A method for the diagnosis of thrombophilia, due to the high activity of coagulation factor viii

 

(57) Abstract:

The invention relates to medicine, namely to the study of blood, and can be used for laboratory diagnosis of thrombophilia, due to the high activity of coagulation factor VIII. The claimed method is to determine the clotting time in two dilutions: 1:20 for the control of plasma and 1:30 for the studied plasma and when the ratio is more than 1.0 diagnosed thrombophilia, due to the high activity of factor VIII. The advantage of the invention is to simplify the method and reducing research time. table 4.

The invention relates to medicine, in particular, the study of blood and can be used for laboratory diagnosis of thrombophilia, due to the high activity of coagulation factor VIII (FVIII).

A known method for the diagnosis of thrombophilia by determining the level of FVIII immunological method [Moritz C., C. Brox, P. L. Turecek et all. Characteristics of a novel assay for the determination of FVIII antigen. Immunozym F VIII:.Ag. // Annals of Hematology. - Supplement II to Vol.74. Abstracts 41th Annual Meeting of the GTH, 1997, Abstr. 232]. The disadvantages of this method include the high complexity in its implementation required nnogo analysis and the need for reagents foreign production.

The closest achieved a positive result (prototype) is a coagulation method of determining the level of FVIII [Standardized one-step method for quantitative determination of factor VIII and IX. Hematology and Transfusiology, 1991, No. 4, S. 33-35].

The prototype method is as follows.

Pre-receive platelet-poor plasma blood (BTP) in the treatment of the patient.

Step 1. Breeding control plasma to obtain five calibration samples needed to construct a calibration curve performed according to the scheme presented in table. No. 1. Then examine the kaolin-kefalevoe the clotting time of the mixture of the diluted calibration samples and plasma deficient in FVIII.

Step 2. The calibration curve. To construct a calibration plot of clotting time on the activity of FVIII use semi-logarithmic coordinate system, and data obtained during the first phase.

Step 3. Studied BTP bred by Michaelis buffer in the ratio 1:10. Then examine the kaolin-kefalevoe the clotting time of the mixture diluted BTP investigated patient and plasma deficient in FVIII (deficit the mu clotting time in this test system depends only on the level of FVIII).

Step 4. The FVIII activity determined using a calibration curve.

A significant disadvantage of this method is that in this system, breeding is possible to determine the activity of FVIII in the plasma only in the range from 6.25 to 100%. Therefore, for determining the activity of FVIII more than 100% in the way that is the prototype, you must perform additional steps.

An additional step in the method prototype is required to determine the enhanced activity of FVIII. If the FVIII activity exceeds 100% to determine the level of FVIII is possible to use one of two options: 1) make an additional calibration dilution of 1:5, the corresponding activity of FVIII 200%, to explore the kaolin-kefalevoe the clotting time of a mixture of additional calibration dilution 1:5 and plasma deficient in FVIII, then to determine the level of FVIII in the calibration curve (however, the so-called “price second” in this case great that significantly reduces the accuracy as it decreases the sensitivity of the test system to the level of the investigated factor, and for determining the activity of FVIII more than 200% need more breeding and the study investigated plasma); 2) to make additional breeding and the 1:20 and plasma deficient in FVIII, then to determine the activity of FVIII in the calibration curve, and the result multiplied by 2. However, if the FVIII activity of more than 200%, you should perform additional dilution of 1:40 and then to explore the kaolin-kefalevoe the clotting time of a mixture of cultivation of the investigated plasma 1:40 and plasma deficient in FVIII. After that you should determine the activity of FVIII in the calibration curve, and the result obtained should be multiplied by 4.

Thus, the major shortcoming of the prototype method should be considered a complex system construct a calibration curve, a large consumption of reagents, the need to perform an additional step to determine the high activity of factor VIII, as well as the need for time-consuming to perform.

The authors propose a simple method based on the comparison of the clotting time in the APTT test is different dissolved study and control plasmas. In the proposed method, determine the clotting time in the APTT test control plasma only in a single breeding Tris-buffer at a ratio of 1:20 (more breeding increases the sensitivity of the test system to the level of the studied factor VIII) and the clotting time in the APTT test investigated plasma at times is very, than in the control only if the activity of FVIII will be more than 150%; clotting time in the study will be longer than in the control only if the FVIII activity will be less than 150% (as breeding study and control BTP differ in 1.5 times). Different breeding study and control plasma allow you to diagnose thrombophilia, due to the high activity of factor VIII in the APTT test without the build phase of the calibration curve. This significantly reduced consumption of reagents, there is no need to perform any additional steps, significantly reduced the research time.

A positive result of the claimed method is to simplify and reduce the time of the study. A positive result is achieved that determine the clotting time in two dilutions: 1:20 for the control of plasma and 1:30 for the studied plasma and when the ratio is more than 1.0 diagnosed thrombophilia, due to the high activity of factor VIII.

The method is as follows:

Reagents and equipment:

1) to 3.8% (0.1 M) solution of sodium citrate. Receive by dissolving 3.8 g of trisodium-citrate in 100 ml of d is P>3) the APTT-e-test (APTT-reagent, lyophilized mixture of Lugovoi acids and phospholipids, the producer of "Technology Standard", Russia);

4) STS-standard-plasma (lyophilized normal control plasma with the certified activity FVIII value, manufacturer Dade-Behring, Germany);

5) Deficient in factor VIII plasma, lyophilized, (producer of "Technology Standard", Russia);

6) calcium Chloride 0.025 M solution (manufacturer "Technology Standard", Russia);

7) Centrifuge ARF-8 (Russia).

Preparation of reagents and plasma samples for research:

Preparation of investigational BTP patient

Blood is obtained from the cubital vein by gravity through a needle into a plastic tube containing 0.1 M solution of sodium citrate (ratio of blood and citrate 9:1). The blood is centrifuged 5 min at a rotation speed of 1000 rpm (200 g). Received platelet-rich plasma re-centrifuged for 20 min at a speed of 3500 rpm (1200 g). Received BTP bred Tris-Hcl buffer at a ratio of 1:30. Such breeding is used to conduct the study.

Preparation of control plasma

In a bottle STS-standard-plasma FVIII activity 100% dobicina 10 min, then diluted in the ratio 1:20. Such breeding is used to conduct the study.

Preparation deficient in factor VIII plasma

In a bottle with a deficient factor VIII plasma FVIII activity less than 1% add 1.0 ml of distilled water, stir without shaking, kept at room temperature (18...20 ° C) for 10 minutes Divorced deficient in factor VIII plasma used for the study.

Breeding APTT-reagent

In a bottle APTT-e-test add 2.5 ml of distilled water, stir without shaking, kept at room temperature (18...20 ° C) for 10 minutes

Course definition:

1) In the first test tube with 0.1 ml of the diluted control plasma, add 0.1 ml deficient in factor VIII plasma, 0.1 ml of APTT reagent, incubate the mixture on a water bath for 3 min at a temperature of C, after which there was added 0.1 ml of a solution of calcium chloride. Record the clotting time (t1) with continuous stirring.

2) In the second test tube with 0.1 ml diluted investigated plasma add 0.1 ml deficient in factor VIII plasma, 0.1 ml of APTT reagent, incubate the mixture on a water bath for 3 min at a temperature of C, then KUB>2).

Evaluation of the obtained results

For the diagnosis of thrombophilia due to the high activity of FVIII calculate the ratio according to the formula

where ratio is the ratio of clotting time in the APTT test is different dissolved study and control BTP;

t1the clotting time in the APTT test mixture diluted 1:20 in the control plasma and deficient in FVIII plasma;

t2the clotting time in the APTT test mixture diluted 1:30 investigated and deficient in FVIII plasma.

If the index ratio greater than 1.0, then diagnosed thrombophilia, due to the high activity of coagulation factor VIII, as FVIII activity more than 150% of the clotting time of the investigated plasma (t2) is shorter than the coagulation control plasma (t1).

The specificity and reproducibility of the method

The inventive method of diagnostics of thrombophilia highly specific, its readings depend only on the levels of coagulation factor VIII and his testimony does not affect other defects of hemostasis, causing thrombotic disorders, because the test system is applied plasma only scarce factor, the NCI reproducibility of the method used determination of the coefficient of variation (in %) during repeated study of the same samples of normal plasma and plasma of patients with high levels of factor VIII on different days. The inventive method showed good reproducibility. The coefficient of variation did not exceed 6%.

Clinical testing of the proposed method

For testing the proposed method for the diagnosis of thrombophilia, due to the high activity of factor VIII was examined 112 patients with thrombotic disorders in age from 17 to 45 years. For the diagnosis of thrombophilia, due to the high activity of coagulation factor VIII, performed the immunological method for determining the level of FVIII, the way the prototype and the proposed method. In addition, he performed other laboratory methods for detection of defects of hemostasis, causing thrombotic disorders. Among the examined patients with thrombophilia, due to the high activity of factor VIII was detected in 11 patients (see table. No. 2). Thus, the prototype method and the inventive method revealed a thrombophilia, due to the high activity of factor VIII in the same 11 patients.

Examples of practical use of the method

Clinical example No. 1.

Patient # 1, 48 years old, ileofemoral thrombosis on the left was developed in November 2001. Was admitted to the vascular surgery Department of the Altai region, the clinically is ambasy in the patient with 39 years of age, three times was pulmonary embolism (age 39 years, 40 years of age and at the age of 43 years). Was implantation navalnogo filter into the inferior Vena cava, percutaneous access level L2-L31996. At the time of inspection General condition is satisfactory, respiratory rate 16 in 1 min, the number of heartbeats 88 min. Blood pressure is 130/80. The left lower limb edema (circumference at the level of the lower third of the left thigh 5 cm more than the same size on the right), its color changed (cherry flavor). According to the echo dopplerography identified impaired blood flow at the level of iliofemoral segment on the left. The study of hemostasis to avoid thrombophilia, due to the high activity of factor VIII (see tab. No. 3). On the basis of complaints, medical history, physical, instrumental and laboratory data, a diagnosis of thrombophilia, due to the high activity of coagulation factor VIII, a complication: acute ileofemoral thrombosis on the left. Condition after recurrent pulmonary embolism (1992, 1993, 1996).

In this clinical example, using the proposed method, was recognized thrombophilia, due to the high activity of factor VIII, so oral thrombosis on the left was developed in March 2000. Was admitted to the vascular surgery Department of the Altai Krai clinical hospital 7.03.2000. Complaints of pain in the left thigh and the left iliac region, swelling of the left thigh, leg and foot.

Thrombotic disorders with 38 years of age. In March 1998 there was an acute thrombosis of superficial veins of the right thigh. Was hospitalized in the surgical Department of the Central district hospital where he was treated for three weeks. In mid-January 1999 developed deep vein thrombosis of the left leg and pulmonary embolism. The treatment was carried out at the Department of vascular surgery of the Altai Krai clinical hospital. Has a disability since August 1999. In the etiology of this thrombotic disease is heredity. Myocardial infarction in older brother developed at the age of 47. Mother died from pulmonary embolism in 55 years of age. Sister died at the age of 25, all of a sudden. In 1999 in the laboratory of pathology of hemostasis was diagnosed with hereditary thrombophilia due to the resistance of factor V by activated protein C.

At the time of inspection General condition is satisfactory, respiratory rate 18 in 1 min, the number of heartbeats 98 MNA 9 cm more than the same size on the right), its color is not changed. According to the echo dopplerography identified impaired blood flow at the level of ileo-femoral segment on the left. The study of hemostasis to avoid thrombophilia, due to the high activity of factor VIII (see tab. No. 4). On the basis of complaints, medical history, physical, instrumental and laboratory data, were diagnosed Thrombophilia is caused by the resistance of factor V by activated protein C. Complication: Acute ileofemoral thrombosis on the left. Condition after PE (1999).

In this clinical example, using the proposed method, was excluded thrombophilia due to the high activity of factor VIII, because the ratio is less than 1.0 (0,95).

A method for the diagnosis of thrombophilia, due to the high activity of coagulation factor VIII, by determining the activated partial thromboplastin clotting time of the mixture deficient in factor VIII plasma and analyzed blood plasma, characterized in that to determine the clotting time in two dilutions: 1:20 for the control of plasma and 1:30 for the studied plasma and when the ratio is more than 1.0 diagnostics

 

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FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the suggested studying should be carried out on the glass simultaneously with several inductors by applying minimal inter-taking antilogarithms concentrations of aggregation inductors which correspond at double combination of inductors: ADP 5.0 x 10-8 M, adrenaline 3.0 x 10-9, collagen - dissolving the main suspension 1:8, thrombin 0.075 U/ml; at triple combination of inductors: ADP 10-9 M, adrenaline 10-9, collagen - dissolving the main suspension 1:9, thrombin 0.060 U/ml. The development of aggregation means thrombocytic activation in patients with arterial hypertension at metabolic syndrome. The method enables to evaluate the changes of thrombocytic functional state with combination of inductors more probably present in area of vascular lesion by applying minimal necessary concentrations that develops real conditions at hemostatic initiation in human vessels.

EFFECT: higher efficiency of studying.

3 dwg, 3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method involves checking consciousness, blood coagulation state, peripheral blood leukocytes number, K+ ions, bilirubin, fibrinogen, hemolysis and hemoglobinuria availability, prothrombin index and exotoxic shock development. Each value is calculated in points as follows. Lucidity is evaluated as -2 points; depression - +3 points; coma - +6 points; lack of changes in blood coagulation system - -2 points; coagulation availability without clinical injuries - +2 points; coagulopathy with clinical manifestation signs - +19 points; K+ ions concentration being less than 3.0 mmole/l - +3 points, from 3.1 to 3.5 mmole/l - -5 points, from 3.6 to 5.0 mmole/l - 0 points, greater than 5.0 points - +7 points, failure in determining K+ ions concentration - 0 points; hemolysis availability - +6 points, its lack - -3 points; hemoglobinuria availability - +8 points, its lack - -1 points; leukocytes number being less than 12.0x109/l - -2 points, from 12,1 to 18.0x109/l - 0 points, higher than 18.0x109/l - +8 points; hourly urine output being less than 30 ml/h - +6 points, greater than 30 ml/h - -2 points; bilirubin content being less than 31 mcmole/l - -2 points, from 30.1 to 50.0 mcmole/l - 0 points, greater than 50.0 mcmole/l - +2 points, failure in determining bilirubin content due to hemolysis being available -+6 points; prothrombin index being equal to or less than 60% - +3 points, greater than 60% - 0 points, failure in determining prothrombin index due to hemolysis being available - +12 points; fibrinogen concentration in blood plasma being less than 2.1 g/l - +4 points, from 2.1 to 4.0 g/l - -1 point, from 4.1 to 6.0 g/l - +1 point, failure in determining fibrinogen concentration due to erythrocyte hemolysis being available - +13 points; exotoxic shock development - +9 points, its lack - -1 point. The points are summed up. The value being greater than +13, admission for treatment in resuscitation department is indicated. The value being less than -13, admission for treatment in therapeutics department is indicated. The value being from -13 to +13, resuscitation expert consultation is advised.

EFFECT: high evaluation accuracy.

3 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: one should evaluate the time for clotting of plasma under testing in phospholipid-dependent test, moreover, one should apply high- and low-sensitive thromboplastin reagents to lupus anticoagulant to calculate the ratio of indices of prothrombin time prolongation and at its value being either equal to or above 1.1 one should diagnose APS.

EFFECT: shortened terms of research.

1 ex, 4 tbl

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EFFECT: high accuracy and objectiveness in differentiating syndrome severity degrees.

1 tbl

FIELD: medicine, diagnostics.

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EFFECT: higher accuracy and efficiency of diagnostics.

4 ex, 2 tbl

FIELD: medicine, obstetrics.

SUBSTANCE: the present innovation deals with predicting disadaptive processes in women in dynamics of menstrual cycle. During menstrual cycle beginning since the 1st d to the 21st d one should detect the dynamics for alteration in coefficient of activity of syntoxic adaptation programs (CASAP), calculated by the following formula:

where CST - concentration of blood serotonin, AAT-III - activity of antithrombin III, Aaoa - total antioxidizing activity of plasma, CCD8+ - concentration of T-suppressors, Cad - concentration of blood adrenalin, Cα2MG - concentration of α2-macroglobulin, CMDA - concentration of malonic dialdehyde, CCD4+ - concentration of T-helpers. Moreover, normally CASAP value alters two-fold against the first day of the cycle - since 0.70 up to 1.40 on the 21st d of the cycle, at no alterations in CASAP value one should diagnose female disadaptive alterations leading to failed pregnancy. The innovation enables to perform diagnostics of disadaptive processes in women in dynamics of menstrual cycle followed by prognostic conclusion upon future pregnancy.

EFFECT: higher accuracy of diagnostics.

2 ex

FIELD: medicine.

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EFFECT: high accuracy of diagnosis.

2 tbl

FIELD: medicine.

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1 dwg

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EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, clinical neurology, neurosurgery.

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EFFECT: higher accuracy of prediction.

2 ex, 1 tbl

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