SU1471132A1 - Method of diagnosing active phase of rneumatic diseases - Google Patents

Abstract

This invention relates to the field of medicine, in particular, to cardiology. The aim of the invention is to improve the accuracy. For this, platelet-free blood plasma is treated with protamine sulfate. The mixture is stirred and maintained at a temperature of 25 ° C for 30 minutes, then centrifuged, the precipitate washed with sodium chloride solution, dissolved in acetic acid and spectrophotometrized at 280 and 320 nm. The concentration of the soluble fibrin / fibrinogen complex in mg / l is then calculated taking into account the volume of blood collected, the volume of the spectrophotometry solution, the difference in extinction of the solution at 280 and 320 nm and the extinction coefficient of fibrinogen in an acidic medium and, if present, diagnose the active phase of rheumatic disease. The method allows to increase the accuracy by increasing by 43% the frequency of positive results in the active phase of this disease and reducing by 33% false-positive results in the inactive phase of the disease. 3 tab.

Description

one

The invention relates to laboratory diagnostics and can be used to diagnose the active phase of rheumatic diseases.

The purpose of the invention is to improve the accuracy of the method.

The goal is achieved by the fact that. in the plateletless plasma of venous blood, the fibrin - fibrinogen complex is precipitated with protamine sulfate, centrifuged, the precipitated complex is washed, followed by its dissolution and spectrophotometry, after which the amount of soluble fibrin is determined from the ratio of fibrin

to draft 1: (2.80-2.92), when a soluble fibrin was detected, the active phase of rheumatic diseases was diagnosed.

The method is carried out as follows.

0.5 ml of a 1% aqueous solution of protamine sulfate in 0.06 M phosphate buffer, pH 7.4, M 0.15 were added to 0.5 MP of the investigated plaqueless plasma of venous blood. The mixture is stirred and left for 30 minutes at 25 ° C, after which it is centrifuged for 15 minutes at 2500 rpm. The precipitate is washed twice with 0.9% solution hpo

Yu

After the second washing, the tube was removed in an inverted video for 5 min. Then the precipitate was dissolved in 3 ml of a 1.5% aqueous solution of acetic acid and spectrophotometrically at 280 and 320 nm. The calculation of soluble fibrin concentration is from the ratio of fibrin to sediment 1: (2.80-2.92).

The calculation of the concentration of fibrin (mg / l produced by the formula:

& „V 10,000

z v K

f

V - V

th

280

where C is the concentration of soluble fibrin-fr1brinogen mg / l;

To 2.86 - coefficient, taking into account the ratio of protein in the sediment to fibrin; volume of plasma taken, mm; volume of the spectrophotometric solution, mm; difference of extinction of the solution at wavelengths of 280 and 320 nm; 15,0 - coefficient of exclusion

fibrinogen in an acidic environment For example, if V 0 ,, V 3.0 ml, then U.8 0,290-0,024 0,266.

Therefore, C 372 mg / l. When detecting soluble fibrin in the blood plasma, an active phase of rheumatic diseases is diagnosed,

In all 28 examined healthy individuals, soluble fibrin in the blood plasma was not determined,

Example 1 Patient L ,, 32 years old Diagnosis: Rheumatoid arthritis, seronegative, PR rapidly, its course, I1T degree activity} with multiple lesions of small and large joints of the upper and lower extremities, FIS II degree admission to the hospital: To 0.3 ml of the citrate plasma of the venous blood of a patient devoid of thrombocytosis, centrifuging at 3000 rpm for 20 minutes, added 0.5 ml of 1% solution of the solution taminesulfate in 0.06 M phosphate buffer, pH 7.4, M 0.15. The mixture was stirred and incubated for 30 minutes at 25 ° C, after which they were cementrified for 15 minutes at 2500 rpm. The precipitate was washed twice with a .0.4% solution

sodium chloride, then kept the tube for 5 minutes in an inverted form, the precipitate was dissolved in 3 ml of a 1.5% acetic acid solution and spectrophotometrized at 280 nm. After spectrophotometry, the extinction magnitude of the solution was 0.405 with a length of 280 nm for the volga and a correction for the turbidity of 0.063 at a wavelength of 320 nm. The amount of soluble fibrin was calculated by the formula

WITH

 V 10,000

Z-V

TO

IOj OBZ) SHOOO G15.0 2.86

 478 mg / l.

The amount of soluble fibrin is significantly lower. Other laboratory data: ESR 48 mm / h, C-reactive serum protein 3 mm, sialic acid concentration 0.390 mmol / l, c-2. serum globulins 12.4%, Y-globulins 28.6%.

In the example given, the clinical data and the known laboratory data correspond to the activity of inflammatory prop, CT degree. A sharp increase in the concentration of soluble fibrin in plasma is adequately clinical and well-known laboratory data. The diagnosis of the III degree of activity of rheumatoid arthritis is confirmed,

PRI mme R 2. The same patient as .and in example 1. The study after two weeks of hospital treatment, accompanied by a significant clinical improvement. The course of the study is identical to that described in the example. After spectrophotometry, the extinction value of the solution was 0.210 at a wavelength of 280 nm and a correction for turbidity of 0.020 at a wavelength of 320 nm.

Calculation of the amount of soluble fibrin in the plasma under study

.. i2000

r-v to.

WITH

-. (Oj.2iOz0 020} j 3 0000. 0.5 15 2.86

 266 mg / l.

Thus, the concentration of soluble fibrin in the blood plasma of the patient significantly decreased, which

Corresponds to clinical improvement. At the same time, the patient's ESR in the patient continued to increase sharply (46 mm / h), the C-reactive protein is still 3 mm, and the concentration of | f - serum globulin increased to 31.4%. The known laboratory indicators of rheumatic activity were less dynamic than the soluble concentration. fibrin in the blood.

PRI me R 3. Patient B., 39 years old. Clinical diagnosis: Osteoarthritis (osteoarthritis) of the knee joints, FTS I degree,

 The course of the study is the same as in the previous examples. After spectrophotometry, the extinction value of the solution was 0.050 at a wavelength of 280 nm and a correction for turbidity of 0.050 at a wavelength of 320 nm. The calculation of the concentration of soluble fibrin in the plasma:

WITH

L U YuOOr

s: -V K

 ()

075-15, 86

There was no detectable fibrin in the test plasma, which made it possible to exclude the diagnosis of osteoarthritis (i.e., the active phase of the inflammatory process) and confirm the diagnosis of osteoarthrosis (ie, the inactive phase of the inflammatory process).

To check the diagnostic value of the proposed method, parallel studies of soluble fibrin and PRF (a known method) were performed in blood plasma in 28 healthy individuals, 49 patients with rheumatic diseases in the active phase (21 points rheumatoid arthritis, 13 osteoarthritis, 9 systemic red lupus, .2 - systemic scleroderma, 2 hemorrhagic vasculitis 2 - intestinal polyarthritis) and in 18 patients with rheumatic diseases in the inactive phase (4 patients with rheumatoid arthritis, 13 with osteoarthrosis, one patient with rodermiey). As can be seen from Table 1, the frequency of detection of soluble fibrin in plasma in patients with rheumatoid arthritis, osteoarthritis and rheumatic diseases in general significantly (by 48-77%) exceeds

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 frequency of detection of PRF in the same individuals. Consequently, the diagnostic accuracy with the use of the predictable method outlined by comparison with the known definition.

In tab. 2, the frequency of detection of soluble fibrin and PRF in the blood plasma of the same 10 patients with rheumatic diseases depends on the processability (49 active patients and 8 patients in the inactive phase).

15Thus, the presence of soluble fibrin in the blood is significantly (by 33-43%) a more accurate criterion of the activity of the process in rheumatic diseases than the presence of 20 PRFs.

Table 3 compares the absolute values of the concentration of soluble fibrin, PRP, and plasma plasma fibrinogen concentration in 12 patients with rheumatoid arthritis before treatment and 2 weeks after the start of treatment. Fibrinogen concentration is selected as a known indicator of rheumatic activity, 30

From the data presented in Table 3, it follows that the concentration of soluble fibrin in the blood is a more labile criterion of the activity of rev-2g matoid arthritis than the concentration of fibrinogen and PRF. In one of the 12 patients in whom the two-week treatment was ineffective, the concentration of soluble fibrin existed- 40 but did not change (respectively 508 and 483 mg / l). In the remaining II patients, the concentration of soluble fibrin in the blood decreased significantly, but did not reach the norm, since the activity of the inflammatory process with the effective treatment of rheumatoid arthritis decreases, but is not eliminated in two weeks. A decrease in the concentration of soluble fibrin in the blood, 0 thus, is an early laboratory sign of a decrease in the activity of the rheumatic process and can serve as a reliable objective criterion of the effectiveness of the treatment being performed. 5 J has an undoubted practical value.

The use of the proposed method provides an increase in the accuracy of laboratory diagnosis of rheuma7 .14

diseases (compared with the known) due to an increase (by 43%) in the frequency of positive results in the active phase of these diseases, yNieHbmeHHH (by 33%) - almost eliminating false positive results in the inactive phase of diseases and more adequate (statistically) reflection of the dynamics activity of the rheumatic process.

Claims (1)

Formula isobuty A method for diagnosing the active phase of a rheumatic disease by biochemical examination of blood, characterized in that, in order to improve the accuracy of the method, platelet-free plasma is separated, treated with protamine sulfate, the mixture is centrifuged, the precipitate is washed with a solution eight sodium chloride, dissolved in acetic acid, spectrophotometrized at 280 and 320 nm, then calculate the soluble content of the complex fibrin - fibrinogen by the formula d i v: ipoop to WITH where G is the concentration of the complex, mg / l, V is the volume of taken plasma, mm; V - volume spectrophotometry-. l solution, mm; L Е25о -5X0 difference of extinctions of the solution at 280 and 320 nm; 21 15.0 - the extinction coefficient of fibrinogen in an acidic environment; To 2.86 - coefficient taking into account the ratio of protein in the sediment to fibrin, and upon detection of soluble fibrin, the active phase of rheumatic disease is diagnosed, Table 1 21 13 21 13 15 14 49 48 Rheumatic diseases in the active phase Rheumatic diseases in the inactive phase 49 48 92 ± 249 27 55 ±, 001 0 + 6186 33 ± 11. 0.05 21 13 11 3 52 ± 1 1 23 + 12 0.001 g: 0.001 93171513 87 ± 9 0.1 98 ± 24927 55i7 0, OQ Table 2 0 + 6186 33 ± 11. 0.05 - comparison of the corresponding values with a group of healthy individuals - comparison of the average values of the corresponding indicator before and after treatment,, 1471132 10 Table 3