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RESEARCH of SYSTEM of the HEMOSTASIS

The blood sampling was made by a dry sterile needle from an ulnar vein in a plastic test tube in the ratio with anticoagulant 9:1. As anticoagulant used 3,8 % a solution of three-replaced Sodium citratum.

Necessary processing and performance of urgent tests were spent within 2 hours after a blood capture. The consecutive centrifugation of blood and plasma allowed to receive plasma rich with thrombocytes. For reception of plasma rich with thrombocytes blood tsentrifugirovali at 1000 rpm within 5 minutes. For preparation beztrombotsitarnoj plasmas blood tsentrifugirovali at 3000 rpm within 10 minutes.

1. Definition of concentration of complexes Thrombinum-ANTITHROMBIN (TAT) by means of firm set Enzygnost-TAT (Behringwerke, Germany). The research principle consists in definition of concentration of complexes TAT by an immunoenzymatic method in a blood plasma with the help

"Sandwich" - technicians.

During the first incubation complexes TAT being in the investigated sample contact antibodies to an antithrombin, fixed on a surface of a plastic test tube. During the further reaction at addition of antibodies to human AT III, konjugirovannyh with peroksidazoj, the last are bound with free (yet not bound) determinants AT of III complexes TAT. Excess of antibodies, konjugirovannyh with peroksidazoj, leaves at a lavage. Then it is measured enzimaticheskaja activity of bond. Enzimatichesky transformations of peroxide of hydrogen and a chromogen are braked by addition in a test tube a divorced chamois of acid. Then fotometricheski intensity of a staining which is proportional to concentration of complexes TAT is measured. Clinical value of definition of concentration of complexes TAT is bound to possibility of early diagnostics trombofilicheskogo conditions as complexes TAT are early markers of a thrombophilia and the beginning of intravascular coagulation of blood.

2. A method of an estimation of an intravascular thrombogenesis - definition D-dimera by means of latex-test Dimertest (Agen, Australia) which is based on interaction vysokospetsifichnyh antibodies to D-dimeru, fixed on lateksnyh particles. D-dimer characterises two-dimensional polymerisation of fibrin in the course of intravascular coagulation of blood; is one of the most specific tests of diagnostics of the IDCS, a thrombophilia and a clottage.

3. Research of a plasma link of system of a hemostasis.

Definition activated partial tromboplastinovogo time (ACHTV), characterising total activity of factors

Internal way of coagulation (except FVII and FVIII) in the conditions of standard activation of factors of contact (FXII and FXI) Kaolinum and the standard maintenance fosfolipidov (a partial thromboplastin) with use of commercial sets Stago, France.

Estimation of indicators of a hemostasis on the device tromboelastograf

«Hellige» (Germany): “r+k” (a chronometric indicator), “ma” (the maximum amplitude) "ITP" (an index trombodinamicheskogo potential) for the purpose of an estimation of chronometric and structural parametres of coagulation.

4. Definition of quantity of thrombocytes.

The control of quantity of thrombocytes in peripheric blood was spent on automatic counter "Тrombocounter", France.

5. Research of functional activity of thrombocytes.

Research of aggregation of thrombocytes spent on agregometre Рауtоn (USA) on method Вогn (106), with graphic registration of intensity and dynamics of aggregation of thrombocytes at their hashing with aggregation stimulators in a ditch agregometra.

The method principle is based on photo-electric registration of dynamics of change svetopropuskanija (optical density) the sample of plasma rich with thrombocytes at hashing with aggregation inductors: a solution adenozindifosfata (ADF) final concentration 1 ´ 10-3М, 1 ´ 10-5М, 1 ´ 10-7М; collagen suspension, an adrenaline solution - 1 ´ 10-3М; arachidonic acid 1 ´ 10-5М.

The tentative estimation agregatometra was spent depending on types of curves: "irreversible aggregation", "reversible aggregation" (disaggregation) and diphasic aggregation (fig. 6). The quantitative estimation of parametres of aggregation of thrombocytes was spent after a tentative estimation on types of curves, by means of calculation of following parametres of aggregation: Tma, Tва, TDA, and t lp (fig. 7).

The characteristic of parametres of aggregation: Tma (%) - size maximum

Aggregations, it is defined by the relation of size of change svetopropuskanija the sample of investigated plasma to interval size svetopropuskanija from 0 % to

56

100 %, intensity of aggregation characterises. Tva (%) - the size of secondary aggregation, is defined by the change relation svetopropuskanija the sample of plasma at a stage of secondary aggregation to size of an interval from 0 % to svetopropuskanija 100 %, characterises intensity of secondary aggregation (only for diphasic curves agretogrammy). Tda (%) - the disaggregation size, is defined by the change relation svetopropuskanija the sample of investigated plasma at a stage of formation of disaggregation (only for reversible aggregation). tлп (sec) - the latent period collagen - of aggregation and aggregation under the influence of arachidonic acid, from the moment of addition of a stimulator prior to the beginning of reaction of liberation of endogenous stimulators and synthesis ThAz, characterises time of the beginning of reaction of liberation of endogenous stimulators of aggregation and synthesis cyclic endoperoksidov - Prostaglandinums and tromboksana А2 in thrombocytes.

T

1 2 3

t

Drawing 4. Types of curves agregatogrammy.

1 irreversible aggregation; 2-diphasic aggregation; 3 reversible (disaggregation) aggregation.

ma

t the Moment of addition of an inductor of aggregation

Drawing 5. Key parametres agregatogrammy.

6. Definition antifosfolipidnyh antibodies. Main principles of their revealing.

Revealing antifosfolipidnyh antibodies (AFA) was based on references of the International Society on a clottage and a hemostasis, published in materials of XVI World congress on a clottage and a hemostasis (Florence, Italy; July, 1997) and XV International congress on a clottage (Antalia, Turkey; October, 1998). These diagnostic approaches began to be used at diagnostics AFS on obstetrics and gynecology chair m/p faculty MMA of And. M.Setchenov (the manager. Chair professor Makatsarija A. D) since September 1997г.

Definition of lupoid anticoagulant (VA) included 3 stages:

1) screening – tests

2) correctional assay

3) confirming assay with fosfolipidami

Simultaneously definition of anticardiolipin antibodies (AKA) was carried out ELISA - a method and included isotype and antiserum capacity revealings.

Variants of the received results were the following: AKA - VA -

AKA + VA +

AKA + VA - AKA - VA +

The special place in research occupied revealing VA as thrombophilia factor. Thus

1) skriningovye methods were carried out by means of following fosfolipid-dependent tests:

- ACHTV with the low maintenance fosfolipidov (RTT - LA; STAGO, France).

- Time of a divorced poison of a viper of Russell (Stago, France; dRVVT)

- Prothrombin time with a divorced thromboplastin (TTI, STAGO, France).

If fosfolipid-dependent tests were normal skriningovaja assay on revealing VA was considered as the negative.

Elongation of fosfolipid-dependent tests (one or several)

Dictated necessity of carrying out of correctional assay:

2) correctional assay was carried out by mixing of investigated plasma with normal plasma in the ratio 1:1; 1:4 and 4:1 according to the purpose of an exception of deficiency of factors.

If at addition of normal plasma fosfolipid-dependent tests remained extended, the tests confirming an orientation of circulating inhibitors against fosfolipidov were carried out.

3) confirming assay - for this purpose lysates of thrombocytes (PNP, STAGO, France) and geksagonalnyj fosfolipid (Staclot, Stago, France) were used.

Shorting ACHTV to norm testified about antifosfolipidnoj to the nature of circulating inhibitors.

At IV stage differentiation of other causes of infringement of coagulability of blood - deficiencies of separate factors and other specific anticoagulants in some cases was spent.

The algorithm of procedure of definition is more low resulted VA at use different fosfolipid - dependent skrinirujushchih tests (fig. 8).

We consider as the extremely important correct interpreting of results of research and an exception of false positive results. Therefore it is necessary to consider, that

1) elongation ACHTV and kaolinovogo time can be at:

- Reception of direct and indirect anticoagulants;

- Deficiency of factors of an internal way of coagulation;

- Circulation of specific and nonspecific anticoagulants;

- Deficiency of vitamin K as consequences malabsorbtsii and a long antibioticotherapia;

- To mechanical icterus;

- Consumption coagulopathies;

-Hyperfibrinolysis.

Drawing 6. Algorithm of definition VA. With use ACHTV

ACHTV it is extended Trombinovoe time

The normal is extended

korrektsionaja assay heparin neutralisation


Correction

(Deficiency of factors)

There is no correction

There is no correction

(Anomalies of a fibrinogen)

Correction

(Heparin)

Confirming assay with fosfolipidami


There is no correction

(A specific inhibitor)

Correction

(Lupoid anticoagulant)

The help of time of a divorced poison of a viper of Russell (dRVVT)

dRVVT it is extended correctional assay

There is no correction correction

Inhibitors deficiency of factors (V or)

Confirming assay with fosfolipidami

There is no correction correction an inhibitor of the factor V lupoid anticoagulant

2) elongation dRVVT can be at:

- Circulation VA;

- Circulation of an inhibitor of the factor V;

- Deficiency of factors V, H and I;

- Reception of direct and indirect anticoagulants.

3) prothrombin time elongation in the test with a divorced thromboplastin can be at:

- Reception of direct and indirect anticoagulants;

- Deficiency of factors of an external way of coagulation;

- Circulation of specific and nonspecific anticoagulants;

- Deficiency of vitamin K;

- To mechanical icterus;

- Consumption coagulopathies.

Also definition of concentration AFA (IgA, IgG, IgM) by an immunoenzymatic method (Stago, Asserachrom APA) was spent. An average caption – 20-40 GPlU/ml; high-> 40.

7. To all patients with taped mutation МТHFRС677Т

Concentration definition gomotsisteina in a blood plasma was spent,

Immunoenzymatic method with use of reactants Axis® of firm Axis - Shield AS, Norway on device ANTOS 2020, the USA (tab. 11).

Table 11. Degree definition gipergomotsisteinemii.

Indicator

(Mkmol/l)

gipergomotsisteinemija
11 30

31-100

> 100

Easy degree Average degree Serious degree

8. The global estimation of functioning of system of a protein With was carried out koagulometricheskim by a method with use of commercial sets "Sail" firm "Technology-standard"-test, Barnaul, Russia for the device «START 4» (Stago, France).

9. Levels PAI-1, AT III and a protein With were defined by a method of synthetic chromogenic substrates Stago, France on a spectrophotometer with a wavelength of 405 nanometers on device ST 88 Diagnostica Stago.

10. Revealing of genetically caused forms of a thrombophilia the Molecular analysis of genetic defects of hemostasis FV Leiden

/1691G-A/, gene mutations folatzavisimogo enzyme metilentetragidrofolatreduktazy MTHFR C677T, and also the mutation in gene Pt G20210A was carried out by a method polimeraznoj chain reaction (PTSR).

The basic stages of revealing of genetic defects of a hemostasis included: DNA-allocation;

-Amplification (method PTSR);

-restriktsiju.

The molecular analysis of genetic defects FVLeiden/1691G-A/, MTHFRC677T, Pt G20210А was carried out by statement polimeraznoj the chain

Reactions. This method of amplification of DNA in vitro provides revealing

Even minor alterations in genes. Principle PTSR consists in a cyclic denaturation with formation of matrix chains, hybridizations of oligonucleotides, komplementarnyh synthesised DNA and so forth

1. Mutation definition in gene С677Т in a gene 5,10 metilentetragidrofolatreduktazy (MTHFR) the person method PTSR.

Mutation С677Т in gene MTHFR of the person represents rest replacement tsitozina in position 677 on the thymine rest.

As a result of a mutation the sequence of amino acids polypeptide enzyme chains that does its thermolabile changes, leads to deficiency cytoplasmatic MTHFR and to level rising gomotsisteina in a blood plasma because of suppression of its metabolism. By means of PTSR amplification of a site of DNA of the surveyed patient changed under the influence of a mutation is made. Thus the maintenance amplifitsiruemogo DNA fragment in assay is enlarged in ~108 times in comparison with the initial. Amplification process is made at repeated cycles of a temperature denaturation of DNA, otzhiga oligonukleotidnyh prajmerov on komplementarnyh sequences of DNA and the subsequent completion polinukleotidnyh chains from these prajmerov by thermostable DNA-polimerazoj. Mutation С677Т leads to formation new sajga restriktsii for restriktazy HinfI. Therefore restriktaza can split in this place mutant, but not normal product PTSR. Upon termination of PTSR inkubirujut with restriktazoj HinfI and reaction products analyze the formed fragment of DNA an electrophoresis in agaroznom gel. In the presence of a mutation find out formation of two low-molecular strips formed under the influence of enzyme. Thus full splitting of product PTSR testifies to presence in analyzed DNA of the homozygous form of mutation C677Т, and partial - heterozygotic (fig. 8).

Products PTSR become visible in 3 % agaroznom gel after prokrashivanija bromid etidiem thanking their fluorescences in ultra-violet light. As a result polimeraznoj chain reaction (38

Cycles) the fragment of DNA long 205 nukleotidnyh a steam is formed. The incubation of product PTSR with restriktazoj HinfI is not accompanied by its splitting in the absence of mutation С677Т (paths 2 and 5 fig. 9), full splitting with formation of fragments of DNA in length of 116 and 79 pairs nucleotides (a path 8), occurs in the presence of the homozygous form of a mutation and at a heterozygotic mutation - partial rashcheplenie (paths 1, 3, 4, 6, 7, 9).

Drawing 7. The molecular analysis of mutation MTHFRC677T.

2. Mutation definition in a gene of the factor V Leiden coagulating system of blood a method polimeraznoj chain reaction.

Mutation Leiden in a gene of the factor V represents replacement of the rest of adenine in position 1691 on the thymine rest. As a result of a mutation there is replacement Arg-506jGln in polypeptide chains FV. As a result of PTSR amplifitsiruetsja the site of investigated DNA changed under the influence of a mutation. Mutation Leiden breaks a site restriktsii for restriktazy Мп1. Therefore restriktaza can split in this place normal, but not mutant product PTSR. Upon termination of PTSR the formed fragment of DNA inkubirujut with restriktazoj Мп1. In the absence of a mutation after an electrophoresis two low-molecular strips formed under the influence of enzyme find out. Full fastness of product PTSR to restriktaze testifies about

Presence in analyzed DNA of a homozygous mutation, and partial – the heterozygotic form (fig. 10).

Products PTSR are found out in 3 % agaroznom gel after prokrashivanija bromid etidiem on fluorescence in ultra-violet light. At carrying out PTSR in the assays containing DNA of the person, the fragment of DNA long 205 nukleotidnyh a steam is formed. The incubation of product PTSR with restriktazoj MnI is accompanied by its full splitting in the absence of mutation Leiden. Splitting is absent in the presence of the homozygous form (a path 3 and 5, fig. 10), at the heterozygotic form of a mutation – partial splitting (paths 7 and 13).

Drawing 8. The molecular analysis of mutation FVLeiden/1991G-A/.

3. Definition of mutation G20210А in a gene of a prothrombin a method polimeraznoj chain reaction.

Mutation PtG20210А represents replacement of the rest of a guanine in position 20210 on the rest of the adenine localised in its trailer not coding part. As a result of a mutation the sequence of amino acids polypeptide prothrombin chains does not change, its stability m-rnk however is enlarged, that is accompanied by rising of level of synthesis of a prothrombin and its concentration in a blood plasma. Mutation PtG020210А breaks in PTSR - a product an artificial site restriktsii for restriktazy Таq I, missing which nucleotides are entered in PTSR - a product by means of one of prajmerov. Therefore restriktaza can

To split in this place normal, but not mutant amplifitsirovannyj DNA fragment. In the presence of a homozygous mutation initial product PTSR is not split by enzyme. Full splitting of product PTSR testifies to absence of mutation PtG20210А in analyzed DNA, and partial - the heterozygotic form of a mutation (fig. 11).

Products PTSR find out in 4 % agaroznom gel after prokrashivanija bromid etidiem on fluorescence in ultra-violet light.

Though in world practice the homozygous form of mutation PtG20210А is described, we had been taped mainly heterozygotic forms.

The incubation of product PTSR with restriktazoj TaqI is accompanied it polnymrasshchepleniem pr absence of a mutation, partial in the presence of a heterozygotic mutation (a path 1,4 and 8).

Drawing 9. The molecular analysis of mutation PtG20210A.

In the course of PTSR-DIAGNOSTICS of polymorphism GP Ia it was used restriktaza HinfI, GP IIIa «1565 T/S» - MspI, a fibrinogen «455 G/A» and a receptor of angiotensin of II type 1 «1166 A/S» – HaeIII, PAI-1 «675 4G/5G» – Bsc4I.

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A source: Egorova Elena Sergeevna. Basic principles of management of pregnant women with anemia and thrombophilia. Thesis for the degree of candidate of medical sciences. Moscow 2014. 2014

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