 rpm for 10 minutes. Then, to start the contact pathway of activation of plasma hemostasis of citrated plasma, optimal concentrations of calcium chloride solutions, equal volumes of distilled water and veronal buffer (VBS) with pH 7.4 are added to the wells of immunological plates with a flat bottom. Mix the samples thoroughly and measure the absorbance at 450 nm on an ELISA photometer (0 min - blank A ₄₅₀ ), then the samples are incubated for 120 min at 37 ° C and the degree of coagulation is measured at 450 nm. (Experiment A ₄₅₀ ). The calculation of the content of fibrinogen is carried out according to the formula:

FG, g / l = ΔA ₄₅₀ × 4.9

Where:

ΔА ₄₅₀ - change in the optical density of the sample during plasma coagulation - (А _{450 (experiment)} - А _{450 (blank)} );

4.9 - coefficient for converting changes in the optical density of the sample during plasma coagulation in g / l.

The dysfunction of fibrinogen is determined as the difference between the amounts of fibrinogen determined by this method and by the Clauss method in%, and if the difference is more than 5%, it is defined as the impaired functionality of fibrinogen.

Selection of the optimal osmolarity in the tested samples by adding distilled water to enhance the coagulation of fibrinogen during the recalcification of citrated plasma. Increasing amounts of distilled water (from 10 to 100 μl) are added to 50 μl of the pooled citrate plasma in the wells of 96-well immunological plates with a flat bottom, the volume in the experimental samples is adjusted with VBS buffer to 100 μl, 50 μl of 25 mM CaCl _{2 is} added, carefully mix and incubate for 60 min at 37 ° C and measure the optical density of samples at a wavelength of 450 nm every 5 min, including 0 min (blank) to determine the degree of plasma coagulation and continue for 60 min to assess the completion of the process. The results are shown in Table 1.

As can be seen from the data presented in Table 1, plasma coagulation during recalcification increases with a decrease in osmolarity in the tested samples when distilled water is added to the samples. The maximum coagulation of fibrinogen is observed when the plasma is diluted by a factor of 2 (0.075 M NaCl) and reaches 147% compared to the control of the pooled plasma without dilution. A decrease in plasma osmolarity does not lead to a change in the rate of fibrinogen coagulation (10 min), but causes the formation of a denser fibrin clot, which is manifested by a higher turbidity of experimental samples during recalcification of citrated plasma. At 60 minutes of incubation, 50 to 90 μL of distilled water in the sample slightly increases the density of the fibrin clot (by about 4%). Therefore, in further studies, 50 μl of distilled water was added to 150 μl of the test system.

Determination of fibrinogen by the proposed method and the Clauss method

In the wells of 96-well flat-bottomed plates, 50 μl of citrate plasma, VBS buffer, distilled water (total volume - 150 μl) are sequentially added and mixed thoroughly. The coagulation reaction of citrated plasma is started by adding 50 μl of 25 mM Ca ²⁺ . The samples are thoroughly mixed and incubated for 0-300 min. The change in the optical density of samples during plasma coagulation (formation of a fibrin clot) is tested turbidimetrically at a wavelength of 450 nm from 0 to 300 min at certain incubation intervals. At the same time, Clauss fibrinogen is determined in the original citrate plasmas using commercial kits "Fibrinogen" (Hemosil, Italy) on an automatic hematological analyzer ACL 8/9/1000 System (Instrum. Lab. Company, USA). The results are shown in Table 2.

As can be seen from the data presented in Table 2, complete plasma coagulation is observed during incubation for 120 minutes. Further incubation insignificantly (at the level of 5%) leads to an increase in the optical density of the samples. To translate changes in the optical density of the sample during coagulation, the coefficient (factor) was calculated as the ratio of the FG content in citrated blood samples, determined by the Clauss method, to the change in optical density (ΔА450) at 120 minutes of incubation. The average value of the factor was 4.90 ± 0.45. Hence, the experimental error is less than 10% of the fibrinogen concentration.

Determination of fibrinogen during plasma recalcification in the presence of PEG-4000 (prototype) and the Clauss method. In the same samples of citrated plasma, FG was determined according to the known prototype, i.e. with plasma recalcification in the presence of an activator of fibrin-stabilizing factor, PEG-4000. For this purpose, 50 μl of citrate plasma, 84 μl of VBS buffer, 16 μl of 10% PEG-4000 and 50 μl of 25 mM Ca ²⁺ are sequentially introduced into the wells of 96-well flat-bottomed ^plates . The samples are thoroughly mixed and incubated for 0-180 minutes. Determine the degree of plasma coagulation turbidimetrically by the change in turbidity in the samples at a wavelength of 450 nm. The coefficient for converting changes in optical density of samples into the amount of FG in g / l is calculated as the ratio of the content of FG, determined by the Clauss method, in citrated blood samples to the change in optical density ΔA450 at 120 minutes of incubation. The results are shown in Table 3.

As can be seen from the data presented in Table 3, complete plasma coagulation is observed during incubation for 120 minutes. Further incubation slightly increases the density of the fibrin clot less than 5%, with the exception of sample No. 17. Perhaps this is due to hypocoagulation associated with low generation of thrombin, rather than fibrin-stabilizing factor, which is activated by PEG. To translate changes in plasma optical density during coagulation (during recalcification), the coefficient was calculated as the ratio of the FG content determined by the Clauss method in citrated blood samples to the change in optical density (ΔА450) at 120 minutes of incubation. The mean value of the factor was 4.90 ± 0.41. Thus, the calculated conversion factor turned out to be equal to the factor that was obtained for the proposed method for determining fibrinogen (see above, table. 2). Considering that both tests use the same principle for detecting plasma coagulation during the formation of a fibrin clot, the turbidimetry method, the same mean value of the conversion factor (recalculation of changes in the optical density of samples during coagulation into the amount of FG in g / l) confirms the reliability of both tests for quantitative determination of fibrinogen.

Example 1. Comparative studies of the determination of FG by the developed method and the Clauss method for correlation analysis and assessment of the functionality of FG.

Comparative studies of the content of fibrinogen by the developed method and the Clauss method in 101 samples of citrated plasma were carried out. The results are shown in FIG. 1.

As seen from the correlation analysis graph in FIG. 1, there are noticeable outliers from the linear trend of the FG data only in 3 samples of citrated plasma. If we exclude them from the calculation (see Fig. 2), then we obtain an even higher agreement between the results of determining the GF by two methods.

However, as noted above, the main disadvantages of the method for determining FG according to Clauss is the sensitivity of the results to hypo-, dis- and hyperfibrinogenemia, as well as to fibrin degradation products, which affect the polymerization of fibrin monomers and can cause both false low and falsely increased results. The presented graph of the correlation analysis just experimentally confirms this fact. As noted above, with an increase in the oxidation state of fibrinogen, the rate of its conversion to fibrin under the action of thrombin decreases. This effect in the method for determining the GF according to Clauss should give an underestimated result, which we have shown in two independent ways. The data obtained indicate that only in one sample of the investigated samples (101 in total) the Clauss method gives an underestimated result (3.9 g / l), while the proposed method - 5.9 g / l. The pathophysiological effect in this case is the prevention of thrombosis by reducing the conversion of FG to fibrin by thrombin.

In 5 samples of citrated plasma, where the Clauss method overestimates FG in comparison with the proposed method, it indicates that oxidation (minimal) in vivo of FG causes a rapid conversion of FG to fibrin under the action of thrombin. This discovered experimental fact indicates the opposite pathophysiological effect - this is hypercoagulation and, as a consequence, thrombosis. It should be noted that these effects are also observed at a normal FG content, i.e. when there is no inflammatory process in the body as the cause of the oxidative modification of FG. In these individuals, dysfibrinogenemia may be due to genetic mutations.

Thus, the determination of FG by two methods (proposed and according to Clauss) expands the laboratory arsenal for both quantitative assessment of the level of FG and dysfunctional FG (Table 4).

As can be seen from the data presented in Table 4, in 14 samples (44%), determination of FG by the Clauss method gave underestimated values (fluctuations from 7% to 22%) FG. In 6 samples, overestimated FG results (19%) were obtained. Fluctuations ranged from 11 to 23%. In total, FG dysfunction was detected in 63% of the tested citrated blood samples. As noted above, the oxidative modification of FG causes a decrease in blood clotting due to the fact that oxidized FG is less activated by thrombin. Taking into account the age contingent of the clinic's patients, it can be concluded that there is a compensatory reaction of hemostasis with age, i.e. the body protects itself from thrombosis. In every fifth test, FG dysfunction is due to the increased sensitivity of oxidized FG to the action of thrombin. This fact can lead to a tendency to thrombosis and requires correction of hypercoagulation to prevent thrombosis. Moreover, in all 6 samples, the content of FG according to Clauss does not exceed the normal value.

Thus, the study of FG dysfunction expands the diagnostic value of FG screening using new methods in laboratory practice for early prevention of vascular catastrophes.

Claims (1)

A method for the determination of fibrinogen (FG) during recalcification of citrated plasma and its functionality, including activation of the contact path of coagulation in polystyrene 96-well flat-bottomed immunological plates by mixing citrated blood plasma with calcium chloride followed by photometric registration of coagulation, characterized in that to enhance coagulation of fibrinogen osmolarity in the experimental sample by adding 50 μl of distilled water to 150 μl of the test system, in the control sample, instead of water, add 50 μl of VBS buffer, start the coagulation reaction by adding 50 μl of 25 mM CaCl ₂ to the sample, mix thoroughly, measure the optical density of the samples, then incubate in for 120 min at 37 ° C, the determination of plasma coagulation is carried out photometrically by the change in the turbidity of the samples at a wavelength of 450 nm with measurement intervals of 0 and 120 min, the content of FG is determined in the initial plasma by the Clauss method, an individual coefficient is calculated for converting the changes in optic density in the sample during coagulation as the ratio of FG according to Clauss to the change in optical density of the sample (ΔА ₄₅₀ ), calculate the average value of the coefficient, the content of fibrinogen is determined by the formula: ФГ (g / l) = ΔА ₄₅₀ × 4.9, where: ΔА ₄₅₀ - change in the optical density of the sample during plasma coagulation; 4.9 - the average value of the coefficient for converting changes in the optical density of the sample during plasma coagulation in g / l of fibrinogen, determine the dysfunction of fibrinogen as the difference between the amounts of fibrinogen determined by this method and the method.

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