RU2239197C1 - Method for detecting lupus anticoagulant - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the present innovation deals with diagnostic methods: at detecting normal values of activated partial thromboplastin time (APTT) and kaolin time (KT) in patient's plasma one should detect factors for blood VIII and IX clotting and at increased activity of these factors patient's plasma should be mixed with plasma deficient by corresponding factor at ratio to provide normal activity of these factors in final mixture. The mixture should be incubated at 37 C for 2 h, one should repeatedly detect there APTT value and at excess of normal time for clot development and its normalization after incubating the mixture with phospholipids concentrate at 37 C for 5 min it is possible to diagnose availability of lupus anticoagulant.

EFFECT: higher accuracy of diagnostics.

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Description

The invention relates to medicine, namely to the diagnosis of thrombotic conditions, for example, recurring thrombosis, thromboembolism, persistent miscarriage of pregnancy, etc., due to the presence in the blood plasma of patients with lupus coagulant.

Currently, there is a method recommended by the International Subcommittee for Standardization of Lupus Anticoagulants (SSC) (1), according to which the determination of lupus anticoagulant is based on sequential measurement of the values of activated partial thromboplastin time (APTT) and kaolin in samples of test platelet-free blood plasma. With an increase in the time of formation of a blood clot (the norm accepted by international standards is 35 ± 5 sec and 60 ± 10 sec, respectively), a subsequent treatment of the test plasma with normal donor plasma is performed and the APTT and CV are re-measured. While maintaining elevated values in these reactions, the test serum is depleted with a standard preparation of phospholipid concentrate and, with normalization of the APTT values after a second study, it is concluded that there is a lupus anticoagulant.

The disadvantage of this method is its low specificity, which complicates the diagnosis and the development of adequate treatment tactics for patients with recurrent thrombosis, thromboembolism, persistent miscarriage, and others, who have increased activity of factors of the internal blood coagulation pathway (factors VIII and IX), masking the elongation of time APTTV and HF.

The objective of the invention is to develop a highly specific method for determining lupus anticoagulant in the plasma of patients with thrombotic conditions.

The essence of the invention lies in the fact that the patient being examined draws blood from the ulnar vein, centrifuges it twice for 10-15 minutes at 2000 × g, and then for 15-20 minutes at 3000 × g, blood plasma containing platelets in amount not exceeding 10 × 10 ⁹ cells / liter. Then, the APTT is determined, for which 50 ± 1 μl of a kefalin-kaolin mixture is added to the test blood plasma in a volume of 50 ± 1 μl (the error is provided by the instructions for standardized devices, for example, coagulometer cuvettes), heated for 3 min at 37 ± 2 ° C, add 50 ± 1 μl of 0.025 ± 0.0005 M calcium chloride solution having a temperature of 37 ± 2 ° C, and fix the time of clot formation. The results obtained are compared with the APTT of normal plasma equal to 35 ± 5 sec. To determine KB in a coagulometer cell, 50 ± 1 μl of the analyzed plasma is mixed, 50 ± 1 μl of a kaolin suspension, heated for 3 minutes at a temperature of 37 ± 2 ° C, 50 ± 1 μl of a 0.025 ± 0.0005 M calcium chloride solution having a temperature of 37 ± 2 ° C, and the time of clot formation is recorded. The result obtained is compared with a normal plasma KB value of 60 ± 10 sec. At normal values of APTT and KB in the plasma of patients with a clinically determined suspicion of thrombophilia (recurring thrombosis, thromboembolism) or in the presence of persistent miscarriage, to eliminate false-negative results due to increased activity of blood coagulation factors VIII or IX, these factors are determined using a unified single-stage method quantitative determination (2). In this case, 50 ± 1 μl of the investigated plasma, previously diluted 5-10 times with imidazole buffer, is introduced into the coagulometer cuvette, 50 ± 1 μl of substrate is deficient in factor VIII or IX plasma, 50 ± 1 μl of kefalin-kaolin mixture and 50 ± 1 μl of 0.025 ± 0.0005 M calcium chloride solution having a temperature of 37 ± 2 ° C, and the clot formation time is recorded. The activity of factor VIII or IX is determined by a pre-built calibration line constructed using the International Standard for the Activity of these factors. When detecting increased activity in the studied plasma of factors VIII and IX, 100 ± 2 μl of the studied plasma is mixed with plasma deficient in the corresponding factor in a ratio that ensures the normal level of activity (100%) of this coagulation factor in the final mixture. The mixture is incubated at 37 ± 2 ° C for 2 hours and the values of APTT and CV are determined. If these values remain within normal values, then they conclude that there is no lupus anticoagulant in the studied plasma. In case of prolongation of the APTT and / or KB values above normal values, it is concluded that the reason for their prolongation under the conditions of normal activity of factors VIII and IX (factors of the internal coagulation pathway) is the presence of anticoagulants. Normalization of the APTT value after incubation of 100 ± 2 μl of the plasma mixture at 37 ± 2 ° C for 5 min with 100 ± 2 μl of phospholipid concentrate obtained from erythrocyte membranes and providing depletion of the lupus anticoagulant allows us to conclude the presence of lupus anticoagulant.

The technical result of using the invention is the high specificity of determining the presence of lupus anticoagulant, independent of the activity of factors VIII and IX and allowing the patient to conduct the correct course of treatment.

The method is as follows. Blood is taken from the cubital vein from the patient, centrifuged twice for 10-15 minutes at 2000 × g, and then blood plasma containing platelets in an amount not exceeding 10 × 10 ⁹ cells is taken for 15-20 minutes at 3000 × g / l Then, the APTT is determined, for which 50 ± 1 μl of a kefalin-kaolin mixture is added to the test blood plasma in a volume of 50 ± 1 μl (the error is provided by the instructions for standardized devices, for example, coagulometer cuvettes), heated for 3 min at 37 ± 2 ° C, add 50 ± 1 μl of 0.025 ± 0.0005 M calcium chloride solution having a temperature of 37 ± 2 ° C, and fix the time of clot formation. The results obtained are compared with the APTT of normal plasma equal to 35 ± 5 sec. To determine KB in a coagulometer cell, 50 ± 1 μl of the analyzed plasma is mixed, 50 ± 1 μl of a kaolin suspension, heated for 3 minutes at a temperature of 37 ± 2 ° C, 50 ± 1 μl of a 0.025 ± 0.0005 M calcium chloride solution having a temperature of 37 ± 2 ° C, and the time of clot formation is recorded. The result obtained is compared with a normal plasma KB value of 60 ± 10 sec. At normal values of APTT and KB in the plasma of patients with a clinically determined suspicion of thrombophilia (recurring thrombosis, thromboembolism) or in the presence of persistent miscarriage, to eliminate false-negative results due to increased activity of blood coagulation factors VIII or IX, these factors are determined using a unified single-stage method quantitative determination. In this case, 50 ± 1 μl of the investigated plasma, previously diluted 5-10 times with imidazole buffer, is introduced into the coagulometer cuvette, 50 ± 1 μl of substrate is deficient in factor VIII or IX plasma, 50 ± 1 μl of kefalin-kaolin mixture and 50 ± 1 μl of 0.025 ± 0.0005 M calcium chloride solution having a temperature of 37 ± 2 ° C, and the clot formation time is recorded. The activity of factor VIII or IX is determined by a pre-built calibration line constructed using the International Standard for the Activity of these factors. When detecting increased activity in the studied plasma of factors VIII and IX, 100 ± 2 μl of the studied plasma is mixed with plasma deficient in the corresponding factor in a ratio that ensures the normal level of activity (100%) of this coagulation factor in the final mixture. The mixture is incubated at 37 ± 2 ° C for 2 hours and the values of APTT and CV are determined. If these values remain within normal values, then they conclude that there is no lupus anticoagulant in the studied plasma. In case of prolongation of the APTT or KB value above normal values, it is concluded that the reason for their prolongation under the conditions of normal activity of factors VIII and IX (factors of the internal coagulation pathway) is the presence of anticoagulants. Normalization of the APTT value after incubation with 100 ± 2 μl of the plasma mixture at 37 ± 2 ° C for 5 min with 100 ± 2 μl of phospholipid concentrate indicates the presence of lupus anticoagulant in the studied plasma.

Example 1

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

To measure APTT in a coagulometer cuvette, 50 μl of plasma obtained from venous blood are mixed by centrifugation twice (12 minutes at 2000 × g and 17 minutes at 3000 × g) and containing 8 × 10 ⁹ cells / l, 50 μl of kefalin-kaolin mixture is heated within 3 minutes at 37 ° C, add 50 μl of pre-heated at 37 ° C 0.025 M calcium chloride, determine the time of clot formation and compare it with the APTT value of normal plasma, equal to 35 ± 5 seconds. To determine the kaolin time, 50 μl of the analyzed plasma, 50 μl of a kaolin suspension are mixed in a coagulometer cuvette, heated at 37 ° C for 3 minutes, 50 μl of 0.025 M calcium chloride preheated at 37 ° C are added, the clot formation time is recorded, and the result obtained is compared. with the size of kaolin time of normal plasma (60 ± 10 seconds). The values of APTT and kaolin time were 33 and 57 seconds, respectively, which did not differ from the norm, but left open the question of the causes of thrombosis. Therefore, in the future, the activity of factor VIII is determined. Add 50 μl of the patient’s plasma previously diluted 5 times with imidazole buffer to the coagulometer’s cuvette, add 50 μl of a substrate that is deficient in factor VIII plasma, 50 μl of kefalin-kaolin mixture, 50 μl of a 0.025 M calcium chloride solution heated at 37 ° С, record the time the formation of a clot and using a previously constructed calibration curve using plasma, certified for the activity of factor VIII using the International Standard, determine the activity of factor VIII, which was equal to 250%, which suggests the possibility of incorrect hydrochloric interpretation hemostasiological status of the patient. To eliminate this possibility, 50 μl of patient B plasma is mixed with 50 μl of factor VIII deficient plasma (1: 1 ratio), incubated for 2 hours at 37 ° C, and after incubation, the APTT and kaolin time values are found to be 47 and 85 seconds respectively. In this case, the norms of APTT and kaolin time are 35 ± 5 seconds and 60 ± 10 seconds. Exceeding the values of these indicators under the conditions of normalization of the activity of factor VIII indicates the presence of a coagulological anomaly in patient B. To identify the possibility of the presence of a lupus anticoagulant in the patient, 100 μl of the studied plasma mixture of patient B and a deficiency plasma of factor VIII are mixed with 100 μl of phospholipid concentrate and incubated for 5 minutes at 37 ° C. The APTT value determined in this mixture was 38 sec, which falls within the normal range of this indicator. Normalization of this indicator in the presence of an excess of phospholipids indicates the presence in the studied plasma of antibodies specifically to phospholipid-protein complexes, i.e. lupus anticoagulant. Based on the results obtained, a conclusion was drawn about the presence in the plasma of patient B of 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 and thrombosis. London, Blackwell Scientific Publications, Third Ed., 1984.

Claims (1)

A method for determining lupus anticoagulant in the plasma of patients with thrombophilia, including measuring the values of activated partial thromboplastin time (APTT) and kaolin time (KB), characterized in that at normal values of APTT and KB, blood coagulation factors VIII and IX are determined and with increased activity of these factors, the patient’s plasma is mixed with plasma deficient in the corresponding factor in a ratio that ensures the normal activity of these factors in the final mixture, the mixture is incubated at 37 ° C in t chenie 2 hours, re-determined in this mixture and the APTT value in excess of normal clot formation time and his normalization after incubation mixture with a concentrate of phospholipids at 37 ° C for 5 min diagnose the presence of lupus anticoagulant.