Heparin concentration determination method

FIELD: analytical methods in medicine.

SUBSTANCE: invention concerns hematological procedures and, in particular, can be used in heparin treatment practice. Method of invention is based on measuring rate of thrombin-mediated hydrolysis of a chromogenic substrate, thrombin having activity 0.5-0.6 unit/ml and chromogenic substrate being z-Ala0Ala-Arg-pNA·HBr.

EFFECT: extended assortment of home reagents, simplified preparation procedure, and increased sensitivity of method.

1 dwg, 3 tbl, 2 ex

 

The invention relates to medicine, in particular to the study of blood, and can be used for monitoring heparin treatment.

It is known that heparin is used to treat a large number of diseases. Thrombosis and embolism, disseminated intravascular coagulation syndrome, glomerulonephritis, ischemic heart disease - this is an incomplete list of diseases that should be used this medicine. However, uncontrolled use of this medication decreases the efficiency of its use, causes life-threatening complications.

The known method of controlling the concentration of heparin by determining the activated partial thromboplastin time (APTT). It is believed that therapeutic concentration of 0.3 U/ml) heparin causes elongation of the APTT 2-3 times of the original value. However, when the number of pathological conditions that require the use of high doses of heparin, this method is unsuitable, for example, during heart surgery using cardiopulmonary bypass when using very high concentrations of this drug. In addition, commercial preparations of the APTT-reagent have different sensitivity to heparin, as part of their different (using kaolin, ellagic acid, oxides of silicon, vegetable phospholipids, Catalina and others) [Thompson J.M. The control of heparin therapy by the activated partial thromboplastin tie: Results of collaborative studies. Scandinavian Journal of Haematology. - 1980. 25 (supl, 27). - P.73-83].

The closest achieved a positive result (prototype) is a method of determining the concentration of heparin in the rate of hydrolysis of a chromogenic substrate by thrombin [violation of the reaction of formation of thrombin. Edited Ruhlman, Per. s angl. - M - Medicine. - 1988. - S-199]. In the method prototype used imported chromogenic substrate S2238, which limits its use. The disadvantages of this method include its low sensitivity (sensitivity of not more than 0.1 IU/ml). In addition, the prototype method has the additional step of preparation of thrombin (preparation of stock solution of thrombin to a concentration of 100 U/ml, freezing and thawing of the aliquot, the breeding of the aliquot to the activity of 8 U/ml).

The inventive method based on the use of domestic chromogenic substrate, devoid of these shortcomings: no additional stage of breeding uterine thrombin and freeze aliquot that reduces the time; the sensitivity of the proposed method are higher due to the use of low activity of thrombin.

A positive result of the proposed method is an extension of Arsenal domestic reagents for determining the concentration of heparin, the simplification of procedures for preparation of reagents and increase case the reality of the way.

A positive result is achieved in that for determining the concentration of heparin used thrombin activity of 0.5-0.6 U/ml and as a chromogenic substrate using the substrate z-Ala-Ala-Arg-pNA.HBr.

The method is as follows.

Reagents and equipment:

1. 3,8% (0,1M) solution of sodium citrate. Receive by dissolving 3.8 g of trisodium-citrate in 100 ml of distilled water.

2. Tris-HCl Buffer, pH 7.4 (manufacturer "Technology Standard", Russia).

3. Chromogenic substrate (z-Ala-Ala-Arg-pNA.HBr) 9 mg in vial, (producer of "Technology Standard", the reagent from the diagnostic kit of reagents Chromium-Those-Antithrombin), Russia*. For preparation of working substrate solution in a bottle to add to 6.0 ml of distilled water, stir until dissolved.

4. Thrombin, 5 ED, producer of "Technology Standard", Russia. To prepare a working solution of thrombin to dissolve the vial in 10 ml of distilled water.

5. Acetic acid, 30%solution in distilled water.

6. Reference normal plasma (RNP), lyophilized, producer of "Technology Standard", Russia.

7. Heparin, 25000 IU in vial, the producer of "Berlin-Chemi", Germany.

8. Centrifuge ARF-8 (Russia).

9. The polystyrene tubes 15 ml, calibrated blood collection, manufacturer Sardstedt, Switzerland.

10. Photometer R-50, manufacturer Voeg, Germany (or similar with a wavelength of 405 Nm, and the optical path length of the cuvette 10 mm).

* - was Previously presented a method for determining the concentration of anti-thrombin III, based on the use of chromogenic substrate z-Ala-Ala-Arg-pNA.HBr [Lousina TL, Makarov V.A., Neverova PU, Thrush N.N., Momot A.P. Method for determining the activity of anti-thrombin III. The decision to grant patent No. 2000130498, priority from 06.12.2000]. However, the presented method is intended solely for determining the level of anti-thrombin III, but is not intended to determine the concentration of heparin.

Sample preparation platelet-poor plasma (BTP)

Preparation of investigational BTP: Blood obtained from the cubital vein silikonizirovannoj needle into a test tube containing 3.8%solution of sodium citrate (ratio of blood and citrate 9:1). The blood is centrifuged 5 min at 1000 rpm (200 g). Received platelet-rich plasma re-centrifuged for 20 min at 3500 rpm (1200 g). Received BTP use for research.

Preparation of sample BTP

Prior to analysis to 0.1 ml of the investigated BTP add 3 ml of buffer Tris-HCl.

Preparation of RNP

For the preparation of the RNP into the vial to make 1.0 ml of distilled water and dissolve the contents at room temperature (+18...+25° (C) and a light rocking in 3 minutes

Preparation of a working sample of RNP

To prepare a working sample to RNP RNP 0.1 ml add 3 ml of Tris-Hcl buffer Tris-HCl.

Preparation of calibration samples

For preparation of standard heparin solution to 0.1 ml of heparin add to 9.9 ml of distilled water (concentration of heparin in the standard solution, 50 U/ml).

1. Preparation of the calibration sample No. 1 (5 U/ml). To 0,90 ml RNP add 0.1 ml of a standard solution of heparin.

2. Preparation of the calibration sample No. 2 (2.5 U/ml). To 0,90 ml RNP add 0.05 ml of a standard solution of heparin and 0.05 ml of distilled water.

3. Preparation of the calibration sample No. 3 (1.0 U/ml). To 0,90 ml RNP add 0,02 ml of a standard solution of heparin and 0.08 ml of distilled water.

4. Preparation of the calibration sample No. 4 (0,5 IU/ml). To 0,90 ml RNP add 0.01 ml of a standard solution of heparin and 0.09 ml of distilled water.

5. Preparation of the calibration sample # 5 (of 0.05 U/ml). To 0,90 ml RNP add 0.1 ml of heparin solution with a concentration of 0.5 U/ml

6. Preparation of the calibration sample No. 6 (0,0 IU/ml). To 0,90 ml RNP add 0.1 ml of distilled water.

All calibration samples (No. 1 to 6) add 3 ml of buffer Tris-HCl.

The process of determining

The study of the concentration of heparin performed in the following sequence (see course definitions).

P is obliku to make a 0.1 ml sample BTP (or 0.1 ml of the calibration sample for calibration curve), 0.3 ml of a working sample of PPR, mix thoroughly. Then add to the mixture, 0.4 ml of a working solution of thrombin. The mixture is incubated at a temperature of +37°With 3 minutes After incubation, add 0.4 ml of chromogenic substrate. After 120 seconds (exactly!) to make 1.0 ml 30%solution of acetic acid, mix well. After 5-10 min after application of acetic acid solution to determine the optical density (at a wavelength of 405 nm) sample plasma against physiological solution of sodium chloride.

The calibration curve

To construct a calibration curve on a linear calibration graph in the y-axis of lay values of optical density, and the abscissa axis lay concentrations of heparin. Points that were obtained in the study of calibration samples, build the dependence of optical density on the concentration of heparin.

Using the data obtained in the determination of the optical density of the sample BTP, find the concentration of heparin in the calibration curve.

The reproducibility of the method

To assess the reproducibility of the method used to calculate the coefficient of variation (%). As with all methods using chromogenic substrates, this method showed very high reproducibility. The coefficient of variation of this test in the study of the different days does not exceed 3%.

The sensitivity of the method

To assess the sensitivity of the method used 10 plasma samples with known levels of heparin. For this purpose, added to the normal BTP 10 healthy people heparin with the certified value to a concentration of 0.05 U/ml In all 10 samples BTP with heparin was determined by the level of heparin proposed method and the method of the prototype. The level of heparin determined by the method of the prototype, was equal to the average of 0.10±0.01 Units/ml (coefficient of variation equal to 7%). The level of heparin defined by the claimed method was equal to the average 0,051±0,002 IU/ml (coefficient of variation equal to 3%), and did not differ from the certified concentration of heparin (to 0.05 U/ml) in samples BTP (see drawing). Thus it is shown that the sensitivity and reproducibility of the proposed method is higher than the method prototype. Higher sensitivity to heparin in the test is achieved by application of low concentrations of thrombin in the inventive test system.

Clinical examples

1. The patient PS, age 23 years, turned in the Altai Hematology center with complaints of recurrent miscarriage. The first miscarriage was in the gestation of 20 weeks, the second was the period of 32 weeks. In 2001 was identified false positive analysis on RW. Upon physical examination of respiratory abnormalities are found. Border relative is uposti hearts are not changed. Heart sounds are clear, correct rhythm, heart rate of 78 per minute. Abdomen palpation soft, painless. Liver and spleen are not enlarged.

These laboratory methods of examination:

b - 125 g/l; ROHE 17 mm/h; Leukocytes - 5,4×109/l; WBC: B - 1% e - 2% P - 1% - 56% L - 32% - 8%; Biochemical analysis of blood: Creatinine 73 µmol/l; urea 4.8 mmol/l; total bilirubin 15 µmol/l, straight 6 µmol/l, indirect 9 µmol/l; glucose 4.1 mmol/l; CRP +; total protein - 78,9 g/l, albumin - 50%, α1 - 5,0%, α2 - 11,0%, β - 15,0%, γ - 19,0%. The study of hemostasis before and during treatment with heparin is presented in table 1.

On the basis of clinical and laboratory data formulated diagnosis

Primary antiphospholipid syndrome, miscarriage.

Treatment: heparin 5000 Units 5 times a day, the course discrete plasma-Teresa, disaggregants. On the background of treatment, the patient appeared uterine bleeding, so temporarily (for 2 days) was terminated heparin to eliminate bleeding. The study of hemostasis before and after the use of heparin (at a dose of 5000 IU 5 times a day) are presented in table 2. As follows from the table, the patient with the ASF traditional way of monitoring heparin therapy being to achieve a 2-3-fold lengthening of the APTT test, it is not possible to control the oxygen the radio heparin in the blood, as the patient with the ASF was the original hypocoagulation in the APTT test, due to lupus anticoagulant in the blood of the patient. Therefore, the uncontrolled use of heparin caused this patient is one of the most frequent complications of heparin therapy is bleeding. Using the proposed method was determined the exact concentration of heparin in the blood. It turned out that the cause of the bleeding was more than double the overdose of heparin, although the APTT was extended less than 2 times. Therefore, after identifying the high level of heparin by the claimed method of treatment heparin continued to decline by half dose of 2500 IU 5 times a day, continuing the treatment of the underlying disease without hemorrhagic complications. The results of the quantification of heparin and indicators of hemostasis after stopping uterine bleeding and correction doses of heparin are presented in table 2.

As follows from the table, after clarification of the concentration of heparin in the blood by the claimed method was achieved concentration of heparin needed to treat the underlying disease that is not caused hemorrhagic complications in the future. Determining the concentration of heparin was performed by the claimed method and the method of the prototype. Differences in the testimony of the proposed method and the prototype method is not found.

2. Patient K. (age 42) 5.05.2000 turned in Altai Krai clinical hospital (ACCB) with complaints of swelling of the legs and hips to the left, pain in the left lower limb at rest, increasing with little exertion. Ill for weeks with 28.04.2000. Treated independently heparin ointment and dipyrone. A positive effect of this treatment has not notes. Called the ambulance, which brought the patient in ACCB diagnosed with acute ileofemoral thrombosis on the left. My father had thrombosis at 30 years of age, his father died of a pulmonary embolism at the age of 49 years. Upon physical examination of respiratory abnormalities are found. Borders of relative dullness of the heart is not changed. Heart sounds are clear, correct rhythm, heart rate is 86 / min. Abdomen palpation soft, painless. Liver and spleen are not enlarged. Left lower leg and left thigh edema, discoloration of the skin is not detected, the superficial veins to the left varicose expanded. Palpation of the calf muscles on the left painful.

These laboratory methods of examination:

b - 140 g/l; ROHE 22 mm/h; Leukocytes - 9,9×109/l; WBC: B - 1% e - 2% P - 1% - 59% L 32% M 5%; Biochemical analysis of blood: Creatinine 77 µmol/l; urea 5.3 mmol/l; total bilirubin 15 µmol/l, direct 10 µmol/l, indirect 5 mmol/l; glucose 4.5 mmol/l; the RB OTP; total protein - to 68.4 g/l, albumin - 56%, α1 to 3,0%, α2 - 10,0%, β and 12.0%, γ - 19,0%. The study of the hemostatic system are presented in table 3.

On the basis of clinical, laboratory data and analysis of hemostasis formulated diagnosis:

Hematogenous thrombophilia. Resistances of factor Va by activated protein C sharp ileofemoral thrombosis on the left.

Treatment: heparin 5000 IU 4 times a day, Detralex, trental and other Study of hemostasis before and on the background of the application of heparin are presented in table 3. As follows from the table, the patient is the traditional way of monitoring heparin therapy, which consists in achieving a twofold extension of the APTT test, it is not possible to determine therapeutic concentrations of heparin in the blood. It is impossible to conclude that a sufficient level of heparin in the blood, because different reagents for the determination of the APTT show different results. However, using the proposed method, was precisely the concentration of heparin in the patient's blood that was allowed to continue treatment of a patient with thrombotic disorders anticoagulants. Determining the concentration of heparin was performed by the claimed method and the method of the prototype. Differences in the testimony of the proposed method and the prototype method is not found.

Table 1

The study of hemostasis in the patient PS, 23 years in the treatment of heparin at a dose of 5000 IU 5 times a day
Research methodsBefore the treatmentOn the background of treatment with heparinControl
The APTT,509135 (N30-40)
Time divorced, thromboplastin,627744 (N33-52)
Test mixing with plasma donor (1:1)no correctionno correction-
Prothrombin,202014 (N14-17)
Liblocale time536636 (N19-38)
Ehitatava time353334 (N19-41)
Thrombin time15>10015 (N13-17}
Antithrombin III, %1109978-122
Protein, %10711070-130
Fibrinogen, g/l5,53.52-4
; Fibrin monomer complex, mg%1883.5
Platelets, ×109/l299303170-80
XIIα - fibrinolysis, min344610
Heparin concentration, the method of the prototype, IU/ml01,100,2-0,4
The concentration of heparin, the claimed method, IU/ml01,110,2-0,4

110
Table 2

The study of hemostasis in the patient PS, Le in the treatment of heparin at a dose of 2500 IU 5 times a day
Research methodsBefore the treatmentOn the background of treatment with heparinControl
The APTT,506934 (N30-40)
Time times. Trombon,626943 (N33-52)
Test mixing with plasma donor (1:1)no correctionno correction-
Prothrombin,202014 (N14-17)
Liblocale time535637 (N19-38)
Ehitatava time353534 (N19-41)
Thrombin time15>10015 (13-17)
Antithrombin III, %9478-122
Protein, %1079070-130
Fibrinogen, g/l5,53.62-4
; Fibrin monomer complex, mg%1873.5
Platelets, ×109/l299366170-380
CPα - fibrinolysis, min343910
Heparin concentration by the method of the prototype, IU/ml00,300,2-0,4
The concentration of heparin by the claimed method, IU/ml00,310,2-0,4

Table 3

The study of hemostasis in a patient K.N., in the treatment of heparin at a dose of 5000 IU 4 times per day
Research methodsBefore the treatmentOn the background of treatment with heparinControl
The APTT, with (test performed with reagent Decalin, manufacturer DiamedAG, Switzerland)293628 (N25-35)
The APTT, with (test performed with reagent Actimat, manufacturer BioMerieux, France)3010831 (27-39)
The APTT, with (test performed with Rea is entom APTT-e-test, producer 000 “Technology-Standart”, Russia)336030 (N30-40)
Prothrombin,141414 (N14-17)
Liblocale time333635 (N19-38)
Ehitatava time333236 (N19-41)
Thrombin time15>10015 (13-17)
Antithrombin III, %1009678-122
The resistance of factor V to APS0,64-0,80-1,20
Protein, %10210770-130
Fibrinogen, g/l4,33,22-4
; Fibrin monomer complex, mg%1573.5
Platelets, ×109/l250200170-380
CPα - fibrinolysis, min352010
Heparin concentration by the method of the prototype, IU/ml00,330,2-0,4
The concentration of heparin by the claimed method, IU/ml00,320,2-0,4

The method for determining the concentration the AI heparin, based on the determination of the rate of hydrolysis of a chromogenic thrombin substrate, characterized in that used the thrombin activity of 0.5-0.6 U/ml, and as a chromogenic substrate using the substrate z-Ala-Ala-Arg-pNA.r.



 

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2 tbl, 4 ex

FIELD: medicine, diagnostics.

SUBSTANCE: the present innovation deals with blood sampling, separating plasma against erythrocytes, moreover, in plasma on should detect activity of antithrombin III, proteins C and S, XIIa-dependent fibrinolysis and concentration of plasminogen obtained results should be expressed as relative units followed by calculating integral parameter that characterizes the state of anticoagulant-fibrinolytic potential (IPAFP) by the following formula: IPAFP = [(C1 + C2)/(C3 + C4)] x 100, where C1 - the ratio of observed value of antithrombin III activity to the value of inferior border of the range of analogous parameter norm; C2 - the ratio of observed value for the activity of proteins C and S system to the value of inferior border of the range of this parameter norm; C3 - the ratio of the value of inferior border of plasminogen concentration under normal conditions to observed value of analyzed parameter; C4 - coefficient calculated with the help of regression equation: C4 = 0.9 + (0.01 x X), where X - terms of lysis of patient's euglobulin clot/min, and at IPAFP value of 101.4 U and higher one should state anticoagulant-fibrinolytic blood potential to be in norm, in interval of 64.8 - 101.3 -as insufficient, and at 64.7 and below - as critical. The present method simplifies the procedure of evaluating the state of endogenous anticoagulants and activity of XIIa-dependent fibrinolysis.

EFFECT: increased diagnostic value of obtained results.

3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, obstetrics, gynecology.

SUBSTANCE: in the first trimester of pregnancy one should study the content of CD8+CD11b lymphocytes and at their values being either equal or above 2% it is possible to predict gestosis. The present innovation enables to choose correct tactics of treating pregnant women that, in its turn, leads to decreased frequency of this complication of pregnancy and the risk for the development of fetal and neonatal pathology.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves carrying out microscopic examination of blood serum samples taken from femoral vein and cubital vein. Femoral vein sample is taken on injured side. The examination is carried out before and after treatment. The blood serum samples are placed on fat-free glass slide in the amount of 0.01-0.02 ml as drops, dried at 18-30°C for 18-24 h. The set of pathological symptoms becoming larger or not changed after the treatment in comparison to sample taken before treatment, and morphological picture of samples under comparison taken from the cubital vein showing no changes or being changed to worse, the treatment is considered to be effective.

EFFECT: enabled medicamentous treatment evaluation in course of treatment to allow the treatment mode to be changed in due time; avoided surgical intervention (amputation); retained active life-style of aged patients.

4 dwg

FIELD: veterinary medicine.

SUBSTANCE: method involves carrying out blood erythrocyte micro electrophoresis in alternating electric field with sign change frequency equal to 0.3-1 Hz. Electric field having current intensity equal to 4.5-5.0 mA is applied. Treatment is applied at 36-38°C. Oscillation amplitude being equal to 3 mm and less, endotoxicosis is detected.

EFFECT: high accuracy and simplicity of the method.

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