Method for predicting antiphospholipid syndrome

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

 

The invention relates to medicine, in particular to the study of blood, and can be used for laboratory diagnosis of the antiphospholipid syndrome.

It is known that the antiphospholipid syndrome (APS) is a common cause of recurrent thrombosis, embolism, heart attacks, cerebrovascular disease and other thrombotic disorders. The essence of its pathogenesis is the appearance in the blood of antiphospholipid antibodies (APA) to negatively charged phospholipids and protein-phospholipid complexes. To this group of antibodies is ″lupus anticoagulant″ (VA), first identified and described Conley C.L. et al. in 1952, He appears in the blood in many immune diseases and syndromes. There are known cases of detection of VA in patients without a clear manifestations of any other diseases. The emergence of VA in the blood affects the preceding disease often leads to the development of thrombotic complications and is one of the causes of miscarriage. Therefore, rapid and accurate diagnosis of ASF is an important task.

A known method for the diagnosis of ASF by defining in the blood of antibodies to negatively charged phospholipids (immunoglobulins G, M and A, including anticardiolipin) (Harris E.N., Gharavi, A.E., Patel SA, Hughes G.R.V.Evaluation of the anti-cardiolipin test: report of an international workshop held on 4 April 1986. // Clin. Exp. Immunol. - 1987. - 68. - P.215-22).

The disadvantage of this method is the low specificity because it does not detect VA, but only determines the state of the immune system, which allows the presence in the blood of this anticoagulant. The known method is time-consuming, often give false-positive results of analysis requires specialized equipment for enzyme immunoassay and expensive imported reagents.

The closest achieved a positive result (prototype) is a method for the diagnosis of ASF patent No. 2104550 from 10.02.98,, Russian Federation. Priority from 24.05.95, (Method for the diagnosis of antiphospholipid syndrome).

The prototype method is as follows:

Pre-receive poor in platelets normal control plasma and platelet-poor plasma (BTP) of the patient.

Step 1. In parallel to the control samples of normal plasma and BTP patient perform approximate phospholipidosis coagulation tests.

Step 2. When identifying gipokoagulyatsii in any approximate test performs the first correction test. To do this as a sample of plasma, a mixture of BTP patient and normal control plasma in a ratio of 4:1. Elimination gipokoagulyatsii in a mixture with normal control plasma excludes the presence of plasma VA./p>

Step 3. In the absence of correction gipokoagulyatsii in the dough mixing with normal control plasma to perform a second correction test, where BTP patient is mixed with a 3% solution of retropolated. If achieved correction gipokoagulyatsii retropolation, diagnose the ASF.

The disadvantages of the prototype method are as follows:

1. The complexity, a lot of stages and associated significant expenditure of time.

2. The inability of quantitative evaluation of the results obtained.

3. The inability diagnosis of ASF on the background of the application of anticoagulants of indirect action (IDA).

The inventive method does not have these disadvantages: requires two steps, which significantly reduces the time of the study and gives quantitative results on its readings do not affect the ANDES.

The authors offer a highly efficient method for the diagnosis of antiphospholipid syndrome in order to identify the ASF, based on the determination of prothrombin clotting time with two thromboplastin reagents: high-sensitivity and low-sensitivity to the action of VA.

A positive result of the invention is to improve the accuracy, due to the quantitative evaluation of the results of the study, effective diagnosis of ASF on the treatment of the ANDES and the simplification of the way. Pological the effective result is achieved by they use high-sensitivity and low-sensitivity to the lupus anticoagulant thromboplastin reagents, calculate the ratio of the index of elongation of prothrombin time and when the value of the ratio of more than 1.1 diagnose the ASF.

The method is as follows:

Reagents and equipment:

1. Sodium citrate, trehzameshchenny, 3.8% of the solution, obtained by dissolving 3.8 g translesanas sodium citrate in 100 ml of distilled water.

2. Thromboplastin VA+ (Techplast™), the company ″Technology-Standard″, Russia, (ISI=1,1), - thromboplastin reagent with high sensitivity to VA, freeze.

3. Thromboplastin VA-the firm "Technology Standard", Russia, (ISI=1,1) - soluble thromboplastin reagent with a low sensitivity to VA, freeze.

4. RNP-plasma, the firm "Technology Standard", Russia. Reference normal plasma, lyophilized.

5. Centrifuge ARF-8.

6. Coagulometer "minilab 701", manufacturer "Unimed, Russia.

Preparation of investigational BTP patient and normal control plasma (in healthy people)

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 for 5 minutes at a speed of 1000 rpm (200 g). Received rich thrombus is zitani plasma re-centrifuged for 20 minutes at a speed of 3500 rpm (1200 g). Received BTP use for research.

Breeding reagents

1. Dilution of Thromboplastin VA+

In a bottle with thromboplastin add 5 ml of distilled water. Mix thoroughly. Incubate reagent at 37°C for 20 minutes

2. Dilution of Thromboplastin VA-

In a bottle with thromboplastin add 5 ml of distilled water. Mix thoroughly. Incubate reagent at 37°C for 20 minutes

3. In a bottle with RNP-plasma add 1 ml of distilled water. Before application, the plasma must be maintained at room temperature for 15 minutes

The process of determining

Step 1. Parallel samples of the reference normal plasma and BTP patient determine prothrombin clotting time with thromboplastin VA+.

0.1 ml BTP incubated for 1 min in a thermostat coagulometer at +37°C. Then add 0.2 ml of thromboplastin reagent with high sensitivity to VA. Coagulometer registers collapse (the result obtained in the plasma sample of the patient, designated as t1and similar to the result of analysis of the reference normal plasma denote as t2).

Step 2. Parallel samples of the reference normal plasma and BTP examine the patient prothrombin clotting time with thromboplastin ALL.

0.1 ml BTP incubated for 1 min in a thermostat is coagulometer at +37° C. Then add 0.2 ml of thromboplastin reagent with a low sensitivity to VA. Coagulometer registers collapse (the result obtained in the plasma sample of the patient, designated as t3and a similar study reference normal plasma denote as t4).

Calculate the indicator NR (NR is the ratio of the indices of the prothrombin time, which quantitatively assesses hypocoagulation effect when the ASF on results of determination of prothrombin time with different thromboplastins) by the formulas:

where: t1the clotting time BTP patient with thromboplastin VA+;

t2the clotting time normal reference plasma thromboplastin VA+;

t3the clotting time BTP patient with thromboplastin VA-;

t4the clotting time normal reference plasma thromboplastin VA-;

Rxthe rate of elongation clotting time of the patient, in comparison with the control test thromboplastin VA+;

Rythe rate of elongation clotting time of the patient, in comparison with the control test thromboplastin VA-;

An assessment of the results

During the examination of healthy people and patients without APS established that among the it index NR was equal to 0,99 (SD=0,055, m=0,01, n=30)and normal fluctuations (X±2SD) was 0.88-1,10. Therefore, NR in healthy people and patients without APS (in plasma are not VA) is always less than the 1.1. In those cases, where NR equals or exceeds 1.1, diagnose antiphospholipid syndrome.

Clinical testing of the proposed method

Tested the proposed method of diagnosis was conducted on 27 patients with APS, which took place at the background of systemic lupus erythematosus (SLE). The patients were examined prior to the appointment of the ANDES and on the background of therapy with these anticoagulants. For clinical and laboratory diagnosis of SLE used adopted by the rheumatology criteria. In all the examined patients with APS had laboratory signs VA (prolongation of clotting times in phospholipidosis coagulation tests, no correction lengthening clotting time of normal plasma in the presence of correction lengthening clotting time by retropolation).

Control studies were performed on a group of healthy people (30 people) and the group of patients with SLE who had no signs of ASF (25 people).

From table I it is seen that NR patients with APS was significantly higher than in the healthy group and indicator NR patients without APS.

Diagnosis of APS patients receiving ANDES

It is known that the main clinical manifestation of the FS are thrombotic disorders, and so these patients are often be treated ANDES under the control of the international normalized ratio (MPE). therapeutic range MPE strive to maintain between 2.0 and 3.0. The inventive method allows to diagnose APS patients receiving the ANDES. The opportunity is available due to the fact that the index of sensitivity of both thromboplastin (the so-called ISI) to hypocoagulation effects caused by the application of the ANDES, are the same, but the sensitivity to hypocoagulation effect different VA. To confirm the diagnosis of ASF on a background of reception of the ANDES, we conducted a study in 11 patients with APS receiving the ANDES, the results of which are presented in table II. The table shows that the application of the ANDES index NR is not changed.

The specificity of the method and its reproducibility

The inventive method has a high specificity to hypocoagulation effect VA. Other causes elongation of prothrombin time, do not affect the readings of the proposed method as define two parallel prothrombin time (with high-sensitivity and low-sensitivity to VA thromboplastin reagents). In particular, heparin, deficiency of coagulation factors, their immune inhibitors, the effects of the ANDES equally will cause hypocoagulation waste as sensitive, and low-sensitivity to VA thromboplastin reagents.

To assess the reproducibility of the method used, the coefficient of variation CV(%) Coefficient of variation of this test in the study on different days does not exceed 5%.

Example of the practical use of the proposed method:

Patient T., aged 30, is observed in the Altai Hematology center with a diagnosis of primary APS since 1999 During the disease twice mentions thrombotic complications: thrombosis of the left leg in June 1999 and pulmonary embolism in August 2001. Heredity is not burdened. Repeated treatment of the patient in the Altai Hematology center due to subfebrile temperature in the last 2 weeks, headache, weakness. Upon physical examination significant violations have been found. In the blood detected: antibodies (Ig G) to cardiolipin, the level of the CEC increased to 40 uded

Laboratory diagnosis of APS (see table III). On the basis of clinical and laboratory data formulated the diagnosis of Primary antiphospholipid syndrome, status post pulmonary embolism (2001).

Assigned to the ANDES in a dose of 5 mg/day (Warfarin). Again the study of hemostasis (see table III). Two weeks after treatment (including plasmapheresis, the use of antiplatelet and other) condition hurt what about the improved temperature was normalized, stopped headaches, weakness decreased. The patient was again conducted laboratory diagnosis of ASF method prototype and by the claimed method (see .IV).

As follows from the table, after treatment significantly decreased rate NR, while traditional tests for detection of WA remain positive.

In the clinical example illustrates the practical value of the proposed method for the diagnosis of antiphospholipid syndrome. As can be seen from table III, the patient qualitative reactions, revealing VA, positive (kaolin time, clotting time with diluted thromboplastin, rebetolbuy test, test correction with normal plasma does not detect the correction, test correction retropolation eliminates the hypocoagulation in tests). Against the backdrop of the ANDES also has a hypocoagulation, but at the same time (using traditional tests) it is impossible to differentiate the cause gipokoagulyatsii (hypocoagulation effect VA or hypocoagulation effect used anticoagulants). The inventive method diagnoses the ASF and shows stable results NR on the background of the application of the ANDES. After treatment of the patient, as can be seen from table IV, qualitative reactions, revealing VA, remain positive, the degree of elongation clotting time in various test is x different therefore, using the prototype method, it is difficult to objectively evaluate the effectiveness of applied treatments, but the inventive method demonstrates the decrease in NR, which corresponds to the clinical improvement of the patient. Thus, the inventive method allows objectively and quantitatively) to evaluate the effectiveness of applied treatments.

Table I

Method for the diagnosis of antiphospholipid syndrome
The estimated valueHealthyPatients SLE without APS IIPatients of SLE with APS III
NR0,99±0,011,01±0,011,22±0,04
The significance of differences, PPI-II>0,5PII-III<0,001PI-III<0,001

Table II

Indicator NR in healthy people and patients with ASF
Indicators

Healthy

I
Patients with APS before therapy ANDES

II
Patients with APS on a background of reception of the ANDES

III
NR0,99±0,011,23±0,071,24±0,08
The accuracy difference is s, PPI-II<0,001PII-III>0,5PI-III<0,005

Indicator NR in healthy people (n=30) and patients with APS before therapy ANDES and on the backdrop of the ANDES (n=11)

Method for the diagnosis of antiphospholipid syndrome (APS), including the assessment of the clotting time of the investigated plasma in fosfolipidzawisimah test, characterized in that the use of high-sensitivity and low-sensitivity to the lupus anticoagulant thromboplastin reagents, calculate the ratio of the indices of lengthening prothrombin time ratio equal to or more than 1.1, diagnose the ASF.



 

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