RU2021609C1 - Method for determining activity of progressing integumentary forms of lupus erythematosus - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: this method prescribes determining antibodies antagonistic to pre-β-lipoproteins in vitro in patients suffering from lupus erythematosus. EFFECT: higher accuracy, greater simplicity and lower rate of traumatism. 2 cl, 3 dwg, 1 tbl

Description

 The invention relates to medicine, namely to dermatovenerology, can be used to determine the activity of the process of integumental forms of lupus erythematosus (hereinafter ICV).

 Features of clinical manifestations in dermatological forms of lupus erythematosus, a mono- and malosyndromic chronic course, a frequent discrepancy between the disappearance of the clinical manifestations of the disease and the preservation of pathological changes in laboratory parameters justify the need to evaluate the activity of the pathological process.

 The determination of the activity of the ICV process is carried out, first of all, by the nature and dynamics of the clinical manifestations: the severity of erythema, the prevalence of the focus, the appearance of new elements, the frequency of relapses, etc. However, this method is largely subjective and depends on the patient's observation and the experience of the doctor .

 For the prototype of the proposed method for determining the activity of the process of integumental forms of lupus erythematosus, a well-known method for determining the activity of ICV is selected, including the study of the patient’s blood serum before a course of treatment, recording the level of a biochemical indicator, followed by comparison with control data.

 The interdependence of these indicators and activity in patients with erythematosis against the background of ESR and leukocytosis are presented in the table.

 Therefore, the assessment of activity is carried out according to the increased content of indicators. At the same time, in the active period of ICV, an increased content of fibrinogen is observed in 41.7% of patients, haptoglobin in 52.7%, ceruloplasmin in 33.3%, i.e. the accuracy of the method is not high enough, even according to the prototype.

 Studies have shown that this percentage does not exceed 41.2%. The content of glycoproteins largely depends on hepatotropic pathology. When analyzing the data obtained, it is necessary to carry out 3 types of laboratory tests. These analyzes cannot be carried out simultaneously and in parallel from the same portion of blood during the formulation of generally accepted serological reactions without additionally injuring the patient. When interpreting the results of a study of a particular patient, difficulties often arise in connection with the discordant data of three indicators.

 The aim of the invention is to improve the accuracy and simplification of the method, reducing its invasiveness.

This goal is achieved by the fact that the level of antibodies to pre-β-lipoproteins is recorded in the patient's blood serum and the presence of activity is determined when antibodies are detected. Antibodies to β-lipoprotein pre- recorded in immunofluorescence reaction, the pre-mixed solution with patient serum lipoprotein human liver, the mixture was incubated at ³⁷ ° C for 15-30 min, then the mixture was applied to a glass with fixed lymphocytes rats repeatedly incubated in wet chamber, wash the resulting preparation, apply luminescent serum against human globulins, incubate again in a wet chamber at room temperature, and then count the number of luminous lymphocytes to the total The number of cells in the visual field.

 The quantitative ratio of the ingredients is selected experimentally. The results of the experiments are shown in figure 1-3. The abscissa axis: different protein content in the lipoprotein (mg / ml protein), figure 3 - the number of BMP in ml, which corresponds to ml lipoprotein protein: the ordinate axis: the percentage of detected luminous cells.

 The standard protein content in the lipoprotein ampoule is 0.860 mg / ml, and in the experiment performed, in 0.011 ml of the lipoprotein, the protein content is 10 mg / ml, in 0.017 - 15 mg / ml, in 0.023 - 20 mg / ml, in 0.029 - 25 mg / ml In Fig.1, the peak of luminous lymphocyte cells falls on the content of 15 mg / ml in the lipoprotein - more than 80%. Figure 2 shows the results of the first control.

 At a dose of protein 15 mg / ml - the result is negative. In Fig.3, the results of the 2nd control, which are similar to those of the first control.

Antigen-antibody reaction in vitro at ³⁷ ° C is usually complete within a few minutes. For example, a precipitation reaction, in particular the Kahn reaction used in the serological diagnosis of syphilis, is carried out for 10 minutes. In the experiment, the results of combining the antigen with the antibody were evaluated not by the precipitation reaction, but by the change in the cells, i.e., they were dealing with a more complex mechanism for the formation of the antigen-antibody complex. At the same time, one indicator was changed - the indicator of protein in the lipoprotein and allowed that the best time is 15 minutes. The subsequent reaction steps were carried out for 30 minutes, based on the fact that, according to well-known data, immunological reactions at the stage of turning on the indicator system usually take place within 30 minutes.

 The need to use target cells in the formulation of the indirect immunofluorescence reaction of rat lymphocytes is argued that rat lymphocytes have receptors for immunoglobulins and do not contain human proteins, which excludes the possibility of nonspecific luminescence during subsequent processing of labeled antiglobulin serum preparations. There is no need to take into account the group substance of lymphocytes. Features of the morphological composition of peripheral blood, in particular a greater number of leukocytes in 1 mm of blood (5000-25000, on average 12500) than in rabbits (8500-19000) and guinea pigs (5000-16000), as well as the percentage of lymphocytes in the leukocyte formula in rats (65-75) versus 42.2 and 43 in rabbits and guinea pigs, they are more likely to isolate the desired number of lymphocytes.

 Analysis of erythrocyte membrane lipoproteins and healthy rat lymphocytes during electrophoresis in PAGE showed the content of pre-β-lipoproteins in the range of 28.2-73.2%, respectively. Thus, taking into account the fact that the commercial lipoprotein used as an antigen is also represented by pre-β-lipoproteins (tested in PAGE), the use of lymphocytes as target cells is quite justified.

 Figure 1 shows a graph of the detection frequency (%) of fluorescent cells from the content of LPC on protein; figure 2 is a graph of the dependence of the activity of LPC on protein on the frequency of detection of fluorescent cells (%); in FIG. 3 is a graph of the frequency of detection of fluorescent cells (%) on the content of antilymphocytic antibodies in blood serum diluted in various ratios.

 Rat lymphocytes in the proposed method are first used as target cells in the indicator system.

 A fundamentally new approach has been developed in evaluating the results in the control due to the fact that patients with ICV can detect antibodies to lymphocytes, on the one hand, and, on the other hand, human lipoprotein, combined with human antiglobulin serum, can also give a glow. Thus, the proposed new approach to assessing the results in the control virtually eliminates false-positive results. The approach itself consists in calculating the percentage of luminous cells from the total number of them in the test tube in the absence of luminescence in the control. In known methods, indexing is usually used.

 The optimal reaction conditions were proposed for the first time in this invention and substantiated empirically.

The positive effect is as follows:
improving the accuracy of determining the activity of the ICV process (in the prototype, the accuracy is 33.3-52.7% according to the obtained data, not higher than 41.2%, in the proposed method 77.5%);
reducing the invasiveness of the method by eliminating the need for multiple blood sampling;
simplification of the determination of ICV activity by reducing the number of analyzed indicators (in the prototype 3, in the proposed method 1).

The method is as follows. A suspension of rat lymphocytes is prepared and applied to a glass slide. To quench autofluorescence, the preparations are fixed in chemically pure acetone for several minutes (acetone is poured onto the preparations and immediately drained). Blood is taken from the patient and serum is obtained in the usual way, for example, as for the Wasserman reaction. 0.02 ml of the patient’s blood serum and 0.017 ml of commercial human liver lipoprotein (15 mg / ml protein) are poured into the test tube. 0.02 ml of the patient’s blood serum and 0.017 ml of the veronal-medial buffer were added to the first control tube: pH = 7.2 (BMB), and to the second, 0.02 ml of commercial lipoprotein and 0.017 ml of BMB. The tubes were incubated for 15-30 min in an oven at ³⁷ ° C, pre-closing stoppers avoid drying. Then the contents of the tubes are applied to the prepared preparations of lymphocytes and incubated in a humid chamber for 30 minutes and thermostat at a temperature of 37 ^about C. Washed 3 times in a VMB at a dilution of 1: 5. Lightly dried and applied luminescent serum against human globulins, previously titrated and depleted by rat erythrocytes. Incubated for 30 min at room temperature in a humid chamber. Washed 3 times in VMB, dried. Results are recorded using a Lumam type luminescent microscope by counting luminous lymphocytes from the total number of cells. For example, a total of 100 cells in the field of view (in an experimental test tube) of which 8 have a glow. Then the results are expressed as a percentage - 8%. Therefore, antibodies to pre-β-lipoproteins were detected. When recording the results of control tubes, there is no glow; there are only shadows of lymphocytes.

 PRI me R 1. Patient K., 32 g., Medical history N 3373. Diagnosis: discoid lupus erythematosus. The duration of the disease is about 28 years. Of the illnesses noted colds. Concomitant disease: vasomotor rhinitis. Family history is not burdened. The study of blood serum showed the content of haptoglobin within normal limits (1.45 g / l with a norm of 0.86 g / l to 1.65 g / l), ceruloplasmin 30.5 mg% (versus 30.0 + 0.36 in control), ESR 40 mm / h. The results for the detection of antibodies to lipoprotein were positive. The latter and ESR testified to the activity of the process in a patient with ICV. Indeed, on the skin of the face there is a bright erythema with peeling, atrophy. The performance of the proposed method correlated with clinical signs of ICV activity, while the prototype parameters were discordant: ESR was increased, haptoglobin content was normal.

 PRI me R 2. Patient N., 29 years old, medical history N564. Diagnosis: discoid lupus erythematosus. The duration of the disease does not exceed 1 year. Of the past illnesses, the patient notes flu, tonsillitis, there are no concomitant diseases. The patient is examined in the stage of clinical remission, i.e. there are no manifest signs of ICV on the skin of the face. In the blood serum, the content of haptaglobin is determined within the normal range (0.95 g / l), ESR 6 mm / h. Laboratory and clinical data indicated the absence of an active ICV process. At the same time, the detected antibodies to lipoprotein in the blood serum allow us to make a judgment about the ongoing active destructive processes. Indeed, a month later there were clinical signs of exacerbation of skin manifestations: bright elevated erythema with clear boundaries, itching appeared, although the content of haptoglobin was within normal limits (1.2 g / l), ESR 2 mm / h. Thus, the proposed method turned out to be more informative for assessing the activity of the process with ICV.

 PRI me R 3. Patient U., 29 years old, medical history N1366. Was in the clinic about discoid lupus erythematosus. The duration of the disease is 4 years. Of the illnesses noted, acute respiratory infections. There are no concomitant diseases. Before admission to the hospital was not treated. A study of serum showed the content of haptoglobin within normal limits (1.55 g / l), ESR 5 mm / h. Antibodies to lipoprotein were not found. However, on the skin of the face there were 3 foci represented by elevated erythema. The patient is worried about a burning sensation. From the anamnesis it was possible to find out that there was no independent regression of manifestations, a new outbreak appeared in the last 6 months. Anamnestic and clinical signs indicate an exacerbation of the skin process, at the same time, the proposed method and the prototype do not reveal signs of activity of the process.

 Using the proposed method in the clinic of the department of skin and sexually transmitted diseases GMI them. S. M. Kirova, on the basis of the Nizhny Novgorod Research Institute of Skin and Venereal Diseases of the Ministry of Health of the Russian Federation, 40 patients with integumental forms of lupus erythematosus were examined. Among the patients, there were 6 women and 34 men aged 20-70 years (mean age 39.5 + 2.9). The duration of the disease ranged from 4 months to 20 years (an average of 8.6 + 1.5).

 Clinical manifestations in the form of erythema, follicular hyperkeratosis, atrophy were observed in the majority of patients on the skin of the face and were limited in 4 of them skin changes had a common form: chest, back, scalp.

 Some patients had a concomitant pathology: rubromycosis (1), allergic rhinitis (1), acariasis (2), vegetovascular dystonia (2), asthma bronchitis (1), lumbar ischalgia (1).

Studies have shown that antibodies to pre-β-lipoproteins using the proposed method in the presence of an exacerbation of the process were found in 77.5% of patients, while an increased content of one of the prototype indicators - haptoglobin - in 41.2% (X ² = 10 , 2; P <0.005), ceruloplasmin - in 27.3% (X ² = 11.05; P <0.001).

Comparison of the results of the detection of antibodies from the presence of concomitant diseases showed the absence of significant differences both in the frequency of their detection (X ² = 0.8; P> 0.05) and in average indicators 26.0 + 10.0% - in the presence of concomitant diseases diseases, 16.0 + 9.0% - the absence of the latter, P> 0.05.

Antibodies to pre-β-lipoproteins were also determined by the enzyme-linked immunosorbent assay on a solid-phase support, however, the sensitivity of this method was lower than the proposed one: 48.8% and 77.5% of positive results, respectively (X ² = 7.3; P <0.01) .

Antibodies were determined in patients during the period of regression of clinical symptoms, after the elimination of exacerbation of the skin process. At the same time, a significant decrease in the detection rate of antibodies was 20.0%, X ² = 8.6; P <0.01 compared with data with exacerbation of ICV).

 A correlation analysis of the results of the proposed method and data on the detection of antibodies to lymphocytes and DNA revealed a moderate relationship between the studied parameters (r = 0.4; r = -0.4, respectively).

 Thus, the presence of antibodies to pre-β-lipoproteins correlates with the activity of skin manifestations in patients with ICV. The proposed method is more sensitive (accuracy) during the exacerbation of the process compared to previously known methods, which are indicators of haptoglobin, ceruloplasmin. The presence of the association of antibodies to pre-β-lipoproteins with anti-lymphocyte antibodies and DNA, i.e. to the components of the cell suggests the same nature of the origin of antibodies as a result of destructive processes. In other words, antibodies to pre-β-lipoproteins are objective reflectors of the pathogenetic nature of ICV.

 The use of this method is possible in parallel with other serological studies, for example, for HIV infection, syphilis, i.e. when implementing the method does not require special preparation of the test material, processing tools, additional venipuncture, sophisticated equipment. The reaction can be carried out in any laboratory with a luminescent microscope.

Claims (2)

 1. METHOD FOR DETERMINING THE ACTIVITY OF THE PROCESS OF THE INTEGRATED FORMS OF RED LUPUS, including the study of the patient's blood serum before the course of treatment, registration of the level of the biochemical indicator, followed by comparison with the control data, characterized in that, in order to increase the accuracy and simplify the method, reduce its morbidity, register the level of antibodies to pre-β-lipoproteins and, when antibodies are detected, the presence of activity is determined. 2. The method according to claim 1, characterized in that antibodies to pre-β-lipoproteins are recorded in an immunofluorescence reaction, wherein the patient’s serum is pre-mixed with a solution of human liver lipoprotein, the mixture is incubated at 37 ^{° C.} for 15-30 minutes, then the mixture is applied to a glass slide with rat lymphocytes fixed on it, re-incubated in a wet chamber, the resulting preparation is washed, luminescent serum against human globulins is applied to it, again incubated in a wet chamber at room temperature, and so count the number of luminous lymphocytes to the total number of cells in the visual field.

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