RU2181203C2 - Method for determining lupus erythematosus anticoagulant availability - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves making use of normal cell membrane fragments procoagulation activity level reduction property in patients having lupus erythematosus anticoagulant in plasma. Donor cell membrane fragments are to be added to blood plasma under study with phospholipid cell membrane fragments being removed from the plasma. Kaolin coagulation time is measured. R2 index is calculated as ratio between the kaolin coagulation time in plasma with phospholipid cell membrane fragments being removed and the kaolin coagulation time in plasma with donor phospholipid cell membrane fragments added. R2 value being less than 1.5, lupus erythematosus anticoagulant availability is considered to be determined. EFFECT: improved specificity of test; simplified and accelerated test method. 4 dwg, 2 tbl

Description

 The invention relates to medicine, in particular blood testing, and can be used for laboratory diagnosis of thrombophilia due to the presence of lupus anticoagulant (VA) in the blood.

Thrombophilia is a group of diseases characterized by an increased tendency of blood to thrombosis. They are widespread in clinical practice and are mostly caused by impaired blood properties, and therefore they are called "hematogenous thrombophilia." One of the common types of thrombophilia is antiphospholipid syndrome (APS), which is caused by the circulation in the blood of an inhibitor of phospholipid matrices involved in blood coagulation reactions. This phospholipid matrix inhibitor is called lupus anticoagulant. There are many ways to identify the effects of VA, based on the detection of elongation of coagulation of platelet-poor plasma (BTP) in phospholipid-dependent coagulation tests. For this purpose often used kaolin clotting time (KB), activated partial thromboplastin time (APTT) reagent Platelin ^®, diluted with test thromboplastin et al. [Brandt JT, Triplet DA, Alving V., Scharrer I. Criteria for the Diagnosis of Lupus Anticoagulants: An Update. On behalf of the Subcommittee on Lupus Anticoagulant / Antiphospholipid Antibody of the Scientific and Standardization Committee of the ISTH. // Thromb. Haemost. - 1995. - V. 74. - 4. - P. 1185-1190.]. In addition, the hemocoagulating effects of snake venoms are used to detect VA [Thiagarajan R., Pengo V., Shapiro SS The use of the dilute Russell's Viper Venom Time for the diagnosis of lupus anticoagulant. Blood 68: 869-874, 1986]. A confirmatory test is the elimination of detected abnormalities by the emulsion of normal destroyed platelets [Ex T., Triplett DA, Taberner D., Machin SJ Guidelines for Testing and Revised Criteria for Lupus Anticoagulants. SSC Subcommittee for Standartization of Lupus anticoagulants. // Thromb. Haemost. - 1991. - 65. - P. 320-322].

 The disadvantages of the known methods are: 1) their non-specificity, i.e. there are no tests that would prove direct inhibition of phospholipid matrices by the lupus anticoagulant (therefore, to identify VA, it is necessary to carry out the whole complex of proposed methods, which leads to an extension of the study time); 2) the non-standard nature of a number of reagents produced by different companies (cephalins and their analogues, thromboplastins, snake venoms, etc.); 3) lack of availability of reagents and their high cost.

 The closest (prototype) is a method for determining the activation of blood plasma coagulation by fragments of cell membranes (patent 2058031). The method allows to determine the membrane activation of blood coagulation (MACK) in the test plasma by determining the time of recalcification of a mixture of fragments of cell membranes of the test plasma and substrate plasma, followed by the assessment of MACK by the calibration curve. However, the known method does not allow to detect lupus anticoagulant in the studied plasma.

 A positive result of the proposed method is the identification of lupus anticoagulant by determining the inhibition of normal phospholipid fragments of cell membranes (FCM) in the filtered plasma of patients with APS.

 A positive result is achieved by the fact that in the presence of VA, a small shortening of KB is detected after the addition of normal PCM to the patient's plasma and incubation due to inhibition of the procoagulant activity of normal PCM by a lupus anticoagulant. In a healthy plasma (and in a patient’s plasma without VA), shortening of KB after adding normal FCM is more pronounced, since there is no inhibition of the procoagulant activity of normal FCM. The determination scheme of VA is shown in FIG. 1.

 The method is as follows.

 Reagents and equipment: 1. 0.1 M sodium citrate solution; 2. Michaelis buffer, pH 7.4 (after preparation, filtered through a filter with a pore diameter of 0.2 μm); 3. Kaolin (light fraction), the company "Technology Standard", a concentration of 0.025 mg / ml; 4. A solution of calcium chloride, 0.025 M; 5. Filters with a pore diameter of 0.2 μm "Minisart-RS15 SM177610Q", the company "Sartorius" (Germany), in a polypropylene frame with syringes for filtration; 6. Centrifuge OPN-8.

Platelet-poor plasma preparation
Blood is obtained from the cubital vein with a silicone needle in a plastic tube containing a 0.1 M sodium citrate solution (blood to citrate ratio 9: 1). Blood is centrifuged for 5 min at a rotation speed of 1000 rpm (200g). The resulting platelet-rich plasma is re-centrifuged for 20 minutes at a speed of 3500 rpm (1200g). The resulting platelet-poor plasma (donor and patient) is used to conduct the study.

 Preparation of test plasma samples.

 1. Obtaining donor plasma lacking FCM. For this purpose, 1.0 ml of BTP of the donor is filtered through a filter with a pore diameter of 0.2 μm (using a Minisart-RS15 SM177610Q filtering device). By filtration, PCM removal from the donor plasma is achieved. (Donor plasma without PCM is designated as sample 1).

 2. Obtaining donor plasma with a normal PCM content. For this purpose, the filter used in preparing sample 1 (with precipitated PCM from donor plasma) is washed with 3 ml of Michaelis buffer (to remove residual plasma). Then the filter is turned over and the filtration (in the opposite direction) of the plasma of the same donor in a volume of 1 ml is washed out of the normal PCM deposited on the filter. This is how the normal PCM deposited on the filter returns to the filtered plasma of the donor. (Sample 2 control).

 3. Obtaining the plasma of a patient lacking FCM. For this purpose, 1.0 ml of BTP of the test patient is filtered through a filter with a pore diameter of 0.2 μm. By filtration, PCM removal from the patient's plasma is achieved. (Plasma of the patient without FCM is designated as sample 3).

 4. Adding the FCM of the donor to the plasma of the examined patient lacking FCM. To do this, filter 1.0 ml of BTP of the donor through a filter with a pore diameter of 0.2 μm (by filtering the PCM of the donor is deposited on the filter). To remove plasma residues, the filter with precipitated FCM donor is washed by filtering 3 ml of Michaelis buffer. Turn the filter over and wash the normal PCM with the patient’s plasma in a volume of 1.0 ml by filtration. This is how the normal PCM deposited on the filter is added from the donor plasma to the filtered plasma of the patient. (Sample 4).

The course of determination. All four samples were incubated for 120 min at +20 ^° C, and then the kaolin clotting time was determined in these samples. For this, 0.1 ml of the test sample is mixed with 0.1 ml of kaolin suspension and incubated for 3 min in a water bath at + 37 ^° C. Then, 0.1 ml of calcium chloride solution is added and the clotting time is recorded.

1. Determine the CV in sample 1 (the result is designated as t ₁ ).

2. Determine the KB in sample 2 (the result is designated as t ₂ ).

3. Determine the KB in sample 3 (the result is designated as t ₃ ).

4. Determine the KB in sample 4 (the result is designated as t ₄ ).

Evaluation of the results. Indexes are calculated
R ₁ = t ₁ / t ₂ ;
R ₂ = t ₃ / t ₄ .

For normal plasma, the ratio of R ₁ is in the range of 1.7-2.0.

In patients with the presence of plasma lupus anticoagulant, the ratio of R _{2 is} always less than 1.5.

results
We studied the clinical data and indicators of the hemostatic system of 17 patients with antiphospholipid syndrome, which proceeded against the background of systemic lupus erythematosus (SLE). The criteria adopted by rheumatologists were used to diagnose SLE. All 17 patients with SLE had classical laboratory signs of VA (prolongation of coagulation time in phospholipid-dependent coagulation tests, lack of correction of coagulation time extension by normal plasma, correction of coagulation time extension by normal and destroyed platelets). Comparison was made with a group of healthy people (20 healthy donors) and a group of patients with SLE (15 patients) who did not have lupus anticoagulant in their blood.

After filtration (sample 1), there are no PCMs in normal plasma, since they are larger than the pore diameter of the filter; therefore, KB after removing the donor PCM (t ₁ ) is extended to 160.1 + 4.9 s. After the donor PCM returns to the donor plasma (sample 2), the kaolin clotting time (t ₂ ) is shortened to 87.5 ± 3.1 s. Accordingly, the index R ₁ was equal to 1.83 ± 0.06 (see figure 2).

KB in filtered plasma of patients with VA (t ₃ ) averaged 210.2 ± 10.5 s. After introducing the PCM donor into the filtered plasma of a patient with VA and incubation, KB (t ₄ ) is shortened, according to average data, to 154.3 ± 9.1. Accordingly, the R ₂ index in patients with VA is on average 1.36 ± 0.04.

Low values of the R ₂ index were found only in the presence of lupus anticoagulant (in all 17 patients with VA, R ₂ was less than 1.5). To prove that this indicator decreases due to the influence of VA, and not due to any other reasons, this index was determined in patients with SLE who did not have VA in the blood. No decrease in the R ₂ index was found in patients with SLE without VA. The R ₂ index in this group was equal to an average of 1.86 ± 0.09 (There were no patients out of 15 with an R ₂ index less than 1.74). This means that the addition of the same normal FCM to the filtered plasma of other patients with SLE (but without VA in the blood) leads to a significant shortening of KB (i.e., the reproduction of the normal procoagulant activity of FCM), but the addition of the same normal FCM to the plasma of a patient with VA leads to a small shortening of KB (which indicates a low procoagulant activity of normal PCM in the filtered plasma of a patient with VA).

 The inventive method is highly specific, since we investigated the degree of inhibition of the procoagulant activity of normal phospholipid fragments of cell membranes by a lupus anticoagulant. Other inhibitors that specifically inhibit the activity of coagulation factors do not inhibit phospholipid matrices; therefore, they do not affect the test readings.

 The method has good reproducibility. When examining the plasma of a patient with VA on different days, the coefficient of variation of the test is not more than 6%.

Clinical examples
1. Patient K., 37 years old (case history number T-1222), was admitted to the therapeutic department of the city hospital in Barnaul city on 12/22/95 with complaints of fever to febrile numbers, pain in the knee, hip, shoulder joints, and hyperemia in the area of the zygomatic arches and nose bridge, headache, weakness, fatigue. Sick for four years (is on the "D" account for SLE). Right popliteal artery thrombosis was in 1995. She took prednisone at a dose of 15 mg / day, non-steroidal anti-inflammatory drugs (ibuprofen), plankvenil. Last deterioration since early October 1995. When viewed from the face, erythematous rashes in the form of a “butterfly” in the area of the zygomatic arches and nose bridge. Passive and active movements in large joints are painful. A physical examination of the respiratory system revealed no pathological changes. The boundaries of relative dullness of the heart are not changed. Heart sounds are clear, the rhythm is correct, heart rate is 66 per minute. The abdomen on palpation is soft, painless. The liver and spleen are not enlarged.

Data from laboratory examination methods:
Hb - 124 g / l; ROE 43 mm / h; White blood cells - 4.5 x 10 ⁹ / l;
Leukocyte formula: B - 1%; E - 3%; P - 3%; C - 56%; L - 30%; M - 7%.

Biochemical analysis of blood: Creatinine 68 µmol / l; urea 5.0 mmol / l; total bilirubin 14 µmol / l, direct 3.5 µmol / l, indirect 10.5 µmol / l; glucose 4.0 mmol / l; total protein - 73.0 g / l, albumin - 58%, α1-3.7%, α2-9.9%, β-11.1%, γ-17.3%.
RW - omp., LE cells 10: 1000 were detected, antibodies to native DNA 1/16, antibodies to denatured DNA 1/8, CEC 40 beats. units

 Immunoglobulins: IgG - 20.2 (7.2-16.4), IgM - 1.1 (0.6-1.8), IgA - 12.9 (1.9-5.4). General urine analysis without features. The study of the hemostatic system - see table. 1.

 Diagnosis: Systemic lupus erythematosus, chronic course, activity II, with damage to the skin and joints, secondary antiphospholipid syndrome.

 Prescribed treatment: indomethacin 1 tab x 4 times a day, prednisone (in tablets) 30 mg per day, heparin 2.5 units x 4 times a day (subcutaneously), plasmapheresis 8 sessions (400 ml per session).

 After treatment, improvement was achieved: the temperature returned to normal, joint pain decreased, but erythematous rashes remained in the form of a “butterfly” on the face.

 2. Patient D, 18 years old, was admitted to the rheumatology department of the Altai Regional Clinical Hospital 10.24.95 (medical history 3-117) with complaints of fever to febrile values, joint pain, visual impairment, the presence of a “butterfly” on the face, weakness fatigue. Sick for two years (is on the "D" account for SLE). During the disease, he noted thrombotic complications twice: retinal vein thrombosis in June 1993 and left femoral artery thrombosis in August 1994. Repeatedly in the general analysis of urine found protein and red blood cells. She took prednisolone at a dose of 20 mg / day, non-steroidal anti-inflammatory drugs, plankvenil. Last deterioration since the end of September 1995. When examined on the face of an erythematous rash in the form of a "butterfly" in the zygomatic arches and nose bridge, divergent strabismus. During a physical examination of the respiratory system, deviations were not found. The boundaries of relative dullness of the heart are not changed. Heart sounds are clear, the rhythm is correct, heart rate is 78 per minute. The abdomen on palpation is soft, painless. The liver and spleen are not enlarged.

Data from laboratory examination methods:
Нb - 115 g / l; ROE 47 mm / h; White blood cells - 5.3 x 10 ⁹ / l;
Leukocyte formula: B - 1%; E - 1%; P - 1%; C - 57%; L - 32%; M - 8%.

Biochemical analysis of blood: Creatinine 74 μmol / l; urea 5.9 mmol / l; total bilirubin 13 μmol / l, direct 4 μmol / l, indirect 9 μmol / l; glucose 4.6 mmol / l; CRP +++; total protein - 82.9 g / l, albumin - 50%, α1-5.0%, α2-11.0%, β-14.0%, γ-20.0%.
Found LE cells 8: 1000, antibodies to native DNA 1/16, antibodies to denatured DNA 1/16. Positive antinuclear factor 1/100. CEC 40 beats. units Immunoglobulins: IgG - 20.2 (7.2-16.4), IgM - 1.1 (0.6-1.8), IgA - 12.9 (1.9-5.4). General urine analysis without features. On the ECG blockade of the right leg of the bundle of His. Optometrist's conclusion: Residual effects of retinal vein thrombosis: Atrophy of the optic nerve OS. Visual acuity 1.0 / 0.01. Exotropia. The study of the hemostatic system - see table. 2.

 Diagnosis: Systemic lupus erythematosus, chronic course, activity III, with damage to the skin, joints, heart, secondary antiphospholipid syndrome. Atrophy of the optic nerve OS.

 Prescribed treatment: indomethacin 1 tab x 4 times a day, prednisone (in tablets) 30 mg per day, heparin 2.5 units x 4 times a day (subcutaneously), plasmapheresis 10 sessions (300 ml per session). After treatment, the patient's condition improved: the temperature returned to normal, joint pain stopped, and weakness decreased. Erythematous rashes in the form of a “butterfly” in the zygomatic arches and nose bridge are preserved.

In the given clinical examples, it was shown that in these patients the coagulation tests detecting VA are positive (KB, APTT, coagulation time with diluted thromboplastin, lebetox, mixing and correction tests). The degree of lengthening of clotting time in different tests is different, the R ₂ index is significantly reduced, but this index rises after effective treatment. Thus, the R ₂ index can be used to detect VA and as a criterion for effective treatment.

Claims (1)

A method for determining lupus anticoagulant by plasma filtration and determining kaolin coagulation time, characterized in that phospholipid fragments of the cell membrane of the donor are added to the blood plasma lacking phospholipid fragments of the cell membranes, the index R _{2 is} calculated as the ratio of kaolin coagulation time in the studied plasma lacking phospholipid fragments of cell membranes, to the kaolin coagulation time in the test plasma after adding phospholipid fragments of cell memes the donor brane and with an R ₂ value _of less than 1.5, the test results are considered positive, indicating the presence of a lupus anticoagulant in the test plasma.

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