The method of determination of lupus anticoagulant

 

(57) Abstract:

The invention relates to medicine, namely to diagnostic methods. The essence of the method: when determining normal values of activated partial thromboplastin time (APTT) and kaolin time (KB) in the plasma of the patient carried out the determination of coagulation factors VIII and IX and increased activity of these factors in the patient plasma is mixed with plasma, deficient for the corresponding factor in the ratio which, in a finite mixture of normal activity of these factors. The mixture is incubated at C for 2 hours, re-define in this mixture, the value of APTT and in excess of the normal time of formation of the clot and its normalization after incubation mixture with phospholipids concentrate when C for 5 min diagnose the presence of lupus anticoagulant. The method improves the accuracy of diagnosis.

The invention relates to medicine, namely to the diagnosis of thrombotic conditions, such as recurrent thrombosis, thromboembolism, sustainable nevynashivanii pregnancy, etc. due to the presence in plasma of patients with lupus coagulant.

Currently am the ommittee for the Standardization of Lupus Anticoagulants) (1), according to which definition of lupus anticoagulant is based on successive measurement values of the activated partial thromboplastin time (APTT) and kaolin time (KB) in the samples tested estramboticos blood plasma. With increasing time of formation of blood clot (adopted by the international standards norm equal to 35±5 sec and 60±10 seconds respectively) conduct post-processing of test plasma normal donor plasma and re-measure the APTT and KV. While maintaining high performance in these reactions Deplete the tested serum standard drug concentrate phospholipids and normalization of APTT after repeated studies conclude that the presence of lupus anticoagulant.

The disadvantage of this method is its low specificity, which complicates diagnosis and the development of an adequate treatment of patients with recurrent thrombosis, embolism, sustainable nevynashivaniyu pregnancy, and others, have increased the activity of the internal factors of the path of blood clotting factors VIII and IX), masking prolonged APTT and KV.

The objective of the invention is the development that accounts for the institutions of the state.

The essence of the invention lies in the fact that the examined patient take blood from the cubital vein, centrifuged her twice for 10-15 min at 2000 g, and then for 15-20 min at 3000 g selected blood plasma containing the platelets in the amount not exceeding 10109CL/L. Then spend the determination of APTT, for which the tested plasma in a volume of 50±1 mm (error provides guidelines for standardized instruments, such as automatic pipettes, kootam of coagulometer) add 50±1 µl cephalin-kaolin mixtures were heated for 3 min at 37±2C, add 50±1 mm 0,025±0,0005 M solution of calcium chloride having a temperature of 37±2C, and record the time of the formation of a clot. The obtained results are compared with the value of APTT normal plasma equal to 35±5 sec. To determine the KB in a cell of coagulometer mix 50±1 ál of the test plasma, 50±1 μl of the suspension of kaolin, heated for 3 min at a temperature of 37±2C, add 50±1 mm 0,025±0,0005 M solution of calcium chloride having a temperature of 37±2C, and record the time of the formation of a clot. The obtained result is compared with the value of KB normal plasma, 60±10 seconds. Under normal APTT values and KB in the plasma of patients with KLINICHESKOI nevynashivanii pregnancy to exclude false-negative results, due to the increased activity of coagulation factors VIII or IX, spend the definition of these factors on the standardized one-step method for quantitative determination (2). 50±1 µl sample of plasma, pre-diluted 5-10 times imidazolium buffer, making the cell of coagulometer add 50±1 µl of the substrate deficient in factor VIII or IX plasma, 50±1 µl cephalin-kaolin mixture and 50±1 mm 0,025±0,0005 M solution of calcium chloride having a temperature of 37±2C, and record the time of the formation of a clot. The activity of factor VIII or IX is determined by a pre-built calibration straight line is built using the International standard activity of these factors. When detecting increased activity in the study of plasma factors VIII and IX mix 100±2 µl sample plasma plasma, deficient for the corresponding factor in the ratio which, in a finite mixture of normal activity level (100%) of the coagulation factor. The mixture is incubated at 37±2C for 2 hours and determine the values of APTT and KV. If these values remain within normal values, then make a conclusion about the absence of the studied plasma lupus anticoagulant. In clunia in the normal activity of factors VIII and IX (factors internal path coagulation) is the presence of anticoagulants. Normalization of APTT values after incubation, 100±2 µl of the mixture of plasmas at 37±2C for 5 min with 100±2 μl of the concentrate of phospholipids derived from erythrocyte membranes and providing depletion on the lupus anticoagulant, allows to make a conclusion about the presence of lupus anticoagulant.

The technical result of the use of the invention is the high specificity of detection of lupus anticoagulant, is not dependent on the magnitude of activity of factors VIII and IX and allowing the patient the correct treatment.

The method is as follows. The patient take blood from the cubital vein, centrifuged her twice for 10-15 min at 2000 g, and then for 15-20 min at 3000 g selected blood plasma containing the platelets in the amount not exceeding 10109CL/L. Then spend the determination of APTT, for which the tested plasma in a volume of 50±1 mm (error provides guidelines for standardized instruments, such as automatic pipettes, kootam of coagulometer) add 50±1 µl cephalin-kaolin mixtures were heated for 3 min at 37±2C, add 50±1 mm 0,025±0,0005 M solution of calcium chloride having a temperature of 37±2C, and fix Vremya definitions KB in a cell of coagulometer mix 50±1 ál of the test plasma, 50±1 μl of the suspension of kaolin, heated for 3 min at a temperature of 37±2C, add 50±1 mm 0,025±0,0005 M solution of calcium chloride having a temperature of 37±2C, and record the time of the formation of a clot. The obtained result is compared with the value of KB normal plasma, 60±10 seconds. Under normal APTT values and KB in the plasma of patients with clinically due to suspected thrombophilia (recurrent thrombosis, embolism) or in the presence of a stable nevynashivanii pregnancy to exclude false-negative results caused by increased activity of coagulation factors VIII or IX, spend the definition of these factors on the standardized one-step method for quantitative determination. 50±1 µl sample of plasma, pre-diluted 5-10 times imidazolium buffer, making the cell of coagulometer add 50±1 µl of the substrate deficient in factor VIII or IX plasma, 50±1 µl cephalin-kaolin mixture and 50±1 mm 0,025±0,0005 M solution of calcium chloride having a temperature of 37±2C, and record the time of the formation of a clot. The activity of factor VIII or IX is determined by a pre-built calibration straight line is built using the International standard is 100±2 µl sample plasma plasma scarce on the corresponding factor in the ratio which, in a finite mixture of normal activity level (100%) of the coagulation factor. The mixture is incubated at 37±2C for 2 hours and determine the values of APTT and KV. If these values remain within normal values, then make a conclusion about the absence of the studied plasma lupus anticoagulant. In the case of elongation values of APTT KB or above normal values conclude that the reason for their prolongation in the normal activity of factors VIII and IX (factors internal path coagulation) is the presence of anticoagulants. Normalization of APTT values after incubation, 100±2 µl of the mixture of plasmas at 37±2C for 5 min with 100±2 μl of the concentrate phospholipids svidetelstvo about the presence of lupus anticoagulant in the tested plasma.

Example 1

In the plasma of the patient B with recurrent thrombotic complications (venous thrombosis of the lower extremities with edema and pain episodes) determine the values of APTT and kaolin time.

To measure the APTT in a ditch of coagulometer mix 50 µl of plasma was obtained from venous blood twice by centrifugation (12 minutes at 2000 g and 17 minutes at 3000 g) and preheated at S 0.025 M calcium chloride, determine the time of formation of a clot and compares it with the value APTT normal plasma equal to 35±5 seconds. To determine kaolin time in the cell of coagulometer mix 50 ál of the test plasma, 50 μl of the suspension of the kaolin is heated at C for 3 minutes, add 50 ál of pre-warmed at S 0.025 M calcium chloride, record the time of the formation of a clot and the obtained result is compared with the value of kaolin time of normal plasma (60±10 seconds). Values of APTT and kaolin time was equal to 33 and 57 seconds respectively, from the norm did not differ, but left open the question about the causes of thrombosis. Therefore, to further determine the activity of factor VIII. In the cell of coagulometer contribute 50 ál plasma of the patient, pre-diluted 5 times imidazolium buffer add 50 ál of the substrate deficient in factor VIII plasma, 50 μl of cephalin-kaolin mixture, 50 μl of warmed when S 0.025 M solution of calcium chloride, record the time of the formation of a clot and pre-constructed calibration curve using plasma, certified by the activity of factor VIII using International Standard, determine the activity of factor VIII, which was equal to 250% of that pricey opportunities 50 μl of patient plasma B is mixed with 50 μl of plasma, deficient in factor VIII (in the ratio 1:1), incubated for 2 hours at C and after incubation to determine the values of APTT and kaolin time, which was equal to 47 and 85 seconds, respectively. The rates APTT and kaolin time is 35±5 sec and 60±10 seconds. Excess quantities of these indicators in terms of achieved normalization of the activity of factor VIII indicates the presence koagulologicescoe anomalies in a patient B. To identify the presence in a patient with lupus anticoagulant 100 µl sample mixture plasma of the patient B and deficient in factor VIII plasma is mixed with 100 μl of the concentrate phospholipids and incubated for 5 minutes at S. Defined in this mixture the APTT value was 38 seconds, which is within the normal values of this indicator. The normalization of this index in the presence of excess phospholipids indicates the presence in the plasma of antibodies to phospholipid-protein complexes, i.e. lupus anticoagulant. On the basis of the obtained results the conclusion about the presence in the plasma of the patient B lupus anticoagulant.

LITERATURE

1. Exner T. et al. Thromb. Haemost.,1991, v.65, p.320-322.

2. Biggs R., Rizza C. Human blood coagulation, haemostasis'an is selected with thrombophilia, including the measurement values of the activated partial thromboplastin time (APTT) and kaolin time (KB), characterized in that the normal values of APTT and KB performed the determination of coagulation factors VIII and IX and increased activity of these factors in the patient plasma is mixed with plasma, deficient for the corresponding factor in the ratio which, in a finite mixture of normal activity of these factors, the mixture is incubated at C for 2 h, define again in this mixture, the value of APTT and in excess of the normal time of formation of the clot and its normalization after incubation mixture with phospholipids concentrate when C for 5 min diagnose the presence of lupus anticoagulant.

 

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