Research fibrinolysis
For the clinics is extremely important as the identification of increasing the fibrinolytic activity of blood, and its reduction, that may be due either to heavy consumption of plasminogen activators and when activation and fixation in the bunch, and thrombi, either increase antiplazminovoy antiaktivatornoy and activity of plasma. It should always be remembered, that intense fibrinolysis in bunches and thrombi, detectable to improve the content of FDP in plasma, logically combined with the reduction and reserve plasminogen activators in plasma (circulating blood).
The status of natural clot lysis
An overview of the state of the natural clot lysis can provide a method Kotovshchikova-Kuznik modification in. P. Baluda or modification E. P. Ivanova.
The principle of the method is, that given the hematocrit, and red cell content of the blood is determined by the number of the last, dropped out of the bunch in a deposit for a certain period of incubation. By increasing the volume of blood fibrinolytic activity of this fraction (third) increased erythrocyte. In the case of deep-anticoagulation and hypofibrinogenemia, which produce defective and looser bunches, these methods can give misleading results.
In a modification procedure Bidwell D. IN. Andreenko fibrinolysis assessed by loss of fibrin clots.
The study fibrinolytic activity euglobulin fractions of plasma
Research fibrinolytic activity euglobulin plasma fractions is the most important basic test, which is confirmed by the Commission of the European Society on Thrombosis.
To do this, use either classical methods of determining euglobulin clot lysis, a definition of lytic activity euglobulin fraction on fibrin plates (in the latter case, the effect of different amounts of fibrin test readings leveled). Lysis in these tests is due to the, in euglobulin fraction was isolated and plasminogen activators, whereas inhibitors of fibrinolysis, the bulk of deleted. Therefore, using the test measured euglobulin Plasma levels of plasminogen activators and.
In the preparation of blood under basal metabolism, t. it is. in the morning on an empty stomach, before lifting the patient from the bed, tissue plasminogen activator in the blood is almost no. Hence, in such conditions is determined primarily by the internal state (proper blood) Activation of plasminogen.
This process can be greatly accelerated by pre-contact activation of factors XIIa-kallikrein-kininogen VM kaolin, resulting in reduced euglobulin lysis with 2.5-3 hours to 2-10 minutes. This principle is based contact activation methods for determining the XII-dependent fibrinolysis.
The ability of vascular endothelial secreted into the bloodstream external activator fibrinolysis (tissue-type activator, Bridging Digital Divide) It is estimated to accelerate euglobulin lysis after compressing vessels cuff from the unit for measuring blood pressure (sample Oyvina-Chekalinoy), or after dosed physical load on the cycle ergometer, or pharmacological stimulation (vasopressin derivatives - desmopressin or adiuretinom).
In the literature, many modifications of these techniques.
So, eg, during compression test on classical technique cuff pressure is maintained at 10,7 kPa (80 mm Hg. Art.) or a 1,3 kPa (10 mm Hg. Art.) higher diastolic for 15-20 minutes, and in more recent modification of the test in. P. Ball - 3 min at compression on 1,3 kPa (10 mm Hg. Art.) higher systolic blood pressure. In both cases, the second time to study the blood taken from the pinched vessel to remove the cuffs.
With such samples can be studied the effect of compression not only fibrinolysis, and other anti-thrombotic properties of vascular walls (anticoagulant, antiaggregant and so forth.).
Stress tests standardized way, to normal euglobulin lysis accelerated in 2 or more times (not doing research in a basal metabolism leads to a very large random errors, therefore the patient before the study began in the morning should not get out of bed, venipuncture should be carried out in the House).
The weakening of the reaction to compression load or indicate a lack of reactivity of the fibrinolytic system and increased risk of thrombogenic, verified that the number of studies.
The content of plasminogen in plasma
The content of plasminogen in plasma It may be semi-quantitatively assessed by adding euglobulin faction specific dose of streptokinase. According to the procedure Slobozhankinoy - Fedorova, chosen such activity streptokinase, which provides lysis euglobulin for 9-11 minutes, However, it is possible to use large concentrations (standard values are displayed in each laboratory separately). At deficiency of plasminogen lysis time in this test is extended to the more, the smaller this proenzyme.
The study clot lysis plasma
A parallel study of clot lysis plasma by the same procedure at stimulation of streptokinase allows for differences resulting figures to estimate the activity of antiplasmin in the test plasma, because euglobulin fraction -antiplasmin virtually no, and coagulated in whole plasma it has.
The amount is calculated at a rate of antiplasmin
(Plc SAND)/SAND;
where PLC — time in the presence of plasma clot Lysis streptokinase,
ELS - euglobulin clot lysis time in the presence of the same dose of streptokinase.
Further, Using this factor, antiplasmin activity is determined as percentage of pre-dilution curve compiled (obtained in mixed samples of normal blood plasma).
More precisely, yet easy to implement research methods fibrinolysis by splitting azofibrina, representing a compound fibrin, covalently svyazannogo with hromogennыm substratom (azopeptidom)
The principle of the method is, that three tubes Pipette 10 mg azofibrina, mixed with buffer (pH 7,4), then two of them Pipette 0,15 ml of blood plasma and studied further in one of these tubes - 10000 U / ml of streptokinase.
After incubation 1 h, the solution was filtered and photometric spectrophotometer at wavelength 440 nm or medical colorimeter at blue light filter.
Plasmin activity is determined by the plasma in the sample without streptokinase Change in optical density of the filtrate compared with the contents of third tubes, and plasma levels of plasminogen - in the sample with streptokinase. Incubation of plasmin and the study of the plasma on the same substrate is determined antiplazminovaya Activity. Benchmarks, obtained in studies of normal plasma, accepted for 100 %.
Thus, with the help of a relatively small set of simple laboratory-functional methods can get an idea about the state of the various mechanisms of fibrinolysis, content in the plasma and released from the vascular walls plasminogen, its activators and inhibitors.
As in the study of blood coagulation, These data can be significantly supplemented immunological determination of components of the fibrinolysis system and degradation products of fibrinogen and fibrin.
Methods for detection of "witnesses" and molecular markers of intravascular coagulation and fibrinolysis
In the process of intravascular coagulation and associated intense fibrinolysis occurs, one side, consumption and a more or less marked reduction in the blood of some components of the hemostatic system, on the other hand - the appearance of parts, fragments and metabolites, which in normal plasma contains no or a small amount of.
Identifying these "witnesses", and activation markers of intravascular coagulation and fibrinolysis is of great diagnostic value in disseminated intravascular coagulation, thromboembolic diseases of different origin and mikrotrombovaskulitah (infectious, Immune, tumor etc.. d.).
Markers consumption and activation of platelet hemostasis
For consumption and activation markers platelet hemostasis are thrombocytopenia, raising the plasma components of the granules - antigeparinovogo factor 4 Platelet, (3-thromboglobulin et al., as well as the preservation of functional circulation less active and less aggregation of platelets.
Key markers of damage and functional disability vascular endothelium - increase plasma levels of von Willebrand factor, relaxation response euglobulin lysis vessels during compression cuff, physical exercise or administration of vasopressin analogues.
Hypercoagulation
Hypercoagulation, if available, may indicate the activation of blood coagulation. She revealed by the general coagulation tests and imaging studies (thromboelastography), and clinic - based on the difficulty of obtaining blood from a vein - thrombosis of both the lung vessel, and needle, and incomplete stabilizing blood by stirring it with citrate (Education in vitro macro- and microclots).
This partially clotted blood is completely unsuitable for further study of the hemostatic system, so be sure to centrifuge the blood should be checked, whether it clots. In the presence of blood clots is not subject to further study (but it can be used for preparing serum and a series of biochemical analyzes). Experience shows, it is not enough adherence to this recommendation is often a source of serious errors in the study of the hemostatic system.
Hypercoagulation in some tests still no evidence of the danger of thrombogenic. Furthermore, A large number of thrombophilia, which are characterized by the initial hypo-, instead hypercoagulability. These include thrombophilia, caused by deficiency of factor XII, prekallikrein and kininogen VM, disfibrinogenemiyami, deficiency of protein C, etc.. d. Clotting virtually unchanged in the propensity to thrombosis due to increased hematocrit index (often while there hypocoagulation), thrombocythemia, inferiority of fibrinolysis, and so. d.
On the basis of the reasons given, can not be equated with hypercoagulable and thrombogenic risk, whereby the feature is currently among the stigmata thrombophilia not included.
Activation of the "bridge" between the factors XIIa kallikrein and Factor VII
Activation of the "bridge" between the factors XIIa kallikrein and Factor VII, that is recognized by examining the prothrombin time with bovine thromboplastin and after cooling to study blood plasma for 18-24 h at 4 ° C (cold test activation). In a number of thrombophilia after cooling observed decrease in prothrombin time 25-50 %. Other thromboplastin, except bovine, for this study are unsuitable.
The presence in the plasma of free peptide A
The presence in the plasma of free peptide A, detected by immunological methods, It is direct evidence thrombinemia, primarily because thrombin splits off from the fibrinogen molecule peptides A. The technique of limited availability due to difficulties in obtaining the immune anti-A Serum.
Besides, increase in plasma peptide usually transient, since it is rapidly eliminated from the circulation system of mononuclear phagocytes. In this regard, the level of peptide A informative only when increasing, which is a reliable sign of the presence of active thrombin in the blood.
The stratification and the formation of a pool of fibrinogen in the plasma soluble fibrin monomer complexes (RFMK)
In healthy people, the properties of the fibrinogen, Electrophoretic mobility and sensitivity to thrombin, completely coagulated in the thrombin assay by the addition of 12-14-second thrombin.
In contrast, when activation of the intravascular clotting (trombinemii) and fibrinolysis occur bundle fibrinogen pool, Education SFMC, binding of the fibrinogen (Locked fibrinogen), and, perhaps, early product of fibrinolysis (fragments X and V), and early and late FDP free state. SFMC and associated fibrinogen or thrombin influenced not coagulated (remained in the serum), or coagulate slowly and only when exposed to high concentrations of thrombin (3-seconds). To detect plasma and serum SFMC and other inert components fibrinogen pool, the following tests paracoagulation.
Beta-test naftolovyj
Beta-test naftolovyj (former name - the definition of fibrinogen B) insufficiently specific, so now rarely used.
Its essence lies in the fact, that the blood plasma solution was added β-naphthol in 50 % ethanol (5 drops to 1 ml). If after 10 min with shaking, precipitates in the form of coarse flakes, the sample is considered positive.
Ethanol test
Ethanol test - legkovypolnim, for it to 0,4 ml plasma investigated, stabilized sodium citrate mixed with aminocaproic acid (to block fibrinolysis), add 0,15 ml 50 % ethanol (concentration checked Alcoholmeters).
Education through 10 min gentle indicates the presence of a clot in blood plasma SFMC. Specific test, but reveals only part of the SFMC (basically - krupnomolekulyarnyh), It gives a positive result in DIC, thrombosis and other types thrombinemia. In stage III the engine on the background of severe hypofibrinogenemia (less than 0.5-0.7 g / l), as for heparinization, ethanol test often becomes negative.
Protaminsulfatny test
Protaminsulfatny test is based on the deposition of the early products of fibrinolysis and of the protamine sulfate SFMC, obtained from the milk of fish. The test is quite specific, but for his conduct suitable only certain types of protamine sulfate.
The ability to inactivate heparin and protamine sulfate cause paracoagulation unrelated. By this, the samples protamine sulphate, used to test paracoagulation, We must first be tested on a solution or a fibrin monomer, or a normal plasma, to which is added a small amount of pre-thrombin and streptokinase (or just streptokinase).
When this test becomes positive, protamine sulfate is suitable for diagnostic use. In many proprietary diagnostic kits includes control (Reference) plasma, giving positive test protaminsulfatnogo. It is used for testing reagent.
It should be taken into account, what using ethanol and protaminsulfatnogo tests identified different components fibrinogen pool, in connection with which their results do not always coincide with each other. So, in some patients with DIC both tests are positive, others - only one of them. Therefore, for a reliable diagnosis is necessary to perform both tests.
Ortofenantrolinovy test
Ortofenantrolinovy test (OFT) - Highly informative, It allows for qualitative and quantitative determination of SFMC in plasma with a small amount of platelets (further centrifuged 20 minutes at 4000 / min).
For carrying out the test only suitable hydrochloride ortofenantrolin. The solution ortofenantrolina 0,033 M (0,78 %) It is mixed at room temperature for equal amounts (by 0,1 ml) to study plasma on a slide and rocking determined the time of appearance of the first flakes mixed.
Recording is done for 2 m.
Results (in seconds) converted via the calibration curve in the amount SFMC. A calibration curve was prepared by adding various concentrations fibrinmonomera, obtained by the method Radzevich-Hodorova, a pool of blood plasma of healthy humans (Donor). Normally flakes appear in 120 from; the faster they appear, the more SFMC contained in the test plasma.
Test reliable disease, than ethanol and protaminsulfatny.
Identification of SFMC erythrocyte agglutination, pokrыtыh fibrin-monomerami (FM-test)
I human erythrocytes(O) Groups covered by the fibrin monomers are used in DIAGNOSTICUM.
Blood plasma is mixed with glass test erythrocytes. The appearance of agglutination (estimated by + to +++) It indicates the presence of SFMC, interacting with fibrin monomers on the surface of red blood cells.
The test is highly sensitive, reveals a concentration above SFMC 10 mg / ml.
Full identification of the coagulation of the fibrinogen and SFMC pool using poison ephah sand
The solution poison sand ephah or coagulating fraction (ehitoks, ekarin) fully coagulated fibrinogen, All products SFMC and early fibrinolysis, in connection with which it can be used to assess the total amount of fibrinogen coagulation components in the blood plasma pool and the number of SFMC and fibrinogen locked, remaining after the initial collapse in serum, obtained after clotting thrombin.
Use the concentration of the poison, which causes coagulation of normal plasma for 20-30.
In the presence of the sample SFMC poison plasma fibrinogen revealed more of, than in the test with thrombin, in serum, obtained after a 15-second clotting thrombin, with the addition of venom produced a second clot, which goes all the SFMC and associated locked fibrinogen, as well as the earlier PDF.
These phenomena are typical for thrombinemia and massive intravascular coagulation (DIC, thromboembolism, and et al.), whereas in the serum of healthy test persons reveals no poison SFMC and locked fibrinogen. After coagulation poison fibrinogen and macromolecular derivatives have not found any chromatography, audio via test adhesive staphylococci.
In addition to the above, to determine the SFMC and early cleavage products of fibrinogen (or fibrin) plasmin can be applied following Tests.
Test adhesion of staphylococci
Test adhesion of staphylococci (TSS, Staphylococcal klamping test) - A simple and highly informative method of detecting serum SFMC and early product of fibrinolysis (фрагментов X, as well as the associated locked fibrinogen).
It based on the ability of certain strains of staphylococcus (Neumann Д2S и др.) due to the presence on their surface a particular factor klamping undergo agglutination influenced remaining serum, SFMC after clotting and early PDF (фрагментов X).
Blood for Research is taken on the stabilizing solution mixed with aminocaproic acid preventing fibrinolysis in vitro.
The test is performed on the glass or on plates 74 nests by mixing equal volumes of diagnostic tools and the study of blood serum at dilutions 1:2, 1:4, 1:8 and t. d.
Taken into account the final dilution, which still determined agglutination. In healthy people, the content of SFMC and early FDP in serum does not exceed 0,002 g / l (2 ug / ml).
Immunological determination of the early and late FDP in serum
Immunological determination of the early and late FDP in serum is based on the ability to produce immune serum antifibrinogenovoy precipitation components fibrinogen pool (Sensitivity is determined by serum dilutions of fibrinogen).
When immunoelectrophoresis due to various mobility SFMC, фрагментов X, AND, D and E are perhaps indicative of their determination. Quantitative evaluation is performed by examining the different dilutions of fibrinogen, plasma and serum. More specific tests with specific antisera anti-D, anti-D dimer, etc..
PDF latex test
Test bonding of latex particles, loaded with anti-D, анти-E, антидимер D и др. (PDF latex test), is determining using diagnostic kits listed PDP gluing the latex particle by mixing different dilutions of serum.
Determination of plasma activated factor XIII as an indicator of thrombin in the blood circulation
Фактор XIII, activatable by thrombin in the presence of calcium ions, A chain is split into, obladayushtuyu fibrin-stabiliziruyushtey aktivnostyyu, and inertial chain S.
Separate determination of their content (on the degree of stabilization of fibrin oligomers and immunological) to diagnose intravascular coagulation of blood by the presence thrombinemia. Method complex, It is used mainly for scientific research. Are being developed more affordable methods for determining factor XIIIa.
Detection of activation of prothrombin
When activated prothrombin factor Xa cleaves the substrate fragment F1-2, determined immunologically. The increase in the content of the latter in the plasma indicates the activation of blood coagulation.
Identification of cellular activation markers of intravascular coagulation
The phenomenon of damage (Fragmentation) erythrocyte
When DIC and other types of blockades microcirculation in small vessels red blood cells are exposed to injury, in connection with which the blood smears increases the number of fragmented cells (more than 7 10%).
More specifically, this phenomenon is determined by the separation of blood cells in a density gradient ficoll-verografin (density 1,077) as described in, used in immunology for the isolation of lymphocytes and monocytes. The normal leukocyte interphase ring is whitish opalescent color and its counting chamber contains less than Goryaeva 400 erythrocytes 1 l (more frequently at most 200).
When DIC number of pop-up (Damaged) red blood cells increases dramatically (for 1000-40 000 in 1 l and more), in connection with which the ring is painted in pink or red. Quantitatively, the phenomenon is estimated by counting the content of red blood cells in the interphase (chamber Goryaeva).
The method allows to differentiate between DIC and the blockade of microcirculation thrombosis of major vessels, in which damage to the red blood cells slightly.
The phenomenon of thromboplastin production by monocytes
Some types of DIC (especially the immune and infectious origin) monocytes are activated and acquire the ability to produce tissue thromboplastin.
To detect this phenomenon blood cells are separated by density gradient (Ficoll-verografinovaya mixture, density 1,077) to obtain a fraction, containing a large number of monocytes, briefly cultivated, and then the slurry is determined by clotting activity before and after disrupting the cells. In severe infectious-septic process, this function can be suppressed by monocytes.
Identifying lowering plasma (consumption) components of the coagulation, fibrinolytic, and kallikrein-kinin systems
In acute and subacute DIC observed increased consumption and metabolism of a number of components of the hemostatic system, therefore the level of plasma is significantly reduced. Especially pronounced inhibition of factor VIII, V, антитромбина III, белков C и S, fibronectin, prekallikreina, high molecular weight kininogen, plasminogen.
Determining some of these parameters is important not so much for the diagnosis, how to assess the severity of the process and effectiveness of substitution therapy.
The level of factor I (fibrinogen) often reduced, what, However, initially masked by a high content of its plasma in infectious and immune diseases, toxemia of pregnancy, and other forms of pathology.
Identify neoantigenov
During the activation of coagulation factors and fibrinolysis and subsequent formation of their complexes with physiological antagonists arise new antigenic markers, there is no separate into constituent parts of these pairs. So, eg, complex of thrombin-antithrombin III form antigen, is not characteristic of "individually or thrombin, ни антитромбину III.
Similarly, formed in neoantigen pairing plasmin - antiplasmin and so on. d. In the presence of antisera to these neoantigenam easy to establish the presence of activated blood clotting key enzymes (Thrombin) and fibrinolysis (plasmin).
Modern clinic has a sufficiently large set of tests, identify intravascular activation of blood coagulation and fibrinolysis.
Many of these tests are publicly available, quick and easy to implement. Using them in combination with some other studies provides reliable laboratory diagnosis of DIC and other types of massive intravascular coagulation. Watching markers of intravascular coagulation it is set for the correct assessment of the dynamics of the pathological process, the effectiveness and adequacy of therapy.
